Zaw Win

Long-term vascular contractility assay using genipin-modified muscular thin films.

Vascular disease is a leading cause of death globally and typically manifests chronically due to long-term maladaptive arterial growth and remodeling. To date, there is no in vitro technique for studying vascular function over relevant disease time courses that both mimics in vivo-like tissue structure and provides a simple readout of tissue stress. We aimed to extend tissue viability in our muscular thin film contractility assay by modifying the polydimethylsiloxane (PDMS) substrate with micropatterned genipin, allowing extracellular matrix turnover without cell loss. To achieve this, we developed a microfluidic delivery system to pattern genipin and extracellular matrix proteins on PDMS prior to cell seeding. Tissues constructed using this method showed improved viability and maintenance of in vivo-like lamellar structure. Functional contractility of tissues fabricated on genipin-modified substrates remained consistent throughout two weeks in culture. These results suggest that muscular thin films with genipin-modified PDMS substrates are a viable method for conducting functional studies of arterial growth and remodeling in vascular diseases.

Guidelines for the Isolation and Characterization of Murine Vascular Smooth Muscle Cells. A Report from the International Society of Cardiovascular Translational Research

Vascular smooth muscle cells (VSMCs) play important roles in cardiovascular disorders and biology. Outlined in this paper is a step-by-step procedure for isolating aortic VSMCs from adult C57BL6J male mice by enzymatic digestion of the aorta using collagenase. The plating, culturing, and subculturing of the isolated cells are discussed in detail along with techniques to characterize VSMC phenotype by gene expression and immunofluorescence. Traction force microscopy was used to characterize contractility of single subcultured VSMCs at baseline.

Microfluidic genipin deposition technique for extended culture of micropatterned vascular muscular thin films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues. Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

Investigation of human iPSC-derived cardiac myocyte functional maturation by single cell traction force microscopy

Recent advances have made it possible to readily derive cardiac myocytes from human induced pluripotent stem cells (hiPSC-CMs). HiPSC-CMs represent a valuable new experimental model for studying human cardiac muscle physiology and disease. Many laboratories have devoted substantial effort to examining the functional properties of isolated hiPSC-CMs, but to date, force production has not been adequately characterized. Here, we utilized traction force microscopy (TFM) with micro-patterning cell printing to investigate the maximum force production of isolated single hiPSC-CMs under varied culture and assay conditions. We examined the role of length of differentiation in culture and the effects of varied extracellular calcium concentration in the culture media on the maturation of hiPSC-CMs. Results show that hiPSC-CMs developing in culture for two weeks produced significantly less force than cells cultured from one to three months, with hiPSC-CMs cultured for three months resembling the cell morphology and function of neonatal rat ventricular myocytes in terms of size, dimensions, and force production. Furthermore, hiPSC-CMs cultured long term in conditions of physiologic calcium concentrations were larger and produced more force than hiPSC-CMs cultured in standard media with sub-physiological calcium. We also examined relationships between cell morphology, substrate stiffness and force production. Results showed a significant relationship between cell area and force. Implementing directed modifications of substrate stiffness, by varying stiffness from embryonic-like to adult myocardium-like, hiPSC-CMs produced maximal forces on substrates with a lower modulus and significantly less force when assayed on increasingly stiff adult myocardium-like substrates. Calculated strain energy measurements paralleled these findings. Collectively, these findings further establish single cell TFM as a valuable approach to illuminate the quantitative physiological maturation of force in hiPSC-CMs.

Empirically determined vascular smooth muscle cell mechano-adaptation law

Cardiovascular disease can alter the mechanical environment of the vascular system, leading to mechano-adaptive growth and remodeling. Predictive models of arterial mechano-adaptation could improve patient treatments and outcomes in cardiovascular disease. Vessel-scale mechano-adaptation includes remodeling of both the cells and extracellular matrix. Here, we aimed to experimentally measure and characterize a phenomenological mechano-adaptation law for vascular smooth muscle cells (VSMCs) within an artery. To do this, we developed a highly controlled and reproducible system for applying a chronic step-change in strain to individual VSMCs with in vivo like architecture and tracked the temporal cellular stress evolution. We found that a simple linear growth law was able to capture the dynamic stress evolution of VSMCs in response to this mechanical perturbation. These results provide an initial framework for development of clinically relevant models of vascular remodeling that include VSMC adaptation.

188. Functional Analysis of Human Induced Pluripotent Stem Cell-Derived Cardiac Myocytes

Recent and exciting advances in stem cell biology have made it possible to quickly and efficiently derive induced pluripotent stem cells (iPSCs) from human somatic cell types, as well as differentiate pluripotent cells to committed lineages, such as cardiac myocytes (hiPSC-CMs). The nature of these cells makes them ideal potential candidates for cell therapy treatments, such as implantation of hiPSC-CMs following myocardial infarction. Use in therapy requires optimization of these cells physiologically, which in turn requires appropriate assays to determine cell function beyond gene expression profiles. Difficulty in accurately characterizing hiPSC-CM function comes from large variability in cell size and shape, as well as isotropy of axis of contraction. We utilize here micropatterning techniques, whereby single cell-sized (2500 μm2) rectangles of fibronectin are applied to glass cover slips. The hiPSC-CMs are then cultured on the cover slip at a low density such that one cell occupies the rectangular shaped space. This persuades the cells to assume a rectangular shape, similar to that of an adult myocyte, and causes them to contract along the long axis of the rectangle. By normalizing size, shape, and alignment of hiPSC-CMs, microscopy can be used to determine and compare kinetics of contraction and relaxation, utilizing the IonOptix software and an inverted microscope. Additionally, this technique can be used for traction force microscopy. Here we use fibronectin patterns that are micropatterned onto a polyacrylamide hydrogel, and the hiPSC-CMs are cultured on top of it. Next, 0.2 μm diameter fluorescent beads are embedded in the hydrogel such that when the cell contracts, it pulls on the hydrogel, and movement of the beads is then tracked using confocal microscopy. An ImageJ program analyzes movement of the beads and uses the known Youngs modulus of the hydrogel to calculate traction force generated by the individual cells. Preliminary results show that iPSC-CMs will morphologically take the shape of the fibronectin stamp and will contract along the long axis of the rectangle. Additional quantification of these data will be presented and discussed.

Microfluidic Device for Spatial Control of Cell Seeding in Engineered Tissues

In vivo tissues have finely controlled hierarchical structure that is often difficult to mimic in vitro. Microfabrication techniques, such as microcontact printing, can be used to reproduce tissue structure in vitro by controlling cell shape and orientation [1]. Several recent results suggest that cellular organization and structure can influence tissue function in engineered tissues [2–4]. For example, using microcontact printing and muscular thin film technology, we recently demonstrated that engineered vascular tissues whose smooth muscle cells possessed more elongated spindle-like geometries, similar to in vivo structure, exhibited more physiological contractile function [5]. In these studies, cells were seeded using traditional imprecise seeding methods. But recent results have shown that cell-cell coupling plays a significant role in functional contractility [6], suggesting that not only cellular geometry, but cell-cell organization, within a tissue is important to reproduce in engineered tissues to mimic in vivo function.© 2013 ASME

High-Throughput Microtissue Contractility Assay for In Vitro Analysis of Vascular Mechanics

Vascular smooth muscle (VSM) plays a key role in regulation of vascular mechanics through modulation of contractile tone. Studies suggest that mechanical or biochemical perturbation can lead to dysfunctional VSM behavior [1, 2]. This aberrant contractility may play a role in vascular dysfunction ranging from cerebral vasospasm to aneurysm genesis.© 2013 ASME