Y.H. Martin

The effect of isolation and culture methods on epithelial stem cell populations and their progeny—toward an improved cell expansion protocol for clinical application

BACKGROUND AIMS The use of cultured epithelial keratinocytes in the treatment of burns and skin graft donor sites is well established in clinical practice. The most widely used culture method for clinical use was originally developed by Rheinwald and Green 40 years ago. This system uses irradiated mouse dermal fibroblasts as a feeder cell layer to promote keratinocyte growth, a process that is costly and labor-intensive for health care providers. The medium formulation contains several components of animal origin, which pose further safety risks for patients. Improvements and simplification in the culturing process would lead to clear advantages: improved safety through reduction of xenobiotic components and reduction in cost for health care providers by dispensing with feeder cells. METHODS We compared the Rheinwald and Green method to culture in three commercially available, feeder-free media systems with defined/absent components of animal origin. RESULTS During the isolation process, short incubation times in high-strength trypsin resulted in increased numbers of liberated keratinocyte stem cells compared with longer incubation times. All three commercially available media tested in this study could support the expansion of keratinocytes, with phenotypes comparable to cells expanded using the established Rheinwald and Green method. Growth rates varied, with two of the media displaying comparable growth rates, whereas the third was significantly slower. DISCUSSION Our study demonstrates the suitability of such feeder-free media systems in clinical use. It further outlines a range of techniques to evaluate keratinocyte phenotype when assessing the suitability of cells for clinical application.

A novel system for expansion and delivery of human keratinocytes for the treatment of severe cutaneous injuries using microcarriers and compressed collagen

Cell therapy with autologous or allogeneic keratinocytes applied as a single‐cell suspension is well established in clinical practice in the treatment of severe burn injuries to augment epithelial barrier restoration. Yet, the application of cell sprays can lead to significant cell loss owing to lack of adhesion of cell suspension to the wound bed. The development of a robust and controllable method of transplanting cells onto the wound bed is yet to be established. The ability to control adhesion and distribution of cells by using a cell carrier embedded in a biodegradable scaffold could significantly improve the treatment of cutaneous wounds with keratinocyte cell therapy. Several microcarrier‐based systems for expanding keratinocytes already exist. A new method for expansion of human keratinocytes in a feeder‐free, defined medium system on microcarriers has been developed. The cells retained their basal, proliferative phenotype after rapid expansion in a clinically relevant time‐frame. The cell‐laden microcarriers were further incorporated into collagen scaffolds fabricated by plastic compression. When cultured in vitro, cells continued to proliferate and migrate along the surface of the collagen scaffold. Using an in vitro wound bed model, cells were observed to form mostly single cell layers and in some areas multiple cell layers within 8 days, while retaining their basal, proliferative phenotype, indicating the suitability of this cell transplantation method to improve epithelial barrier restoration. This advanced cell expansion and delivery method for cutaneous cell therapy provides a flexible tool for use in clinical application. Copyright