In vitro cytocompatibility evaluation of a thermoresponsive NIPAAm-MMA copolymeric surface using L929 cells.

Scaffold free tissue constructs are preferred in tissue engineering as they overcome all the problems associated with scaffolds. Stimuli responsive polymers enable generation of scaffold free multilayered tissue constructs which would in turn reduce the use of biomaterials in vivo. In this study, we investigated cytocompatibility and thermoresponsiveness of a copolymer of N-Isopropylacrylamide and Methyl Methacrylate. Thermoresponsive surfaces were prepared by coating tissue culture polystyrene with the copolymer solution in isopropanol. Mammalian fibroblast cells (L929 cells) readily adhered on the copolymer. The viability and cellular activity was ensured through Neutral red staining, MTT assay, Tritiated thymidine uptake assay and Immunofluorescent staining for cytoskeletal organisation. Incubation under lower critical solution temperature of copolymer resulted in intact detachment of cells. To conclude, in-house synthesized cytocompatible smart culture substrate intended for tissue engineering was developed using a cost effective and simple technique. Moreover, presence of methyl methacrylate in the copolymer reduced the lower critical solution temperature facilitating extended in vitro manipulation time. As the copolymer is insoluble in water, the copolymer could be polymerised without additional crosslinkers.

Optimization of culture conditions for an efficient xeno-feeder free limbal cell culture system towards ocular surface regeneration.

Ex vivo expansion of limbal stem cells from a small biopsy and its subsequent transplantation is the golden choice of treatment for limbal stem cell deficiency. Use of murine 3T3 feeder layer is a prerequisite for this ex vivo expansion. There is an ever-increasing demand for feeder free cultures to avoid xenotoxicity and transmission of xeno-diseases to human system. This study was aimed to establish an efficient xeno-feeder free limbal culture system towards ocular surface regeneration. To study the effect of initial dispase treatment and culture system used, migratory distance of cells from explants was analyzed from phase contrast images using "interactive measurements" of Qwin software (Leica). Expression of p63 in different culture systems was studied by immunofluorescent staining, followed by quantitative confocal microscopy (Carl Zeiss). Results showed dispase treatment was not necessary for establishing limbal explant culture. A combination of Iscove's modified Dulbecco's medium and Panserin 801 resulted in formation of autofeeder layer with maintenance of progenitor characteristics, thus mimicking natural tissue architecture. Further analysis of this culture system showed that cells could be cultured till confluency. Immunofluorescent staining of ABCG2 revealed presence of stem cell marker in the confluent cell layer. Scanning Electron Micrographs demonstrated homogenous population of tightly packed cells in this culture system. Replacement of bovine serum with autologous serum did not affect morphology or growth of cells in this culture system. This study will be a major step in the development of xeno-feeder free epithelial equivalents towards ocular surface reconstruction.