Umbilical cord matrix derived-mesenchymal stem cell production in microcarrier- based culture systems
January 15-19, 2017
Umbilical cord matrix (UCM)-derived mesenchymal stem/stromal cells (MSC) are promising therapeutic candidates for regenerative medicine settings. UCM MSC have advantages over adult cells as these can be obtained through a noninvasive harvesting procedure and display a higher proliferative capacity. However, the high cell doses required in the clinical setting make large-scale manufacturing of UCM MSC mandatory. In this work, we established two different microcarrier-based culture systems for the scalable production of UCM MSC. In the first approach, we established a spinner flask culture system combining gelatin-based Cultispher®S microcarriers and xeno-free culture medium for the expansion of umbilical UCM MSC. This system enabled the production of 2.4 (±1.1) x105 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.
In a second approach, a commercially available human platelet lysate-based culture supplement (UltraGROTM, AventaCell BioMedical, under a collaboration agreement with iBB) (5%(v/v)) was tested to effectively isolate UCM MSC and to expand these cells under (1) static conditions, using planar culture systems and (2) stirred culture using plastic microcarriers in a spinner flask. The MSC-like cells were isolated from UCM explant cultures after 11 ± 2 days. After five passages in static culture, UCM MSC retained their immunophenotype and multilineage differentiation potential. The UCM MSC cultured under static conditions using UltraGROTM-supplemented medium expanded more rapidly compared to UCM MSC expanded using a previously established protocol . Importantly, UCM MSC were successfully expanded under dynamic conditions on plastic microcarriers using UltraGROTM-supplemented medium in spinner flasks. Upon an initial 54% cell adhesion to the beads, UCM MSC expanded by >13-fold after 5–6 days, maintaining their immunophenotype and multilineage differentiation ability. By using this human platelet lysate supplement we were able to establish an easily scalable integrated culture platform based on for the effective isolation and expansion of UCM MSC in a xenogeneic-free microcarrier-based system. Overall, both strategies suited the intrinsic features of UCM-derived MSC and represent an important advance towards a GMP compliant large-scale production platform for these cells.
 Carmelo JG, Fernandes-Platzgummer A, Diogo MM, da Silva CL, Cabral JM. “A xeno-free microcarrier-based stirred culture system for the scalable expansion of human mesenchymal stem/stromal cells isolated from bone marrow and adipose tissue.” Biotechnol J. 2015 Aug;10(8):1235-47.
Ana Fernandes-Platzgummer, António M. de Soure, Yi-Feng Huang, William Milligan, Amanda Mizukami, Kamilla Swiech, Cláudia L. da Silva, and Joaquim M.S. Cabral, "Umbilical cord matrix derived-mesenchymal stem cell production in microcarrier- based culture systems" in "Scale-up and Manufacturing of Cell-based Therapies V", Tom Brieva, Celgene Cellular Therapeutics William Miller, Northwestern University Chris Mason, University College London Eds, ECI Symposium Series, (2017). http://dc.engconfintl.org/cellbasedtherapies_v/71
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