Large-Scale Cell Production Based on GMP-Grade Dissolvable Porous Microcarriers.

Researchers in the cell and gene therapy (CGT) industry have long faced a formidable challenge in the efficient and large-scale expansion of cells. To address the primary shortcomings of the two-dimensional (2D) planar culturing system, we innovatively developed an automated closed industrial scale cell production (ACISCP) platform based on a GMP-grade, dissolvable, and porous microcarrier for the 3D culture of adherent cells, including human mesenchymal stem/stromal cells (hMSCs), HEK293T cells, and Vero cells. To achieve large-scale expansion, a two-stage expansion was conducted with 5 L and 15 L stirred-tank bioreactors to yield 1.1 x 1010 hMSCs with an overall 128-fold expansion within 9 days. The cells were harvested by completely dissolving the microcarriers, concentrated, washed and formulated with a continuous-flow centrifuge-based cell processing system, and then aliquoted with a cell filling system. Compared with 2D planar culture, there are no significant differences in the quality of hMSCs harvested from 3D culture. We have also applied these dissolvable porous microcarriers to other popular cell types in the CGT sector; specifically, HEK293T cells and Vero cells have been cultivated to peak cell densities of 1.68 x 107 cells/mL and 1.08 x 107 cells/mL, respectively. This study provides a protocol for using a bioprocess engineering platform harnessing the characteristics of GMP-grade dissolvable microcarriers and advanced closed equipment to achieve the industrial-scale manufacturing of adherent cells.

GMP-grade microcarrier and automated closed industrial scale cell production platform for culture of MSCs.

Efficient and large-scale expansion of mesenchymal stem/stromal cells (MSCs) has always been a formidable challenge to researchers in cell-based therapies and regenerative medicine. To reconcile major drawbacks of 2D planar culturing system, we innovatively developed an automated closed industrial scale cell production (ACISCP) platform based on GMP-grade microcarrier for culture of umbilical cord-mesenchymal stem/stromal cells (UCMSCs), in accordance with the criteria of stem cell bank. ACISCP system is a fully closed system, which employs different models of vivaSPIN bioreactors (CytoNiche Biotech, China) for scale-up cell culture and vivaPREP (CytoNiche Biotech, China) for automated cell harvesting and cell dosage preparation. To realize industrial scale expansion of UCMSCs, a three-stage expansion was conducted with 1 L, 5 and 15 L vivaSPIN bioreactors. Using 3D TableTrix® and ACISCP system, we inoculated 1.5 × 107 of UCMSCs into 1 L vivaSPIN bioreactor and finally scaled to two 15 L bioreactor. A final yield of 2.09 × 1010 cells with an overall expansion factor of 1975 within 13 days. The cells were harvested, concentrated, washed and prepared automatically with vivaPREP. The entire process was realized with ACISCP platform and was totally enclosed. Critical quality attributes (CQA) assessments and release tests of MSCs, including sterility, safety, purity, viability, identity, stability and potency were performed accordingly. The quality of cells harvested from 3D culture on the ACISCP and conventional 2D planar culture counterpart has no significant difference. This study provides a bioprocess engineering platform, harnessing GMP-grade 3D TableTrix® microcarriers and ACISCP to achieve industrial-scale manufacturing of clinical-grade hMSCs.

Large-Scale Expansion of Umbilical Cord Mesenchymal Stem Cells with Microcarrier Tablets in Bioreactor.

Mesenchymal stem cells show great potential in tissue engineering and cell-based therapies. This protocol demonstrates the use of 3D TableTrix® microcarrier tablets for large-scale manufacturing of human umbilical cord mesenchymal stem cells (hUCMSCs) in a 5-L stirred tank bioreactor. 3D TableTrix® microcarrier tablets readily disperse into tens of thousands of porous microcarriers to simplify cell seeding, and 3D FloTrix® vivaSPIN bioreactor could automate, monitor, and control the entire culture process. 3D TableTrix® microcarriers could also be fully dissolved upon adding dissolution reagent to gently harvest the expanded cells at a high recovery rate. With this protocol, more than 109 cells could be produced in a 5-L bioreactor.

Dispersible and Dissolvable Porous Microcarrier Tablets Enable Efficient Large-Scale Human Mesenchymal Stem Cell Expansion.

Human mesenchymal stem cells (hMSCs) have wide applications in regenerative medicine but their clinical translation is largely hindered by limited production capacity of current cell expansion regime. This study utilizes a novel dispersible and dissolvable porous microcarrier tablet, 3D TableTrix™, in stirred bioreactor to demonstrate a scalable expansion protocol for industrial manufacturing of hMSCs. The 3D TableTrix is a ready-to-use tablet that disperses into 10s of 1000s porous microcarriers upon contact with culture media, eliminating the need to prepare microcarriers before cell seeding, hence simplifying operation process. We demonstrate over 500 times expansion of adipose-derived hMSCs using serum-free culture medium in 11 days with bead-to-bead transfer for a partial scale-up from laboratory-scale spinner flasks to a 1-L bioreactor system. A final yield of 1.05 ± 0.11 × 109 hMSCs was achieved, and yield of over 3 × 109 with an overall expansion factor of 1530 could theoretically be realized with full scale-up. Cells were harvested by dissolving microcarriers with 98.6% ± 0.1% recovery rate. Cells retained their immunophenotypic characteristics, trilineage differentiation potential, and genome stability with low indications of senescence phenotype. This study illuminates the potential of industrializing clinical-grade hMSC production using 3D TableTrix microcarrier tablets and stirred tank bioreactors. Impact statement The 3D TableTrix™ is a newly available microcarrier ingeniously designed as dispersible and dissolvable porous microcarrier tablets for human mesenchymal stem cell (hMSC) expansion. This eliminates the need of tedious preparation work usually required for microcarriers and its dissolvable nature allows for high cell recovery rate of 98.6% ± 0.1%. Over 500 times expansion of adipose-derived mesenchymal stem cells in serum-free culture media using a 1-L bioreactor system demonstrates its tremendous potential for industrial production of hMSCs.