Lin Yang Research Summary Ucsf

Lin Yang 2010 UCSF Biomedical Sciences PhD My most significant research project was undertaken at Duke University as part of the Pratt Research Fellowship. I was attached to a cardiac tissue engineering lab headed by Dr. Nenad Bursac, and worked on the optimization of in vitro culture methods for the production of 3-dimensional (3D) cardiac bio-artificial muscle (BAM). These BAMs consisted of a mix of primary cardiomyocytes and cardiac fibroblasts embedded within a porous and highly hydrated 3D matrix (a ‘hydrogel’) supplemented with extracellular matrix proteins such as fibrin and collagen. After extended periods in culture, the enmeshed cells interact with the fibrin or collagen fibers to remodel and compact the matrix, thus creating bio-artificial muscles which could potentially mimic the biological behavior of native tissue. While the methods of tissue engineering are robust enough to give rise to tissues with some degree of functionality (for example, engineered cardiac tissue recapitulate key characteristics of the myocardium such as the ability to propagate action potentials and twitch spontaneously), the application of these tissue in regenerative medicine is limited by poor cell viability within the 3D matrix due to the hypoxia and nutrient deprivation. The aim of my project was to find novel methods to improve cell survival in 3D hydrogels with the long term goal of increasing the thickness and functionality of bio-artificial cardiac muscle. To tackle this problem, I first tested the effect of introducing hormones such as insulin to the culture media. Insulin is a key regulator of cell metabolism, and is thought to promote cell survival by participating in the PI3K/Akt pro-survival pathway to enhance the efficiency of glucose uptake by cells. I thus hypothesized that the delivery of insulin to engineered tissue during in vitro culture might be able to reduce the amount of cell death. After quantitatively assessing cell viability using the lactate dehydrogenase (LDH) assay, I discovered that the percentage of viable cells within BAMs was indeed increased with the addition of insulin. While I had ascertained the effects of insulin in promoting cell survival, the overall performance of the system was still unsatisfactory. This led me to approach the problem from another perspective. I found out that cardiomyocytes making up the muscle fibers in the native myocardium were highly aligned along one axis, and have been shown to survive better in the presence of static stretch. In contrast, hydrogels are isotropic materials and, as such, demonstrate limited abilities to mimic the architecture of native heart muscles. This inspired me to introduce conditions of passive loading during the biosynthesis process by fixing Velcro pieces to the walls of the hydrogel molds. I also supplemented the extracellular matrix with additional collagen in hopes that the collagen fibers would assist with the transmission of tension throughout the tissue. These modifications significantly improved cell viability as assessed by the LDH assay. Using immunostaining followed by confocal microscopy, I found that cells within the hydrogel were stretched out along the axis of tension as seen from their elongated cell nuclei. More importantly, immunostaining for the cardiac gap junction protein connexin-43 revealed that the cells were well coupled to each other in the 3D environment. This project has been an intense but very enriching research experience – it was not only my first independent project, but also the first time in which I had to design and optimize my own research protocols. One of the most valuable lessons I took away with me was

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Lin Yang 2010 UCSF Biomedical Sciences PhD the realization that successes in research come from keeping an open mind and daring to try unconventional methods. Indeed, had I limited myself solely to the screening of small molecules, the idea of using mechanical cues to modulate cell behavior and improve cell survival might never have occurred to me. I believe that my conscious efforts to dissect problems from different perspectives will guide me towards future successes in scientific research.

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