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

Abstract

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.

Keywords: Cryoprotection; Ice recrystallization; Molecular weight; Permeability; Poly(ethylene glycol).

Fig. 1

Fluorescence images of MSCs recovered from cryopreservation at –196 °C for 7 days, without post-thaw culture (a) and with 24 h post-thaw culture at 37 °C (b). The scale bar is 100 µm. PEGs as cryoprotectants varied their molecular weight over 400, 600, 1 K, 1.5 K, 5 K, 10 K, and 20 K Da at a concentration 10 wt.%. 0 h, 2 h, and 4 h indicate preincubation time of MSCs in the presence of PEGs at 37 °C before cryopreservation. c) Quantitative analysis of cell recovery for the post-thaw culture systems at 37 °C for 24 h. ** indicates p < 0.01. N = 3

Fig. 2

a Live/dead images of PEG-treated cells adhered to the culture plate (A) and suspended in the medium (S). The scale bar is 100 μm. b Quantitative assay of cell viability for adhered cells and suspended cells. The relative cell viability in DMEM in the presence of PEGs compared with in the absence of PEGs (100%). Cell viability was assayed using the CCK-8. ** indicates p < 0.01, N = 3

Fig. 3

Inhibition of growth and nucleation of ice crystals. a Microscopy images of ice crystals in the presence of PEGs solutions (10 wt.%) in DMEM. The scale bar is 100 μm. b MLGS of PEG solutions relative to that of DMEM without PEGs. c) Fraction frozen of the droplets as a measure of INI activity of aqueous PEG solution (10 wt.%). d) Nucleation temperature at which 50% of the 20 droplets was frozen. * and ** indicate p < 0.05 and p < 0.01, respectively. N = 3

Fig. 4

Membrane stabilization of MSCs by PEGs. a Changes in fluorescence intensity in the medium containing PEGs. Control is the medium containing neither DMSO nor PEGs. DMSO 20% and DMSO 10% indicate the medium containing and 20% and 10% of DMSO, respectively. b Comparison of the fluorescence intensity at 180 min from plot a). **: p < 0.01. N = 3

Fig. 5

Internalization of PEGs into the MSCs in the presence of inhibitors. PEG 600, PEG 1.5 K, and PEG 20 K were used. Control is the data in the absence of an inhibitor. ns: nonsignificant. *: p < 0.05. N = 3

Fig. 6

a Fluorescence images exhibiting proliferation of MSCs recovered from cryopreservation at –196 °C for 7 days. PEG 400* and PEG 600* indicate that the recovered cells had been preincubated for 2 h at 37 °C before cryopreservation. DMSO indicates the traditional system using DMSO 10% for cryopreservation of MSCs. The scale bar is 100 μm. b Quantitative analysis of cell proliferation relative to day 0 (100%) assayed by the CCK-8

Fig. 7

Differentiation of recovered MSCs into osteocytes, chondrocytes and adipocytes. The cells were stained by alizarin red (osteogenic; O), alcian blue (chondrogenic; C), and oil red O (adipogenic; A) staining, respectively. The scale bar is 100 μm