Title: Biomaterial-based Cell Culture Platform for Podocyte Phenotype Study with Shape and Substrate Rigidity Control
Description: Cells sense and interact with their microenvironment to retrieve signals which include cell-matrix and cell-cell contacts. These signals account for the influence of culturing conditions and often control the local cellular phenotype and global functions of tissues. Here, I sought to understand if there is any information processed by cells in guiding cellular phenotype given the control of cell shapes and substrate rigidities. If there is, would these phenotypic changes achieve biomedical purposes? What is the strategy to engineer platforms that can handle the longstanding challenges in those fields? In this dissertation, the first chapter serves as an introduction which involves the origin of motivations, which mainly came from current challenges in biomedical researches of kidney podocytes. I have attempted to understand if it is possible to control podocyte differentiation through cell shape control which mimics their in vivo morphology. On the other hand, I have tried to reveal if it is possible that tissue stiffness can affect podocyte phenotype as a result of stiffness sensing.

These two topics were rarely investigated for kidney podocytes, which is the critical component of human filtration barrier to perform renal functions. The effort that addresses the question how shape and substrate rigidity as in- formation repositories affect kidney podocytes phenotype has profound meaning in the understanding of renal physiological system and pathological mechanisms. The second chapter will focus on the methods to achieve successful long-term shape control on cells. Engineered cell-device interface using cross-linking biomaterial SU-8 plays a key role in this study. Compared with other previously used approaches summarized in this chapter, SU-8 provides various advantages both in the fabrication of micro- pattern architecture as well as its sustaining effectiveness in controlling cell shape. This approach has been proved very efficient and economic to facilitate single cell level manipulation. The chapter will describe in details the interface micro-fabrication and encountered technical challenges.

The results that kidney podocytes were in good compliance with the micro-pattern proved the successful application of this technique. The third chapter will then transfer from micro-fabrication to biological experiments, which discusses in details how in intro kidney podocytes responded to their shapes by enforcing protein localization which characterizes a phenotype found in vivo. This phenotype among in vitro podocytes was further verified that it may contribute to podocytes differentiation and physiological functions. The information processed by shape was proved independent of tension-related processes and thus shape and tension could be regarded as separate contributors in cellular development. The interpretation of shape’s contribution could be referred to my previous publication in the journal of Cell: ”Decoding Information in Cell Shape”. In this study, the motifs of research were applied to other cell lines (Human vascular smooth muscle cell) as a step to generalize the ubiquity of shape’s contribution to cell differentiation.

The study here was to differentiate shape and tension through investigating the difference between two major mechanosensors: β3 and β1 integrin receptors. The difference in cell phenotypes through integrin inhibition experiments demonstrated critical but unique role of integrin-based shape sensing in vitro. This chapter in dissertation covers most of the content in a previously submitted paper to Nature Cell Biology. In the fourth chapter, I further carried out a study that investigated if stiffness sensing can influence kidney podocyte phenotype. The fourth chapter will basically review the techniques in the fabrication of hydrogel-based cell culture platforms. In a similar manner to previous study using biomimetic shape for podocytes and find its phenotype, the target of this analysis was to use hydrogel-based biomimetic substrate with renal physiological stiffness and find if there is a differentiation phenotype. Since numerous materials have been reported in hydrogel studies, I will focus on the introduction to representative ones that have been most widely used.

Their characteristics will be compared with the demands of kidney podocyte reasearch. Methodologies were the key to a successful research, and in this chapter I will describe in details what choices I made in choosing experimental methods that improved the efficiency and quality of cell culture platforms. A natural enzyme (microbial transglutaminase) cross-linked gelatin hydrogel was adopted here to provide ideal substrate rigidity control for podocytes. This method has demonstrated high efficiency and stability in making large cell culture surface. Moreover, it provides the hydrogel platform with an ideal range of elastic moduli suitable for renal tissue culture. The results will be discussed in detail in the fifth chapter. I successfully found a differentiation phenotype for podocytes cultured on the hydrogel platforms with a physiological stiffness. Similar phenotype, on the contrary, were not found in podocytes on platforms which were either too soft or too stiff. These resutls have formed one of my submitted paper to Scientific Report.

The differentiation phenotype for kidney podocytes was characterized by up-regulation of differentiation markers. These findings were in a similar manner to a series of stem cells differentiation guided by regulated substrate stiffnesses. This phenotype of kidney podocytes was verified by microarray technique which confirmed the stiffness sensing using transcription factors. The enrichment analysis of kinases also showed significant response of Src, Fyn etc, of which the activities have been shown critical for podocytes to preserve their physiological functions. These results have successfully suggested the close relations between stiffness changes of glomeruli basement membrane (GBM) and progressive podocyte dysfunction. In summary, this dissertation covers interdisciplinary researches that decoded the information processed by cells from two separate aspects: shape and stiffness sensing. The details in each chapter cover a broader scope than the content selected for publications. Through this dissertation, readers will get in touch with the technique developed for plat- form and their applications to biomedical researches. I wish this will help people new in the field to get my hands-on experience.