Serum-free primary human fibroblast and keratinocyte coculture.
Research has shown that the inclusion of a fibroblast cell support layer is required for the isolation and expansion of primary keratinocytes. Recent advances have provided keratinocyte culture with fibroblast-free alternatives. However, these technologies are often undefined and rely on the incorporation of purified proteins/components. To address this problem we developed a medium that used recombinant proteins to support the serum-free isolation and expansion of human dermal fibroblasts and keratinocytes. The human dermal fibroblasts were able to be isolated serum free by adding recombinant human albumin to a collagenase solution. These fibroblasts were then expanded using a serum-free medium containing recombinant proteins: epidermal growth factor, basic fibroblast growth factor, chimeric vitronectin:insulin-like growth factor-I protein, and recombinant human albumin. These fibroblasts maintained a typical morphology and expressed fibroblast markers during their serum-free isolation, expansion, and freezing. Moreover, these fibroblasts were able to support the serum-free isolation and expansion of primary keratinocytes using these recombinant proteins. Real-time polymerase chain reaction and immunofluorescence analysis confirmed that there were no differences in expression levels of p63 or keratins 1, 6, and 10 when keratinocytes were grown in either serum-supplemented or serum-free medium. Using a three-dimensional human skin equivalent model we demonstrated that these keratinocytes also maintained their ability to reform an epidermal layer. In summary, the techniques described provide a valuable alternative for culturing fibroblasts and keratinocytes using recombinant proteins.
Mujaj
Sally
S
School of Life Science, Queensland University of Technology , Brisbane, Australia . s.mujaj@student.qut.edu.au
Manton
Kerry
K
Upton
Zee
Z
Richards
Sean
S
D016475:3T3 Cells; D000418:Albumins / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000379:methods*; D016895:Culture Media, Serum-Free / Q000737:chemistry*; D017931:DNA Primers / Q000235:genetics; D004815:Epidermal Growth Factor / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D015603:Keratinocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D007633:Keratins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D011994:Recombinant Proteins / Q000494:pharmacology; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism; D019096:Vitronectin / Q000494:pharmacology
D000418:Albumins; D016895:Culture Media, Serum-Free; D017931:DNA Primers; D011994:Recombinant Proteins; C115051:TP63 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D019096:Vitronectin; D016222:Fibroblast Growth Factor 2; D004815:Epidermal Growth Factor; D007334:Insulin-Like Growth Factor I; D007633:Keratins