Agent Based Modelling Helps in Understanding the Rules by Which Fibroblasts Support Keratinocyte Colony Formation

Abstract

BackgroundAutologous keratincoytes are routinely expanded using irradiated mouse fibroblasts and bovine serum for clinical use. With growing concerns about the safety of these xenobiotic materials, it is desirable to culture keratinocytes in media without animal derived products. An improved understanding of epithelial/mesenchymal interactions could assist in this.Methodology/Principal FindingsA keratincyte/fibroblast o-culture model was developed by extending an agent-based keratinocyte colony formation model to include the response of keratinocytes to both fibroblasts and serum. The model was validated by comparison of the in virtuo and in vitro multicellular behaviour of keratinocytes and fibroblasts in single and co-culture in Greens medium. To test the robustness of the model, several properties of the fibroblasts were changed to investigate their influence on the multicellular morphogenesis of keratinocyes and fibroblasts. The model was then used to generate hypotheses to explore the interactions of both proliferative and growth arrested fibroblasts with keratinocytes. The key predictions arising from the model which were confirmed by in vitro experiments were that 1) the ratio of fibroblasts to keratinocytes would critically influence keratinocyte colony expansion, 2) this ratio needed to be optimum at the beginning of the co-culture, 3) proliferative fibroblasts would be more effective than irradiated cells in expanding keratinocytes and 4) in the presence of an adequate number of fibroblasts, keratinocyte expansion would be independent of serum.ConclusionsA closely associated computational and biological approach is a powerful tool for understanding complex biological systems such as the interactions between keratinocytes and fibroblasts. The key outcome of this study is the finding that the early addition of a critical ratio of proliferative fibroblasts can give rapid keratinocyte expansion without the use of irradiated mouse fibroblasts and bovine serum.

Nonirradiated Human Fibroblasts and Irradiated 3T3-J2 Murine Fibroblasts as a Feeder Layer for Keratinocyte Growth and Differentiation in vitro on a Fibrin Substrate

“…Irradiated HFs, cultured on a plasma polymer surface, have been reported to be effective in sustaining keratinocyte expansion in serum-free culture media [Higham et al, 2003;Bullock et al, 2006]. However, keratinocyte growth has been demonstrated to be more efficiently supported by nonirradiated HFs [Sun et al, 2008]. coworkers [2004, 2008] developed serum-free coculture systems with HKs and nonirradiated HFs grown on different substrates, such as tissue culture plastic, plasma polymer surfaces or non-tissue culture polystyrene surfaces.…”

Skin Equivalent Tissue-Engineered Construct: Co-Cultured Fibroblasts/ Keratinocytes on 3D Matrices of Sericin Hope Cocoons

“…Recently co-cultured grafts comprising of dermal and epidermal modules capable of autologous skin replacement are of prime interest due to their accelerated healing processes [8]. Skin wound healing basically comprises epidermal tissue regeneration regulated by interaction of cytokines and growth factors with controlled functional behaviour of keratinocytes and dermal fibroblasts [9-11]. Keratinocytes proliferation in co-culture model is found to be based on active interplay between both cell types in a double paracrine mechanism, cell-matrix interactions and direct cell-cell contact signals [9,12,13].…”

Skin Stem Cell Hypotheses and Long Term Clone Survival – Explored Using Agent-based Modelling

“…Computer models on the other hand, are ideal tools for investigating individual cell behaviour by combining laboratory data and the existing literature. Agent-based models have been frequently used for studying a group of entities (or agents)123, such as keratinocytes, each with their unique properties45. The behaviour of each agent is defined using a set of rules based on the experimental literature.…”

“…The modelling framework has been developed in the Sheffield group in parallel with a well-established in vitro tissue-engineered human skin model in our laboratory2122. The modelling technique has been well validated against experimental data on studies of keratinocyte colony formation4, the role of fibroblasts in supporting keratinocytes5, and the role of TGF-β1 in wound healing17.…”