Hypothermia and nutrient deprivation alter viability of human adipose-derived mesenchymal stem cells

Abstract Purpose Adipose-derived mesenchymal stem cells (MSCs) are attractive biological agents in regenerative medicine. To optimize cell therapies, it is necessary to determine the most effective delivery method for MSCs. Therefore, we evaluated the biological properties of MSCs after exposure to various temperatures to define optimal storage conditions prior to therapeutic delivery of MSCs. Design Prospective observational study. Methods and materials Adherent and non-adherent MSCs were incubated at multiple temperatures (i.e., 4, 23 and 37 °C) in Lactated Ringers (LR) solution lacking essential cell growth ingredients, or in culture media which is optimized for cell growth. Cells were assessed either after the temperature changes (4 h) or after recovery (24 h). Metabolic activity of MSCs, cell number and expression of representative mRNA biomarkers were evaluated to assess the biological effects of temperature. We monitored changes in mRNAs expression related to cytoprotective- or stress-related responses (e.g., FOS, JUN, ATF1, ATF4, EGR1, EGR2, MYC), proliferation (e.g., HIST2H4, CCNB2), and extracellular matrix production (ECM; e.g., COL3A1, COL1A1) by quantitative real time reverse-transcriptase polymerase chain reaction (RT-qPCR) analysis. Results Our study demonstrates that storing MSCs in Lactated Ringers (LR) solution for 4 h decreases cell number and metabolic activity. The number of viable MSCs decreased significantly when cultured at physiological temperature (37 °C) and severe hypothermia (4 °C), while cells grown at ambient temperature (23 °C) exhibited the least detrimental effects. There were no appreciable biological differences in mRNA markers for proliferation or ECM deposition at any of the temperatures. However, biomarkers related to cytoprotective- or stress-responses were selectively elevated depending on temperature or media type (i.e., LR versus standard media). Conclusion The biological impact of nutrient-free media and temperature changes after 4 h exposure persists after a 24 h recovery period. Hence, storage temperature and media conditions should be optimized to improve effective dosing of MSCs.