Irradiated Human Dermal Fibroblasts Are as Efficient as Mouse Fibroblasts as a Feeder Layer to Improve Human Epidermal Cell Culture Lifespan

Abstract

A fibroblast feeder layer is currently the best option for large scale expansion of autologous skin keratinocytes that are to be used for the treatment of severely burned patients. In a clinical context, using a human rather than a mouse feeder layer is desirable to reduce the risk of introducing animal antigens and unknown viruses. This study was designed to evaluate if irradiated human fibroblasts can be used in keratinocyte cultures without affecting their morphological and physiological properties. Keratinocytes were grown either with or without a feeder layer in serum-containing medium. Our results showed that keratinocytes grown either on an irradiated human feeder layer or irradiated 3T3 cells (i3T3) can be cultured for a comparable number of passages. The average epithelial cell size and morphology were also similar. On the other hand, keratinocytes grown without a feeder layer showed heavily bloated cells at early passages and stop proliferating after only a few passages. On the molecular aspect, the expression level of the transcription factor Sp1, a useful marker of keratinocytes lifespan, was maintained and stabilized for a high number of passages in keratinocytes grown with feeder layers whereas Sp1 expression dropped quickly without a feeder layer. Furthermore, gene profiling on microarrays identified potential target genes whose expression is differentially regulated in the absence or presence of an i3T3 feeder layer and which may contribute at preserving the growth characteristics of these cells. Irradiated human dermal fibroblasts therefore provide a good human feeder layer for an effective expansion of keratinocytes in vitro that are to be used for clinical purposes.

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“…23 Irradiated human dermal fibroblasts provide a suitable human feeder layer that has similar efficiency as mouse fibroblasts for effective expansion of keratinocytes in vitro, but keratinocytes grown without a feeder layer may stop growth after a few passages. 19 Researchers showed that coculture of follicular keratinocytes with dermal fibroblasts treated with mitomycin C supported the proliferation of keratinocytes. They noted that it is easier to handle human fibroblasts than 3T3 cells, and homologous fibroblasts better mimic the physiologic state.…”

“…Keratinocytes usually are cultured for 3 to 4 passages for clinical applications. 19 We could culture these cells up to 3 passages. When the keratinocytes could be cultivated after cryopreservation, it was evident that growth of cells with normal morphology was possible on feeder cells in serum-free medium.…”

“…19,20 The key to obtaining suitable keratinocytes is optimization of the culture system to support keratinocyte growth. We compared various conditions such as the presence of a feeder layer and type of medium on the growth of these cells.…”

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“…16,17 However, application of 3T3 cells for keratinocyte expansion is associated with the risk of contamination with infections and animal antigens. 18 At present, the focus of research is to discover an optimal protocol for keratinocyte culture.…”

Comparison between the cultures of human induced pluripotent stem cells (hiPSCs) on feeder-and serum-free system (Matrigel matrix), MEF and HDF feeder cell lines

“…HDF feeder could be used for iPSCs culture and provides suitable base to support the stem cells. HDF feeder in low passages (P 1-3) could decrease the immune response, easy handling, removing the contamination with animal pathogens, and maintains for long-term periods in the culture (Richards et al 2002;Tecirlioglu et al 2010;Bisson et al 2013;Ghasemi-Dehkordi et al 2014). In recent years, feeder-free culture such as BD Matrigel™ using MEF conditioned medium has been reported to be efficient for culture of stem cells.…”