Mesenchymal stem cells (MSCs) are plastic adherent spindle shaped cells that express stem cell markers CD73, CD90 and CD105, while lacking leukocyte, endothelial and haematopoietic markers such as CD34 and CD45. They can differentiate into a range of different cell types. Compared to embryonic stem cells, they are easier to purify and can be used for allogenic therapy. As of 2021, over 900 trials have taken place, primarily involving tissue repair, treatment of immune disorders and more recently, COVID-19. Cryopreservation damages cells, while current non-freezing methods require cold temperature, expensive special materials or serum, and have not been tested beyond 7 days. The project aims to improve on current work by using a simple and chemically defined medium to maintain cell survival, consisting of a DMEM base to provide the necessary substrates, vitamins, salts and amino acids, insulin supplement (ITSE) to keep cells viable in the absence of serum and a low bicarbonate buffer to maintain physiological pH at atmospheric CO2.

The medium was developed at 37°C using hTERT MSC monolayers. When hTERT human MSCs were cultured in the insulin based preservation medium, cells proliferated between day 6 and 12, while in DMEM basal medium cell death occurred. Cell monolayers in ITSE based medium displayed higher levels of oxidative stress, but no significant difference in autophagy compared to cells in expansion media (DMEM/FBS). hTERT MSCs in ITSE based media displayed lower levels of glucose consumption, lactate production and proliferation, and displayed a wire like morphology. When seeded in DMEM/FBS after 12 days in ITSE based media, metabolic and morphological changes were reversed, and cells retained proliferation and differentiation potential.

The ITSE based medium was then adapted to work at room temperature by lowering the bicarbonate concentration from 44 mM to 0.9 mM. The majority of hTERT human and primary bovine MSCs remained viable as monolayers by day 12. hTERT MSCs retained proliferation potential, but primary bovine MSCs remained permanently anchored to the flask. When preserved as 3D structures in alginate beads, at 12k cells/bead and 360k cells/mL of ITSE based media, both hTERT and primary MSCs remained viable and functional for 8 days. At this point the limiting factor was lack of serum and pH acidity. In alginate beads at atmospheric conditions, hTERT MSCs still underwent low levels of glycolysis over 6 days. For primary MSCs, metabolite changes over 6 days were too low to measure.