Nonirradiated Human Fibroblasts and Irradiated 3T3-J2 Murine Fibroblasts as a Feeder Layer for Keratinocyte Growth and Differentiation in vitro on a Fibrin Substrate

Abstract

The standard method for producing graftable epithelia relies on the presence of a feeder layer of lethally irradiated 3T3-J2 murine fibroblasts (Rheinwald and Green technique). Here, we studied a new keratinocyte culture system, which envisages the utilization of nonirradiated human fibroblasts embedded into a fibrin substrate, in cultures destined for a future clinical application. We tested this culture system using keratinocytes grown on a fibrin gel precoated with 3T3-J2 murine fibroblasts as a control. In order to evaluate the new technology, we compared the clonogenic potential and the proliferative, differentiative and metabolic characteristics of keratinocytes cultured on the fibrin gel under the two culture conditions. The results demonstrated that the proposed technology did not impair the behavior of cultured keratinocytes and revealed that cells maintained their proliferative potential and phenotype under the experimental conditions. In particular, the demonstration of stem cell maintenance under the adopted culture conditions is very important for acute burn treatment with skin substitutes. This work is a first step in the evaluation of a new keratinocyte culture system, which has been studied in order to take advantage of an additional human cell population (i.e. nonirradiated, growing fibroblasts) for future transplantation purposes in acute and chronic wounds. Additional research will allow us to attain (1) the removal of murine cells in the initial phase of keratinocyte cultures, and (2) the removal of other potentially dangerous animal-derived materials from the entire culture system.

Optimization of human keratinocyte culture to develop an artificial human skin model: cell alternatives as feeder layer of Advanced Therapies

“…Se ha demostrado que con los fibroblastos dérmicos humanos subletalmente irradiados como capa alimentadora, los queratinocitos presentan una tasa de duplicación equiparable a la obtenida con el feeder 3T3i. Estos resultados indican que los fibroblastos dérmicos humanos representan un sistema de capa alimentadora adecuada para soportar el cultivo de queratinocitos humanos primarios (27) (28).…”

Ultrastructural evaluation of human keratinocyte growth and differentiation on a fibrin substrate

“…Furthermore, it allows a significant reduction of the costs of cultured autografts and eliminates problems related to handling and transportation 14,18 . However, to obtain these results, it has been emphasized that the presence of a "feeder layer" of lethally irradiated mouse fibroblasts is an absolute requirement 14 ; and because fibrinolysis occurs rapidly as soon as epidermal cells or irradiated 3T3 cells are added 13 , until now there are no long-term evaluations of the in vitro differentiation process in the epidermis reconstituted under this condition.…”

“…Moreover, considering the possibility of late graft loss due to antigenic response originated by xenogeneic fibroblasts 12 , there are numerous research efforts to avoid the use of a "feeder-layer" 13 .…”

Overexpression of YAP1 induces immortalization of normal human keratinocytes by blocking clonal evolution

“…The fibroblast-fibrin supports were prepared using the fibrin sealant ''TISSUCOL'' from Baxter, as described in Panacchia et al (2010). Briefly, human fibroblasts were resuspended in Ham's F12 Medium (0.32 9 10 6 cells/ml) and then mixed with thrombin and fibrin solutions previously diluted in saline solutions.…”

“…This technology has been demonstrated to be suitable to obtain stratified cultures since it does not impair the behavior of cultured keratinocytes and maintain their proliferative potential (Panacchia et al 2010). Two independent experiments for each strain were performed.…”