Bringing Safe and Standardized Cell Therapies to Industrialized Processing for Burns and Wounds

Abstract

Cultured primary progenitor cell types are worthy therapeutic candidates for regenerative medicine. Clinical translation, industrial transposition, and commercial implementation of products based on such cell sources are mainly hindered by economic or technical barriers and stringent regulatory requirements. Applied research in allogenic cellular therapies in the Lausanne University Hospital focuses on cell source selection technique optimization. Use of fetal progenitor cell sources in Switzerland is regulated through Federal Transplantation Programs and associated Fetal Biobanks. Clinical applications of cultured primary progenitor dermal fibroblasts have been optimized since the 1990s as "Progenitor Biological Bandages" for pediatric burn patients and adults presenting chronic wounds. A single organ donation procured in 2009 enabled the establishment of a standardized cell source for clinical and industrial developments to date. Non-enzymatically isolated primary dermal progenitor fibroblasts (FE002-SK2 cell type) served for the establishment of a clinical-grade Parental Cell Bank, based on a patented method. Optimized bioprocessing methodology for the FE002-SK2 cell type has demonstrated that extensive and consistent progenitor cell banks can be established. In vitro mechanistic characterization and in vivo preclinical studies have confirmed potency, preliminary safety and efficacy of therapeutic progenitor cells. Most importantly, highly successful industrial transposition and up-scaling of biobanking enabled the establishment of tiered Master and Working Cell Banks using Good Manufacturing Practices. Successive and successful transfers of technology, know-how and materials to different countries around the world have been performed. Extensive developments based on the FE002-SK2 cell source have led to clinical trials for burns and wound dressing. Said trials were approved in Japan, Taiwan, USA and are continuing in Switzerland. The Swiss Fetal Transplantation Program and pioneer clinical experience in the Lausanne Burn Center over three decades constitute concrete indicators that primary progenitor dermal fibroblasts should be considered as therapeutic flagships in the domain of wound healing and for regenerative medicine in general. Indeed, one single organ donation potentially enables millions of patients to benefit from high-quality, safe and effective regenerative therapies. This work presents a technical and translational overview of the described progenitor cell technology harnessed in Switzerland as cellular therapies for treatment of burns and wounds around the globe.

Keywords: GMP manufacturing; burns; cell therapy; chronic wounds; clinical cell banking; fibroblasts; progenitor cells.

Copyright © 2020 Laurent, Lin, Scaletta, Hirt-Burri, Michetti, de Buys Roessingh, Raffoul, She and Applegate.

Figures

Figure 1

Technological advantages of appropriate whole-cell bioprocessing for skin progenitors. From one single organ donation (FE002, 2009), various samples yielding differentiated tissue-specific progenitor cells were bioprocessed for isolation using the proprietary non-enzymatic method. Intrinsic cellular identity and characteristics were therein optimally maintained throughout the transposition to adherent monolayer culture. Inherent technical and clinical advantages are attributed to the specific choice of the cell source. Optimized and consistent biobanking allowed for establishment of extensive GMP cell banks. Thorough testing throughout manufacturing along with the consistency of the cell source guarantees optimal homogeneity and safety of the biological starting material for therapeutic products. Hundreds of millions of treatments can be produced based on the available stocks.

Figure 2

FE002-SK2 stock projections and product manufacture. Assuming maximal expansion of all available cells, the theoretical number of final products is quantified in dozens of billions. Quality and safety testing are highly consumptive in materials throughout the GMP manufacturing process, but further optimization of bioprocessing and biobanking protocols would reasonably allow for such numbers of effective treatments to be produced, using one dedicated organ donation. To manufacture end-products, cryopreserved FE002-SK2 cells at specific passages are thawed and seeded on sterile bio-compatible scaffolds, which are applied to patients following defined clinical protocols.

Figure 3

FE002-SK2 GMP biobank establishment and validation. After the FE002-SK2 PCB was declared fit for GMP manufacturing, expansion campaigns were conducted for the establishment of extensive and consistent GMP FE002-SK2 Master (P2), Working (P4), and End of Production (P12) cell banks. All the progeny cells were stored in liquid nitrogen vapor phase. Several vials of each production batch were initiated for recovery studies and quality release testing. The EOPCB materials served for extensive safety and stability assays, both in vitro and in vivo. The complete battery of tests comprised (a) isoenzyme tests, (b) sterility tests, (c) mycoplasma detection, (d) karyology, (e) transmission electron microscopy (viruses, virus-like particles, mycoplasma, yeasts, fungi, bacteria), (f) in vitro adventitious viruses testing (picornavirus, orthomyxovirus, pramyxovirus, herpesvirus, adenovirus, reovirus, West Nile virus), (g) in vivo adventitious viruses testing (suckling mice, adult mice, guinea pigs, embryonated eggs), (h) Q-PCR (HepB/C, HIV-1/2, HTLV-1/2, HHV-6/7/8, EBV, hCMV, SV40, B19 parovirus) and (i) in vivo tumorigenicity testing in nude mice.

Figure 4

FE002-SK2 tiered biobanking and GMP tec-transfers. The robust biobanking protocols devised for the FE002-SK2 progenitor fibroblasts allowed for successful technology transfers to BioReliance (Scotland), Transwell Biotech Co. Ltd. (Taiwan), and the CHUV CPC (Epalinges/Lausanne, Switzerland). Varying banking nomenclatures exist between the different groups, the pertinent passage numbers being those used by BioReliance. Quantities of vials listed exclude those consumed for recovery studies and release testing. Pre-clinical safety testing was performed on the EOPCB by BioReliance. Clinical applications comprise the ongoing use of PBBs in the CHUV Burn Center, the pending Priority Project Bru_PBB and both clinical trials sponsored in Asia by TWB.

Figure 5

Representative imaging of migrated primary human keratinocytes (DAPI staining) in Transwell® migration assay (left and middle columns). Representative imaging includes middle fields of 3 wells/group. Contamination of Transwells® by FE002-SK2 progenitor fibroblasts was negligible (right column). Mean cell counts are listed in Table 1.

Figure 6

Epithelial migration in the presence of FE002-SK2 conditioned media and sham media. Quantitative results of three experimental repetitions are compared (A–C). At the 9- and 18-h timepoints in the second and third replicates, respectively (B,C), the gaps treated with FE002-SK2 conditioned media were completely filled. Representative imaging of epithelial migration from the first experiment is shown (D). Negligible filling was detectable at the 3-h timepoint. Imaging data was acquired under 40X optical magnification.

Figure 7

Representative imaging of keratinocyte proliferation in the absence and presence of FE002-SK2 cell extracts. Adjunction of sham medium or cell extracts on Day 0 did not stimulate keratinocyte proliferation and did not ensure maintenance of cell populations (A–F,J–L). Adjunction of cell extracts on Day 0 and Day 2 resulted in augmented keratinocyte proliferation (H,I,N,O). Imaging data was acquired under 40X optical magnification. Mean harvesting total cell counts are listed in Table 2.

Figure 8

Proliferation of primary keratinocytes in the presence of mitomycin C-treated FE002-SK2 progenitor fibroblasts (MMC-FE002-SK2 cells). DK-SFM is deficient in extracellular matrix (ECM) proteins. Considering that FE002-SK2 fibroblasts can secrete ECM proteins for cell attachment, both collagen-coated (A–I) and un-coated surfaces (J–S) were comparatively tested. No cell proliferation was observed in the single-population cultures of keratinocytes (D–F,J–L) or MMC-FE002-SK2 cells (A–C). Keratinocyte proliferation was observable in co-cultures (G–I,M–P). Selected wells were also stained with anti-CK-14 antibodies (P–S). MMC-FE002-SK2 progenitor fibroblasts appeared negative for immunostaining (S), while proliferating cells in co-culture wells appeared positive (Q). Staining data confirmed that proliferating cells were keratinocytes. Imaging data was acquired under 40X optical magnification and fluorescence microscopy.

Figure 9

Representative imaging of primary keratinocyte and FE002-SK2 progenitor fibroblast proliferation in single- and co-cultures. Initial viable seeding densities are indicated for each FE002-SK2 group. Initial viable seeding density of keratinocyte was of 2,000 cells/cm2. No proliferation was observed in the single-population cultures of keratinocytes (A–D) or FE002-SK2 fibroblasts (E–L). High proliferation was observable in the co-cultures (M–T). Imaging data was acquired under 40X optical magnification. Mean harvesting cumulated cell counts are listed in Table 3.

Figure 10

Representative and evolutive imaging of porcine wound models. Data were acquired from pig N°052. All 4 experimental groups are presented at different timepoints ranging from wound creation (Day 0) up until sacrifice (Day 14). Treatment groups [A–D] consisted in the sham control, hydrogel scaffold alone, and hydrogel scaffolds with low and high doses of FE002-SK2 progenitor fibroblasts, respectively.

Figure 11

Representative imaging of tissue sections isolated for histological examination. Sections were isolated on Day 14 and stained using HE. Treatment groups [A–D] consisted in the sham control (A), hydrogel scaffold alone (B) and hydrogel scaffolds with low (C) and high doses (D) of FE002-SK2 progenitor fibroblasts, respectively. Arrows in (A,B) indicate observable epidermal detachment.