Immortalization of Human Oral Keratinocytes with Defined Genetic Elements in the Development of Organotypic Oral Culture
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Primary cell culture is limited by the increase in cellular levels of p16INK4a in response to an in vitro culture environment and, in conjunction with telomere shortening following cell division, presents a barrier to cellular proliferation. The use of transformed cell lines is limited for studies wherein the aim is to generate data akin to an in vivo environment as commonly such cell lines achieve their immortal benefits by down regulating important tumour suppressive mechanisms and inhibiting cell cycle checkpoints. Normal Human Oral Keratinocyte (NHOK) cells expressing shp16+hTERT were generated and compared to NHOK cells expressing Bmi1 +hTERT using an optimized retroviral transduction protocol and compared simultaneously to an epidermal control. Population doubling assessment of cell lines revealed that shp16+hTERT was not sufficient to extend replicative lifespan in the absence of p53 whilst cell lifespan extension was observed not only in cells expressing Bmi1+hTERT, but also in cells transduced with Bmi1 alone. Upon characterization, cells showed expression of p53 and responsiveness to UVB-induced apoptosis as demonstrated by an increase in p53 expression. NHOKBmi1+hTERT displayed adaptability to serum free culture when weaned into keratinocyte serum free media (KSFM) and retained the ability to stratify into multiple layers when supported by feeders on polycarbonate membrane inserts. The cell line NHOKBmi1+hTERT will be beneficial for in vitro studies looking to utilise an alternative to transformed or spontaneously derived cell lines and holds potential for further development and optimization into a well characterized SSE in a user friendly, and reproducible system for the testing for oral products.
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