Rediscovery of poly(ethylene glycol)s as a cryoprotectant for mesenchymal stem cells

Background: A medium containing dimethyl sulfoxide (DMSO) (10% v/v) is most widely used for cell cryopreservation at –196 °C. However, residual DMSO consistently raises concerns because of its toxicity; thus, its complete removal process is required. Method: As biocompatible polymers approved by the Food and Drug Administration for various biomedical applications for humans, poly(ethylene glycol)s (PEGs) with various molecular weights (400, 600, 1 K, 1.5 K, 5 K, 10 K, and 20 K Da) were studied as a cryoprotectant of mesenchymal stem cells (MSCs). Considering the cell permeability difference of PEGs depending on their molecular weight, the cells were preincubated for 0 h (no incubation), 2 h, and 4 h at 37 °C in the presence of PEGs at 10 wt.% before cryopreservation at –196 °C for 7 days. Then, cell recovery was assayed. Results: We found that low molecular weight PEGs (400 and 600 Da) exhibit excellent cryoprotecting properties by 2 h preincubation, whereas intermediate molecular weight PEGs (1 K, 1.5 K, and 5 K Da) exhibit their cryoprotecting properties without preincubation. High molecular weight PEGs (10 K and 20 K Da) were ineffective as cryoprotectants for MSCs. Studies on ice recrystallization inhibition (IRI), ice nucleation inhibition (INI), membrane stabilization, and intracellular transport of PEGs suggest that low molecular weight PEGs (400 and 600 Da) exhibit excellent intracellular transport properties, and thus the internalized PEGs during preincubation contribute to the cryoprotection. Intermediate molecular weight PEGs (1 K, 1.5 K, and 5 K Da) worked by extracellular PEGs through IRI, INI, as well as partly internalized PEGs. High molecular weight PEGs (10 K and 20 K Da) killed the cells during preincubation and were ineffective as cryoprotectants. Conclusions: PEGs can be used as cryoprotectants. However, the detailed procedures, including preincubation, should consider the effect of the molecular weight of PEGs. The recovered cells well proliferated and underwent osteo/chondro/adipogenic differentiation similar to the MSCs recovered from the traditional DMSO 10% system. Graphical Abstract: [Figure not available: see fulltext.].