Comparison of different epidermal keratinocyte media for preservation of keratinocyte stem cells in serial cultures and epidermal transplants
Authors
Löhberg L, Derow E., Schuler G, Hartmann A
Institution
Universitätsklinik, Hautklinik, 91052 Erlangen
Country
Germany
Year
2009
Journal
Experimental Dermatology, Abstract
Abstract
Hair follicle and epidermal basal layer human keratinocyte precursor cells play a central role in tissue homeostasis and wound repair and gain increasing interest as cell source for tissue engineered skin reconstructs. Whereas a high yield of stem cells would be favourable for integrity and long-term stability, they were lost during serial passages under generally used culture conditions. In order to generate standardized cell culture protocols for preservation of keratinocyte precursor cells we focused our attention on different types of commercially available epidermal keratinocyte media. Interfollicular keratinocytes (KC) isolated from the basal membrane (BM) of human skin biopsies as well as follicular KC isolated from the outer root sheath (ORS) of plucked anagen hair follicles were maintained in serial cultures using CNT-57- andCNT-07 medium, (CellnTec), KG-medium (KGM, Clonetics, Lonza) andKG2-medium (KGM 2, Promocell) under serum-free culture conditions. In parallel cultures using the different cell sources and media, cell morphology was analyzed, viability and cell division rate were determined. By immunocytochemistry expression of markers of stem and transient amplifying cells (CK15, follistatin, CD71, p63,) as well as differentiation markers (involucrin) was determined. By clonality assays proliferative capacity, clonality and clonal conversion were compared. Multilayered epidermal transplants using both cell types under different culture conditions were reconstructed on feeder cells and characterized immunohistochemically. For ORS-KC CNT-57 as well as KGM-2 media were superior to KGM with respect to cell viability, cell division - and passage rate. They support colony forming efficiency in serial cultures as well as preservation of stem cells in epidermal transplants. For epidermal BM-KC CNT 57/ 07 media were most favourable allowing a high cell viability and passage rate and a serial propagation of holoclones. In contrast, KGM medium supports differentiation and paraclone formation of isolated keratinocytes thereby leading to a lower passage rate. Based on these findings, the selection of adequate progenitor targeted keratinocytemedia seems mandatory to protect epidermal stem cells and to prevent clonal conversion under serial propagation.
Product use
Isolation and cultivation for a comparison with KGM, Clonetics, Lonza and KGM 2, Promocell. cell morphology was analyzed, viability and cell division rate were determined. By immunocytochemistry expression of markers of stem and transient amplifying cells
Tissue type
Epidermal
Tissue info
Interfollicular keratinocytes isolated from the basal membrane of human skin biopsy and follicular KC isolated from the outer root sheath (ORS) of plucked anagen hair follicles