Low-Molecular-Weight PEGs for Cryopreservation of Stem Cell Spheroids

Abstract

Stem cell spheroids (SCSs) are a valuable tool in stem cell research and regenerative medicine. SCSs provide a platform for stem cell behavior in a more biologically relevant context with enhanced cell-cell communications. In this study, we investigated the recovery of SCSs after cryopreservation at -196 °C for 7 days. Prior to cryopreservation, the SCSs were preincubated for 0 h (no preincubation), 2 h, 4 h, and 6 h at 37 °C in the presence of low-molecular-weight poly(ethylene glycol) (PEG) with molecular weights of 200, 400, and 600 Da. The recovery rate of SCSs was markedly affected by both the PEG molecular weight and the preincubation time. Specifically, when SCSs were preincubated with a PEG200 solution for 2 to 6 h, it significantly enhanced the recovery rate of the SCSs. Internalization of PEG200 through simple diffusion into the SCSs may be the cryoprotective mechanism. The PEG200 diffuses into the SCSs, which not only suppresses osmotic pressure development inside the cell but also inhibits ice formation. The recovered SCSs demonstrated both fusibility and capabilities for proliferation and differentiation comparable to SCSs recovered after dimethyl sulfoxide 10% cryopreservation. This study indicates that PEG200 serves as an effective cryoprotectant for SCSs. A simple preincubation procedure in the presence of the polymer greatly improves the recovery rate of SCSs from cryopreservation.

Fig. 1.

(A) Preparation of SCSs. Phase-contrasted images and live/dead images of SCSs are shown in white/black and color, respectively. The green fluorescence indicates live cells. (B) Cryopreservation process of harvested SCSs. The SCSs were suspended in DMEM solutions containing low-molecular-weight PEGs and preincubated for 0 to 6 h before cryopreservation. They were slowly cooled to –80 °C at a cooling rate of –1 °C/min and incubated at –80 °C for 12 h. Then, they were quenched into liquid nitrogen and cryopreserved at –196 °C for 7 days, followed by fast thawing at 37 °C.

Fig. 2.

(A) Live/dead images of SCSs recovered from cryopreservation at –196 °C for 7 days. Live and dead cells are shown in green and red, respectively. 0 h, 2 h, 4 h, and 6 h indicate the preincubation time of SCSs in the presence of PEGs (10 wt.% in DMEM) at 37 °C before cryopreservation. The scale bar is 100 μm. (B) Quantitative analysis of spheroid recovery using the CCK-8 kit. Recovery rate of SCSs from cryopreservation in DMSO 10% was assigned as 100%. N = 3. The asterisks * and ** indicate P < 0.05 and P < 0.01, respectively.

Fig. 3.

Cytotoxicity assay of PEGs for SCSs. (A) Live/dead images of SCSs in DMEM containing PEGs. The SCSs were suspended in the solution and incubated at 37 °C for 0 h, 2 h, 4 h, and 6 h. The scale bar is 100 μm. (B) Cell viability assayed using the CCK-8 kit. The relative cell viability was compared with 0 h (100%).

Fig. 4.

(A) Ice crystal images formed from PEG aqueous solution (10.0 wt.% in DMEM). The images of ice crystals formed from DMEM without PEG were also shown as a control. (B) Mean largest grain size (MLGS) of PEG aqueous solutions (10 wt.% in DMEM). The MLGS of ices formed from DMEM without PEG was assigned to be 100%. (C) INI measurements of PEG aqueous solutions (10 wt.%). Twenty droplets were used for determination of INI activity. (D) Ice nucleation temperature at which 50% of the droplets were frozen.

Fig. 5.

Internalization of PEG200 (A) and PEG20K (B) into the SCSs. SCSs were incubated for 6 h in the PEG solutions (10 wt.%) in DMEM in the presence of inhibitors. The control indicates the same protocol in the absence of inhibitors. N = 3. No statistical significance in the internalization of PEG200 among the treated systems in (A). The asterisk * in (B) indicates P < 0.05.

Fig. 6.

(A) The simulated internalization for PEGs compared to PEG200 assuming passive diffusion. The simulated data were obtained using Fick’s law. The observed actual PEG200 and PEG20K internalization is shown (aPEG200 and aPEG20K) in comparison. aPEG and sPEG indicate actual and simulated PEG, respectively. (B) Simulated relative loading of PEGs in SCS of equivalent diameters versus relative time (t^#) assuming non-dimensional diffusion coefficients with a ratio of 1/0.76/0.65/0.16 for PEG200/PEG400/PEG600/PEG20K.

Fig. 7.

F-actin (green) staining of SCSs recovered from cryopreservation. The scale bar is 100 μm. The PEG200/6 h indicates the SCSs preincubated at 37 °C for 6 h before cryopreservation.

Fig. 8.

Fusibility of the recovered SCSs using PEG200, PEG400, and PEG600 preincubation for 0 to 6 h followed by cryopreservation at –196 °C for 7 days. SCSs were put together on a non-adhesive dish for fusibility, and their fusion was observed in 3 days. The scale bar is 100 μm.

Fig. 9.

Proliferation of SCSs recovered from cryopreservation at –196 °C for 7 days. (A) Fluorescence images of cells 3 days after the proliferation. The scale bar is 100 μm. (B) Quantitative analysis of cell proliferation relative to day 0 (100%) assayed by the CCK-8. N = 3. The asterisks * and ** indicate P < 0.05 and P < 0.01, respectively.

Fig. 10.

Differentiation of recovered SCSs into osteocytes, chondrocytes, and adipocytes. (A) The cells were stained in red, blue, and brown by oil red O (adipogenic), alcian blue (chondrogenic), and alizarin red (osteogenic), respectively. (B) Semiquantitative expression of the images using ImageJ software.