Comparability Exercise of Critical Quality Attributes of Clinical-Grade Human Mesenchymal Stromal Cells from the Wharton's Jelly: Single-Use Stirred Tank Bioreactors Versus Planar Culture Systems.

Background aims: The increasing demand of clinical-grade mesenchymal stromal cells (MSCs) for use in advanced therapy medicinal products (ATMPs) require a re-evaluation of manufacturing strategies, ensuring scalability from two-dimensional (2D) surfaces to volumetric (3D) productivities. Herein we describe the design and validation of a Good Manufacturing Practice-compliant 3D culture methodology using microcarriers and 3-L single-use stirred tank bioreactors (STRs) for the expansion of Wharton's jelly (WJ)-derived MSCs in accordance to current regulatory and quality requirements. Methods: MSC,WJ were successfully expanded in 3D and final product characterization was in conformity with Critical Quality Attributes and product specifications previously established for 2D expansion conditions. Results: After 6 days of culture, cell yields in the final product from the 3D cultures (mean 9.48 x 10(8) +/- 1.07 x 10(7) cells) were slightly lower but comparable with those obtained from 2D surfaces (mean 9.73 x 10(8) +/- 2.36 x 10(8) cells) after 8 days. In all analyzed batches, viability was >90%. Immunophenotype of MSC,WJ was highly positive for CD90 and CD73 markers and lacked of expression of CD31, CD45 and HLA-DR. Compared with 2D expansions, CD105 was detected at lower levels in 3D cultures due to the harvesting procedure from microcarriers involving trypsin at high concentration, and this had no impact on multipotency. Cells presented normal karyotype and strong immunomodulatory potential in vitro. Sterility, Mycoplasma, endotoxin and adventitious virus were negative in both batches produced. Conclusions: In summary, we demonstrated the establishment of a feasible and reproducible 3D bioprocess using single-use STR for clinical-grade MSC,WJ production and provide evidence supporting comparability of 3D versus 2D production strategies. This comparability exercise evaluates the direct implementation of using single-use STR for the scale-up production of MSC,WJ and, by extension, other cell types intended for allogeneic therapies. (c) 2023 International Society for Cell & Gene Therapy. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)