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Rapid culture of human keratinocytes in an autologous, feeder-free system with a novel growth medium

Published:October 11, 2022DOI:https://doi.org/10.1016/j.jcyt.2022.09.003

      Abstract

      Background aims

      The ability to culture human keratinocytes is beneficial in the treatment of skin injury and disease, as well as for testing chemicals in vitro as a substitute for animal testing.

      Results

      We have identified a novel culture medium for the rapid growth of keratinocytes from human skin. “Kelch's medium” supports keratinocyte growth that is as rapid as in the classical Rheinwald and Green method, but without the need for cholera toxin or xenogeneic feeder cells. It enables keratinocytes to out-compete co-cultured autologous fibroblasts so that separation of the epidermis from the dermis is no longer required before keratinocyte culture. Enzymatic digests of whole human skin can therefore be used to generate parallel cultures of autologous keratinocytes, fibroblasts and melanocytes simply by using different cell culture media.

      Conclusions

      This new keratinocyte medium and the simplified manufacturing procedures it enables are likely to be beneficial in skin engineering, especially for clinical applications.

      Key Words

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      References

        • Hickerson WL
        • Remmers AE
        • Recker DP.
        Twenty-Five Years' Experience and Beyond with Cultured Epidermal Autografts for Coverage of Large Burn Wounds in Adult and Pediatric Patients, 1989-2015.
        J Burn Care Res. 2019; 40: 157-165
        • Wood FM
        • Kolybaba ML
        • Allen P.
        The use of cultured epithelial autograft in the treatment of major burn wounds: eleven years of clinical experience.
        Burns. 2006; 32: 538-544
        • Boyce ST
        • Lalley AL.
        Tissue engineering of skin and regenerative medicine for wound care.
        Burns Trauma. 2018; 6: 4
        • MacNeil S.
        Progress and opportunities for tissue-engineered skin.
        Nature. 2007; 445: 874-880
        • Watt SM
        • Pleat JM.
        Stem cells, niches and scaffolds: Applications to burns and wound care.
        Adv Drug Deliv Rev. 2018; 123: 82-106
        • Wood FM.
        Skin regeneration: the complexities of translation into clinical practise.
        Int J Biochem Cell Biol. 2014; 56: 133-140
        • Rheinwald JG
        • Green H.
        Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells.
        Cell. 1975; 6: 331-343
        • Bullock AJ
        • Higham MC
        • MacNeil S.
        Use of human fibroblasts in the development of a xenobiotic-free culture and delivery system for human keratinocytes.
        Tissue Eng. 2006; 12: 245-255
        • Nanba D.
        Human keratinocyte stem cells: From cell biology to cell therapy.
        J Dermatol Sci. 2019; 96: 66-72
        • Hynds RE
        • Bonfanti P
        • Janes SM.
        Regenerating human epithelia with cultured stem cells: feeder cells, organoids and beyond.
        EMBO Mol Med. 2018; 10: 139-150
        • Boyce ST
        • Ham RG.
        Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture.
        J Invest Dermatol. 1983; 81: 33s-40s
        • Meuli M
        • Hartmann-Fritsch F
        • Huging M
        • Marino D
        • Saglini M
        • Hynes S
        • et al.
        A Cultured Autologous Dermo-epidermal Skin Substitute for Full-Thickness Skin Defects: A Phase I, Open, Prospective Clinical Trial in Children.
        Plast Reconstr Surg. 2019; 144: 188-198
        • Boyce ST
        • Simpson PS
        • Rieman MT
        • Warner PM
        • Yakuboff KP
        • Bailey JK
        • et al.
        Randomized, Paired-Site Comparison of Autologous Engineered Skin Substitutes and Split-Thickness Skin Graft for Closure of Extensive, Full-Thickness Burns.
        J Burn Care Res. 2017; 38: 61-70
        • Supp DM
        • Hahn JM
        • Combs KA
        • McFarland KL
        • Schwentker A
        • Boissy RE
        • et al.
        Collagen VII Expression Is Required in Both Keratinocytes and Fibroblasts for Anchoring Fibril Formation in Bilayer Engineered Skin Substitutes.
        Cell Transplant. 2019; 28: 1242-1256
        • Lenihan C
        • Rogers C
        • Metcalfe AD
        • Martin YH.
        The effect of isolation and culture methods on epithelial stem cell populations and their progenyࣧtoward an improved cell expansion protocol for clinical application.
        Cytotherapy. 2014; 16: 1750-1759
        • Terunuma A
        • Limgala RP
        • Park CJ
        • Choudhary I
        • Vogel JC.
        Efficient procurement of epithelial stem cells from human tissue specimens using a Rho-associated protein kinase inhibitor Y-27632.
        Tissue Eng Part A. 2010; 16: 1363-1368
        • Strudwick XL
        • Lang DL
        • Smith LE
        • Cowin AJ.
        Combination of low calcium with Y-27632 rock inhibitor increases the proliferative capacity, expansion potential and lifespan of primary human keratinocytes while retaining their capacity to differentiate into stratified epidermis in a 3D skin model.
        PLoS One. 2015; 10e0123651
        • Scott TL
        • Christian PA
        • Kesler MV
        • Donohue KM
        • Shelton B
        • Wakamatsu K
        • et al.
        Pigment-independent cAMP-mediated epidermal thickening protects against cutaneous UV injury by keratinocyte proliferation.
        Exp Dermatol. 2012; 21: 771-777
        • Michel M
        • Torok N
        • Godbout MJ
        • Lussier M
        • Gaudreau P
        • Royal A
        • et al.
        Keratin 19 as a biochemical marker of skin stem cells in vivo and in vitro: keratin 19 expressing cells are differentially localized in function of anatomic sites, and their number varies with donor age and culture stage.
        J Cell Sci. 1996; 109: 1017-1028
        • Zhou X
        • Li G
        • Wang D
        • Sun X
        • Li X
        Cytokeratin expression in epidermal stem cells in skin adnexal tumors.
        Oncol Lett. 2019; 17: 927-932
        • Lowa A
        • Vogt A
        • Kaessmeyer S
        • Hedtrich S.
        Generation of full-thickness skin equivalents using hair follicle-derived primary human keratinocytes and fibroblasts.
        J Tissue Eng Regen Med. 2018; 12: e2134-e2e46
        • Hybbinette S
        • Bostrom M
        • Lindberg K.
        Enzymatic dissociation of keratinocytes from human skin biopsies for in vitro cell propagation.
        Exp Dermatol. 1999; 8: 30-38
        • Bottcher-Haberzeth S
        • Biedermann T
        • Klar AS
        • Widmer DS
        • Neuhaus K
        • Schiestl C
        • et al.
        Characterization of pigmented dermo-epidermal skin substitutes in a long-term in vivo assay.
        Exp Dermatol. 2015; 24: 16-21
        • Okada N
        • Kitano Y
        • Ichihara K.
        Effects of cholera toxin on proliferation of cultured human keratinocytes in relation to intracellular cyclic AMP levels.
        J Invest Dermatol. 1982; 79: 42-47
        • Takahashi H
        • Honma M
        • Miyauchi Y
        • Nakamura S
        • Ishida-Yamamoto A
        • Iizuka H.
        Cyclic AMP differentially regulates cell proliferation of normal human keratinocytes through ERK activation depending on the expression pattern of B-Raf.
        Arch Dermatol Res. 2004; 296: 74-82
        • Pontiggia L
        • Biedermann T
        • Meuli M
        • Widmer D
        • Bottcher-Haberzeth S
        • Schiestl C
        • et al.
        Markers to evaluate the quality and self-renewing potential of engineered human skin substitutes in vitro and after transplantation.
        J Invest Dermatol. 2009; 129: 480-490
      1. Administration FaD. Manufacturers of Biological Products [Internet].https://fda.report/media/76350/Letter-to-Manufacturers-of-Biological-Products–Recommendations-Regarding-Bovine-Spongiform-Encephalopathy.pdf 2000.

        • Rowan MP
        • Cancio LC
        • Elster EA
        • Burmeister DM
        • Rose LF
        • Natesan S
        • et al.
        Burn wound healing and treatment: review and advancements.
        Crit Care. 2015; 19: 243
        • Hirsch T
        • Rothoeft T
        • Teig N
        • Bauer JW
        • Pellegrini G
        • De Rosa L
        • et al.
        Regeneration of the entire human epidermis using transgenic stem cells.
        Nature. 2017; 551: 327-332