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

Background aims: Current methods of mesenchymal stromal cell (MSC) cryopreservation result in variable post-thaw recovery and phenotypic changes caused by freezing. The objective of this investigation was to determine the influence of exvivo cell expansion on phenotype of MSCs and the response of resulting phenotypes to freezing and thawing. Methods: Human bone marrow aspirate was used. 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 h after thaw. Results: Viability did not differ significantly between samples before freeze or after thaw. Senescence increased over time in pre-freeze culture and was significantly higher in one sample that had growth arrest both before freeze and after thaw. Freezing resulted in similar initial post-thaw recovery in all samples, but 48-h post-thaw growth arrest was observed in the sample with high senescence only. Conclusions: High pre-freeze senescence appears to correlate with poor post-thaw function in MSC samples, but additional studies are necessary to obtain a sample sizes large enough to quantify results.