Functional and phenotypic characterization of human keratinocytes expanded in microcarrier culture

Skin cells for transplantation are routinely prepared by growing patient keratinocytes in a semi-defined cocktail of growth factors, including serum and feeder cells. However, these reagents require substantial risk remediation and can contribute to transplant rejection. Microcarrier culture is an emerging technology that may allow the elimination of feeder cells whilst facilitating expansion of cultured keratinocytes. However, the behavior of keratinocytes in microcarrier culture and the potential of these cells to form an epidermis have been poorly defined. We characterized freshly isolated human keratinocytes cultured on CultiSpher-G microcarriers in the absence of murine feeder cells and assessed the potential of the keratinocytes to form an epidermis in an in vitro model. In a single passage, keratinocytes multiplied 44.9-fold in microcarrier-bioreactor culture in 17 days, whereas two-dimensional cultures reached confluence in 9 days and only expanded 7.4-fold. Histological characterization of keratinocytes on the microcarriers revealed that the cells were randomly distributed within these porous structures, however, not all pores contained cells. High-resolution microcomputed tomography imaging of the microcarriers confirmed limited interconnectivity of the pores. Immunoreactivity of specific epidermal markers was confirmed during cell expansion via immunohistochemistry. Despite the expression of differentiation markers, microcarrier-expanded keratinocytes retained the capacity to form an epidermis, as was evaluated using an in vitro human skin equivalent model. The epidermis formed by microcarrier-expanded keratinocytes in this model exhibited morphology similar to native skin. Significantly, the microcarrier technique successfully eliminates the need for a feeder cell layer and hence facilitates development of an improved culture system.