Friday, June 22 – 3:00 PM
Biomaterials / Drug Delivery
Using real-time fluorescence measurements to monitor the structural dynamics of lipid nanocarriers during simulated gastric and intestinal digestion
Daniel A. Bricarello, Nitin Nitin
Efficient oral delivery of drugs and food bioactives remains a critical challenge due to limited bioavailability and solubility of bioactive ingredients such as omega-3 fatty acids, small molecules such as curcumin, retinol, tocopherol, and also pharmaceuticals such as anti-cancer, anti-inflammatory and anti-microbial drugs. While various micro and nanoscale encapsulation strategies have been proposed, there is limited understanding of how engineered structures influence the delivery and bioavailability of bioactives. To address these needs, our research is focused on characterizing the dynamics of micro- and nano-scale materials during simulated gastrointestinal digestion processes and to design these structures to enable efficient delivery of bioactives. In the work reported here, the dynamics of both the lipid structure interface and core is monitored during digestion using Foerster Resonant Energy Transfer (FRET) for synthetic high density lipoprotein, liposomes and nanoemulsions. Monitoring the ratio of donor/acceptor emission of dual labeled systems shows that lipid nanostructure is an important factor in the kinetics of digestion. Bile salts interact immediately with liposomes and reconstituted HDL to disrupt packaging of fatty acids as evidenced by the loss of the FRET signal of the initial structure. In contrast, phospholipase has little effect on the structure of these nanocarriers. Phospholipase activity in nanoemulsions shows a two-phase breakdown, with an initial dramatic molecular rearrangement followed by a slow steady separation of donor/acceptor. In the simulated gastric environment, low pH and active pepsin have little effect on the three lipid structures. Further research is on going to investigate the influence of these nanoscale dynamics on release of encapsulated bioactives.
Quantifying the Effects of Transient Cellular Characteristics Using a Novel Streaming Potential Method
Nicole Carvajal, Prashanthi Vandrangi, David D. Lo and Victor G.J. Rodgers
Streaming potential captures the cellular electrostatic characteristics in biological systems. Studying electrostatic properties of cells and cellular layers will help in effectively designing drug delivery vehicles for in-vivo vaccination. Tangential flow devices are widely employed to measure the streaming potential of cells. This, however, is ineffective as it does not take into account the areas between the cells (leaky junctions), the extracellular matrix synthesized by the cells, and the effect of cellular processes such as microvilli, lamellipodia or filopodia etc. Moreover, tangential flow systems also displace the cells from the membrane on which they are seeded. Our laboratory has developed a novel device that measures the streaming potential of cells using normal flow. Our device not only captures the electrostatic properties of the areas between the cells, but prevents the displacement of cells. Our previous work also developed a mathematical equation using Helmholtz-Smoluchowski and Darcy’s law that extracts the charge contribution of a confluent cellular layer from a cell-membrane configuration. In this study, we will be employing the proposed device and methodology to quantify the effect of cellular features on the overall streaming potential of cells. We will capture the streaming potential and the corresponding pressure and flux across the cell-membrane configuration. To quantify the electrostatic contribution of microvilli growing on epithelial cells and the contribution of extracellular matrix synthesized by endothelial cells, we will use a variant of epithelial colorectal cells (Caco-2) and human endothelial cells (EA926) respectively. The streaming potential will be measured for Caco-2 cells and for EA926 cells at 2, 6, and 14-day time intervals. We expect that these results will provide us a better understanding of the electrostatic contributions of cellular features which can be used to efficiently design drug delivery vehicles.
Targeted Delivery of Drug‐Loaded Nanoparticles with a Transferrin Variant
Ricky Y.T. Chiu1, Takuma Tsuji2, Christina T. Liu1, Johnny Wang1, Anne B. Mason3, Daniel T. Kamei1
1Department of Bioengineering, UCLA;
2Department of Materials, Physics, and Engineering, Nagoya University, Japan;
3Department of Biochemistry, University of Vermont College of Medicine
Transferrin (Tf) has been investigated for several years to target drugs to cancer cells, since many cancer cells overexpress Tf receptors on their cell surfaces. However, these approaches have been limited, since native Tf spends a short time inside the cell. To increase the time Tf spends with the cell, i.e., increase the cellular association of Tf, our group previously investigated a Tf variant. This Tf variant was found to exhibit an increase in cellular association, which translated into an improved ability to deliver a conjugated toxin. Although molecular Tf-drug conjugates can be used for cancers that are treated locally, they cannot be administered systemically due to competition from the high concentration of endogeneous Tf. To address this challenge, we proposed to use the Tf variant to target nanoparticles (NPs), since NPs have the ability to accumulate at a tumor site via the enhanced permeability and retention effect. In this project, we investigated the performance of drug-loaded NPs decorated with the Tf variant and polyethylene glycol (PEG). The cellular association of Tf-conjugated polystyrene nanoparticles (Tf-PNPs) was assessed by radiolabeling Tf with 125I and measuring the amount of Tf internalized in PC3 and A549 human cancer cell lines as a function of time. Doxorubicin (DOX)-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (DP) were prepared by nanoprecipitation. The DPs were conjugated to maleimide-PEG10000-amine via the carboxyl groups on the surfaces of the particles, and subsequently, thiolated Tf molecules were attached to the extended maleimide groups on PEG to form Tf-PEG-DP (TPDP). An increase in cellular association was observed for the variant Tf-PNPs relative to the native Tf-PNPs. This increase in cellular association translated into increased potency for TPDP, where less drug was required to achieve the same level of growth inhibition for variant TPDP than native TPDP.
In Vitro Evaluation of Novel Magnesium-Zinc-Strontium Alloys for Next-Generation Biodegradable Medical Implants
Aaron F. Cipriano1, Ren-Guo Guan2, Ian Johnson1, Zhan-Yong Zhao2, Salvador Garcia3,4, Tong Cui2, and Huinan Liu1,4,5 *
1Department of Bioengineering, University of California, Riverside CA 92521 USA; 2 School of Materials and Metallurgy, Northeastern University, Shenyang 110004, China; 3 Department of Biology, California State University, San Bernardino, San Bernardino CA 92407 USA; 4 Stem Cell Center, University of California, Riverside, CA 92521 USA; 5 Materials Science and Engineering Program, University of California, Riverside, CA 92521; *Corresponding Author
Magnesium (Mg) alloys hold great promise for next-generation medical implant and device applications due to their unique biodegradable capability and desirable mechanical properties. Controlling the degradation rate of Mg alloys has been the critical issue of current research. Carefully engineered degradation rates will ensure that the structural integrity of the implant is maintained when load bearing is required, while at the same time promote tissue regeneration.. Cellular responses and tissue integration around the Mg-based implants are equally important design considerations as these will enhance clinical success at later stages. In this study, four distinct Magnesium-Zinc-Strontium (ZSr41) alloys were designed and produced. The degradation behavior of ZSr41 alloys and their interactions with human embryonic stem cells (hESC) were studied. H9 hESC culture was used as an in vitro cytocompatibility model due to the higher sensitivity of hESCs to known toxicants which allows to potentially detect toxicological effects of new biomaterials at an early stage. Alloy degradation was characterized by measuring total weight loss of samples, pH change in the cell culture media due to Mg degradation products (i.e. hydroxide ion), and the concentration of Mg ions released into cell culture media through degradation. Surface microstructure and composition before and after culturing with hESCs were also characterized using field emission scanning electron microscopy (FESEM) and energy dispersive X-ray spectroscopy (EDS). The results showed that the Mg-Zn-Sr alloy with 0.15 wt.% Sr provided slower degradation and improved cytocompatibility as compared with pure Mg control. This comparative study has identified the key ZSr41 alloy composition, which is promising for medical applications. Future studies of this alloy in an animal model are needed for eventual clinical translation.
Investigation of a Dynamic Biomimetic Apatite Nanoparticle Delivery System for Targeted Non-viral Gene Transfection
Debobrato Das1, Eric Tsang1, Benjamin M. Wu1,2,3*
University of California at Los Angeles, 1Department of Bioengineering, 2Department of Materials Science and Engineering, 3Division of Advanced Prosthodontics, Biomaterials and Hospital Dentistry, Los Angeles, CA 90095
*Corresponding Author. Department of Bioengineering, University of California, Los Angeles. 410 Westwood Plaza, Engineering V. Los Angeles, CA 90095. Fax: (310) 794-5956. E-mail address: firstname.lastname@example.org (B.M. Wu).
Common approaches for gene delivery use nanomedicines that may produce in vivo toxicity and immunogenicity, and are limited in the variety of transportable nucleic acids. Moreover, these common high-cost systems illustrate low efficacy of gene-capsule cell internalization, followed by inadequate release and weak intracellular stability of the viral sequences. This study aims to maximize transfection efficiency and minimize adverse host responses by utilizing biocompatible properties of calcium phosphate (CaP). CaP nanoparticles (NPs) present a unique class of non-viral vectors, which can serve as efficient alternatives for targeted gene delivery. In this project, CaP NPs were constructed from simulated body fluids (SBF), which have historically been used to create apatite coatings that elicit in vivo bioactivity, decreased immunogenicity, and facilitate osteogenic differentiation of adipose stem cells. SBF-CaP NPs co-precipitated with RNAi and pDNA were administered to MC3T3 cells to assess biocompatibility, cellular uptake of CaP-siRNA NPs, and modulation efficiency of intracellular protein and gene expression. As expected, CaP NPs induced consistently less cytotoxicity than traditional transfection reagents such as Lipofectamine®. Cells cultured with CaP-GAPDH siRNA NPs (Fig. 1) demonstrate knockdown of GAPDH activity. Preliminary results indicate comparable up-take efficiencies among miRNA, siRNA, and pDNA sequences using the SBF-CaP, suggesting that this methodology can be applied to deliver a variety of engineered genetic therapeutics. Current efforts focus on the detailed characterization of the NP- RNA/DNA interactions, and cell-NP-RNA/DNA interactions.
Forming Stable, Multi-Component Peptide Amphiphile Micelles Targeted to Atherosclerotic Plaques
Laurie B. Drews, Daniel Krogstad, Mohit Jethi, Matthew V. Tirrell
Micelles formed from the self-assembly of peptide amphiphiles have been demonstrated as potential drug delivery vehicles. Peptide amphiphiles are formed by conjugating a hydrophilic peptide headgroup to a hydrophobic lipid tail. Targeting of micelles formed from peptide amphiphiles can be achieved through the use of specific peptides that have been discovered to bind to sites of interest in the body. Our approach is to use two peptides that can target both early stage inflammation markers of atherosclerosis as well as late stage markers of atherosclerotic plaques. In this work, we will present both an early and late stage targeting peptide that when attached to a diC16 tail, a hydrophobic tail formed from two alkyl chains each sixteen carbons in length, form different structures. One peptide amphiphile micelle forms spherical shaped micelles approximately 10 nm in diameter, whereas the other micelle formed is cylindrical. Using these micelles with varying shape, we can begin to observe the extent of mixing of the two peptide amphiphile monomers when forming multi- component micelles using both transmission electron microscopy (TEM) and Forster Resonance Energy Transfer (FRET), using two dye-labeled peptide amphiphile monomers. In addition, we can tailor the hydrophobic tail to form more stable micelles that increase the micelle’s half life in vitro as well as lower the concentration at which micelles begin to form to 1 μM. Finally, we will show the in vitro binding capabilities of micelles formed from these early and late stage targeting peptides. The early stage targeting peptides have been shown to bind to endothelial cells that upregulate markers of inflammation, specifically VCAM-1. Late stage targeting peptide amphiphile micelles will also be shown that can bind to fibrin, formed from the creation of a plasma clot.
Function and viability of explantation of human islet alginate sheets following xenotransplantation
Jonathan RT Lakey1,3, Morgan Lamb1, Rick Storrs2, Ouwen Liang1, Kelly Laugenour1, Randy Dorian2, David W Chapman1, Michael Alexander1, David Imagawa1, Clarence Foster III1, Scott King2
1Department of Surgery, 3 Department of Biomedical Engineering, University of California Irvine, Orange, CA, 2Islet Sheet Medical LLC, San Francisco, CA
Background: Islet encapsulation in macroscopic polymer devices offers a means to protect transplanted islets and offers the ability to retrieve or replace the device if necessary. This study specifically assessed the viability and function of human islets encapsulated in alginate sheets and transplanted into the subcutaneous space, and explanted up to 8 weeks post transplant.
Methods: Human islets were isolated from cadaveric organ donors at University of California, Irvine using purified collagenase HA and BP Protease dissociation and continuous Ficoll-UWD purification. After overnight tissue culture, human islets were encapsulated in alginate sheets and either transplanted subcutaneously into Lewis rats or maintained in tissue culture (37oC/5%CO2). At 1, 2, 4 and 8 weeks, islet sheets were retrieved from the subcutaneous implant site and assessed for viability using FDA/PI and function using a static glucose stimulated insulin release test.
Results: Encapsulated human islets were 95±0.2% viable (mean±SEM) after 1 week in tissue culture. The initial stimulation index (SI, ratio of insulin produced in high over low glucose) was 3.8±0.2 and the maximum secretion (MX, ratio of high glucose + IBMX over high glucose), was 2.0±0.1. At 1-week post transplant the sheet was intact and viability was 90± 4%. Islet function was maintained with a SI=3.0±0.5 and MX=2.1±0.6 (p=ns,t-test). At 2-weeks post transplant, explanted sheets were 85±0.8% viable and maintained glucose responsiveness with a SI of 3.0±0.4, MX of 1.8. At 4 and 8 weeks post transplant, explanted sheets remained both viable (86.0±2.2%, 73.0±1.5%; respectively) and continued glucose responsiveness (SI=1.5±0.5 and MX=1.5±0.1, SI=1.5±0.3 and MX=1.6±0.6; respectively).
Conclusion: Our research goal is to continue the development of this technology towards clinical trials indiabetic patients. Results demonstrate that encapsulated human islet sheets can survive and maintain islet viability and function in vivo as demonstrated function studies on explanted islet containing sheets.
Detecting Multiple Cell Secreted Proteins Using Aptasensors and Electrochemical Spectroscopy
Ying Liu and Alexander Revzin*
Cell function analysis is increasingly being used in disease diagnostics. This is particularly true in immunology where cellular production of such cytokines as interferon (IFN)-γ and tisse necrosis factor (TNF)-α is used to determine disease specific cellular responses. Our laboratory has been developing aptamer-based biosensors for detection of cell-secreted cytokines; focusing on continous detection at the site of a small group of cells. In this presentation we describe an analytical method – electrochemical spectroscopy – for detection of multiple cytokines using the same miniature electrode placed next to cells. The populations of aptamers specific to IFN-γ and TNF-α are labeled with different redox reporters and then co-assembled on the same gold electrode using terminal thiol moieties. When resultant aptamer electrodes are analyzed by square wave voltammertry, scanning the voltage from -0.8 to 0 V vs. Ag/AgCl, distinct redox peaks are observed due to the presence of different aptamer species on the same electrode. Incubation with either IFN-γ or TNF-α caused a decrease in redox current associated with one of the cytokines. When challenged with a mixture of the two cytokines, both redox peaks decreased. The linear range of this dual aptasensor was between 1 to 80 ng/ml and 10 to 120 ng/mL, with the detection limits of approximately 60pM and 1nM corresponding to IFN-gamma and TNF-alpha, respectively. Utility of this biosensor was further demonstrated in a proof of concept experiments using primary human leukocytes. In this experiment, micropatterned gold electrodes were functinoalized with aptamers and integrated into a microfluidic device. The device was infused with RBC-lysed blood and leukocytes were captured on anti-CD45 antibody in proximity of the sensing electrodes (Figure 1A). Cytokine production from captured cells was triggered by mitogenic activation and detected at the aptamer modified electrodes using as shown in Figure 1B. The strategy of using redox labels to create electrochemical signatures for specific aptamer populations is particulalry appealing for multianalyte sensing where signal may be read out using voltammetry. Such electrochemical spectroscopy may be used in the future to increase multiplexing power of the biosensors employed for cell function analysis.
Co-Precipitation of Composite Magnetic Particles for Drug Delivery and Tissue Engineering Applications
Jaclyn Lock, Huinan Liu
Targeted drug delivery and tissue engineering can be improved through the use of biodegradable magnetic iron oxide composite particles. Such magnetic particles are highly advantageous for biomedical applications due to their magnetic properties coupled with low toxicity and biocompatibility.1 We synthesized poly(vinyl alcohol)-ferrite magnetic particles using the co-precipitation method and characterized their microstructure and composition using scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) analysis. Furthermore, we showed that the amount of ferrite encapsulated in the resulting particles is dependent upon the concentrations of: (1) poly(vinyl alcohol) polymer, and (2) iron chloride. The SEM micrographs demonstrated that these magnetic particles have nano-structural features, which have been previously shown to improve cell adhesion and function.2 We observed that these particles were non-toxic to rat bone marrow stromal cells, i.e., cells adhered and spread-out on the magnetic particles after a 24-hour incubation period. These magnetic particles can be functionalized with drugs or bioactive factors and magnetically delivered to the targeted defect site. Thus, we believe that these synthesized magnetic particles serve as promising platform for applications in targeted drug delivery and tissue engineering.
1. Pankhurst, Q. A.; Connolly, J.; Jones, S. K.; Dobson, J., Applications of magnetic nanoparticles in biomedicine. Journal of Physics D-Applied Physics 2003, 36 (13), R167-R181.
2. Liu, H.; Webster, T., Enhanced biological and mechanical properties of well-dispersed nanophase ceramics in polymer composites: from 2D to 3D printed structures. Materials Science and Engineering 2010.
Molecular Dynamics Simulations of Peptide Self-Assembly Under Confinement
Cade B. Markegard, Iris W. Fu, and Hung D. Nguyen
Molecular dynamics simulations of self-assembled peptide aggregates have commonly been investigated under bulk conditions, however to more accurately represent the crowded, heterogeneous environment of a cell, it is necessary to perform studies of protein folding and aggregation under confinement. In this work, we look at three peptide sequences (KA14K, WQVQVEVQVEVQVQVQV, & WQVEVQVQVQVQVEVQV), to study their individual aggregation processes starting from a random configuration when confined between two surfaces. In general, we investigate how confinement influences the kinetics of aggregation compared to bulk. Since long-range interactions especially those between charged groups are most affected by confinement, we examine how the location of charged amino acid residues modify the kinetics and final self-assembled structures. To achieve this we use an extended version of a coarse-gained amino acid model, PRIME (Protein Intermediate Resolution Model), and utilize Discontinuous Molecular Dynamics in order to obtain the long time scales needed to study aggregation. Our results indicate that confinement increases the propensity to form beta-structures. Moreover, the position of the charged residues determines the location and length of a beta-strand. In addition, we propose general guidelines for how the placement of electrostatic residues in a peptide affects the assembly of ordered structures.
Improving Survival Following Cerebral Edema Using a Hollow Fiber-Hydrogel Device
Devin W. McBride1, Mike S. Hsu2, B. Hyle Park1, Devin K. Binder,2 and V. G. J. Rodgers1
1 Department of Bioengineering, University of California, Riverside
2 Division of Biomedical Sciences, University of California, Riverside
Cerebral edema, an increase in brain tissue water content, is responsible for significant morbidity and mortality in many disease states, including traumatic brain injury (TBI), stroke, infection, tumor, and chemical and metabolic intoxications. TBI is particularly important from a public health standpoint since it is the foremost cause of morbidity and mortality in persons under 45 years of age worldwide. In the U.S., about 200,000 victims of TBI need hospitalization annually, and approximately 52,000 U.S. deaths per year result from TBI. Affected individuals experience delayed onset of cerebral edema following head injury which can lead to raised intracranial pressure, brain herniation, and death.
Significant secondary injury to the brain could be avoided if cerebral edema could be treated early.The most widely used current treatments of cerebral edema are osmotherapy, ventriculostomy, and craniectomy. While often effective, even combinations of these therapies may have limited success in treating severe edema.
A new method of directly removing water from brain tissue would potentially circumvent some limitations of current therapies. For therapeutic benefit in treatment of cerebral edema, the ideal medical device would have the capacity to remove water from brain tissue in a controlled fashion, have the flexibility for deployment on the surface of the brain, not require brain tissue penetrationm, and not do any harm to the underlying brain. Here, we have developed a direct surface-contactbased treatment using a novel hollow fiber-hydrogel device (HFHD) and successfully enhanced survival in mice with severe cerebral edema. Animals treated with the HFHD survived up to five hours longer than animals treated with craniectomy only (p<0.001) or no treatment (p<0.001). Animals treated with the HFHD had a survival rate of 80% within the observation period (360 minutes), whereas no animals treated with craniectomy only or no treatment survived longer than 50 and 33 minutes, respectively.
Doxorubicin Loading in Novel Block Copolypeptide Vesicles
Uh-Joo Choe, April R. Rodriguez, Brian S. Lee, Garrett L. Mosley, Timothy J. Deming, and Daniel T. Kamei
University of California, Los Angeles, Department of Bioengineering, Los Angeles, CA
Investigation of polymeric vesicles as novel drug delivery vehicles is an emerging area of research and shows great promise. With respect to this field, our focus has been on developing amino acid-based nanomaterials for drug delivery. We previously developed vesicles composed of either lysine-leucine (poly(L-lysine)60-block-poly(L-leucine)20, K60L20) or glutamate-leucine (poly(L-glutamate)60-block-poly(L-leucine)20, E60L20) polypeptides. This presentation describes our studies that were performed to characterize the encapsulation and leakage properties of these vesicles using the small molecule drug doxorubicin (DOX). A pH gradient method was used to load DOX inside the K60L20 vesicles (Figure 1). When DOX is incubated with the vesicle, the gradient allows DOX to be trapped inside the aqueous core of the vesicle. To load DOX inside the E60L20 vesicles, a pH ion gradient method was used, where, in addition to the pH gradient, a high concentration of sulfate ions was established within the vesicle. This allows the positive DOX inside the vesicle to form a neutral salt complex with the negative sulfate ion. The K60L20 vesicles encapsulated a high concentration of DOX with a slow drug release profile, which are both favorable properties for systemic delivery applications. However, these vesicles can become toxic at high concentrations. In contrast, although the E60L20 vesicles demonstrated undesirable drug release profiles when compared to the K60L20 vesicles, a recent cytotoxicity assay demonstrated that they were much less cytotoxic, which is an important feature for a drug carrier. Hence, our labs are currently focusing on further improving the use of E60L20 vesicles as drug delivery vehicles.
Degradation of Magnesium Alloy Screws in Phosphate Buffered Saline for Orthopedic Applications
Tejas Patel, Maria Iskandar, Huinan Liu
Department of Bioengineering, the Materials Science and Engineering program, University of California, Riverside
Current metallic orthopedic implants are made out of stainless steel, titanium alloys, or cobalt-chromium based alloys. Although these materials are effective in securing fractures or tissue grafts, they also cause many clinical problems. They can release toxic ions due to implant wear, which lead to local inflammation. Moreover, they require a second surgical procedure for removal after they have served their purpose. Magnesium (Mg) alloys are a great alternative to these permanent metallic implants. They are biocompatible, biodegradable, and can promote new bone growth. Moreover, their light weight and mechanical resemblance to bone make them an ideal material for orthopedic implant applications. However, their rapid degradation in the physiological environment is a major obstacle. In this study, interference screws made of pure Mg, AZ31 (3% aluminum, 1% zinc), and Mg-4Y (4% yttrium) were tested to determine their rate of degradation. Each screw was submerged in 3mL of phosphate buffered saline (PBS) solution. 1x PBS was used for the first 15 days of the study. 10x PBS was used from 17 days through 31 days to accelerate the degradation. Of these three Mg alloys, Mg-4Y screw was the first to completely degrade in PBS. Large pieces of the Mg-4Y screw degraded, making it the fastest degrading alloy. AZ31 screw showed the slowest degradation in PBS, followed closely by pure Mg. This degradation study provided important guidelines for designing next-generation biodegradable Mg-based implants.
Delivery of Small and Large Molecules From A Hydrogel Via Light
Jessica L. Schlosser 1, Donald R. Griffin 2, Thi H. Nguyen 3, Heather D. Maynard 2,3,4 and Andrea M. Kasko 1,2,4
UC Los Angeles
Hydrogel scaffolds are used in biomedicine to study cell differentiation and tissue evolution, where it is critical to be able to deliver chemical cues (therapeutics and nutrients) in a spatially and temporally defined manner. Covalently tethering therapeutic agents into a hydrogel through a degradable linkage allows their predictable and controlled release. Hydrolyzable and enzymatically degradable linkages have been reported, but lack external control. In contrast, photodegradation is externally controlled, allowing user-dictated release. We synthesized and characterized macromers with photodegradbale ortho-nitrobenzyl (o-NB) groups linked to activated disulfides. These macromers were copolymerized into hydrogel depots. The activated disulfide allows post- polymerization conjugation of thiol-containing molecules (such as peptides and proteins containing accessible cysteine residues) into the hydrogel depot via disulfide exchange. Using this approach, we fabricated hydrogels depots with photoreleasable peptide (glutathione) and protein (modified lysozyme), and quantified the release of the peptide/protein as a function of light exposure (365 nm, 10 mW/cm2).
Electrochemical peptide-based biosensors for detection of cell-secreted proteases
Dong-Sik Shin, Ying Liu, Alexander Revzin
Matrix metalloproteinases (MMPs) play a key role in metastasis and development of cancer due to their ability to degrade surrounding extracellular matrix (ECM) proteins. Existing bioanalytical approaches for detection of MMP expression from cancer cells include immunoassay and gelatinase zymography that are based on the end point analysis. Here, we describe a method for monitoring protease activity using redox-labeled peptides functionalized onto gold electrodes. The biosensor consists of a micropatterned surfaces with miniature electrodes positioned next to antibody-containing cell capture sites (Figure 1A). Protease molecules secreted by captured cells cleave peptide segment with redox label, causing the electrochemical signal to decrease. This principle was used to construct methylene blue (MB)-modified peptides specific to MMP9, then capture monocytes (U937 cell line) and monitor protease release/activity from the cells (Figure 1B, C). These novel biosensors will have wide ranging applications given the importance of protease production in matrix remodeling and cell migration associated with tissue injury, regeneration, wound healing and cancer.
Microstructure of a Collagen Scaffold
Argus Sun, Samuel S Murray
UC Los Angeles
California holds close to 12.4 percent of the United States population and led the nation in hospital discharges with 901,000 in 2009. While hospital use shows steady decline, the percentage of patients admitted with lower back pain and the proportion of those patients treated with spinal fusion operations remains unchanged. With 24% of California hospitals located in Los Angeles county, it is estimated that close to 250 spinal fusion procedures were performed in the region in 2009. Currently, bioresorbable collagen sponges are used in spinal fusion procedures as drug delivery devices, but after implantation can also serve as sites for cell attachment. Characterization of response to mechanical and fluid forces within the scaffold allows prediction of both mass transport of payload molecule and prediction of cellular response. This predictive ability can save time adjusting conditions via experimentation. Using micro computed tomography (μCT), we imaged a section of collagen sponge treated with a chemical contrast agent. Reconstruction yielded a 31.25 micron resolution three-dimensional image. This showed microstructure details previously not visible. We used a software meshing algorithm to transform the image into a finite-element (FE) mesh. A section of scaffold measuring 1.78 x 2.06 x 0.75mm required 814,710 tetrahedral elements. Fluid flow was then modeled using the generated mesh.
Integration of photoactivated membrane proton pump into silicon nanowire bio- nanoelectronic devices
Ramya Tunuguntla, Kyunghoon Kim, Mangesh Bangar, Pieter Stroeve, Costas P. Grigoropoulos, Caroline Ajo-Franklin, Aleksandr Noy
Membrane proteins represent an interesting and promising extension of the bionanoelectronic toolkit because of the many important functions that they perform in the living cells. Integrating membrane proteins with nanoelectronics requires a versatile biocompatible matrix that can preserve the protein functionality. We accomplish this task by using hierarchical assembly of lipid molecules and membrane proteins into a nanowire transistor to create a nano-bioelectronic device that can convert photoactivated proton transport events into electrical signals. Our devices use a photoactivated proton pump, proteorhodopsin, to create a silicon nanowire FET that is sensitive to green light excitation. This presentation will discuss the device preparation, characterization, and its performance.
Photoresponsive Dispersion and Acutation of Graphene‐Elastin Hybrid Materials
Eddie Wang, Jin‐Woo Oh, & Seung‐Wuk Lee
In the field of bioengineering, graphene‐derivatives (GDs) have emerged as promising nanomaterials for incoporation into sensors, therapeutics, and biomaterials due to their mechanical strength, thermal/electrical conductivity, optical properties, and low cost. However, as a consequence of their poor colloidal stability and lack of inherent specificity towards specific targets, GDs generally require covalent or non‐covalent modifications before they can function in biological systems. Here we describe the one‐step non‐covalent functionalization of GDs, i.e. graphene oxide (GO), and reduced graphene oxide (rGO) with a genetically engineered, protein‐based polymer. Specifically, we utilzed elastin‐like polypeptides (ELPs) due to their tunable thermoresponsiveness (reversible aggregation above a critical temperature), biocompatibility, and mechanical properties. ELPs genetically engineered to display a phage‐display derived graphene‐binding petpide motif were shown to bind to both GO and rGO and stabilize their dipersion in aqueous and organic solvents. The ELP‐GD hybrids retained a stimuli‐responsive behavior both in solution, and when incorporated into hydrogels resulting in reversible aggregation/dispersion and swelling/deswelling, respectively. Furthermore, these responses could be induced remotely by infrared light illumination as a result of the GDs’ ability to convert IR to heat. In the case of hydrogels, local IR stimulation allowed for controlled actuation due to non‐uniform deswelling. Finally, we demonstrated that further engineering of ELP‐GDs with integrin‐ binding motifs allows for efficient cell adhesion onto GD surfaces. GD functionalization with genetically engineered ELPs provides a facile and tunable method for imparting multiple functions and stimuli‐ responsiveness. This strategy can be utilized in the future for theranostics, sensing, and tissue engineering, as well as be adapted for use with other nanoparticles.
Thermal Stress of Supported Lipid Bilayer Induces Uniform Radius Tubules
Kimberly L. Weirich and Deborah K. Fygenson
Biomolecular Science & Engineering Program and Physics Department, University of California, Santa Barbara, 93106
Biological membranes spatially transform in response to environmental stresses such as area expansion, osmotic pressure, and protein insertion. It is not understood how adhesion to a support, such as a surface or cytoskeletal network, influences this remodeling. Supported lipid bilayer (SLB) is a model membrane system in which a bilayer is adsorbed to a solid (glass) support. We use SLB to quantitatively investigate fluid bilayer transformations from planar to tubular morphologies. A large mismatch in thermal expansivities causes the bilayer to enlarge more than its support upon heating.
Small increases in temperature (~1°C) relax via bilayer creep on the support. Larger increases (~5°C) result in an irreversible bilayer transformation, in which semi-flexible filaments (worms) extrude from the fluid SLB. Individual worms are <1 μm in diameter, but can reach hundreds of microns in length. At high ionic strength, the sub-resolution worms are adsorbed to the SLB, enabling the measurement of their radius to within ±5 nm using fluorescence microscopy. We demonstrate that the worms are tubular and report the effects of flow-induced tension on their radii.
Characterizing Phenotype of Hepatocytes Cultured on Heparin Hydrogels of Varying Stiffness
Su-A Park1,2, Jungmok You1, Dong-Sik Shin1, Alexander Revzin1*
1 Department of Biomedical Engineering, University of California, Davis, California 95616, United States, 2 Nano Convergence & Manufacturing Systems Research Division, Korea Institute of Machinery & Materials, Daejeon, Korea
There is an increasing interest in examining how mechanical properties of the substrate contribute to cell function and phenotype. In hepatic tissue engineering, it is frequently challenging to maintain differentiated primary hepatocytes in culture as these cells tend to de-differentiate and loose hepatic phenotype. Our laboratory has been exploring the use of heparin-containing hydrogels as matrices for cultivation of functional hepatocytes [1,2]. This study focused on the effects of heparin gel stiffness on the hepatic function. Softer and harder heparin gels were fabricated on glass slide via UV light induced thiol-ene coupling reaction of thiolated heparin (Hep-SH) and PEGDA precursors . Figure 1(a) shows the different elastic modulus of heparin gels. Analysis of mechanical properties revealed that high concentration of precursor solution resulted in strongly networked gel with an elastic modulus of ~ 110 kPa. The molar ratio of Hep-SH and PEGDA was adjusted to equimolar addition. As described in Figure 1(b)~(e), the presence of heparin in gel as well as the amount of heparin in gel were confirmed by toluidine blue O staining. Immunostaining for intracellular albumin of primary hepatocytes cultured on softer and stiffer heparin gels were carried out to measure the hepatic function. Figure 1(f) shows strong albumin signal in the hepatocytes cultured on soft heparin gel compared to stiff heparin gel. Suggesting that softer surface was more conducive to maintenance of hepatic phenotype. In conclusion, our findings point to the importance of substrate mechanical properties on hepatic function. In the future we intend to investigate convergence of growth factor incorporation and substrate mechanical properties on hepatic phenotype maintenance and stem cell to hepatocyte differentiation.
Biomechanics / Mechanobiology
Intestinal Permeability Increase During Ischemia Is Reduced by Enteral Serine Protease Inhibitors Angd and Cyclokapron
Angelina E. Altshuler, Alexander H. Penn, Jessica A. Yang, Itze Lamadrid, Diana Li, Stephanie Ma, Leena Kurre, Lynn Han, and Geert W. Schmid-Schönbein
UC San Diego
Objective: During hypotension, the intestine becomes ischemic resulting in hemorrhagic necrosis, especially in the distal ileum, and inflammatory mediators are released into the peritoneal space, lymph, and circulation. Digestive serine proteases and lipases may contribute to the increased intestinal permeability and inhibitors to these enzymes serve to preserve the structure of the intestine in experimental shock. However, little is known about the effect of serine protease or lipase inhibition in the lumen of the intestine on intestinal permeability.
Methods: To produce complete intestinal ischemia, the proximal jejunum to the distal ileum was excised from male Wistar rats, flushed with saline, and sectioned into 8 equal segments. Segments were cannulated with tubing connectors, filled with 20 μg/ml fluorescein mixed with either saline, the serine protease inhibitors ANGD, aprotinin, or cyclokapron, and the lipase inhibitor orlistat, and sealed before placing in conical tubes filled with saline (N=6/group). The fluorescence intensity in the exterior fluid was determined at times 0, 30, 60, 90, and 120 minutes.
Results: Intestinal permeability increased during ischemia and reached higher levels in the distal ileum than the jejunum. ANGD and cyclokapron administration significantly reduced the rate of permeability compared to saline treatment, while orlistat and aprotinin, alone or in combination, were not effective.
Conclusion: During ischemia, permeability across the intestinal wall is increased in the distal ileum where hemorrhagic necrosis develops during ischemia. The rise of permeability was attenuated with ANGD and cyclokapron in the lumen of the intestine. These inhibitors may reduce the transport of inflammatory mediators from the lumen of an ischemic intestine. Supported by GM-85072.
A 3D Model of Reach Loads for Workstation Design
Matt Camilleri, Alan Barr, Dennis Baum, Robert Duarte, Ira Janowitz, David Rempel, Justine Woo
The purpose of this project is to present the development plan for a reach model for workstation design that incorporates shoulder moment, shoulder posture, anthropometry, task duration, and duty cycle. Phase I of the project identifies existing software and literature with which to estimate shoulder strength and reach envelope. Phase II integrates task duration with shoulder strength to define volumes in the reach envelope in which a task can be performed continuously for 2 hours (green), performed for 2 minutes or less every 10 minutes (red), and performed for intermediate durations (yellow). Ultimately, the model will be incorporated into CAD design software, via mannequins of different anthropometry and gender, to allow workstation designers to consider human capabilities in order to reduce pain and fatigue and increase the work quality in the design process.
Tumor cell motility is directed by fibrillar ECM architecture
Luke Cassereau, Matt Rubashkin, Chris Dufort, Valerie Weaver
UC San Francisco
Tumor progression is traditionally linked to the sequential accumulation of genetic mutations. However, we have shown that tumor development is also strongly influenced by physical cues such as extracellular matrix (ECM) organization and stiffness. To understand how ECM mechanics and architecture regulate tumor progression, we conducted a comprehensive analysis of the interplay between ECM tension and topology and tumor development. Second harmonic generation (SHG) imaging and atomic force microscopy (AFM) measurements revealed that ECM stiffening in developing mammary cancers is functionally linked to collagen linearization and cross-linking that generate heterogeneous oriented matrix bundles that influence tissue architecture and mechanics. We hypothesize that this aligned ECM architecture creates a mechanically anisotropic environment, which fosters directed tumor cell invasion and motility. To explore the molecular mechanisms through which anisotropic ECM stiffness regulates tumor cell motility, our groups among others have performed experiments using ECM protein laminated and mechanically-tuned 2D gel systems. In 2D we demonstrated that ECM tension gradients significantly promote the directional migration, or durotaxis, of oncogenically transformed breast cells. Yet, 2D surfaces do not recapitulate the complex, heterogeneous, and 3D in vivo microenvironment. To address this issue, we have designed a system in which we cross-link collagen hydrogels to a stretchable PDMS membrane, and by applying a uniaxial strain on the membrane, create a mechanically loaded and aligned fibrillar collagen gel. SHG imaging revealed that our mechanically loaded collagen gels recapitulate the architectural heterogeneity and highly fibrillar collagen structure of the ECM associated with breast tumors in vivo. Utilizing this system and real time two-photon imaging we could show that the structure and organization of oncogenically-transformed mammary organoids adjacent to mechanically loaded collagen fibers were highly disrupted (Figure 1). Consistent with results from our 2D assays, we found that in 3D, persistent invasion was favored following oncogene transformation, epidermal growth factor (EGF) stimulation, and with the addition of tethered fibronectin to the collagen gels. Blocking integrin-fibronectin binding or preventing EGFr activation prevented acini disruption, invasion, and cell motility (Figure 1). Studies are now und erway using our fibrillar collagen gel system in combination with transgenic mouse models to explore how dimensionality and ECM architecture in combination with oncogenic transformation could modify MEC mechanotransductive signaling to promote invasion. Work supported by: PSOC U54CA143836-01, R01 CA138818-01A1, DOD W81XWH-05-1-0330 to VMW.
Isoform specific Contributions of Non muscle Myosin II to Viscoelastic Properties of Stress Fibers
Ching Wei Chang, Sanjay Kumar
Stress fibers (SFs) are bundles of F-actin, actin-binding proteins, and non-muscle myosin II (NMMII) that enable cells to generate traction forces against the extracellular matrix. It has recently been shown that two isoforms of NMMII (NMMIIA and NMMIIB) play distinct roles in orchestrating cellular contractile forces, yet the specific contribution of each isoform to stress fiber viscoelastic properties remains incompletely understood. To address this open question, we used laser nanosurgery to disrupt single SFs in living cells in the setting of isoform-specific NMMII suppression. We used lentiviral shRNAs to knock down the heavy chains of NMMIIA and NMMIIB in U373 MG human glioma cells, severed individual mCherry LifeAct-labeled SFs, and tracked viscoelastic SF retraction kinetics. The retraction of the two severed ends of the SF was recorded and the retraction behavior was fit to a Kelvin-Voigt model described by a viscoelastic time constant (τ) and a plateau retraction distance (L0). Our results demonstrate that NMMIIA and NMMIIB have different contributions to cell morphology and SF viscoelastic properties. NMMIIA suppression produced a dose-dependent morphology change and also produced slightly faster SF retraction kinetics. By contrast, NMMIIB suppression to a greater extent reduced the time constant associated with viscoelastic retraction, consistent with our previous findings with myosin light chain kinase inhibition (Tanner et al., Biophys J 2010). This may suggest a model in which NMMII isoforms provide different levels of resistance in SF retraction. To our knowledge, this represents the first attempt to resolve isoform-specific contributions of NMMII to stress fiber mechanics and provides new insight into the roles specific myosin II isoforms play in the mechanobiology of human glioma cells. Given recent observations that NMMII can promote glioma cell invasion by generating high contractile forces, this study may also lend insight into the molecular biophysical basis of tumor invasion.
Stress-Induced Whitening occurs in Demineralized Bone
1,2Hardisty, M R; 3Garcia-Nolen T; 2Choy S; 2Dahmubed J; 3Stover, S; 1,2Fyhrie D P
Introduction: With increasing average population age, the occurrence of fracture has the potential to adversely affect a patient’s quality of life. Our goal is to understand how fracture risk is caused by changes in bone tissue quality. Bone visually whitens during loading immediately prior to failure. Bone whitening during early damage is very similar to polymer crazing. In crazing, microvoids open, absorb energy, increasing toughness. We hypothesize that stress-induced whitening of bone is a property of the organic matrix and that whitening will be dependent upon strain rate and hydrogen bonding.
Methods: 12 demineralized dog-bone shaped cortical bone beams (2x8x30mm) were prepared from MC3 bones of necropsied thoroughbred horses. Specimens were loaded in tension to failure (MTS) at strain rates from 0.05/s-0.25/s. Hydrogen bonding’s effect on mechanical properties and crazing was probed by bathing specimens in solutions of differing Hansen’s hydrogen bonding energy (δh): 100% ethanol: δh=19.4(J/cm3)1⁄2, 0.9% saline: δh=42.3(J/cm3)1⁄2. Stress induced whitening was quantified by semi-automated segmentation of high-speed (1000fps) videos. (ImageJ & Matlab)
Results: Whitening was observed in the organic matrix of bone without the presence of the mineral phase for all cases. Stress-induced Whitening of the organic matrix was significantly increased with increasing δH (p<p=0.007). Decreasing δH by Ethanol immersion increased elastic moduli (290MPa vs 130MPa, p=0.012 ) and showed a trend of reduced ultimate strain (0.19 vs 0.12, p=0.05). After specimens failed, whitening was greatly reduced implying the process was partially reversible. Conclusion: Stress induced whitening was observed in demineralized bone, contradicting previous suggestions that whitening observed in whole bone tissue is caused by the mineral. This work established a connection between hydrogen bonding and bone whitening. Crazing and hydrogen bonding may contribute to bone toughness independent of mineral content; this may help to explain increased fracture risk due to aging, independent of mineral content.
CD44-based Adhesion and Mechanotransductive Signaling on Engineered Hyaluronic Acid Matrices
Yushan Kim, Badri Ananthanarayanan, Sanjay Kumar
Glioblastoma multiforme (GBM) is the most malignant primary brain tumor and is characterized by diffuse infiltration of glioma cells into brain parenchyma. We present new evidence that glioma cells are also sensitive to the stiffness of hyaluronic acid (HA)-based matrices not functionalized with adhesive peptides, suggesting that HA receptors such as CD44 are involved in the sensing of glioma cells to their biophysical environment.
Hydrogels were synthesized by functionalizing HA with methacrylate groups. Michael Addition reactions were performed to crosslink methacrylate groups with dithiothreitol, and in some gels to biofunctionalize with cysteine-containing RGD peptides. To determine glioma cell response to 2D matrix parameters, stiffness was modulated either in the absence or presence of RGD functionalization. Morphometric analysis and time-lapse imaging demonstrated that cell spreading and random migration speed increase with HA stiffness. Cells adopt a strikingly different morphology from that of typical adherent culture, forming thin filopodia-like protrusions rich in CD44. Cell adhesion on pure HA surfaces is decreased by addition of CD44-targeting antibody, but not addition of untethered RGD to the cell media, demonstrating that integrins are not involved in adhesion to pure HA hydrogels. Finally, we describe genetic loss-of-function experiments in which we stably transduce human glioma cells with anti-CD44 shRNAs and examine effects on HA regulation of cell adhesion, stiffness-dependent cell motility, and spreading.
While the mechanosensing mechanisms of integrins and cadherins are widely studied, these findings reveal a previously underappreciated role of cancer cell adhesion and mechanotransduction via CD44, and possibly other HA-specific receptors. This has broad implications on the field’s fundamental understanding of how glioma cells interact with the tumor microenvironment, and potentially reveals new strategies for therapeutic interventions.
Computationally Evaluating Effects of Osteoconductive Bulking Agents on Stress Distribution in Reconstructed Mandible
R KozakaA, H BhaktaA, P VandrangiA, J YalungB, JM CarusoB, and VGJ RodgersA
Previous studies of thirteen Rhesus Macaque monkeys were used to evaluate different forms of an osteoconductive bulking agent (compression-resistant matrix (CRM)) in a critical-sized mandibular defect. Our research focuses on a finite element model which examines the efficiency of the CRM complex. We computationally model these matrix complexes using Comsol Multiphysics (Module: Plane Stress; Version 3.5) to capture the von Mises stress as well as the deformation along the maxillary jaw. We have successfully implemented a 2-D mesh into the simulated maxillary jaw consisting of 8093 mesh elements. The model mimics jaw clenching by applying a force on the tip (689475 N/m) of the tooth and a rotational force at the hinge of the jaw. Subdomain conditions such as the density, Young’s Modulus, and the Poisson’s ratio were physiologically set. A separate subdomain within the jaw was modelled to represent the regenerative CRM complex defined with its physical properties. Preliminary computational analyses illustrate that the density and the Young’s Modulus of the regenerative subdomain plays a role in the von Mises stress as well as the deformation in the jaw. To predict the shear stress distribution of the regenerated mandible, we will further import images from Amira MRI scans (Version: 5.2.2) of the performed experimental trials . We will also examine which regenerative CRM complex withstands the physiological loading force of jaw clenching by defining their mechanical properties.
 A S Herford, M Lu, A N Buxton, J Kim, J Henkin, P J Boyne, J M Caruso, K Rungcharassaeng, J Hong. Recombinant Human Bone Morphogenetic Protein 2 Combined With an Osteoconductive Bulking Agent for Mandibular Continuity Defects in Nonhuman Primates. J Oral Maxillofac Surg. 2011
Experimentally Validated Computational Simulation of Lumbar Spine Intervertebral Disc Puncture
Kristen Lipscomb, Nesrin Sarigul-Klijn
Back pain is a debilitating medical condition affecting a patient’s work and lifestyle. Discography of the intervertebral disc (IVD) is often used to diagnose pathology of the disc and determine if it is a source for chronic back pain. Due to its invasive nature, discography may lead to disc degeneration, and has been a cause of controversy among spine care physicians. An anatomically accurate L3-L5 adult spine model was developed. Compressive loads of 100N-800N were applied to the superior surface of L3, holding the inferior surface of L5 fixed. IVD material properties were manipulated to validate the computational model by comparing disc displacements to data from a cadaveric experimental model. Discography was then simulated computationally as needle puncture affecting the disc. The material properties of the nucleus pulposus (NP) were adjusted to match results of post- discography experimental displacement testing with the model run under identical boundary and loading conditions. Our data show puncture of the IVD leads to increased disc deformation compared to the intact IVD (Figure 1). The maximum principal stress within the disc increased from 4.37MPa to 4.56MPa, concentrated within the annulus fibrosis (AF), resulting from a loss of pressure within the NP due to disc puncture. More specifically, as the load on the IVD increased, pressure loss in the NP increased. These mechanical effects may cause disc degeneration through initiation of AF fiber rupture, lowered water content and decreasing disc height. While previous studies suggest NP pressure loss as a result of disc puncture, this pressure change has never before been evaluated. Our data show a direct relationship between disc depressurization and applied load. Although discography has been utilized since the 1960s to determine if the IVD is a source of back pain, the potential long- term degenerative effects of the procedure are only now coming into light.
Identification of a Mechanoresponsive Promoter Region in the Human Cartilage Oligomeric Matrix Protein Gene
Lu, Jeffrey C. , Haudenschild, Dominik R.
Chondrocytes in cartilage respond to cyclic stresses and strains in their environment. Mechanical overloading of joints contributes to osteoarthritic cartilage degradation, whereas extended mechanical unloading results in cartilage atrophy. How mechanical forces regulate chondrocyte gene expression is important to understanding how healthy cartilage is maintained; however the DNA elements within a cartilage gene promoter that confer sensitivity to mechanical forces remain largely unknown. Primary human chondrocyte cell strains seeded in a three-dimensional hydrogel are mechanically loaded by a multiaxial bioreactor to accurately control the compressive, shear, and tensile forces on the hydrogel to simulate the various physical forces of walking and running. To assess fold changes of gene expression between mechanically stimulated samples and free-swell controls repeatedly and non-destructively, we designed a secreted luciferase reporter construct driven by the 3KB proximal Cartilage Oligomeric Matrix Protein (COMP) promoter and five distal Evolutionarily Conserved Regions (ECR). This cassette was sub-cloned into a lentiviral vector and transduced into primary human chondrocytes. We have previously found that the 3.0KB promoter is responsive to cyclical compression in both human stem cells and chondrocytes, resulting in a two to three fold upregulation of the COMP gene. Controls using a non-responsive promoter did not show any response to mechanical stimuli, and dsDNA quantification showed no difference in cell proliferation between compressive and free-swell samples. The response of the promoter sequence to mechanical loading proves the presence of a mechanoresponsive element within the sequence. However, the response was not of the same magnitude as in vivo loaded cartilage. We are currently working on increasing the responsiveness of the system through the inclusion of: 1. More physiological mechanical loading incorporating shear as well as compression, 2. Additional ECR regions within the COMP gene, and 3. A stiffer hydrogel for more precise force transduction to the embedded cells.
LABEL-FREE BIOMECHANICAL SINGLE CELL MICROFLUIDIC MEASUREMENTS FOR DIAGNOSIS OF CANCER STEM CELLS IN PATIENT TISSUES
Michael Masterman-Smith, Danny Gossett, Henry Tse, Dino Di Carlo
UC Los Angeles
Introduction: The exact cellular origin of many tumors is a topic of intense investigation. A fraction of cells with stem cell properties, typically termed cancer stem cells (CSCs), are thought to have true tumorigenic potential and may be responsible for tumor initiation and maintenance, making some cancers incredibly difficult to diagnose and treat. Progress into understanding these unique cells has come from taking patient cancer tissues and culturing them in vitro which selects and enriches for CSCs. The ability of these human-derived models of cancer to be cultured in vitro is highly predictive of reduced patient survival. However, determining ‘stem-like’ status in this manner is labor intensive and not suitable for clinical decision-making. Identifying these cells in a simpler manner could eventually serve as a clinical diagnostic which can define the degree of malignancy based on CSC subtypes.
Materials and Methods: We have previously introduced a microfluidic-based label-free high throughput deformability and size measurement technology for single cells. These new capabilities allow for high sensitivity, reproducible measurements which can dissect cellular heterogeneity based on biomechanical properties of these cells (Figure 1).
Results and Discussion: Embryonic stem cells appear highly deformable but become rigid upon differentiation. Hypothetically, brain CSCs derived from malignant patient brain tumors may yield similar deformability signatures. Analyzing a disaggregated primary malignant brain tumor revealed a large population of small, rigid cells and a subset of highly deformable cells that, when passaged in vitro, become larger and suggest a cancer stem cell phenotype. We are investigating a series of malignant brain tumors, their CSC enriched and differentiated progeny, molecular characteristics, and collecting patient outcome to compare to our biomechanical readouts.
Conclusion: Microfluidic-based label-free biomechanical methods have the potential to understand signatory properties of cancer stem cells and transform our understanding of biology, diagnosis, prognosis and treatment management of these cells.
Predicting the Crowded Protein Osmotic Pressure of Non-Interacting Binary Solutions
Harpal S. Sagoo, Devin W. McBride, and Dr. Victor G. J. Rodgers
Macromolecular crowding describes the encumbrance of large molecules in solution. Observed in many cells, it contributes to the osmotic pressure which effects membrane transport and cell swelling. The physics which describes the osmotic pressure of crowded environments are not fully understood. Various osmotic pressure models exist and attempt to describe the osmotic pressure non-idealities observed in near- saturation concentrations by assuming solute-solute and other interactions. However, of the myriad of proteins within a cell, many of them only have a limited number of interactions; thus, the assumption that solute-solute interactions alone are the cause of the non-ideal osmotic pressure is questionable. Recently, a free-solvent model was developed to account for solute-solvent interactions, namely protein hydration and protein-ion binding, and has been shown to provide excellent predictive power for the osmotic pressure of near-saturation protein solutions. Here, we extend the FSM to an array of binary protein solutions for bovine serum albumin (BSA) and ovalbumin (OVA) which do not interact. The osmotic pressure of this binary system is measured for a range of molar ratios (1:3, 1:1, 3:1 BSA:OVA) in 0.15 M NaCl at several pH (4.5, 5.4, 7.0 and 7.4) for concentrations up to near-saturation using an osmometer. The osmotic pressure of the single protein solutions was used to obtain the hydration and ion binding values by non-linear regression of the experimental data. The regressed hydration and ion-binding values for the individual proteins were used in the FSM to predict the osmotic pressure for the binary protein solutions. The predicted binary protein osmotic pressure is in good agreement with the measured osmotic pressure. Ultimately, this model may be used to explain the role of macromolecular crowding and describe the physics which may contribute to membrane transport and cell swelling
On-axis vs. Off-axis Strength Behavior of Human Trabecular Bone
Arnav Sanyal and Tony M. Keaveny
While trabecular bone is primarily adapted to sustain compressive loads along its principal material orientation (on-axis loads), traumatic activities such as falls can generate off-axis compressive loads that can lead to hip fractures or wrist fracture. The purpose of this study was to compare and relate the strength of human trabecular bone under on-axis and off-axis loads using micro-CT based finite element modeling and explore the underlying failure mechanisms. On-axis (n=39) and off-axis (n=33, 450 off-axis) human trabecular bone specimens (5mm cube) were taken from four anatomic sites. For the on-axis specimens, micro-CT based non-linear finite element analysis was performed for five loading cases – longitudinal compression and tension, transverse compression and tension and pure shear. In addition, the off-axis specimens were analyzed in compression loading. The apparent yield strength was calculated from the nonlinear stress-strain response and the amount of failed tissue and the mode of failure at tissue-level (compressive vs. tensile) were calculated at the apparent yield point. Using Mohr’s circle stress transformation from the offaxis compressive stress state to an on-axis stress state, the Tsai-Wu quadratic failure criterion was used to predict the off-axis compressive strength from the five on-axis strengths. The off-axis compressive strength (4.88±4.77 MPa) was significantly lower than the on-axis compressive strength (10.00±9.04 MPa) (see figure). While the off-axis compressive strength was predicted reasonably well by the Tsai-Wu quadratic criterion, its magnitude varied between the transverse compressive strength and shear strength. There was more tensile tissue failure under off-axis compression due to bending of the trabeculae similar to shear and transverse compression. These results demonstrate that human trabecular bone is much weaker under non-habitual off-axis loads and the failure could be dominated by shear or transverse compression. The Tsai-Wu criterion can provide a reasonable estimate of the off-axis strength of trabecular bone.
Detection of ischemia using osmotic pressure data of patient blood samples. A Computational approach
Noriko U. Sausman, Devin McBride, and Victor G. J. Rodgers
During cardiac ischemia, circulating albumin near the damaged tissues is exposed to oxygen free radicals that alter the protein’s structure (protein fragmentations and amino acid modifications), especially the N-terminus of albumin: the metal ion binding site. This ischemia-modified albumin (IMA) has been used as a biomarker for detection of ischemia by cobalt binding assays in conjunction with troponin tests and non-diagnostic electrocardiogram. However, we hypothesize that an osmotic pressure assay of the concentrated plasma may provide an inexpensive and more rapid diagnostic alternative that will be especially useful in low resource settings. Here we analyze the feasibility of this novel approach using our previously developed free solvent osmotic pressure model.
The free-solvent osmotic pressure model has been used to show that the deviation of osmotic pressure of protein solutions from ideality at high concentrations is coupled to two biologically significant and extremely sensitive parameters; solvent accessible surface area (SASA) and salt ion binding. During cardiac ischemia, these parameters may be altered since protein fragmentations will change the SASA and disruption of the metal ion binding capacity of N-terminus of albumin (consisting of N-Asp-Ala-His-Lys) will alter the electrostatic potential distribution in the local region, ultimately affecting salt ion binding capacity. Here, we computationally investigate the sensitivity of the osmotic pressure method in detecting IMA proteins and its practical limitations. For protein fragmentation, we analyze selective fragmented cases and show their impact on osmotic pressure For modifications of the N-terminus, we computationally determine the isopotential contour of IMA, integrate the charge distribution, correlate these results to changes to ion binding, and, ultimately, osmotic pressure. In addition, because the concentration of IMA is typically 1-2 % in healthy individuals but increases to 6% after ischemia, we determine the potential of the osmotic pressure method in observing these changes in whole samples.
Synthesis of hyaluronic acid hydrogels for investigating the mechanobiology of glioblastoma multiforme
Gurshamnjot Singh, Badriprasad Ananthanarayan, Yushan Kim, and Sanjay Kumar
This study investigated the impact of regulating matrix stiffness on the proliferation and motility of glioblastoma multiforme (GBM). GBM cells are glioma cell lines capable of aggressively invading neural parenchyma. Such pervasiveness is partly attributed to the nature of biochemical and biophysical cross-talk between the glioma cells and the neural extra-cellular matrix (ECM). The brain ECM consists of an elaborate biochemistry, governed by the presence of various glycosaminoglycans and glycoproteins, that differs significantly from the chemical makeup of collagen-based ECMs. As a result, traditional Collagen and MatriGel scaffolds do not effectively mimic the neural ECM (1). The primary objective of this study was to synthesize hyaluronic acid gels that imitated the biochemical and biophysical make-up of neural tissue. Furthermore, the study delved into investigating the dependence of glioma cell behavior on matrix density, matrix stiffness, and ligand density. The synthesis of methacrylated hyaluronic acid allowed variation of such parameters. Sodium hyaluronate was methacrylated using methacrylic anhydride and then exposed to dithiothreitol (DTT) to achieve crosslinking. The relevant storage and loss moduli of the varying hydrogel formulations were measured through rheology. The particular formulations used in this study displayed storage moduli ranging from 300 Pa to 1.5 kPa. The methacrylated hyaluronic acid was treated with RGD prior to crosslinking in order to promote tumor cell adhesion. The motility of U373 and C6 glioma lines displayed a direct correlation with substrate elasticity. Furthermore, the methodology of glioma invasion depended heavily upon matrix characteristics and glioma cell types. The interdependence between tumor cell behavior and extra-cellular characteristics highlighted the relevance of biophysical interactions in governing glioma cell invasion.
Massively Parallel Polarization and Analysis of Single Cell Behavior under Magnetic Nanoparticle- mediated Mechanical Tension
Peter Tseng, Jack W. Judy, Dino Di Carlo
UC Los Angeles
Mechanical force plays a critical role in a large variety of cellular processes, including cell division, contractility, differentiation, and motility. Understanding the recurring mechanisms and themes in cellular response to force not only enlightens us about single-cell biology, but could provide a tangible method by which to influence cellular function. A fundamental need in the study of cellular mechanics is the on-demand local application of controlled forces over a large population of cells to obtain statistically relevant measurements of noisy biological responses. We demonstrate an approach in which many individual cells are instead brought into uniform alignment with arrays of inducible micro-magnets. This microfabricated platform allows generation of large, coordinated, magnetic nanoparticle mediated mechanical stimuli on tens of thousands of cells at a time. We utilize our technique to generate large asymmetries in cellular behavior. The technique demonstrates the ability to polarize the cell by generating local modulation in important cellular biochemistry (PAK), and control higher order, coordinated cellular responses, such as polarization of cell division axis, and formation of highly localized and directed filopodia.
Our large sample sizes allow new observations of consistent localized effects on actin distribution, including significant asymmetry in filopodia generation across a large percentage of cells at tensions above ~1 nN/µm. Our rapid sample generation allows us to extensively explore the mechanism of force-mediated filopodial induction, as we conduct an array of biochemical and inhibitory studies, discovering its dependence on the protein PAK, which is implicated in mechanotransduction. We additionally find that PAK localizes to regions of high mechanical deformation, forming a distinct band progressing locally around magnetic nanoparticles in the z-dimension. Finally, we find that local nanoparticle force, applied throughout mitosis, can strongly bias the axis of cell division in a manner that competes with extracellular adhesive cues.
Regulation of Growth Factor Dependent Tumor Cell Proliferation by ECM Mechanics
Vaibhavi Umesh, Theresa Ulrich, Sanjay Kumar
The rapid progression of glioblastoma multiforme (GBM), the most common and lethal primary brain tumor, is driven by the diffuse infiltration of tumor cells into the brain. Recent genome sequencing efforts have revealed that many GBM tumors share lesions in the epidermal growth factor receptor (EGFR) pathway, which strongly promotes migration and proliferation. Previously, our laboratory has demonstrated that stiffening the extracellular matrix (ECM) can amplify the proliferation rate of human GBM tumor cells by more than a factor of five (Ulrich et al, Cancer Res 2009), raising the possibility that mechanical inputs can cross‐talk with mitogenic signaling pathways to promote GBM tumor growth. The objective of this study was to directly explore this hypothesis by investigating the extent to which ECM biomechanics can regulate GBM cell cycle progression, chemotherapeutic sensitivity, and growth factor receptor (EGFR)‐dependent signaling. We find that human glioma cells cultured on soft (80 Pa) fibronectin‐coated polyacrylamide gels are more likely to be in G0/G1 and less likely to be in S phase of the cell cycle than cells cultured on stiff (119kPa) ECMs. Consistent with this finding, cells cultured on soft polyacrylamide gels preferentially escape the G2/M arrest normally induced in stiff environments following exposure to taxol. In addition, Western Blot reveals that the expression and phosphorylation of EGFR and its downstream effectors, including Akt and PI3 kinase, depend strongly on ECM rigidity, with EGFR phosphorylation rising with increasing ECM stiffness. Furthermore, EGFR organization is highly rigidity‐dependent (Fig. 1), with EGFR co‐clustering with focal adhesions on stiff substrates and receding into a diffuse distribution as matrix rigidity falls to physiological levels, suggesting that ECM stiffness may promote proliferation by spatially amplifying EGFR signaling. Together, these results support a model in which ECM stiffening acts through mechanotransductive pathways to trigger EGFR‐based mitogenic signaling that promotes proliferation.
Effects of Tissue-level Ductility on Vertebral Bone Strength
Haisheng Yang; Tony M Keaveny
Understanding the failure mechanisms in human vertebra is fundamental to studying the etiology of osteoporotic spine fracture. One key issue is the influence of tissue-level ductility on the overall strength of vertebra since it is well known that aging and drug treatments can affect tissue-level ductility. To provide insight into this issue, our goal was to determine how vertebral strength is altered when the tissue is changed from perfectly brittle to perfectly ductile — the two extremes of possible tissue-level ductility. High-resolution CT scans (30 microns) of one elderly human cadaver T9 vertebra (aged 65 years) were used to generate a 3D finite element model having about 60 million elements, subjected to 1% strain uniform compression loading condition. Fully nonlinear and quasi-nonlinear analyses were performed to simulate perfectly-ductile and perfectly-brittle behavior of vertebral bone tissue, respectively. In the fully nonlinear analysis, all bone tissue was modeled using a rate-independent elasto-plasticity model with higher strength in compression than in tension. The quasi-nonlinear analysis was constituted a series of linear analyses in which the tissue was assumed to fracture as soon as the stress exceeded either the compressive or tensile tissue-level yield stress. The vertebral strength obtained from ductile analysis was almost two times larger than that obtained from brittle analysis. For the ductile analysis, 23.3% of the total tissue failed at the overall yield point of the whole bone. By contrast, for the brittle analysis, 0.84% of the total tissue had failed at the overall ultimate point. These results provide unique insight into the failure mechanisms in the vetebral body. One interesting aspect of these results is the tiny amount of tissue that is required to fail for overall bone failure, particularly if all post yield deformation is suppressed, suggesting a highly localized nature of tissue failure.
Molecular, Cell and Tissue Engineering
Enhancing Osteoconductivity of Fibrin Gels with Apatite‐Coated Polymer Microspheres
Hillary E. Davis1, Bernard Y.K. Binder1, Phill Schaecher1, Dana D. Yakoobinsky1, Archana Bhat1, and J. Kent Leach1,2*
1Department of Biomedical Engineering, University of California Davis, Davis, CA 95616
2Department of Orthopaedic Surgery, School of Medicine, University of California, Davis, Sacramento CA 95817
Fibrin gels are a promising material for use in promoting bone repair and regeneration due to their ease of implant formation, tailorability, biocompatibility, and degradation by natural processes. However, these materials lack necessary osteoconductivity to nucleate calcium, integrate with surrounding bone, and promote bone formation. Polymeric substrata formed from poly(lactide-co-glycolide) (PLG) are widely used in bone tissue engineering. A carbonated apatite layer of bone-like mineral can be successfully grown on the surface of PLG microspheres after a multi-day incubation process in modified simulated body fluid (mSBF). Such coatings improve the osteoconductivity of the polymer, provide nucleation sites for cell-secreted calcium, and enhance the potential osseointegration with host tissue. We examined the capacity of mineralized polymeric microspheres suspended within fibrin hydrogels to enhance the osteoconductivity of fibrin gels and increase the osteogenic potential of these materials. The inclusion of microparticles, both nonmineralized and mineralized, reduced the capacity of entrapped mesenchymal stem cells (MSCs) to contract the gel. When cultured in osteogenic media, we detected a near linear increase in both calcium and phosphate incorporation in gels containing mineralized microspheres and entrapped MSCs. The osteoconductivity of acellular fibrin gels with mineralized and nonmineralized microspheres was assessed in a rodent calvarial bone defect over 12 weeks. Compared to untreated rodent calvarial bone defects, we detected significant increases in vascularization when treated with fibrin gels, with greater vascularization, on average, occurring with gels containing microspheres. We detected a trend for increased bone mineral density in gels containing mineralized microspheres after 12 weeks. These findings demonstrate that the osteoconductivity of fibrin gels can be increased by inclusion of mineralized microspheres, but additional signals may be required to rapidly accelerate bone repair.
The physicochemical rules governing bilayer self-organization in the human mammary epithelium
Alec Cerchiari, James Garbe, Tejal Desai, Mark LaBarge, Zev Gartner
Understanding the intrinsic and extrinsic cues that direct the assembly of cells into tissues is of central importance for understanding morphogenesis, tissue homeostasis, and the onset of disease. We study mixtures of human mammary luminal and myoepithelial cells and their dependency on the microenvironment as they self-organize into bilayered structures reminiscent of the mammary gland. Recently, self-organization was shown to be a lineage-intrinsic property of mammary epithelial cells that heavily relies on the differential expression of cadherins on the cells’ surface. Our preliminary results add to this model but suggest that the microenvironment plays a significant, if not dominant role, in this process. We hypothesize that interactions with basement membrane proteins generate polarity signals that direct cadherin ligation and cell positioning. Successfully characterizing the processes directing self organization of luminal and myoepithelial cells will improve in-vitro 3D culture models of the mammary epithelium and advance our understanding of the physicochemical forces governing structure formation and maintenance of epithelial tissues.
Intermittent hypoxia conditioning of in vitro vascularized fibrin gels
Seema M. Ehsan and Steven C. George
Understanding the role of oxygen during vessel formation will aid the development of thick (>1mm) prevascularized tissues by harnessing the beneficial consequences of hypoxia. Intermittent hypoxia (IH) has been linked to pro-angiogenic gene expression and stress resistance in endothelial cells; however an IH conditioning strategy has not yet been exploited in the development of prevascularized thick tissues. The goal of this work is to test the hypothesis that in vitro capillary networks can be conditioned with IH to adopt a proangiogenic, stress resistant phenotype. A culture chamber system was fabricated to provide programmable control of ambient oxygen levels. This, combined with an in vitro model of angiogenesis, provided an experimental platform to study IH-induced angiogenesis. It was found that tissues exposed to an IH profile alternating 2 hours at 20% O2 and 30 min at 1% O2 for 7 days showed a higher angiogenic capacity compared to 20% O2 conditioned tissues. Furthermore, when fully-grown tissues were exposed to an additional 7 days of hypoxic stress (1% O2), the IH conditioned tissues demonstrated significantly higher vessel stability compared to the 20% O2 conditioned tissues. These results show that IH enhances capillary network development in vitro, and that the network is more resistant to sustained hypoxia. This effect was found to be independent of VEGF concentration in the media. We demonstrate the utility of IH as a promising strategy to not only enhance the prevascularization of engineered tissues, but also to preserve capillary networks following implantation into hypoxic conditions.
Oxygen sensing and control of engineered tissue
Seema M. Ehsan and Steven C. George
To understand the mechanisms behind physiological processes that involve heterogeneous oxygenation conditions, such as tumor angiogenesis and ischemic reperfusion, an experimental platform is needed to properly mimic those processes in vitro. Studies have demonstrated that ambient oxygen concentrations can differ significantly from the concentrations cells actually experience. The inherent diffusion limitation of 3D culture, particularly at clinically relevant dimensions (>1mm), adds an additional hurdle for oxygen transport. We present an experimental platform that offers simultaneous control and measurement of oxygen diffusion through 3D engineered tissues. The control component was achieved using a fabricated culture chamber with programmable oxygen tension in the gas phase. The measurement component consisted of an oxygen sensor patch placed at the base of a fabricated well, and on top of which a cellularized tissue was constructed. The well was then placed inside the oxygen control chamber, where an external oxygen probe non-invasively provided real-time measurements of the oxygen concentration at the base of the tissue. A non-steady state diffusion model was built, with Michaelis-Menten kinetics used to model cellular oxygen consumption. Finite element analysis was then used to predict detailed spatial and temporal oxygen distributions in the tissue. The oxygen control system achieves complete gas equilibration within 60 seconds, and can be programmed to deliver 0-100% O2. The Michaelis Menten parameters were found to be: Vmax= 1.1e-4 mol/m3/s and Km= 0.036 mol/m3. A square wave oxygen exposure profile at the top of the tissue (Figure: dashed line) has a significantly attenuated effect on the oxygenation of the base of a 2mm thick tissue (Figure: solid and dotted lines). While most mathematical models of 3D culture rely on steady state approximations and constant boundary conditions, the present model employs non-steady state analysis and allows for time varying boundary conditions consistent with in vivo observations of intermittent hypoxia.
Self-Assembly of Peptide Amphiphiles Into Hydrogel Via Multiscale Simulations
Iris W. Fu, Cade B. Markegard, and Hung D.
Peptide amphiphiles (PA), which are an emerging class of molecules that have been shown to selfassemble into novel nanostructures such as nanofibers in hydrogel, are of significant interest due to their applications in tissue engineering. A representative PA molecule is comprised of a hydrophobic alkyl tail, a short peptide sequence for intermolecular hydrogen bonding, a group of charged amino acids for enhanced solubility, and a region for bioactive signals to be transduced via cells or proteins. Our studies examine the role of peptide sequences and the conditions on the morphology and mechanical properties of PA molecule assemblies by performing molecular dynamics simulations. Using a newly extended intermediate-resolution protein model that can represent twenty different amino acids with adequate details, we focus on the folding of peptides with or without the alkyl segment as a function of the various conditions such as pH, ion concentrations, or temperature and compare our results with those obtained from all-atom simulations using CHARMM. To examine the kinetic mechanisms involved in PA self-assembly, we perform constant temperature simulations to observe the whole process of PA assembly starting from random configurations of relatively large PA systems. We also perform replica-exchange simulations to delineate a phase diagram characterizing different types of structure exhibited for each sequence as a function of the condition being examined. The findings of this research will guide experimentalists to identify systems of novel biomaterials with advantageous mechanical properties.
3D cardiac microtissue derived from human induced pluripotent stem cells
Or Gadish, SangMo Koo, Zhen Ma, Natalie C. Marks, Micaela Finnegan, Costas P. Grigoropoulos, Kevin E. Healy.
Treatments for cardiovascular diseases are significant unmet needs in the global medical community. The principal long-term objective of this project was to establish an in vitro model of human cardiac tissue based on the reconstitution of synthetic models of human ventricular myocardium with populations of cardiomyocytes derived from human induced pluripotent stem (hiPS) cells. While several groups have successfully differentiated cardiac cells from human pluripotent stem cells, there still remains a paucity of approaches to organize these cardiomyocytes into an aligned 3D structure. The long-term goal of this work is to construct 3D cardiac micro-tissues derived from hips cells on an engineered platform to simultaneously determine the electrical and mechanical properties in a systematic and high-throughput manner. We used lithographic microfabrication to make a platform with multiple pairs of cantilever beams, on which fibers were polymerized to recapitulate the filamentous matrix of the myocardium. We used two-photon-initiated polymerization to create these scaffolds, and then seeded them with hips cell-derived cardiomyocytes (hiPS-CMs) generated using a directed differentiation protocol. Beating hiPS-‐CMs placed on the aligned 3D filamentous matrix attached and aligned according to the directionality of the fibers with elongated contractile proteins. Whereas, these cells plated on a flat substrate did not show the aligned contractile proteins. These preliminary studies indicate that hiPS-‐CMs can be organized into nascent 3D micro-tissues with aligned structure. Furthermore, this platform allows for mechanical characterization via the cantilever beams and visual characterization of the electrical properties using voltage-sensitive dyes. We believe this system can be developed into a powerful human in vitro model of cardiac tissue, useful for identifying the genetic and environmental basis of cardiac disease and to screen potential drug candidates for treatment.
Myosin IIA deficient cells migrate without exerting traction force
Melissa Jorrisch, Wenting Shih, Soichiro Yamada
Cell motility is a cornerstone of embryogenesis, tissue remodeling and repair, and cancer cell invasion. It is generally thought that migrating cells grab and exert traction force onto the extracellular matrix in order to pull cell body forward. Using an array of micron-sized pillars as a force sensor and shRNA specific to each myosin II isoforms (A and B), we analyzed how myosin IIA and IIB individually regulate cell migration and traction force generation. Although myosin II isoforms had similar localization in migrating cells, myosin IIA and IIB preferentially localized to the leading edge where traction force was greatest and the trailing edge, respectively. When individual myosin II isoforms were depleted by shRNA, myosin IIA deficient cells lost actin stress fibers and focal adhesions, whereas myosin IIB deficient cells maintained similar action organization and focal adhesions as wildtype cells. Interestingly, myosin IIA deficient cells migrated faster than wildtype or myosin IIB deficient cells, yet, myosin IIA deficient cells exerted less traction force than wildtype or myosin IIB deficient cells. These results suggest that, in the absence of force generating machinery, cells move by gliding on the surface without traction forces, thus demonstrating the remarkable ability of cells to adapt and migrate.
Analysis of maturing young porcine islet tissue by flow cytometry.
Morgan Lamb1,4, Vanessa Scarfone4, Kelly Laugenour1, Peter Donavan3,4, Clarence Foster III1, Jonathan RT Lakey1,2,4
Departments of Surgery1, Biomedical Engineering2, Developmental and Cell Biology3, Sue and Bill Gross Stem Cell Research Center4, University of California, Irvine.
Background: Porcine pancreatic islets have been explored as source of islets for xenotransplantation into patients with Type 1 diabetes. Methods of islet isolation from market weight pigs is labor intensive, costly and islets tend to fragment resulting in suboptimal yields for transplantation. Methods of islet isolation and maturation of young piglet islets, yield viable function islets and protocols that are scalable for clinical trials. The aim of this study was to further characterize young porcine islets while tracking changes in islet cellular characteristics using flow cytometry and standard isolation endpoints.
Methods: Pancreases from Yorkshire pigs (age 14-20 days) were rapidly recovered and partially digested using low dose VitaCyte enzyme. Pancreatic tissue was then matured in tissue culture (37oC/5%CO2) for up to 7 days. During in vitro maturation tissue samples were analyzed for islet yield (IEQ), purity (dithizone) and cell viability (FDA/PI). In vitro function was assessed using a static glucose stimulated insulin release (GSIR) assay. Samples were characterized for islet cellular composition by dissociating islets and analyzing by immunohistochemistry for antibodies targeted for glucagon(α-cells), C-peptide(β‐cells), and amylase(acinar tissue), using flow cytometery.
Results: The proportion of dithizone positive tissue increased during tissue culture (12.6×103±183 IEQ (mean±sem) to 33.3×103±136 after 7 days of culture (n=6), p<0.05)) with a majority of islets between 50–150 um in diameter (94.52±11% after 7 days). Islet function (GSIR) improved during time in culture (SI=1.3±0.1 at day 0, to 2.6±0.2 after 7 days). During islet maturation proportions of amylase decreased, while beta cell proportion within the islet increased (Table 1) after 2 and 7 days in tissue culture (p<0.05).
Conclusion: During maturation in tissue culture, young porcine islets increase their beta cell proportion while decreasing exocrine portions. Viable functional young piglet islets are achieved using this protocol and these cells are being considered for future transplant studies.
Effects of cold storage duration on young porcine islet isolation and maturation.
Laura Young1, Morgan Lamb1, Kelly Laugenour1, Clarence Foster III1, Jonathan RT Lakey1,2
1Departments of Surgery and 2Biomedical Engineering, University of California Irvine, Orange, CA
Background: Islet transplantation has been demonstrated as a potential therapeutic option for patients with Type 1 diabetes; eliminating hypoglycemic events and demonstrating sustained insulin independence. A major limitation of this technology is the shortage of donor organs for transplantation. Porcine islets are now being explored as a source for transplantation and could become an effective, scalable source of islets. Centralized animal and cell processing facilities are required and currently there is need to transplant organs from a USDA approved pig facility to a FDA approved good manufacturing practice (GMP) facility to process the tissue for transplant. The aim of this study is to determine the maximal duration of cold storage before islet isolation and maturation.
Methods: Pancreases were rapidly resected from Yorkshire pigs (age 15-16 days) and stored in UW solution. Cold storage time was <1 hour for control group pancreases, and 4 or 12 hours for variable groups. Partial enzymatic digestion was performed using low dose VitaCyte Enzyme. Islet tissue was cultured at 37oC, 5% CO2 for 7 days with media changes every 48 hours. Samples were taken at 1, 3, and 7 days during maturation and stained with either dithizone to quantify islet yield (IEQ) and purity, or with Newport-Green and propidium iodide to analyze viability.
Results: Islets isolated from groups up to 12 hours cold storage showed no significant difference in islet yield and purity when compared to control (Table 1). Islet viability was statistically reduced in the 12 hour stored group but still remained >75% viable after 7 days of maturation.
Conclusion: Pancreases from young pigs can be stored for up to 12 hours in UW solution without impacting islet yield and purity, however islet viability is compromised. These results allows for assisting and determining parameters from xenotransplantation trials in the future.
Recruitment of Endogenous Progenitor Cells for In Situ Regeneration of Blood Vessels
Benjamin Li‐Ping Lee, Jian Yu, Aijun Wang, Zhenyu Tang, Jeffrey Henry, Song Li
Small-diameter synthetic vascular grafts have high failure rate due to thrombogenic responses. Tissue- engineered blood vessels have been created in vitro by utilizing cells and scaffolds, but the long development time limits the scalability of the technology. Here we present an in situ tissue engineering approach that recruits two types of endogenous progenitor cells for the regeneration of blood vessels. Heparin was conjugated to microfibrous vascular grafts to suppress thrombogenic responses, and stromal cell-derived factor-1α (SDF-1α) was immobilized onto heparin to recruit endogenous progenitor cells. Heparin-bound SDF-1α was more stable than adsorbed SDF-1α under both static and flow conditions. Microfibrous grafts were implanted by anastomosis at carotid arteries in a rat model. Heparin coating increased the patency, which was further improved by immobilized SDF-1α for at least 3 months. Within 1 week, SDF-1α effectively recruited endothelial progenitor cells (EPCs) to the luminal surface of the grafts. EPCs differentiated into mature endothelial cells (ECs), resulting in a complete coverage of luminal surface within 4 weeks. In contrast, the grafts without SDF-1α showed much slower endothelialization, especially in the middle portion. In addition, SDF-1α increased the recruitment of smooth muscle precursor cells (SMPCs) to the outer surface of the grafts at 1 week, where they differentiated into smooth muscle cells (SMCs). In addition, SDF-1α-immobilized grafts had significantly higher elastic modulus after 3 months. This work demonstrates the feasibility of simultaneously recruiting progenitor cells of ECs and SMCs for in situ blood vessel regeneration. This approach will have wide applications in regenerative medicine.
A Novel Quantitative Fӧrester Resonance Energy Transfer Assay for Protease Kinetics Determination
Yan Liu, Jiayu Liao
Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research, and is a very powerful tool for elucidating protein interactions in either dynamic or steady state. SUMOylation (the process of SUMO (small ubiquitin-like modifier) conjugation to substrates) is an important posttranslational protein modification with critical roles in multiple biological processes. Conjugating SUMO to substrates requires an enzymatic cascade. Sentrin/SUMO-specific proteases (SENPs) act as an endopeptidase to process the pre-SUMO or as an isopeptidase to deconjugate SUMO from its substrate. Defining the kinetics of key regulatory reactions is necessary for understanding the roles of SENPs in the SUMOylation cycle and provide valuable reference for future drug discoveries. Here, we report a novel quantitative FRET-based kinetics assay for SENP2 endopeptidase activity by internal correction in one sample during the assay. The assay is based on the differentiating absolute fluorescence signals contributed by the FRET-induced acceptor’s emission and self-fluorescence emissions from donor and acceptor respectively. The kinetic parameters, kcat, KM, and catalytic efficiency (kcat/KM) of catalytic domain SENP2 toward pre-SUMO1/2/3, were obtained by this novel assay. The general principles of this quantitative FRET-based protease kinetic determination can be applied to other proteases as well. This provides a novel robust technology platform for protease kinetics determinations.
The Statistical Optimization of Bioactive Regimens for Engineering Biomimetic Fibrocartilage
Regina F. MacBarb, Eleftherios A. Makris, Jerry C. Hu, and Kyriacos A. Athanasiou
Fibrocartilage lacks the ability to self-repair following disease- or injury-induced degradation. Comprising the menisci of the knee, the intervertebral discs of the spine, and the disc of the temporomandibular joint (TMJ), fibrocartilages are integral for providing effective load distribution, joint stability, and smooth articulation. Due to the current lack of treatments for degenerated fibrocartilage, the global objective of this work is to tissue engineer a fibrocartilage spectrum representative of the various fibrocartilaginous tissues found in the body. To create this spectrum, co-cultures of meniscus cells (MCs) and articular chondrocytes (ACs) were self-assembled at either 50:50 or 75:25 MC:AC. Constructs were treated with bioactive agents TGF-β1 and chondroitinase-ABC (C-ABC) individually or in combination, each of which have previously been shown to enhance the functional properties of tissue-engineered articular cartilage. It was hypothesized that TGF-β1 and C-ABC would synergistically enhance matrix synthesis and, subsequently, improve mechanical properties in the engineered fibrocartilage.
Self-assembled fibrocartilage constructs were cultured for 5 weeks and analyzed via histological, immunohistochemical, biochemical, and biomechanical assays, as well as via scanning electron microscopy (SEM). Biochemical and biomechanical analysis found similar trends in collagen/WW and the Young’s modulus of the constructs. Collagen/WW was synergistically enhanced in the combined treated constructs, while the Young’s modulus of this group was additively increased over controls (Fig. 1). SEM analysis found the combined treated constructs to have significantly increased collagen fibril diameter and density over control constructs (Fig. 1). These increases were also greater than either treatment alone, signifying a synergistic behavior between these two agents in modifying collagen network organization. Overall, this study has identified two factors that significantly increase the biochemical and biomechanical properties of engineered fibrocartilage, allowing for a spectrum of fibrocartilage tissue to be generated. Such work speaks to the potential of engineering clinically applicable tissues to repair or replace fibrocartilage.
Development of High Sensitive FRET-based Assay to Study Protein-Protein Interactions in NEDD8 Conjugation Pathway
Harbani Kaur Malik-Chaudhry, Frank Lee, Sherman Yaghmaee and Jiayu Liao
Department of Bioengineering, University of California, Riverside, California 92521, USA
Reversible post-translational modifications are widely used to dynamically regulate protein activities in vivo. Small chemical groups, sugars, lipids and polypeptides can modify proteins. The most well known example of polypeptide modifiers is ubiquitin and ubiquitin like proteins (Ubl). One Ubl, Neural precursor cell-Expressed Developmentally Down regulated (NEDD8) has been shown to covalently modify a large number of proteins with important roles in many cellular processes. The understanding of the covalent attachment of NEDD8 to target proteins and its kinetics of conjugation cascade will provide greater insights into this process in normal and pathological conditions. Förster Resonance Energy Transfer (FRET) technology has been used in various biological and biomedical research. FRET is a nonradiative energy transfer that is widely used in the studies of protein-protein interaction. Herein, we report development of novel FRET-based high-sensitive protein assay, to study covalent conjugation of NEDD8 to UBA3 (NEDD8’s E1) and Ubc12 (NEDD8’s E2). Our method is based on fluorescent energy transfer between CyPet and YPet fused with NEDD8 and E1 and E2 respectively. We are able to differentiate between covalent and noncovalent interaction of NEDD8 and UBA3, and NEDD8 and Ubc12. Our FRET-based method is robust to study various protein interactions in NEDDylation pathway and it can be further employed for drug discovery for NEDDylation pathway.
In vitro cardiac diseased tissue model generated from human induced pluripotent stem cells
Natalie C. Marks, Micaela Finnegan, Or Gadish, Zhen Ma, Bruce R. Conklin, and Kevin E. Healy
Cardiovascular diseases are the leading cause of patient morbidity and mortality globally. Long-‐QT syndrome (LQTS) is a cause of cardiac arrest and sudden cardiac death, and yet the understanding of this symptom is hindered due to lacking of appropriate human diseased tissue models. However, with the advent of human induced pluripotent stem cells (hips cells) and advances in biomaterials, it is possible to address these issues directly. The principle long-term goal of this project is to establish an in vitro model of diseased human cardiac tissue for “disease in a dish” studies using, hips cells from patients with LQTS. This in vitro model can be used to study the disease mechanisms and ultimately design and screen patient specific therapeutics prior to clinical trials. Previous studies showed that hips cells derived from LQTS patients exhibited the same pluripotency markers as human hESCs. In this work, cardiomyocytes were generated from LQTS hips cells employing a serum-free matrix‐“sandwich” method differentiation protocol and were characterized by cardiac-specific markers (cardiac troponin T and sarcomeric α-actinin). Currently, our differentiation protocol yields approximately 50% cardiomyocytes in the cell culture population. Our ongoing studies are focused on improving the yield of the cardiogenic differentiation from hips cells using substrata with lower moduli that is consistent with native tissue. To date, our results indicate that LQTS hiPS cells have the potential to be used to develop powerful human in vitro models of cardiac tissue useful for identifying the genetic and environmental basis of cardiac disease and to screen for potential drug candidates for treatment.
Tailoring Biophysical Properties of Fibrin Gels to Promote Bone Formation with Co-Cultured Cells
Kaitlin Murphy, B.S., Matt Mui, M.S., and J. Kent Leach, Ph.D.
UC Davis, Biomedical Engineering Graduate Group
Statement of Purpose: A current priority of bone tissue engineering is to create a biomaterial that can revascularize a bone defect while simultaneously driving cells to participate in bone formation. Mesenchymal stem cells (MSCs) have the potential to directly participate in osteogenesis by differentiating towards the osteoblastic phenotype, or they may indirectly accelerate tissue repair by secreting trophic factors that stimulate capillary formation by endothelial cells. Endothelial colony forming cells (ECFCs) represent a promising cell source for neovascularization in vivo and exhibit robust proliferative and angiogenic potential in ischemic conditions in vitro. Fibrin hydrogels are an exciting platform to implant cells at a defect site, as fibrin naturally occurs in the body as a scaffold for tissue regeneration. Previously, we reported that supplementing the fibrin gel solution with NaCl alters gel stiffness while retaining low concentrations of fibrinogen and thrombin. The purpose of this study is to determine the optimal fibrin gel stiffness that simultaneously supports an osteogenic response from MSCs and an angiogenic response from ECFCs when cocultured.
Methods: Fibrin gels were prepared as we previously described (Davis et al., 2011) and allowed to gel for 1 hr in cylindrical PDMS molds. Cells were added to the pre-gel solution at total concentration of 1 million cells/mL, with monoculture gels containing 1 million MSCs/mL and co-culture gels containing 500,000 cells/mL each of MSCs and ECFCs. We quantified the osteogenic response by measuring alkaline phosphatase (ALP) and total calcium normalized to total DNA content. ECFC sprouting was monitored to evaluate the angiogenic response using GFP-transduced ECFCs and confocal microscopy. Additionally the diameter and distribution of cells within the gels was monitored over time.
Results: DNA content was decreased in monoculture and co-culture gels with increasing NaCl concentration. Normalized calcium content increased with NaCl concentration. While ALP activity in monoculture gels increased with increased NaCl concentration, the coculture gels exhibited decreased ALP activity over the same NaCl range. We observed clear morphological differences between monoculture and co-culture gels, as monoculture gels contracted severely and the cells migrated to the center while coculture gels did not contract and cells remained evenly distributed.
Using Growth Factor-Encoded Surfaces to influence hepatic phenotype expression
Dipali G Patel, Caroline Jones, Nazgul Tuleuova, Elena Foster, Tam Vu and Alexander Revzin
The goal of this study was to investigate epithelial-to-mesenchymal transition (EMT) in primary rat hepatocytes cultured on growth factor (GF) microarrays in vitro. Transforming-growth factor-β1 (TGF-β1) is known to have pro-fibrogenic effects in the liver, contributing to loss of function and epithelial phenotype in hepatocytes. Hepatocyte growth factor (HGF) on the other hand contributes to maintenance of epithelial hepatic phenotype. In this study we created surfaces with imprinted spots of HGF and TGF-β, cultured hepatocytes on these surfaces and demonstrated that the local hepatic phenotype (either epithelial or mesenchymal) could be controlled based on the underlying GF type.
HGF, TGF-β1 and a combination of both GFs were mixed with collagen (I) and robotically printed onto glass slides to create arrays of 500μm diameter spots. Hepatocytes were seeded on the arrays forming clusters corresponding in size to the underlying spots. The distances between the spots were varied. After 4 days in culture, the phenotype and function of the cells were analyzed using albumin ELISA and immunofluorescent staining for albumin, e-cadherin and n-cadherin.
As shown in Figure 1, hepatocytes residing atop HGF spots strongly expressed epithelial markers, e-cadherin and albumin, whereas adjacent hepatocytes on TGF-β spots showed much lower levels of these markers. Additionally, cells on TGF-β spots showed an increase in mesenchymal marker n-cadherin. Interestingly, phenotype expression was distance dependent where the edge-to-edge spacing of 1.5mm and above between HGF and TGF-β spots was associated with different phenotypes. Shorter distances were associated with more pronounced epithelial phenotype expression in cells residing on TGF-β spots, suggesting involvement of paracrine interactions between the cell clusters.
Overall, our results demonstrate that local GF presentation may induce the same cell type residing on the same surface to differentiate towards divergent phenotypes. This platform may be used to investigate mechanisms of EMT in adult hepatocytes.
Engineering Polymer Scaffolds and Activated Nanosensors to Improve Molecular Cancer Diagnostics
Maha Rahim, Sumi Lee, Rajesh Kota, Neal K. Devaraj, and Jered B. Haun
Patient-specific molecular analysis of cancer cells can offer an enhanced understanding of long-term tumor behavior, as well as assist in developing personalized therapeutics. However, current molecular detection techniques lack the sensitivity for molecular characterization of size-limited clinical samples. In a recently developed nanoparticle targeting platform, antibodies against biomarkers of interest were modified with trans– cyclooctene (TCO) and used as scaffolds to couple tetrazine (Tz)-modified nanoparticles onto live cells. This cycloaddition coupling mechanism is rapid, catalyst-free, and supports amplification of biomarker signals, making it superior to alternative targeting techniques. Still, relevant protein markers, while being over expressed in cancer, may be too low for their signal to be detected against the inherent nanosensor background. To date, nanosensor amplification has been supported by the fortuitously large size of antibodies, which act as a support scaffold. In this study, our first goal is to improve detection sensitivity by engineering polymer scaffolds to attain greater control of coupling site placement and density. Poly-lysine scaffolds offer a high density of primary amine functional groups, thus providing numerous attachment sites on the scaffold where nanoparticles can be bound using the bioorthogonal Tz/TCO chemistry. This increases localized binding and achieves higher signal intensity. Additionally, our second goal is to improve detection specificity using liposomes that turn-on after binding to the scaffold. Liposomes containing the conjugated polymer polydiacetylene (PDA) in the bilayer undergo a blue-to-red color change upon different stimuli such as temperature, pH, etc. We are developing novel PDA liposomes that activate upon bioorthogonal reaction with the scaffold. This reduces background signal for the detection technique, and opens the door to lower expression level targets such as phosphorylated signaling molecules. Together, these strategies will improve detection sensitivity and specificity to enable advanced molecular profiling applications.
Tandem zyxin LIM sequences do not enhance the force sensitive accumulation
Amanda N. Steele, Grant M. Sumida, and Soichiro Yamada
The ability to sense mechanical forces is vital to cell physiology. Yet, the molecular basis of mechano- signaling remains unclear. Previous studies have shown that zyxin, a focal adhesion protein, is recruited at force-bearing sites on the actin cytoskeleton and, therefore, identifies zyxin as a mechano-sensing protein candidate . Furthermore, zyxin accumulation at force-bearing sites requires the LIM domain located at the C-terminus of zyxin. The zyxin LIM domain consists of three LIM motifs, each containing two zinc-binding sites. Since individual LIM motifs do not accumulate at focal adhesions or force- bearing sites, we hypothesize that multiple zyxin LIM domains increase force sensitivity. Using a miniature force sensor and GFP-tagged LIM variants, we quantified the relationship between single, tandem dimer and trimer LIM protein localization and traction forces. While the presence of extra LIM domains affected VASP recruitment to focal adhesions, force sensitivity was not enhanced over the single LIM domain. Therefore, zyxin force sensitivity is optimal with a single LIM domain, while additional LIM domains fail to enhance force sensitivity.
Fibroblast and Pre-Osteoblast Cellular Activity on Graded Polydimethylsiloxane
C. S. Tam1, P.T. Nguyen1, T. R. Zink1, P. Loomer2, Y. Zhang2, S. P. Ho1
(1) Department of Preventative and Restorative Dental Sciences, and
(2) Department of Orofacial Sciences, UCSF, CA 94143
Regeneration of hard-soft tissues such as bone-ligament complexes continues to be challenge. In this study, we propose the use of elastically graded materials as a biomimetic solution for forming such tissue interfaces. Cellular activity of three cell lines (NIH3T3 fibroblasts, MC3T3 pre-osteoblasts, human periodontal ligament (hPDL) fibroblasts) as a function of varying polydimethylsiloxane (PDMS) substrate elasticity values was evaluated. By altering PDMS base-curing agent ratios (w/w), a total of five substrates were created and tested: three discrete (stiff/6.5Mpa, intermediate/1.7Mpa, compliant/0.2Mpa) and two graded (ramp, step – both with ratios ranging from 60:1 to 5:1). Implementing timelapse microscopy, hemocytometry, and immunohistochemistry, we evaluated rate of cell adhesion by identifying cell morphology, cell adhesion strength via trypsin study, and cell proliferation rate via fluorescent imaging of cell nuclei and metabolic activity (mitochondria). For discrete PDMS, pre-osteoblasts adhered/grew 3x faster on stiffer substrates, while fibroblasts adhered/grew 2x times faster on more compliant substrates. MC3T3s and NIH3T3s exhibited strong adhesion (2.3% adherent cells, overall cell numbers >1200 on day 6), whereas hPDLs exhibited weak adhesion (0% adherent cells, overall cell numbers <50 on day 6). These results were consistent on graded PDMS, however NIH3T3s and MC3T3s grew seven and four times faster, respectively, by day 6 along ramp graded regions compared to all other areas on other substrates. Cells grown on graded step PDMS showed no difference in proliferation rate at junction regions. Our findings indicate that cells respond to differentials in elasticity based on the mechanical properties of the native extracellular matrices from which they originate. Furthermore, the elasticity of natural bone-ligament interfacial regions resemble functional gradients as opposed to discrete segments. Future studies will include the implementation of stem cells and examination of stiffness graded PDMS on a tissue level.
Exploring Tissue Architecture via Programmed Assembly: Organotypic, Heterotypic, and Multiscale
Michael Todhunter, Noel Jee, and Zev Gartner
UC San Francisco
The structure of biological systems is essential to their function. We are interested in structure at the tissue level, which includes everything from the bilayered acini of the mammary gland to the cortices of the brain. To understand biological systems such as these, we must be able to observe and manipulate them. We endeavor to facilitate these needs via tissue engineering. Using DNA-mediated programmed assembly and microscale direct writing, we have designed a method for the creation of microtissues with exceptionally controlled structure, defining their overall architecture at the centimeter scale all the way down to their cell-cell contacts at the micron scale. Furthermore, we can simultaneously organize multiple cell types to build heterotypic microtissues and embed them in biological matrices for organotypic cell culture. The method involves writing single-stranded DNA on a templating surface and displaying complementary single-stranded DNA on cells. We use DNA hybridization like molecular Velcro to snap these cells to the locations of the written DNA. This process is fast, strong, programmable, and reversible. The method works sufficiently well that, by using it, we have been able to explore otherwise intractable questions in mammary gland biology.
Effect of Biaxial Mechanical Stimulation on Maturation of Human Embryonic Stem Cell-Derived Cardiomyocytes.
David Tran, Claire Robertson, Linda McCarthy, Tim Smith, Steven George
As the heart develops within an embryo, cardiac cells are subject to a transient mechanical environment that changes with maturity. Studies in chick models have shown mechanical loading is essential for normal cardiac morphogenesis. Past work on in vitro cellular response to mechanical stimulation includes uniaxial and radially equiaxial strains, which does not recapitulate the cardiac embryonic environment of anisotropic, biaxial strain. Human embryonic stem cell-derived cardiomyocytes (hESC-CM) have an immature phenotype in regards to beating rate, sarcomeric organization and calcium handling. We hypothesized that anisotropic biaxial strain would stimulate the maturation of hESC-CM.
We have developed a new system capable of applying controlled arbitrary dynamic planar strain regimes to hESC-derived cardiomyocytes cultured as a monolayer. An Instron biplanar axial testing system was adapted to custom machined arms that attach to a flexible polydimethysiloxane (PDMS) culture device. The system delivers controlled magnitudes of stretch to the PDMS device, and thus controlled strain to the cultured cells. Movement within the culture area of the PDMS device is tracked to determine induced strain. The operating strains have been designed to mimic the in vivo embryonic biaxial cardiac environment, delivering 25% strain in the x-axis and 18% strain in the y-axis.
Human embryonic stem cells (H9 transduced with mCherry under control of an α-MHC promoter) were differentiated into cardiomyocytes using serial application of Matrigel, activin A, BMP-4 and bFGF. Differentiation was quantified by FACS analysis of mCherry-‐positive cells. Up to 64% of the cells produced by this protocol were hESC-CM. Once differentiated, hESC-CM were subjected to up to 4 hours of 19% pure uniaxial strain. Beating rate of mechanically stimulated samples remained constant while control hESC-CM increased in beating rate (Fig 1). We conclude that hESC-CM phenotype is sensitive to mechanical stimulation.
Derivation and characterization of endothelial cell subpopulations from embryonic stem cells using serum-free conditions
Lian E. Wong, Alicia A. Blancas, Drew E. Glaser, Kara E. McCloskey
Endothelial cells (ECs) have the capability to be utilized in a wide variety of therapeutic strategies, especially applied to vascular and cardiovascular systems. Embryonic stem cells (ESCs) are used to induce differentiation into ECs because of their pluripotency and self-renewing capabilities. ECs derived from ESCs have also been shown to play a major role in angiogenesis. Recently, specialized ECs called tip, stalk, and phalanx cells have been identified as playing key roles in the formation of new blood vessels. Using our serum-free medium formulations, we set out to generate these distinct EC subphenotypes from ESCs . These ESC-EC derived cells were then extensively characterized for expression of tip, stalk, and phalanx-specific EC markers: Notch-1, DLL4, CXCR4, and Tie-2. The results indicate that we can generate EC populations that include highly angiogenic tip and stalk EC, and we can also purify the EC for the less angiogenic phalanx-type ECs.
References: 1.Blancas, A., Shih, AJ, Lauer, NE, McCloskey KE, Endothelial Cells from Embryonic Stem Cells in a Chemically Defined Medium. Stem Cells and Development, 2011.
Master’s or Undergraduate Capstone Projects
Equine Lameness Monitoring: Sensor Insert for Soft-Ride Boot
Anahid Ebrahimi, Matt Halverson, Derek Pell, Amanda Borer
Laminitis is an inflammatory disease that manifests in the nail beds of horses and can cause extensive hoof pain. The disease often develops due to internal factors (i.e. poor nutrition, intestinal disorders, etc.) and may not be outwardly visible. Signs that horses are suffering from laminitis include frequently shifting from limb to limb, spending less time standing, and favoring unaffected limbs (usually the back two hooves). Dr. Alonso Guedes is an anesthesiologist in the Department of Surgical and Radiological Sciences at the UC Davis School of Veterinary Medicine. Pain relief medications are used in his research on laminitis-affected horses, but there is no satisfactory way of quantifying the success of his treatment. Currently, veterinarians use a subjective rating scale to assess the horse’s improvement in response to the treatment. Although a quantitative solution does exist (Tekscan®’s Hoof System), it is neither affordable nor feasible for long-term use. An objective way to determine pain in these animals is necessary for Dr. Guedes and future veterinarians to continue to improve their understanding and treatment of the disease. Dr. Guedes requested a wireless system that can objectively measure the frequency of weight shifting from limb to limb and the weight distribution across a horse’s hooves, over a period of two weeks. Our solution is to insert a force sensor within a therapeutic horse boot and wirelessly transmit the force readings for data processing. Weight distribution will be given directly by the sensor output, and frequency of weight shifting will be calculated in data reduction. Working prototypes of the device are currently being manufactured and tested.
Functional polyelectrolyte embedded in artificial nanochannel
D. Hwang, A. Wollenberg, B. Vilozny, B. Singaram, N. Pourmand
UC Santa Cruz
Using the characteristic of the artificial nanochannel, charge dependent polyelectrolyte sensor was synthesized and embedded at the tip of the sensing zone allowing us to measure concentration of saccharide. Synthesized from quartz glass, nonbiological nanopores have distinct characteristic of negatively charged wall surface which induce more current flow when negative voltage applied. As a linear voltage is applied from -1 to 1 Volt, the I-V curve of a standard nanopores displays phenomenon of “negative rectification.” Due to the negatively charged wall surface, more negative ions are allowed through the pore compared to positive ion. Utilizing this characteristic, surface chemistry attachment of polymer or antibodies transforms nanopores into sensitive biosensor. As a result, a simple functionlization of a nonbiological nanopore will transform into a label free biosensor without complex chemical synthesis. Integrated with electrical circuits such as amplifier and digitizer, we are able to collect quantitative data and perform analysis. Sophisticated instruments allows detections within picoamp and milisecond range with small voltage applied. Artificial polylelctrolyte is synthesized with boronic acid receptor to detect saccharides. The polylectrolyte synthesized properties of soluble in solvents, poor solubility in water, change in properties in the presence of saccharides, and reversible signal. The overall postive charged polyelectrolyte attach with the negative charged wall to form label free biomarker with simple surface chemistry. Utilizing the difference between aqueous and non aqueous interface, we were able to embed the non aqueous polyelectrolyte in the aqueous solution filled nanochannel forming a gel like interface. As sugar pass through the interface, the positively charged boronic acid on the polyelectrolyte will undergo conformational change become negatively charged. The reverse in rectification indicated successful functionalization, and when saccharides are presents, the signal revert back to negative rectification depending on the concentration. The response time of the saccharide sensor is known to be within minutes. The combinations of the two techniques yield a full electrical integration, label free saccharide sensor with reversible reading. This could lead to miniaturize, reversible, and refined continuous saccharide sensor for the market.
Microfluidic Immunomagnetic Escherichia coli Separation Using Shrink Induced Nano-magnetic Traps
Dharmakeerthi Nawarathna, Nazila Norouzi, Jolie McLane, Scott Strayer, Himanshu Sharma, Aaron Chen, and Michelle Khine.
Immunomagnetic cell separation allows high-throughput sorting of target cells based on surface markers. Currently fluorescence activated cell (FACS) and magnetic-activated cell sorting (MACS) systems have been considered as the gold standards for cell separation [1-4]. However these methods are not suitable for rare cell isolation in dilute samples. Combining MACS with microfluidic technology provide simple but highly advantageous bacteria separation systems with high sorting efficiency [5,6]. This technique combined with the aid of magnetic particles could allow rapid and real-time detection of various Escherichia coli (E.coli) strains with high capture recovery and purity [7,8]. The feasibility in immunomagnetic E.coli separation is demonstrated by creating a rapid and reproducible approach to fabricate nano-wrinkled nickel structures (NWNS) of tunable size on a shape memory polymer and incorporated into a microfluidic channel. The NWNS are formed by depositing a thin layer of nickel on a polymer and heating the polymer substrates to cause the substrates to reduce its area to less than 5% of its original size and therefore induces the stiffer, nonshrinkable metal film to form wrinkles (Figure 1) . When E.coli flows over the externally magnetized magnetic traps, these nano-structures with high magnetic field gradients trap fluorescently labeled E.coli O157:H7 attached to antibody immobilized magnetic beads inside the microfluidic channel. The trapped bacteria are collected by turning off the external magnetic field. Similar concept has been applied to separate 1um magnetic beads from 1 um polystyrene beads with the high purity of 98% and enrichment of 10,000 fold. Conventional detection methods prove to be time-consuming however this device can be employed to separate and detect E.coli attached to magnetic beads in the small sample in minutes and is a useful alternative for routine analyses as a specific diagnostic assay without the need for an expensive setup and well trained technicians .
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A Detachable, Magnetic Grasper for Single Incision Laparoscopic Surgery (SILS TM)
Keiko Amano, Matthew Sander, Yang Zhao, Michael Harrison
We have developed a detachable, magnetically operable grasper for single incision laparoscopic surgery (SILS). It consists of 3 parts: a grasper, which grips and retracts tissue of interest and is anchored to a small magnet; an external rare earth magnet, which engages the grasper magnetically from outside the body, controlling its retraction angle and position; and a handle which deploys, controls, and retrieves the grasper (See Figure 1A). To operate the device, both the grasper and handle are inserted through a surgical incision. Once inserted, the grasper prongs enclose around the tissue of interest. This is accomplished by the conversion of a torque applied at the handle into a linear motion, which elastically deforms the prongs (see Figure 1B). Deformation allows the prongs to tightly squeeze together, gripping an organ or vessel. Engagement with the external magnet allows the organ or vessel to be pulled towards the abdominal wall at various force and vector angles (See Figure 1C). This conceived surgical device has demonstrated initial success at UCSF’s surgical proof of concept lab in artificial human models with 1.8 cm and 2.5 cm thick abdominal walls. Future work includes continual rapid prototyping and iterative design, as well as pre-clinical studies, and submission to the FDA as a class 1 medical device, with an indication of SILS cholecystectomy. We believe ours and similar magnetic technology has the capacity to revolutionize the field of minimally invasive surgery, reducing incisions, and facilitating angles of operation that were previously impossible.
Home Monitoring: Early Detection of Asthma Flares
Charvi Shetty, Vinidhra Mani
Asthma, a disorder causing airway constriction, affects 34.1 million people in the United States. In the clinical setting, this degenerative, heterogeneous disorder with varied triggers is often subject to grossly generalized treatments. This is because very little information exists on patterns and predictability of asthma attacks. Thus, clinicians exhibit a need for more precise and relevant information to properly treat asthma and prevent long-term damage to the respiratory system. Through an extensive needs finding process, it seemed that there is an implicit need for a noninvasive, consistent monitoring of a patient’s asthmatic flares. In order to effectively track a patient’s progression of asthma over time and have the greatest impact, the target group was specified to children ages 8-12 with severe persistent asthma. The solution presented is an inhome, portable asthma monitoring device with components for airflow and chemical measurement, which can be integrated onto a spacer that simultaneously delivers asthma medication to children. The device is easy-to-use and data gathered is clinically relevant, with the potential to predict the onset of an asthma attack. When testing our functional prototype, with spirometry features intact, against a known benchmark, comparable results were obtained but differences between the integrated device and control were statistically significant. Multiple rounds of testing revealed that airflow calibration is very sensitive to changes in ambient pressure. The main consideration in designing a more advanced prototype is a more sensitive differential pressure sensor, which can then be calibrated on the integrated device using a flow syringe. A nitric-oxide level detector would also have to be calibrated before implementation into the device and software would need to be redesigned for compatibility with LabView and other pertinent software interfaces.
Protein Engineering of a Mesophilic DNA Polymerase For Increased Salt Tolerance in Nanopore DNA Sequencing.
Akeson, M., Brar, R., Davidson, T., Erkander, J., Huynh, S., Olsen, H., Shelansky R., Spilman, C., Wells, L., Wescoe, Z.
UC Santa Cruz
Nanopore DNA sequencing technology provides an elegant, innovative platform for accurate, low-cost, rapid whole-genome DNA sequencing. Φ29 DNA polymerase is used as a molecular motor to precisely control translocation of DNA through the nanopore DNA sequencer. Increased salinity of buffers used in nanopore sequencing improves sequencer performance through increased discrimination between individual DNA bases as they transit through the nanopore sequencer. The overall goal of this project is to engineer Φ29 DNA polymerase to retain function in buffer salt concentrations up to 1 M KCl. In the present study, we have used information on differences in known structural motifs between mesophilic and halophilic protein homologs, and DNA sequence/protein structure alignments of halophilic (salt-loving) DNA polymerases with Φ29 DNA polymerase, to identify 17 candidate amino acid mutations for improving Φ29 DNA polymerase halotolerance. To date, ten of seventeen site-directed mutants have been generated, sequence confirmed, and expressed at the protein level in E. coli. Testing of enzymatic properties of these mutants is in progress with two mutants exhibiting polymerase extension activity less than wildtype Φ29 DNA polymerase. Site directed mutagenesis to generate remaining candidate mutations, and enzyme assays for halotolerance are in progress. Increased halotolerance of Φ29 DNA polymerase will contribute to the further improvement in the performance of the nanopore sequencing platform.
Q-Path: A Flow Through High-Throughput Quantitative Histology Platform
Andrew Tan, Armin Arshi, David Kuo, Robert Lee, Elizabeth Ng
UC Los Angeles
A hallmark of transitional cell carcinoma is the presence of primary tumorigenic cells in urine samples of afflicted patients. While cytopathologists exploit simple parameters of these rare cells to make clinical decisions, sample preparation and analysis is often time-consuming, subjective, and low in sensitivity, resulting in a significant number of false negatives and late diagnoses. Here, we report the development of the Quantitative Pathologist, or Q-Path, a high-throughput flow through imaging system that can achieve objective and quantitative analysis of clinical samples. Simulated urine suspensions containing stained cells are processed in a microfluidic device at flow rates of up to 1 mL min-1 and inertially focused to achieve highquality color images of cells, which are then analyzed using a customized MATLAB program. The output of this automated process is a comprehensive profile of cellular parameters of interest to the diagnosing physician, including nuclear-to-cytoplasmic area ratio, nuclear-to-cytoplasmic chromaticity ratio, nuclear area, cytoplasmic area, and nuclear morphological irregularities. As an application, this concatenated platform was used to establish a histology index to quantitatively depict with high statistical confidence morphological differences between three breast cell lines: benign MCF10A epithelial cells, low-grade MCF7 adenocarcinoma cells, and high-grade MCF7 cells modified with 12-O-tetradecanoylphorbol 13-acetate. We envision that such a versatile tool can address many of the deficiencies associated with traditional urine cytopathology and may make available opportunities for low-cost, quantitative, and sensitive screenings to aid clinical decision making.