Optimization of a PDMS-Based Cell Culture Substrate for High-Density Human-Induced Pluripotent Stem Cell Adhesion and Long-Term Differentiation into Cardiomyocytes under a Xeno-Free Condition

Abstract

Despite the numerous advantages of PDMS-based substrates in various biomedical applications, they are limited by their highly hydrophobic surface that does not optimally interact with cells for attachment and growth. Hence, the lack of lengthy and straightforward procedures for high-density cell production on the PDMS-based substrate is one of the significant challenges in cell production in the cell therapy field. In this study, we found that the PDMS substrate coated with a combination of polydopamine (PDA) and laminin-511 E8 fragments (PDA + LME8-coated PDMS) can support human-induced pluripotent stem cell (hiPSC) attachment and growth for the long term and satisfy their demands of differentiation into cardiomyocytes (iCMs). Compared with prior studies, the density of hiPSCs and their adhesion time on the PDMS surface were increased during iCM production. Although the differentiated iCMs beat and produce mechanical forces, which disturb cellular attachments, the iCMs on the PDA + LME8-coated PDMS substrate showed dramatically better attachment than the control condition. Further, the substrate required less manipulation by enabling one-step seeding throughout the process in iCM formation from hiPSCs under animal-free conditions. In light of the results achieved, the PDA + LME8-coated PDMS substrate will be an up-and-coming tool for cardiomyocyte production for cell therapy and tissue engineering, microfluidics, and organ-on-chip platforms.

Molecular imprinting as a simple way for the long-term maintenance of the stemness and proliferation potential of adipose-derived stem cells: an in vitro study

“…4 The in vivo microenvironment provides numerous regulating stimuli not available in conventional cell culture methods. 12 In native tissues, cells do not live alone and continually sense and interact with the extracellular matrix (ECM). 13 The ECM supports most cellular functions chemically, mechanically, and physically, 13,14 and can determine the fate of stem cells.…”

“…[29][30][31][32][33][34] The most often used substrates for in vitro cell culture are tissue culture plates that increase the likelihood of osteogenic differentiation because of the substrate rigidity. 35 So, in stem cell-based regenerative medicine, suitable cell culture platforms providing well-defined biophysical and biochemical properties are needed to mimic the in vivo microenvironments, 12 and simulate identical cell proliferation and differentiation conditions. 36 Nonetheless, designing a substrate that will give the required biological cues to drive cells still is challenging.…”

“…42 Also, against conventional TCP, PDMS has many other advantages such as nontoxicity, adjustable physico-mechanical properties, easy moldability, precision at micro-scale and nanoscale, and low manufacturing costs, which make it desirable as a cell culture substrate. 12 As a substrate to study cell behavior, PDMS provides a better understanding of cell behavior toward stretching, 43 stiffness, 44 mechanical stimulation, 45 and topography. [46][47][48][49] However, the surface hydrophobicity of PDMS has a negative effect on cell adhesion and, consequently, normal cell behaviors and functions.…”

“…[46][47][48][49] However, the surface hydrophobicity of PDMS has a negative effect on cell adhesion and, consequently, normal cell behaviors and functions. 12 Additionally, because cells and especially stem cells do not intrinsically attach to any surface, some surface modifications are mandatory for PDMS to improve cell attachment and spreading. 50 So, many approaches have tried to alter the PDMS surface somehow to overcome its limitations, 51,52 which can be done by plasma treatment, ECM protein coating, or other molecular coatings.…”

“…Despite better cell adhesion and spreading acquired via these modifications, they are not practical for all cell lines, can be influenced by variable conditions, and cannot ensure the expected demand for the qualified cell source for human therapeutic use. 12 This journal is © The Royal Society of Chemistry 2022 Some studies have focused on using cellular patterns as biophysical stimulating factors to direct cellular functions in a specific direction. For the first time, Mahmoudi et al prepared a cell-imprinted substrate by PDMS casting and polymerization on mature chondrocytes to induce chondrogenic differentiation in rabbit adipose tissue-derived mesenchymal stem cells.…”