Use of Human Fibroblasts in the Development of a Xenobiotic-Free Culture and Delivery System for Human Keratinocytes

Abstract

Previous work has shown that keratinocytes can be cultured serum-free on an acid-functionalized, plasma-polymerized surface (for subsequent delivery to patients' wound beds) by inclusion of a fibroblast feeder layer. This study seeks to extend this work by substituting human for murine feeder cells in serum-free culture and examining the performance of keratinocytes expanded in this way to transfer to an in vitro human dermal wound bed model. We compared murine and human fibroblasts (both short-term dermal fibroblasts and a fetal lung fibroblast cell line MRC-5, which has a long history in human vaccine production), alternative methods for growth-arresting fibroblasts, establishing culture of cells serum-free, and the impact of culture with fibroblasts on the differentiation of the keratinocytes. Irradiated human and murine fibroblasts were equally effective in supporting initial keratinocyte expansion, both in the presence and absence of serum. Keratinocytes were significantly less differentiated, as assessed by measuring involucrin expression relative to DNA when grown serum-free with fibroblasts than when grown with serum. Initial cultures of fibroblasts and keratinocytes could be initiated serum-free but were much slower to establish than if serum were used. Transfer of keratinocytes from keratinocyte/fibroblast co-cultures cultured on a plasma polymer surface to a human dermal wound bed model was as successful as from monocultures in both serum and serum-free cultures. In summary, we have revisited a well-accepted methodology for expanding human keratinocytes for clinical use and avoided the use of bovine serum and a mouse fibroblast feeder layer by introducing an irradiated human fibroblast feeder layer.

Epithelialization of hydrogels achieved by amine functionalization and co-culture with stromal cells

“…The approach of co-culture which was introduced in these experiments was based on prior work from our laboratories on skin cell culture, where we have shown that fibroblasts will promote keratinocyte proliferation in the absence of serum on a variety of substrates [25][26][27][28]. The fibroblast feeder layer also reduces the degree of differentiation of keratinocytes, which may be advantageous for initial wound healing, and the fibroblasts can be derived from several sources -murine, human, donor or autologous cells [25].…”

“…This has not yet been achieved to the best of our knowledge. In this paper we describe how we have combined amine modification of the surface of a hydrogel with a knowledge of epithelial/stromal cell dependency (as detailed in [24][25][26][27][28] to control and optimise epithelialization on hydrogel surfaces. In brief our prior experience with stromal cell/epithelial cell co-culture has shown that it is possible to achieve epithelial cell growth (both primary human keratinocytes [25][26][27][28] and primary human corneal epithelial cells) [29] on a range of substrates providing one co-cultures them with stromal cells.…”

“…In brief our prior experience with stromal cell/epithelial cell co-culture has shown that it is possible to achieve epithelial cell growth (both primary human keratinocytes [25][26][27][28] and primary human corneal epithelial cells) [29] on a range of substrates providing one co-cultures them with stromal cells. Cell culture conditions can be achieved where stromal cells act as initial support/feeder cell for epithelial cells so that epithelial cells can be grown serum-free on substrates where they would not previously grow unaided [25][26][27][28][29]. .…”

A Chemically Defined Carrier for the Delivery of Human Mesenchymal Stem/Stromal Cells to Skin Wounds

“…Over the past decade, our group has utilized the technique of plasma polymerization to deposit thin films of acrylic acid on a variety of substrates. We have used these surface modifications to make materials supportive of the attachment and growth of a number of cell types (e.g., keratinocytes, melanocytes, and corneal epithelial cells), [16][17][18][19][20][21] and demonstrated delivery of human keratinocytes 19 and cocultures of keratinocytes and melanocytes 17,18 to in vitro model wound beds. These cell carriers have been used successfully in the clinic to deliver autologous keratinocytes for the treatment of patients with extensive skin loss resulting from burn injury and diabetic foot ulcers.…”

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“…[13][14][15][16] Irradiated human dermal fibroblasts and mouse fibroblasts (3T3 cells) can improve the life span of human keratinocytes. Therefore, the present study was performed to evaluate various conditions such as culture medium and feeder layer use on keratinocyte morphology and proliferation in our cell culture laboratory.…”