Cutaneous Cell Therapy Manufacturing Timeframe Rationalization: Allogeneic Off-the-Freezer Fibroblasts for Dermo-Epidermal Combined Preparations (DE-FE002-SK2) in Burn Care

Abstract

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.

Keywords: FE002 dermal progenitor fibroblasts; autologous keratinocytes; burn center; cutaneous cell therapy; dermal template; dermo-epidermal grafts; early coverage solutions; manufacturing optimization; severe burns; standardized skin grafts.

Figure 1

In vitro comparative functional characterization of primary patient fibroblasts and FE002-SK2 primary progenitor fibroblasts. (A1–A4) Endpoint imaging of Sirius Red staining for both primary fibroblast types following 7 days of vitamin C induction in fibroblast proliferation medium. Scale bars = 400 µm for patient fibroblasts and 200 µm for progenitor fibroblasts. (A5) Time-course of collagen synthesis during vitamin C induction in fibroblast proliferation medium. (B1–B4) Endpoint imaging of Sirius Red staining for both primary fibroblast types following 7 days of vitamin C induction in keratinocyte proliferation medium. Scale bars = 400 µm for patient fibroblasts and 200 µm for progenitor fibroblasts. (B5) Time-course of collagen synthesis during vitamin C induction in keratinocyte proliferation medium. Results of the statistical analyses relative to collagen quantification experiments under vitamin C stimulation are presented in Table S2. CTRL, control group; Vit C, vitamin C.

Figure 2

Schematic process detailing the parallel and sequential phases of DE-FE002-SK2 construct preparation. (A1) Using optimized technical specifications, allogeneic FE002-SK2 fibroblasts are expanded to confluency. (A2) For stimulation of collagen production, fibroblast cultures are treated with vitamin C. (B1) Upon epidermal biopsy reception, autologous keratinocyte isolation is rapidly performed. (B2) Autologous keratinocytes are cultured until the allogeneic dermal template is formed. (C1) The allogeneic dermal template and the autologous epidermal components are combined and further co-cultured in view of finished DE-FE002-SK2 construct formation. (C2) The excess autologous keratinocytes are cryopreserved and may be subsequently used to rapidly prepare new batches of DE-FE002-SK2 constructs. C, vitamin C; D, day; FIB, fibroblast proliferation medium; KER, keratinocyte proliferation medium.

Figure 3

Macroscopic and microscopic records of allogeneic dermal templates and of the DE-FE002-SK2 constructs in formation. (A1) FE002-SK2-based dermal template following vitamin C induction. Scale bar = 5 mm. (A2) Detached FE002-SK2-based dermal template in PBS. Scale bar = 5 mm. (A3) Harris hematoxylin staining of the FE002-SK2-based dermal template. Scale bar = 300 µm. (B1) Fully formed FE002-SK2-based dermal template after 1 day of co-culture with patient primary keratinocytes. Scale bar = 5 mm. (B2) Combined DE-FE002-SK2 construct in co-culture following endpoint MTT staining. Scale bar = 5 mm. (B3) Harris hematoxylin staining of the fully formed combined DE-FE002-SK2 construct, showing stratified epidermal component formation. Scale bar = 300 µm. PBS, phosphate-buffered saline.

Figure 4

Assessment results of CEA and DE-FE002-SK2 construct adhesion capacities on a standardized ex vivo DED model. (A1,A2) Aspect of the DED model following topical CEA construct application and following gentle removal of the transport gauze. Scale bars = 10 mm. (B1,B2) Aspect of the DED model following topical DE-FE002-SK2 construct application and following gentle removal of the transport gauze. Scale bars = 10 mm. (C1) MTT staining of the model from the CEA group after 1 week of air–liquid organoculture, showing inhomogeneous graft take. Scale bar = 7 mm. (C2) MTT staining of the model from the DE-FE002-SK2 group after 1 week of air–liquid organoculture, showing homogeneous graft take. Scale bar = 7 mm. (C3) MTT staining of the model from the DED control group after 1 week of air–liquid organoculture. Scale bar = 7 mm. CEA, cultured epithelial autografts; DED, de-epidermalized dermis.

Figure 5

Functional investigation results for allogeneic dermal templates and fully formed DE-FE002-SK2 constructs on the ex vivo DED model. (A1–A3) MTT staining of the allogeneic dermal template on the DED, the DE-FE002-SK2 construct on the DED, and the DED control group following 1 week of air–liquid organoculture. Scale bars = 5 mm. (B1,B2) H&E staining of the allogeneic dermal template on the DED and the DE-FE002-SK2 construct on the DED. Scale bars = 150 µm. (C) H&E staining of the normal skin control group. Scale bar = 150 µm. (D1,D2) P63 staining of the allogeneic dermal template on the DED and the DE-FE002-SK2 construct on the DED. Scale bars = 150 µm. (E) P63 staining of the normal skin control group. Scale bar = 150 µm. (F1,F2) Ki67 staining of the allogeneic dermal template on the DED and the DE-FE002-SK2 construct on the DED. Scale bars = 150 µm. (G) Ki67 staining of the normal skin control group. DED, de-epidermalized dermis; H&E, hematoxylin and eosin.