Abstract
The current expansion of autologous human keratinocytes to resurface severe wound defects still relies on murine feeder layer and calf serum in the cell culture system. Through our characterization efforts of the human skin basement membrane and murine feeder layer 3T3-J2, we identified two biologically relevant recombinant laminins—LN-511 and LN-421- as potential candidates to replace the murine feeder. Herein, we report a completely xeno-free and defined culture system utilizing these laminins which enables robust expansion of adult human skin keratinocytes. We demonstrate that our laminin system is comparable to the 3T3-J2 co-culture system in terms of basal markers’ profile, colony-forming efficiency and the ability to form normal stratified epidermal structure in both in vitro and in vivo models. These results show that the proposed system may not only provide safer keratinocyte use in the clinics, but also facilitate the broader use of other cultured human epithelial cells in regenerative medicine.
Highlights
The current expansion of autologous human keratinocytes to resurface severe wound defects still relies on murine feeder layer and calf serum in the cell culture system
Based on the knowledge about subepithelial basement membrane (BM) components, we hypothesized that human epidermal keratinocytes (HEKs)-associated laminin matrices present in vivo can support the growth of keratinocytes in vitro and replace feeder cells, essentially as we have shown for pluripotent human embryonic stem cells, which can be expanded from singlecell suspension on a pure LN-511 or LN-521 matrix without the use of feeder cells and Rho kinase inhibitors that inhibit apoptosis[29,30,31]
We confirmed that LN-332, LN-511, and LN-521 are the laminins expressed in the sub-epidermal BM (Fig. 1a, b)
Summary
The current expansion of autologous human keratinocytes to resurface severe wound defects still relies on murine feeder layer and calf serum in the cell culture system. We demonstrate that our laminin system is comparable to the 3T3-J2 co-culture system in terms of basal markers’ profile, colony-forming efficiency and the ability to form normal stratified epidermal structure in both in vitro and in vivo models These results show that the proposed system may provide safer keratinocyte use in the clinics, and facilitate the broader use of other cultured human epithelial cells in regenerative medicine. Assessment and removal strategies of these high-risk AMs, in particular for cholera toxin, remain challenging in today’s good manufacturing practice systems Regulatory agencies such as the Food and Drug Administration and the European Medicines Agency currently classify cultured epithelial autografts produced using the R&G method as xenografts and these are approved only for treatment of severe burns (i.e. above 30% total body surface area) or for compassionate use[1,11]. Based on the knowledge about subepithelial BM components, we hypothesized that HEK-associated laminin matrices present in vivo can support the growth of keratinocytes in vitro and replace feeder cells, essentially as we have shown for pluripotent human embryonic stem cells, which can be expanded from singlecell suspension on a pure LN-511 or LN-521 matrix without the use of feeder cells and Rho kinase inhibitors that inhibit apoptosis[29,30,31]
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