title: Comparison of the effects between animal-derived trypsin and recombinant trypsin on human skin cells proliferation, gene and protein expression.
abstract: Animal-derivative free reagents are preferred in skin cell culture for clinical applications. The aim of this study was to compare the performance and effects between animal-derived trypsin and recombinant trypsin for skin cells culture and expansion. Full thickness human skin was digested in 0.6 % collagenase for 6 h to liberate the fibroblasts, followed by treatment with either animal-derived trypsin; Trypsin EDTA (TE) or recombinant trypsin; TrypLE Select (TS) to liberate the keratinocytes. Both keratinocytes and fibroblasts were then culture-expanded until passage 2. Trypsinization for both cell types during culture-expansion was performed using either TE or TS. Total cells yield was determined using a haemocytometer. Expression of collagen type I, collagen type III (Col-III), cytokeratin 10, and cytokeratin 14 genes were quantified via RT-PCR and further confirmed with immunocytochemical staining. The results of our study showed that the total cell yield for both keratinocytes and fibroblasts treated with TE or TS were comparable. RT-PCR showed that expression of skin-specific genes except Col-III was higher in the TS treated group compared to that in the TE group. Expression of proteins specific to the two cell types were confirmed by immunocytochemical staining in both TE and TS groups. In conclusion, the performance of the recombinant trypsin is comparable with the well-established animal-derived trypsin for human skin cell culture expansion in terms of cell yield and expression of specific cellular markers.
authors: Tissue Engineering Centre, Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latiff, Bandar Tun Razak, 56000, Cheras, Kuala Lumpur, Malaysia.
authors: Maarof
authors: M
authors: Manira
authors: Khairoji
authors: K
authors: Khairul Anuar
authors: Wan Tai
authors: WT
authors: Seet
authors: Abd Wahab
authors: AW
authors: Ahmad Irfan
authors: Min Hwei
authors: MH
authors: Ng
authors: Kien Hui
authors: KH
authors: Chua
authors: Mohd Yunus
authors: MY
authors: Mohd Heikal
authors: Bin Saim
authors: BS
authors: Aminuddin
authors: Bt Hj Idrus
authors: BH
authors: Ruszymah
mesh_terms: D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D024042:Collagen Type I / Q000096:biosynthesis; D024061:Collagen Type III / Q000096:biosynthesis; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D006801:Humans; D053550:Keratin-10 / Q000096:biosynthesis; D053547:Keratin-14 / Q000096:biosynthesis; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000494:pharmacology*; D012867:Skin / Q000166:cytology* / Q000187:drug effects*; D014357:Trypsin / Q000494:pharmacology*
chemical_list: D024042:Collagen Type I; D024061:Collagen Type III; C507493:KRT10 protein, human; C507540:KRT14 protein, human; D053547:Keratin-14; D011994:Recombinant Proteins; D053550:Keratin-10; D014357:Trypsin