Cold storage effects on mitochondrial bioenergetics and protein expression in human mesenchymal stromal cells

A ready-to-use format for cell therapy products, human mesenchymal stromal cells (MSCs) or other progenitor cells, would make their use in acute trauma feasible by the military or in rural community hospitals. In designing a strategy to package MSCs, it was noted that vitality (adenosine triphosphate [ATP] content) fell prior to viability. This study investigated the effects of cold storage on mitochondrial bioenergetics and protein in MSCs.

Commercial MSCs were harvested and resuspended in either a balanced salt solution (PlasmaLyte A) or xeno-free medium (XFM) and then stored at 4°C. Cells were assayed on Days 0, 4, 7, 14, and 21 for cell count, viability, and ATP content, mitochondrial bioenergetics by Seahorse XF24 and Oroboros, and mitochondrial membrane potential by JC1 staining. Levels of proteins involved in mitochondrial function were assayed by Western blots. Proteins assessed included those involved in mitochondrial fusion (OPA1, MFN1, MFN2), fission (FIS1, DRP1, and DRP1 phosphoserine 637), regulation (PINK1 kinase and Parkin ubiquitin-ligase), mitophagy (NDP52 and optineurin), and electron transport chain function (COX IV, SDHB, cytochrome C, and NDUFS1).

Total counts for cells stored in PlasmaLyte A and XFM were similar through Day 21. However, by Day 4, while viability was modestly decreased for cells stored in PlasmaLyte A compared with those in XFM (68% vs. 83%), ATP content plummeted for cells stored in PlasmaLyte A, with only 9.5% of the initial ATP compared with 86% of the initial ATP levels for cells stored in XFM. Both the Seahorse assays and JC1 staining identified further differences between media. JC1 staining revealed that mitochondria were almost completely depolarized by Day 7 following storage in PlasmaLyte A whereas polarized mitochondria were still evident at Day 21 for cells stored in XFM. By Western blot analyses, significant changes in fusion, fission, and mitophagy proteins were observed both for media and over time whereas the electron transport proteins were generally stable. Significant changes in the phosphorylated form of the fission protein DRP1S637 most closely correlated with the ATP data. All parameters were better preserved over time in the XFM.

This study highlighted changes that occur during 4°C storage in the areas of vitality, mitochondrial membrane polarization, and fission. With these targets, research into treatments or additives to a media to improve cold storage and maintain functional cells at 4°C could result in a product that greatly extends the therapeutic use of cellular therapies.