Cells are cultured on platforms made of a variety of materials with selected topographies during studies of cell response and behavior. Understanding the effects of substrates is essential for such applications as developing effective interfaces between body cells and implanted materials and devices. In this study, the effects of substrate surface properties on cell differentiation and alignment on C2C12 myoblasts cultured on conventional or fabricated polymeric cell culture substrates were investigated. Comparisons were made between cells cultured on tissue culture grade polystyrene (TCPS), glass, Permanox, and cured polydimethylsiloxane (PDMS) substrates. Fluorescent immunohistochemistry of cell markers was used to analyse the extent of differentiation. Alignment and guidance of cell growth and spread were studied using patterned platforms. Gratings were made on polystyrene (PS) and PDMS and differentiation was facilitated after 5 days by media exchange. Differences in cell morphology were observed between cells cultured on TCPS and PDMS substrates. Fully differentiated myotubes were observed in highest numbers on TCPS substrates and were non-detectable on PDMS substrates in the time frame of 144 h. Muscle cell alignment and their differentiation followed along the grating patterns on PS and elongated along the pattern length. On the other hand, on PDMS cells formed sheets of tissue and peeled from the substrate. We have revealed the potential for the combinations of surface materials and topography on cell behavior to induce accelerated differentiation and coordinated alignment. The results demonstrate that culture environment can be designed or engineered to modify or regulate muscle cell functions. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1638-1645, 2016.
BackgroundIt is becoming recognised that traditional methods of culture in vitro on flat substrates do not replicate physiological conditions well, and a number of studies have indicated that the physical environment is crucial to the directed functioning of cells in vivo. In this paper we report the development of a platform with cell-like features that is suitable for in vitro investigation of cell activity. Biological cells were imprinted in hard methacrylate copolymer using soft lithography. The cell structures were replicated at high nanometre scale resolution, as confirmed by atomic force microscopy. Optimisation of the methacrylate-based co-polymer mixture for transparency and biocompatibility was performed, and cytotoxicity and chemical stability of the cured polymer in cell culture conditions were evaluated. Cells of an endometrial adenocarcinoma cell line (Ishikawa) were cultured on bioimprinted substrates.ResultsThe cells exhibited differential attachment on the bioimprint substrate surface compared to those on areas of flat surface and preferentially followed the pattern of the original cell footprint.ConclusionsThe results revealed for the first time that the cancer cells distinguished between behavioural cues from surfaces that had features reminiscent of themselves and that of flat areas. Therefore the imprinted platform will lend itself to detailed studies of relevant physical substrate environments on cell behaviour. The material is not degraded and its permanency allows reuse of the same substrate in multiple experimental runs. It is simple and does not require expensive or specialised equipment. In this work cancer cells were studied, and the growth behaviour of the tumour-derived cells was modified by alterations of the cells’ physical environment. Implications are also clear for studies in other crucial areas of health, such as wound healing and artificial tissues.
This work introduces a novel process for the fabrication of polymer cell culture substrates containing physical topography based on timepoint specific cell phenotype replicas. Bioimprinting of human nasal chondrocyte at different cell adhesion time points was used to demonstrate the nanoscale replication process. Atomic force microscopy confirmed morphology progression at 1, 6, 12, and 24 h timepoints corresponding to dedifferentiation of the chondrocytes to fibroblast-like phenotype. Topographical analysis of the imprinted substrates yielded an inverse relationship between surface coverage, increasing from 11% to 87%, and maximum average pattern depth, decreasing from 1.2 lm to 430 nm. Methacrylate bioimprint features were successively replicated into a transitional polydmethylsiloxane mold used as an intermediate for further replication into polystyrene by a highthroughput embossing method. Substrates fabricated with this process have applications in stem cell engineering, regenerative medicine, and implantable devices.
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