Clinical mesenchymal stromal cell products undergo functional changes in response to freezing

Abstract

Background Current methods of MSC cryopreservation result in variable post thaw recovery and phenotypic changes due to freezing. The objective of this investigation is to determine the influence of ex-vivo cell expansion on phenotype of MSCs and the response of resulting phenotypes to freezing and thawing. Methods Human bone marrow aspirate was purchased from Lonza (Walkersville, MD). MSCs were isolated, and cells were assessed for total count, viability, apoptosis, and senescence over 6 passages (8–10 doublings/passage) in ex vivo culture. One half of cells harvested at each passage were re-plated for continued culture, and the other half were frozen at 1°C/min in a controlled rate freezer. Frozen samples were stored in liquid nitrogen, thawed, and reassessed for total cell count, viability, and senescence immediately and 48 hours post thaw. Results Viability did not differ significantly between samples pre freeze or post thaw. Senescence increased over time in pre freeze culture, and was significantly higher in one sample that experienced growth arrest both pre freeze and post thaw. Freezing resulted in similar initial post thaw recovery in all samples, but 48 hour post thaw growth arrest was observed in the sample with high senescence only. Conclusion High freeze senescence appears to correlate with poor post thaw function in MSC samples, but additional studies are necessary to obtain a sample size large enough to quantify results.

Potency Analysis of Mesenchymal Stromal Cells Using a Combinatorial Assay Matrix Approach

“…It has been demonstrated that freezing and thawing affects MSC functionality (Chinnadurai et al, 2016; François et al, 2012; Hoogduijn et al, 2016; Kadekar et al, 2016; Moll et al, 2014, 2016; Nold et al, 2015; Pogozhykh et al, 2017; Pollock et al, 2015; Quimby et al, 2013). Here we further show that thawed MSCs display a defective secretome signature with T cell proliferation.…”

Immune dysfunctionality of replicative senescent mesenchymal stromal cells is corrected by IFNγ priming

“…High prefreeze senescence cell content has been shown to correlate with poor post-thaw function of MSCs, 58 and thus both replicative senescence and thawing from cryopreservation may be additive detrimental determinants of MSC function and potency. In the present study, we have used MSCs that are cultured in fetal calf serum growth–supplemented media akin to the most prevalent culture methods in support of clinical trials.…”

Enhanced viability and neural differential potential in poor post-thaw hADSCs by agarose multi-well dishes and spheroid culture

“…Then, post-thaw MSCs showed poor homing/engraftment and viability. It was hypothesized that poor post-thaw MSC functions might have been responsible for the failure of a clinical trial [55][56][57][58][59]. Therefore, the ways to improve the activity of poor postthaw hADSCs become more crucial.…”