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In recent years, the use of hMSCs, which may be isolated from adipose tissue among others, for the treatment of diseases has increased significantly. The cell quantities required for such therapeutic approaches, between 1012 and 1013, have thus far been predominantly produced using commercially available multi-tray systems, such as the Cell Factory (Thermo Fisher Scientific) or HYPERStack (Corning), which can be purchased with up to 40 layers. However, the handling of these planar multilayer systems is difficult, and process monitoring opportunities remain limited. Here, automated stirred single-use bioreactors provide a viable alternative to the time-consuming multiplication of cells using such planar systems, while still managing to achieve the desired clinically relevant quantities. In these stirred single-use systems, adherent cells are predominantly cultivated in suspension up to pilot scale using carrier materials, also referred to as microcarriers (MCs).This chapter describes the steps which need to be realized to guarantee successful hMSC expansion within a stirred single-use bioreactor (Eppendorf's BioBLU® 0.3c) operated using MCs under serum- and xeno-free conditions at benchtop scale. The cultivations were performed using an immortalized human adipose-derived mesenchymal stem cell (hASC) line, hence referred to as hASC52telo, and a new chemically defined, xeno-free medium, hence referred to as the UrSuppe formulation. Spinner flask cultivations were performed under comparable process conditions.

Human-induced pluripotent stem cells (hiPSCs) can be derived from a variety of biopsy samples and have an unlimited capacity for self-renewal and differentiation into almost any cell type in the body. Therefore, hiPSCs offer unprecedented opportunities for patient-specific cell therapies, modeling of human diseases, biomarker discovery, and drug testing. However, clinical applications of hiPSCs require xeno-free and, ideally, chemically defined methods for their generation, expansion, and cryopreservation. In this chapter, we present a chemically defined and xeno-free slow freezing method for hiPSCs along with a chemically undefined protocol. Both approaches yield reasonable post-thaw viability and cell growth.

Mesenchymal stem cells (MSCs) have been used in therapies owing to their regenerative potential, paracrine regulatory effects, and immunomodulatory activity. To foster commercialization and implementation of stem cells treatments, researchers have recently derived MSCs from human induced pluripotent stem cells (iMSCs). For therapeutic applications, human iMSCs must be produced in xeno-free culture conditions and following procedures that are compatible with the principles of Good Manufacturing Practice.

As stated in Chap. 2, the use of inactivated fibroblasts as feeders for the derivation, expansion and maintenance of pluripotent stem cells (PSCs) is still the predominant method used in most laboratories. These cells hold great promise for clinical utility in the field of regenerative medicine and are useful tools in areas such as toxicology and the development of disease models. However, these applications require defined and refined culture systems, which are essential to ensure that processes and assay systems are reliable, reproducible, robust, efficacious and safe. A feeder-free system also facilitates activities such as the scale-up of processes and high-throughput screening. In the clinical setting, the ultimate goal is to produce a culture system that is xeno-free (free of animal products), in which the culture medium is chemically defined and the cell culture substrate is a defined synthetic matrix. Amongst the advantages of defined xeno-free culture systems is the amelioration of intrinsic batch-to-batch variation introduced into the system via the use of biological products, as well as a reduction in the risk of contamination with viruses associated with animal products.