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

Francis Bisson,Sylvain Guérin,Éloise Rochefort,Carolyne Simard-Bisson,Lucie Germain,Amélie Lavoie,Karine Zaniolo,Danielle Larouche,Odile Damour,François Auger

doi:10.3390/ijms14034684

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.

Highlights

The culture of keratinocytes in vitro has many clinical and research applications such as the treatment of chronic ulcers or burn patients, tissue-engineered skin for wound healing or pharmacological studies [1,2,3]
Keratinocytes grown with feeder layers reached a high number of passages in culture. These results suggest that human fibroblasts prevent the early terminal differentiation of keratinocytes through a molecular pathway involving Sp1, whereas defined media and the absence of a feeder layer lack the ability to maintain Sp1 expression, which correlates with the loss of the keratinocyte proliferative capability
To evaluate whether keratinocyte expansion in vitro, using defined media developed for serum/feeder layer-free keratinocyte culture systems, is equivalent to feeder layer culture systems, keratinocytes were cultured without any feeder layer in complete DME-F12, KGM-2, DKSFM or KGM-CD medium

Summary

Introduction

The culture of keratinocytes in vitro has many clinical and research applications such as the treatment of chronic ulcers or burn patients, tissue-engineered skin for wound healing or pharmacological studies [1,2,3] These epithelial cells, rapidly lose their proliferative capabilities and differentiate early when cultured in vitro under inappropriate conditions [4,5]. In 1975, Rheinwald and Green developed a method to produce cultured epithelial autografts (CEA), consisting of skin epithelial cells cultured with irradiated mouse 3T3 fibroblasts (i3T3) allowing the formation of a thin epithelial sheet suitable for grafting [5] This technique was rapidly applied clinically for the treatment of severely burned patients [6,7]. The molecular effects of the feeder layer on keratinocytes still remain poorly understood

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