Optimizing mesenchymal stem cell therapy: from isolation to GMP-compliant expansion for clinical application

Abstract

Background: Mesenchymal stem cells (MSCs) are promising for cell-based therapies targeting a wide range of diseases. However, challenges in translating MSC-based therapies to clinical applications necessitate standardized protocols following Good Manufacturing Practices (GMP) guidelines. This study aimed at developing GMP-complained protocols for FPMSCs isolation and manipulation, necessary for translational research, by (1) optimize culture of MSCs derived from an infrapatellar fat pad (FPMSC) condition through animal-free media comparison and (2) establish feasibility of MSC isolation, manufacturing and storage under GMP-compliance (GMP-FPMSC).

Methods: FPMSCs from three different patients were isolated following established protocols and the efficacy of two animal component-free media formulations in the culturing media were evaluated. The impact of different media formulations on cell proliferation, purity, and potency of MSCs was evaluated through doubling time, colony forming unit assay, and percentage of MSCs, respectively. Furthermore, the isolation and expansion of GMP-FPMSCs from four additional donors were optimized and characterized at each stage according to GMP requirements. Viability and sterility were checked using Trypan Blue and Bact/Alert, respectively, while purity and identity were confirmed using Endotoxin, Mycoplasma assays, and Flow Cytometry. The study also included stability assessments post-thaw and viability assessment to determine the shelf-life of the final GMP-FPMSC product. Statistical analyses were conducted using one-way ANOVA with Tukey's Multiple Comparisons.

Results: The study demonstrated that FPMSCs exhibited enhanced proliferation rates when cultured in MSC-Brew GMP Medium compared to standard MSC media. Cells cultured in this media showed lower doubling times across passages, indicating increased proliferation. Additionally, higher colony formation in FPMSCs cultured in MSC-Brew GMP Medium were observed, supporting enhanced potency. Data from our GMP validation, including cells from 4 different donors, showed post-thaw GMP-FPMSC maintained stem cell marker expression and all the specifications required for product release, including > 95% viability (> 70% is required) and sterility, even after extended storage (up to 180 days), demonstrating the reproducibility and potential of GMP-FPMSCs for clinical use as well as the robustness of the isolation and storage protocols.

Conclusions: The study underscores the feasibility of FPMSCs for clinical uses under GMP conditions and emphasizes the importance of optimized culture protocols to improve cell proliferation and potency in MSC-based therapies.

Keywords: Good manufacturing practices; Infrapatellar fat pad-derived MSCs; Mesenchymal stem cells; Stem cell therapy; Translational research.

Fig. 1

Colony forming units (CFU) were measured as number of visible colonies visible/cm² (A) Bar plots show mean + SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0005 as indicated, one-way ANOVA with Tukey’s Multiple Comparisons. Images were comprised of 25 stitched images, representative images shown in (B) Scale bar = 4 mm

Fig. 2

MSC marker expression in FPMSCs. MSCs were defined as CD45-, CD73+, CD90+, CD105+, determined via flow cytometry (A) Bar plots show mean + SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0005 as indicated, one-way ANOVA with Tukey’s Multiple Comparisons. (B) shows gating strategy and representative plots for FPMSC-8

Fig. 3

Flow diagram of FP-MSC GMP manufacturing

Fig. 4

Doubling time for three patients (FPMSC-8, FPMSC-11, FPMSC-13) cultured in MEM α (supplemented with 10% FBS), MSC-Brew GMP Medium and MesenCultTM-ACF Plus at passage 2, 3 and 4 (A) Bar plots show mean + SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0005 as indicated, one-way ANOVA with Tukey’s Multiple Comparisons. (B) shows representative images of cell density for FPMSC-8 at passage 2, scale bar = 100 μm

Fig. 5

Cell viability (A) and stem cell marker expression (B) in FPMSCs following cryopreservation