RESUMEN
Autologous cell therapy manufacturing timeframes constitute bottlenecks in clinical management pathways of severe burn patients. While effective temporary wound coverings exist for high-TBSA burns, any means to shorten the time-to-treatment with cytotherapeutic skin grafts could provide substantial therapeutic benefits. This study aimed to establish proofs-of-concept for a novel combinational cytotherapeutic construct (autologous/allogeneic DE-FE002-SK2 full dermo-epidermal graft) designed for significant cutaneous cell therapy manufacturing timeframe rationalization. Process development was based on several decades (four for autologous protocols, three for allogeneic protocols) of in-house clinical experience in cutaneous cytotherapies. Clinical grade dermal progenitor fibroblasts (standardized FE002-SK2 cell source) were used as off-the-freezer substrates in novel autologous/allogeneic dermo-epidermal bilayer sheets. Under vitamin C stimulation, FE002-SK2 primary progenitor fibroblasts rapidly produced robust allogeneic dermal templates, allowing patient keratinocyte attachment in co-culture. Notably, FE002-SK2 primary progenitor fibroblasts significantly outperformed patient fibroblasts for collagen deposition. An ex vivo de-epidermalized dermis model was used to demonstrate the efficient DE-FE002-SK2 construct bio-adhesion properties. Importantly, the presented DE-FE002-SK2 manufacturing process decreased clinical lot production timeframes from 6-8 weeks (standard autologous combined cytotherapies) to 2-3 weeks. Overall, these findings bear the potential to significantly optimize burn patient clinical pathways (for rapid wound closure and enhanced tissue healing quality) by combining extensively clinically proven cutaneous cell-based technologies.
RESUMEN
The standard treatment of burns is early excision followed by autologous skin grafting. The closure of extensive deep burns poses a considerable challenge. Cultured autologous keratinocytes have been used since 1981 in an effort to improve healing. However, the time required to culture the cells and the lack of a dermal component limit the expectations of outcome. Our aim was to compare the duration of hospital stay between patients who were treated with autologous skin grafts and cultured autologous keratinocytes and those who were treated with autologous skin grafting without cultured autologous keratinocytes. In this retrospective study all patients treated with cultured autologous keratinocytes between 2012 and 2015 were matched by size and depth of burn with patients not treated with cultured autologous keratinocytes. Multivariable regression was used to analyse associations between duration of hospital stay and treatment adjusted for age, mortality, size and depth of the burn. Then, we investigated the possibility of differentiation of human bone marrow stem cell line to keratinocyte- like cells as a future direction. The regression analysis showed a coefficient of 17.36 (95% CI -17.69 to 52.40), p= 0.32, for hospital stay in the treatment group, compared with the matched group. Our results showed no difference in the duration of hospital stay between the two treatments. Autologous stem cells should be considered as a future modality of burn management, although further studies are needed.
Le traitement de référence des brûlures est l'excision- greffe précoce, qui est problématique en cas d'atteinte étendue. La culture de kératinocytes autologues est utilisée depuis 1981 dans le but de répondre à cette problématique mais se heure au temps nécessaire à sa mise en oeuvre, ainsi qu'à l'absence de feuillet dermique, génératrice de séquelles. Cette étude a comparé la durée de séjour des patients traité par excision- greffe et culture de kératinocytes à celle des patients traités de manière conventionnelle. Les patients hospitalisés entre 2012 et 2015 ont été comparés à des patients de même surface et profondeur traités conventionnellement, en utilisant une analyse multivariée ajustée sur l'âge, la mortalité, la surface et la profondeur de la brûlure. L'analyse n'est pas significative (coefficient 17,36 ; IC95 -17,69 à 52,4 ; p= 0,32). Il serait utile d'étudier l'utilisation des cellules souches médullaires, différentiées en kératinocytes, dans un protocole de culture.
RESUMEN
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.