Reactor concepts for human mesenchymal stem cell (hMSC) production are introduced. Thereby, special interest is laid on the realization of these concepts as disposables fulfilling the GMP and PAT requirements. The specialty of the hMSC production process is the cell itself being the product. This results in completely different process requirements compared to e.g. protein production in mammalian cells. Thus, great attention has to be given to the shear sensitivity of the cells. The cultivation and the harvest of the cells have to be very gentle to neither influence cell viability nor cell differentiability. Further, the production process should not cause any undesirable cell changes. For hMSC production, cell harvest is the main challenging process step. The reactor concepts should be suitable for hMSC production for clinical trials as ATMPs. Therefore, disposable systems are especially applicable. The review describes more detailed bone marrow-derived hMSC production in a disposable stirred tank reactor as promising reactor concept.

For cell therapy, a high biomass of human mesenchymal stem cells (hMSCs) is required for clinical applications, such as in the form of encapsulated implants. An easy and reproducible microcarrier-based stirred tank reactor cultivation process for hMSCs in 1.68 L scale is described. To avoid medium changes, studies comparing high-glucose DMEM (DMEM-HG) with low-glucose EMEM were performed showing that high-glucose medium has positive effects on cell proliferation and that cell differentiability remains. Studies on the inoculation strategy and cell density, carrier concentration, volume, and stirrer speed were performed and resulted in a set of optimized parameters, inoculation strategy was found to be 45 minutes of static state and 2 minutes of stirring repeated in 4 cycles. The inoculation density was chosen to be 7×10³ cells/cm2, and the carrier concentration of glass surface carrier was 25 g/L. For the described reactor system, a stirrer speed of 120 rpm for the inoculation process and a daily increase of 10 rpm up to 160 rpm were found to be suitable. Process reproducibility was shown by 3 repeated cultivations at the determined set of parameters allowing high biomass values of up to 7×10⁸ cells per batch. With DMEM-HG, no limitation of glucose was found, and lactate and ammonia remained lower than critical inhibitory concentrations. Comparison of the static (T-flask) and dynamic cultures in the stirred tank reactor showed for both cases, that cells were of high vitality and both maintained differentiability. In both cases, encapsulation of the cells resulted in high bead vitality, a basic requirement for cell therapy application.

Cell therapies require the in vitro expansion of adherent cells such as mesenchymal stromal cells (hMSCs) in bioreactor systems or other culture environments, followed by cell harvest. As hMSCs are strictly adherent cells, cell harvest requires cell detachment. The use of hMSCs for cell therapy requires GMP production in accordance with the guidelines for advanced therapeutic medical products. Therefore, several GMP-conform available proteolytic enzymes were investigated for their ability to promote hMSC detachment. An allogeneic hMSC cell line (hMSC-TERT) that is used in clinical trials in the form of alginate cell capsules was chosen as a model. This study investigated the influence of several factors on the outcome of proteolytic hMSC-TERT detachment. Therefore, hMSC-TERT detachment was analyzed in different cultivation systems (static, dynamic) and in combination with further cell processing including encapsulation. Only two of the commercially available enzymes (AccutaseTM, TrypZeanTM) that fulfill all process requirements (commercial availability, cost, GMP conditions during manufacturing and non-animal origin) are found to be generally suitable for detaching hMSC-TERT. Combining cell detachment with encapsulation demonstrated a high impact of the experimental set up on cell damage. It was preferable to reduce the temperature during detachment and limit the detachment time to a maximum of 20 minutes. Cell detachment in static systems was not comparable with detachment in dynamic systems. Detachment yields in dynamic systems were lower and cell damage was higher for the same experimental conditions. Finally, only TrypZeanTM seemed to be suitable for the detachment of hMSC-TERT from dynamic reactor systems.

In cell therapy, the use of autologous and allogenic human mesenchymal stem cells is rising. Accordingly, the supply of cells for clinical applications in highest quality is required. As hMSCs are considered as an advanced therapy medicinal products (ATMP), they underlie the requirements of GMP and PAT according to the authorities (FDA and EMA). The production process of these cells must therefore be documented according to GMP, which is usually performed via a GMP protocol based on standard operating procedures. This chapter provides an example of such a GMP protocol for hMSC, here a genetically modified allogenic cell line, based on a production process in a microcarrier-based stirred tank reactor including process monitoring according to PAT and final product quality assurance.

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