Investigating early detection of delayed onset of apoptosis in cryopreserved mesenchymal stem cells using mass spectrometry

There is currently a growing interest in the use of Mesenchymal Stem Cells (MSCs) in the development of cell therapies for the treatment of many common conditions, including COVID-19. However, cryopreservation, a key process in manufacturing cell therapies, has been found to affect cell viability and function post-thaw. Delayed apoptosis is where cells that appear viable immediately post-thaw undergo apoptosis in a delayed manner. This is a particular area of concern due to the possibility of cells turning apoptotic and losing functionality following administration in patients. Additionally, there is currently no rapid, and non-invasive method to identify such cell populations at an early stage.

The primary aim of this work was to develop a rapid method to detect biomarkers, post-thaw, that could indicate cells undergoing apoptosis. Gas Chromatography / Mass Spectrometry (GC/MS) and Liquid Chromatography / Mass Spectrometry (LC/MS) were used for this work. Additionally, with possible effects on cell function as a result of apoptosis, post-thaw, this project aimed to also investigate the effects of cryopreservation of MSC function, with a particular focus on the immunomodulatory properties of the cells. The properties investigated included the ability of the cells to inhibit T cell proliferation and induce the differentiation of the CD4+ T cells into T regulatory cells (Tregs) following co-culture.

Initially, the MSCs were frozen using different freezing procedures involving varying freezing rates and using different DMSO concentrations in the freezing media, with ‘fresh’ MSCs (post-passage cells) used as a control. Investigating the five day proliferation and viability of the cells post-thaw identified that using DMSO concentrations of 5% and 20% (1°C/min freezing rate) resulted in the best and worst outcomes out of the procedures studied. The ‘20% DMSO’ cells were found to perform particularly badly with a significantly lower number of cells (p < 0.01) at day 5 post- thaw, and a significantly lower viability (p < 0.001) at day 2 post-thaw in comparison to the control, possibly due to apoptosis. It was hence expected that the immunomodulatory properties of the ‘20% DMSO’ cells would be affected post-thaw, with apoptotic biomarkers seen when using mass spectrometry.

While the LC/MS work did not find conclusive results, preliminary GC/MS results identified that the decrease in the concentration of benzaldehyde across 24hrs in the headspace of cell cultures could be used as a marker for cell health. In comparison to headspace samples from blank media (control), a significantly greater decrease in benzaldehyde was seen in the headspace of ‘5% DMSO’ and ‘20% DMSO’ cells (p < 0.001 and p < 0.01 respectively) at 24hrs thaw. A significant decrease in benzaldehyde was not seen in the headspace of cells treated with the apoptosis-inducing agent, Staurosporine. However, further work is required to confirm and validate these findings. While there were differences in the concentrations of compounds released by the cells following cryopreservation, such as benzaldehyde, cryopreservation was not found to affect the immunomodulatory properties of the cells.