Scalable Manufacturing of Human Mesenchymal Stromal Cells in the Vertical‐Wheel Bioreactor System: An Experimental and Economic Approach

Abstract

Mesenchymal stromal cells (MSC) hold great promise for tissue engineering applications and cell‐based therapies. Large cell doses (>1 × 106 cells kg−1) and Good Manufacturing Practices (GMP)‐compliant processes are however required for clinical purposes. Here, a serum‐ and xenogeneic‐free (S/XF) microcarrier‐based culture system is established for the expansion of human umbilical cord matrix (UCM)‐ and adipose tissue (AT)‐derived MSC using the Vertical‐Wheel system (PBS‐0.1 MAG; PBS Biotech). UCM and AT MSC are expanded to maximum cell densities of 5.3 ± 0.4 × 105 cell mL−1 (n = 3) and 3.6 ± 0.7 × 105 cell mL−1 (n = 3), respectively, after 7 days of culture, while maintaining their identity, according to standard criteria. An economic evaluation of the process transfer from T‐flasks to PBS‐0.1 MAG shows a reduction in the costs associated with the production of a dose for an average 70 kg adult patient (i.e., 70 million cells). Costs decrease from $17.0 K to $11.1 K for UCM MSC and from $21.5 K to $11.1 K for AT MSC, proving that the transition to Vertical‐Wheel reactors provides a cost‐effective alternative for MSC expansion. The present work reports the establishment of a scalable and cost‐effective culture platform for the manufacturing of UCM and AT MSC in a S/XF microcarrier‐based system.

Scalable Production of Human Mesenchymal Stromal Cell-Derived Extracellular Vesicles Under Serum-/Xeno-Free Conditions in a Microcarrier-Based Bioreactor Culture System

“…The present work aimed to establish a scalable culture platform for the manufacturing of MSC-EVs in S/XF culture conditions. This was achieved by building on previous work from our group where a S/XF microcarrier-based culture system was implemented in single-use bioreactors (VWBR), employing a hPL culture supplement (UltraGRO TM -PURE) for MSC expansion ( de Sousa Pinto et al, 2019). In the present study, EVs were produced by MSC isolated from 3 different human tissue sources (BM, AT and UCM) in a process that comprises a cell expansion stage and a culture medium conditioning stage.…”

“…In general, MSC expansion in static conditions was performed as previously described ( de Sousa Pinto et al, 2019). In summary, previously isolated BM, AT and UCM MSC were thawed and plated on T-flasks (Falcon), at a cell density between 3000-6000 cell/cm 2 .…”

“…Expansion of human MSC in VWBR was generally performed as previously described ( de Sousa Pinto et al, 2019). In summary, previously isolated and expanded human MSC were inoculated in a PBS 0.1 MAG bioreactor (PBS Biotech Inc.) with a working volume of 100 mL.…”

“…After an initial intermittent agitation regime, a continuous agitation mode was set at 25 rpm, as previously described ( de Sousa Pinto et al, 2019). This agitation rate was always maintained, except for AT MSC culture, which required an increment in the agitation rate to 30 rpm at day 2 or 3 of culture and to 35 rpm at day 4 or 5, due to increased medium viscosity and the subsequent formation of cell aggregates.…”

“…In addition, the Vertical-Wheel TM impeller can fully suspend microcarriers with minimal power input and thus minimize shear stress effects (Croughan et al, 2016). Moreover, this technology is scalable, being available at working volumes that range from 100 mL up to 500 L. Recently, VWBR have been successfully applied in microcarrier-based cell culture processes for the expansion of MSC from multiple sources (Sousa et al, 2015;de Sousa Pinto et al, 2019), as well as for human induced pluripotent stem cells (Rodrigues et al, 2018;Nogueira et al, 2019).…”