Processing and Ex Vivo Expansion of Adipose Tissue-Derived Mesenchymal Stem/Stromal Cells for the Development of an Advanced Therapy Medicinal Product for use in Humans

Abstract

Adipose tissue (AT) represents a commonly used source of mesenchymal stem/stromal cells (MSCs) whose proregenerative potential has been widely investigated in multiple clinical trials worldwide. However, the standardization of the manufacturing process of MSC-based cell therapy medicinal products in compliance with the requirements of the local authorities is obligatory and will allow us to obtain the necessary permits for product administration according to its intended use. Within the research phase (RD), we optimized the protocols used for the processing and ex vivo expansion of AT-derived MSCs (AT-MSCs) for the development of an Advanced Therapy Medicinal Product (ATMP) for use in humans. Critical process parameters (including, e.g., the concentration of enzyme used for AT digestion, cell culture conditions) were identified and examined to ensure the high quality of the final product containing AT-MSCs. We confirmed the identity of isolated AT-MSCs as MSCs and their trilineage differentiation potential according to the International Society for Cellular Therapy (ISCT) recommendations. Based on the conducted experiments, in-process quality control (QC) parameters and acceptance criteria were defined for the manufacturing of hospital exemption ATMP (HE-ATMP). Finally, we conducted a validation of the manufacturing process in a GMP facility. In the current study, we presented a process approach leading to the optimization of processing and the ex vivo expansion of AT-MSCs for the development of ATMP for use in humans.

Keywords: adipose tissue-derived mesenchymal stem/stromal cells; advanced therapy medicinal product; cell-based therapy; good manufacturing practices; regenerative medicine.

Figure 1

Overview of the HE-ATMP manufacturing process. In the diagram, we marked parameters that were assessed during the RD phase and the GMP phase (validation of the manufacturing process). (*) CPP: critical process parameters; (#) stability study was conducted during the validation of the manufacturing process; QCA: quality control attributes.

Figure 2

Optimization of the procedure of isolation of SVF cells from adipose tissue (AT). (a) Washing of AT with PBS did not cause the loss of adherent cells of SFV. Representative images of a few cells isolated from PBS obtained after the washing of AT 3 and 9 days post-seeding. PBS were centrifuged (300× g, 5 min, RT); the oil phase and, subsequently, pellet containing RBCs and tissue sections were resuspended in complete culture medium (DMEM/F12 supplemented with 10% FBS and 1% Penicillin-Streptomycin solution) and cultured for 9 days. (b) Examination of the suitability of selected collagenase concentrations for AT digestion. Yield of isolation is presented as the total number of SVF cells obtained per 1 g of lipoaspirate. Viability of SVF cell is presented in the table. AT was incubated with A1, A2, or A3 concentrations of collagenase for 40 min at 37 °C and, after a washing step, the number of cells isolated was counted. (c) Establishment of primary culture for analyzed collagenase concentrations. SVF cells isolated from AT following digestion with A1, A2, or A3 collagenase concentrations were seeded on culture flasks in complete culture medium (DMEM/F12 supplemented with 10% FBS and 1% Penicillin-Streptomycin solution) and assessed by light microscopy at days 2, 4, and 8 (for A1, A2, A3 collagenase concentrations); day 5 (for A1 and A2 collagenase concentrations); or day 6 (for A3 collagenase concentration). (d) Examination of the influence of an additional step of RBC lysis on the yield of the cell isolation and culture (presented as the number of isolated cells or cells detached from 1 cm² of cell growth area) directly after SVF isolation and two consecutive passages. (e) Effect of RBCs’ lysis on SVF viability. Lysis of RBCs was conducted according to AP no. 1 or 2. (f) Morphology of adherent cells isolated using standard SVF isolation protocol and two alternative protocols (AP no. 1 or 2). Representative pictures were taken prior to the first passage. Scale bars: 50 µm. Results (b,d,e) are presented as means ± SDs. n = 3 (biological replicates, corresponding to three individual human donors) and they were assessed by an ANOVA model with Tukey’s post hoc test. The p values less than 0.05 (p < 0.05) were considered to be significant and labeled by an asterisk p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).

Figure 3

Optimization of the culture conditions of AT-MSCs. (a) Selection of the optimal medium for AT-MSC culture. Morphology of the AT-MSCs cultured in the following culture media: Medium A, Medium B, and Medium C. Efficiency of the AT-MSC culture in tested media presented as the number of cells detached from 1 cm² of the growth area at passages nos. 1, 2, and 3. (b) Analysis of cell viability from the isolation to the third passage. (c) Analysis of the number of AT-MSCs obtained in three consecutive passages. (d) Kinetics of the growth of AT-MSCs seeded on cell culture flasks at concentrations equal to 3000, 4000, 5000, or 6000 cells per 1 cm² at days 2, 3, and 4 post-cell seeding by light microscopy. Scale bars: 50 µm. Results (a–c) are presented as means ± SD. N = 3 (biological replicates, corresponding to three individual human donors), and they were assessed by an ANOVA model with Tukey’s post hoc test. The p values less than 0.05 (p < 0.05) were considered to be significant; p < 0.05 (*), p < 0.01 (**), (*), p < 0.001 (***), p < 0.0001 (****).

Figure 4

Characterization of AT-MSCs according to the criteria defining multipotent MSCs proposed by the International Society for Cellular Therapy and their chondrogenic potential. (a) Representative images of the morphology of AT-MSCs at passage 4 by light microscopy. (b) Trilineage differentiation potential of AT-MSCs. Representative images of AT-MSCs differentiated into osteoblasts, chondroblasts, and adipocytes. AT-MSCs were cultured using a StemPro Osteogenesis Differentiation Kit (for 21 days), StemPro Chondrogenesis Differentiation Kit (for 14 days), or StemPro Adipogenesis Differentiation Kit (for 11 days). Post differentiation, AT-MSCs were fixed with paraformaldehyde and stained with Alizarin Red S (red staining of deposits of calcium phosphate characteristic for osteogenic differentiation) or Alcian Blue (blue staining of sulfated proteoglycans characteristic for chondrogenic differentiation), whereas oil droplets characteristic for adipogenic differentiation were shown in unfixed and unstained AT-MSCs. Scale bars: 50 µm. (c) Antigenic profile of AT-MSCs by flow cytometry. Representative histograms of the expression of MSC-negative markers (CD19, CD14, D45, CD34, CD31), MSC-positive markers (CD44, CD73, CD90, CD105), and HLA-DR antigens on viable (7-AAD⁻) AT-MSCs. (d) Chondrogenic differentiation potential of AT-MSCs in a microenvironment resembling the conditions in human joints. Representative images of AT-MSCs cultured using the StemPro Chondrogenesis Differentiation Kit or standard cell culture medium (Control) in a hypoxic (5% O₂) or normoxic (21% O₂) environment parallel exposed to normal (1 PSI) or high (2 or 5 PSI) pressure in the Avantar System. At 7, 14, and 21 days, AT-MSCs were fixed with paraformaldehyde and stained with Alcian Blue. Scale bars: 50 µm.

Figure 5

Stability of AT-MSCs solution. (a) Viability of AT-MSCs post-product formulation. A total of 10 × 10⁶ AT-MSCs were resuspended in carrier solution, transferred into container closure system, and stored at 2–8 °C. Viability of AT-MSCs was measured every 3 h up to 24 h post-product formulation by an automated cell counter ADAM. (b) Storage temperature of AT-MSC solution during the stability test. Results are presented as means ± SDs, n = 3 (independent validation batches).