Abstract
Automation will likely to play a key role in the development of scalable manufacturing processes for cell-based therapies. In this study, we have compared the effects of manual centrifugation and automated non-centrifugation cell culture process steps, performed using TAP biosystems’ CompacT SelecT automated cell culture platform, upon hMSC morphology, number, viability, surface marker expression, Short tandem repeat (STR) profile, and paracrine function. Furthermore, the comparability between flow cytometry analyses of hMSCs, performed at multiple sites, was investigated. No significant difference in hMSC growth and characteristics was observed between cells cultured using either the manual centrifugation process step or the automated non-centrifugation process step, in which residual dissociation agent is carried over. However, some variability in paracrine activity was observed between hMSCs cultured using alternative process steps. It is also apparent that differences in analytical methods can influence the inter-laboratory reproducibility of hMSC flow cytometry analysis, although differences in culture may also contribute to the variability observed in the expression of 2 of the 8 surface markers examined. This novel investigation into the effects of these two key process steps will help to improve the understanding of the influence of automated cell culture upon various cell culture parameters, as well as upon process comparability.
Highlights
Mesenchymal stromal cells (MSCs) were first described by Friedenstein et al in the 1970s as an adherent, non-haematopoietic cell type, present in the bone marrow, with the capacity to form fibroblastic colonies in vitro, and were assigned the name ‘colony-Abbreviations: BM-MNCs, bone marrow mononuclear cells; Cell line authentication (CLA), cell line authentication; DMEM HG GlutaMAXTM, Dulbecco’s modified eagle’s medium high glucose glutamaxTM; DMSO, dimethyl sulfoxide; Fetal Bovine Serum (FBS), fetal bovine serum; hMSC, human mesenchymal stromal cells; hypoxic pre-treatment’ (HY-PT), hypoxic pre-treatment; IDO, indoleamine 2,3 dioxygenase; inflammatory pre-treatment’ (IN-PT), inflammatory pre-treatment; NT, no treatment; prostaglandin E-2 (PGE-2), prostaglandin E2; Short tandem repeat (STR), short tandem repeat; VEGF, vascular endothelial growth factor.forming unit-fibroblasts’ [1]
These results indicate that no significant difference in the viability of hMSC cultures was observed between centrifugation and non-centrifugation cell culture process steps, which would suggest that the enzyme carryover in the non-centrifugation process step had no detrimental effect upon cell viability
It can be concluded that no significant difference in hMSC morphology, surface marker expression, STR profile, viable cell yield, and viability was observed between hMSCs cultured using either the manual centrifugation step or the automated non-centrifugation process step, in which residual dissociation agent is carried over
Summary
Mesenchymal stromal cells (MSCs) were first described by Friedenstein et al in the 1970s as an adherent, non-haematopoietic cell type, present in the bone marrow, with the capacity to form fibroblastic colonies in vitro, and were assigned the name ‘colony-forming unit-fibroblasts’ [1]. Despite ongoing uncertainty regarding their ‘stemness’, defining surface marker profile, in vivo identity, and tissue source, the ISCT published a definition paper [3], outlining the basic criteria for the identification of MSCs. Despite ongoing uncertainty regarding their ‘stemness’, defining surface marker profile, in vivo identity, and tissue source, the ISCT published a definition paper [3], outlining the basic criteria for the identification of MSCs These included their adherence to culture plastic, their positive expression of CD105, CD73 and CD90, their lack of CD45, CD34, CD14 or CD11b, CD79 ̨ or CD19 and HLADR expression, and their osteogenic, chondrogenic and adipogenic differentiation potential in vitro. This definition may lack accuracy and specificity to the MSC cell type, it represents a step closer to defining the MSC phenotype. Substantial progress is required before in vivo MSC function is fully understood and comprehensive potency assays for each of the MSC putative mechanisms of action can be developed
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