Stirred tank bioreactor culture combined with serum‐/xenogeneic‐free culture medium enables an efficient expansion of umbilical cord‐derived mesenchymal stem/stromal cells

Abstract

Mesenchymal stem/stromal cells (MSC) are being widely explored as promising candidates for cell-based therapies. Among the different human MSC origins exploited, umbilical cord represents an attractive and readily available source of MSC that involves a non-invasive collection procedure. In order to achieve relevant cell numbers of human MSC for clinical applications, it is crucial to develop scalable culture systems that allow bioprocess control and monitoring, combined with the use of serum/xenogeneic (xeno)-free culture media. In the present study, we firstly established a spinner flask culture system combining gelatin-based Cultispher(®) S microcarriers and xeno-free culture medium for the expansion of umbilical cord matrix (UCM)-derived MSC. This system enabled the production of 2.4 (±1.1) x10(5) cells/mL (n = 4) after 5 days of culture, corresponding to a 5.3 (±1.6)-fold increase in cell number. The established protocol was then implemented in a stirred-tank bioreactor (800 mL working volume) (n = 3) yielding 115 million cells after 4 days. Upon expansion under stirred conditions, cells retained their differentiation ability and immunomodulatory potential. The development of a scalable microcarrier-based stirred culture system, using xeno-free culture medium that suits the intrinsic features of UCM-derived MSC represents an important step towards a GMP compliant large-scale production platform for these promising cell therapy candidates.

Impact of the type of microcarrier and agitation modes on the expansion performances of mesenchymal stem cells derived from umbilical cord

“…Until now, WJ-MSC have been successfully isolated and cultivated, in culture scales up to 800 ml. [32][33][34][35] Recently, two studies explored the culture of WJ-MSC on microcarriers in a medium supplemented with hPL, adapted for clinical use. 36,37 Petry et al compared five different microcarriers (Hillex II, ProNectin F-coated, Plastic, Plastic-Plus, and Glass-coated) in static mode and validated the dynamic culture in Spinner flasks with Plastic-Plus, which gave the best growth and attachment performance in static mode.…”

Application of Bioreactor in Stem Cell Culture

“…Thus, serum free culture is a direction in the future standardized production of biological products with bioreactors [51,52]. It is necessary to investigate the metabolic characteristics of SCs in large scale expansion of cells, design the key components in the serum free medium and optimize the physical and chemical properties of the medium, which may develop individualized culture medium [53].…”

The Challenge of Human Mesenchymal Stromal Cell Expansion: Current and Prospective Answers

“…Although this removes the need for cell-carrier separation, a longer incubation time is necessary which can change the immunophenotype of the cells, but this can be reverted during re-cultivation in static cultures [41].…”

“…The most common are spinner flasks, stirred tank reactors (STRs), wave systems [38], fixed-bed reactors (FBRs), fluidized bed and wall-rotating systems [22,[39][40][41] as well as the application of Vertical-Wheel™ technology [42]. Most published studies describe cell expansion in spinner flasks, but these are difficult to scale up and automate, and the only process control options are pH and oxygen concentration monitoring.…”

“…The expansion processes differed according to the source of the hMSCs, such as umbilical cord matrix-derived hMSCs (UCM-hMSCs), highlighting the need for tailored process development. The cultivation of UCM-hMSCs was recently demonstrated using gelatin-based microcarriers and XFM in a controlled STR culture with a working volume of 800 mL [41]. To increase process efficiency, an automated continuous process (e.g., without intervention for media replacement) was developed based on continuous perfusion in a STR and the cells retained their differentiation capacity [95].…”