The Institutional Animal Care and Use Committee of Ajou University School of Medicine reviewed and approved all animal experiments, which complied with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Postnatal day 1 (P1) Sprague–Dawley (SD) rats were purchased from Orient Bio Korea, Inc.

OPCs were isolated from P1 SD rat cerebral cortices through a two-step differential centrifugation process developed and previously reported by the authors (Kim et al., 2024). The isolated OPCs were seeded onto Poly-D-Lysine (PDL, Gibco, #A3890401)-coated T75 flasks (Corning, #430641 U) and proliferated for 5 days with OPC proliferation media: Dulbecco’s Modified Eagle Medium/Ham’s F12 (DMEM/F12, Thermo Fisher Scientific, #11320033) with 1% GlutaMAX (Thermo Fisher Scientific, #35050-061), 1% penicillin/streptomycin (Cytiva, #SV30010), 2% B-27 supplement (Thermo Fisher Scientific, #17504-044), 30 ng/mL platelet-derived growth factor-AA (PDGF-AA, PeproTech, #100-13A), 10 ng/mL fibroblast growth factor (FGF, PeproTech, #100-18B), 10 ng/mL epidermal growth factor (EGF, PeproTech, #AF-100-15) (Kim et al., 2024).

After 5 days of proliferation, confluent colonies of OPCs were observed. The cells were detached with 5 mL/flask of Accutase (Thermo Fisher Scientific, #A11105-01) and pelleted by centrifugation, 200 × g/3 min/room temperature (RT = ~23°C). For the direct subculture of passaged OPCs, the cell pellet was resuspended in 1 mL of OPC stabilization media (=OPC proliferation media minus EGF), counted with a dual-fluorescence cell counter, seeded onto PDL-coated 96-well plates or 9 mm coverslips (SPL, #20009) at 1 × 10⁴ live cells/cm², and maintained for 2 days. For the cryopreservation of passaged OPCs, the cell pellet was resuspended in 2 mL/flask CELLBANKER 2 (Zenogen Pharma, Japan), a serum-free cryopreservation medium, and counted with a dual-fluorescence cell counter (Logos Biosystems, #L20001) utilizing acridine orange/propridium iodide (AO/PI). The cell suspension in CELLBANKER 2 was transferred to cryopreservation vials (Thermo Fisher Scientific, #368632), two vials/flask, 1 mL/vial, and the vials were placed in a cell cryopreservation container (Thermo Fisher Scientific, #5100-0001) utilizing isopropanol. The containers were placed in a −80°C deep freezer, as recommended by the manufacturer, for 1 day, after which the cryovials were taken out of the container and stored at −80°C for 1–6 months. On the day of usage, the vial to be used was thawed in a 37°C water bath, and the thawed OPCs in CELLBANKER 2 were transferred to a 15 mL conical tube. Two milliliters of phosphate-buffered saline (PBS) was added, and the tube was centrifuged at 200 × g/3 min/RT. The pellet was resuspended in 1 mL of OPC stabilization media, and the cell yield and viability were measured with a dual fluorescence cell counter. The thawed OPCs were plated on PDL-coated 96-well plates or 9 mm coverslips at 1 × 10⁴ live cells/cm² in OPC stabilization media and maintained for 2 days. Experimental groups used throughout the article were: (1) control (OPCs that were not frozen), (2) 1–2 months cryopreserved OPCs, (3) 3–6 months cryopreserved OPCs.

After 2 days of maintenance in OPC stabilization media, control and cryopreserved OPCs underwent differentiation for 4 days by exchange of culture media to OL differentiation media: Neurobasal medium (NBM, Thermo Fisher Scientific, #21103049) with 1% GlutaMAX, 1% penicillin/streptomycin, 2% B-27, 1% N2 supplement (Thermo Fisher Scientific, #17502048) and 40 ng/mL triiodothyronine (thyroid hormone T3, Sigma Aldrich, #T6397) (Kim et al., 2024). For in vitro myelination, differentiation was induced on OPCs seeded onto PDL-coated cell culture inserts with aligned nanofibers (Sigma Aldrich, #Z694614-12EA) (Kim et al., 2024; Bechler, 2019).

At the endpoint of OPC stabilization or OL differentiation, cells were fixed with 4% paraformaldehyde for 20 min at RT and stored at 4°C until staining. Blocking was performed for 1 h at RT, with a blocking solution comprising 10% normal goat serum (NGS) and 0.1% Triton X-100 in PBS. Following a PBS wash, the cells were incubated overnight (~16 h) at 4°C with the following primary antibodies suspended in the blocking solution: anti-NG2 (1:200, Merck Millipore, #AB5320), anti-myelin basic protein (MBP) (1:500, Abcam, #AB7349), anti-Olig2 (1:200, Merck Millipore, #MABN50), anti-adenomatous polyposis coli (APC-CC1) (1:200, Merck Millipore, #OP80). The cells were washed with PBS, and secondary antibody incubation was conducted for 1 h at RT, with goat secondary antibodies conjugated with Alexa Fluor 488, 594, and 680 (Thermo Fisher Scientific). Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI, 1 μg/mL in distilled water, Sigma Aldrich, #D9542) for 10 min at RT, and the cells were finally mounted onto glass slides. A Zeiss LSM800 confocal microscope was used to visualize the results.

An EdU Assay/EdU Staining Proliferation Kit (iFluor 488) (Abcam, #ab219801) was used to assess OPC proliferation, and EdU staining was performed according to the manufacturer’s protocol. Control and cryopreserved OPCs were stabilized/proliferated for 2 days, and before sample collection and fixation, the OPCs were incubated with EdU (1:1000) for 1 h. The cells were immunostained for Olig2 according to the steps described in section 2.4, with an additional step for EdU labeling with a reaction mixture containing iFluor 488 azide, 30 min at RT, after secondary antibody incubation and before cell nuclei counterstaining.

Statistical analyses used in the article were conducted with GraphPad Prism, version 10.3.0 (GraphPad Software, Boston, Massachusetts, United States). One-way analysis of variance (ANOVA), Kruskal–Wallis tests with Dunn’s multiple comparison tests, and simple linear regression were performed. Statistical analyses were two-sided, and statistical significance was defined as a p-value of <0.05. Cell counting and positive cell detection were performed with the Positive Cell Detection function in QuPath (Bankhead et al., 2017). Fractal dimension analysis to quantify cell morphological complexity was performed with the box counting method by using the Fractal Box Count function in Fiji/ImageJ (Schindelin et al., 2012). The equations used for image quantification were as such: OL purity = Olig2⁺ & DAPI⁺ cells/DAPI⁺ cells, OPC proliferation (cell count) = Olig2⁺ cells per mm² (measured from 16 images, total 3.17mm² and containing ~1,200 OPCs, per sample), OPC proliferation (EdU) = EdU⁺ & Olig2⁺ cells/Olig2⁺ cells, mature OL proportion = MBP⁺ & DAPI⁺ cells/DAPI⁺ cells, APC-CC1⁺ & DAPI⁺ cells/DAPI⁺ cells.

In previous studies, the authors have demonstrated a method utilizing two-step differential centrifugation to isolate OPCs from P1 SD rats, which we have termed the “E3 (easy, efficient and effective) method” (Kim et al., 2024). After 5 days of proliferation, the isolated OPCs could be passaged and stabilized for an additional 2 days to yield OPC cultures of 98–99% purity (Kim et al., 2024). The cell count at the passaging step was 3–5 × 10⁶ OPCs/pup, and a substantial excess of passaged OPCs were unused and discarded. Cryopreservation was applied to make use of the excess OPCs (Figure 1A). The commercially available, serum-free cryopreservation medium CELLBANKER 2 was selected, as serum is known to influence the fate of OPCs towards type 2 astrocytes (Suzuki et al., 2017; Hu et al., 2010; Raff et al., 1983). A brief pilot study revealed that if combined with cooling rate control using isopropanol, passaged OPCs cryopreserved at −80°C for 1–2 months had a viability of 90–95% when measured with a dual-fluorescence cell counter utilizing AO/PI, and also appeared to successfully reproduce basic in vitro OL characteristics such as OPC proliferation and differentiation when observed with a brightfield microscope.

To confirm the implications that cryopreservation is a viable option for primary OPCs cultured through the E3 method, we devised experiments to validate the viability and purity of cryopreserved OPCs and the extent to which they could recapitulate OL functions observed in vitro, such as OPC proliferation, OL differentiation, and in vitro myelination. Three experiment groups were compared: (1) control = OPCs that were directly subcultured without freezing, (2) OPCs cryopreserved at −80°C for 1–2 months, (3) OPCs cryopreserved at −80°C for 3–6 months. When the viability of OPCs from each group was measured with a dual-fluorescence cell counter, the control group had a viability of ~98%, the 1–2 months cryopreservation group showed ~94%, and the 3–6 months cryopreservation group showed a viability of ~88%. The viability of cells in the control group was significantly different from cells in the 3–6 months cryopreservation group (Figure 1C) (p = 0.005, two-sided Kruskal–Wallis test with Dunn’s multiple comparison test, n = 4 independent experiments per group).

Another critical and frequently reported parameter used to evaluate the quality of OL cultures is the purity of OL lineage cells, as primary OL cultures are initially isolated from brain tissue that contains potential contaminants, such as neurons and other glial cells. We have documented 98–99% purity for the E3 method for OPCs stabilized for 2 days after passaging by quantifying the proportion of Olig2⁺ cells. In concordance with our past reports, all three experimental groups exhibited 97–99% purity (Figure 1D) (p = 0.24, two-sided Kruskal–Wallis test, n = 4 independent experiments per group), and the cells could be discerned as OPCs judged by their expression of NG2 and their bipolar morphology (Figures 1B, 2A).

After validating the viability and purity of cryopreserved OPCs, the three experimental groups were compared for OPC proliferation, an essential function of OPCs as progenitors of the OL lineage. The number of viable OPCs seeded was kept consistent for all three experiment groups. The seeded OPCs were maintained for 2 days in stabilization media containing PDGF-AA and FGF, two mitogens known to synergistically promote OPC proliferation and prevent differentiation into mature OLs (McKinnon et al., 1990; Baron et al., 2000; Hart et al., 1989; McKinnon et al., 1991). Proliferation was assessed through two analyses: (1) measurement of OPC cell density (=Olig2⁺ cells/mm²), (2) proportion of OPCs positive for the proliferation marker EdU (=EdU⁺ Olig2⁺/Olig2⁺ cells). The density of OPCs was ~450 OPCs/mm² for the control and 1–2 month cryopreservation group, and 333 OPCs/mm² in the 3–6 months cryopreservation group (Figure 2B) (p = 0.03, two-sided Kruskal–Wallis test, n = 8 independent experiments per group). The proportion of OPCs positive for EdU was approximately 17% for all groups (Figure 2C) (p = 0.75, two-sided Kruskal–Wallis test, n = 4 independent experiments per group).

Another crucial function of the OL lineage is the differentiation of OPCs into mature, myelinating OLs. After 2 days of OPC stabilization, we induced differentiation with an NBM-based differentiation medium containing thyroid hormone T3, a well-established inducer of in vitro and in vivo OL differentiation (Barres et al., 1994). After 4 days of differentiation, APC-CC1⁺ and MBP⁺ mature OLs could be observed (Figure 3A). Complex, web-like myelin processes, which are characteristic of OLs cultured on two-dimensional surfaces, were evident in all experiment groups. The extent of maturation was evaluated with two parameters: (1) the proportion of mature OLs (=MBP⁺ DAPI⁺/DAPI⁺ cells, APC-CC1⁺ DAPI⁺/DAPI⁺ cells), (2) morphological complexity of MBP⁺ processes (=measurement of fractal dimensionality through the fractal box counting method in Fiji/ImageJ). Cryopreserved OPCs did not differ in terms of the proportion of cells that matured (Figures 3B,C) (p = 0.25 and 0.44, two-sided Kruskal–Wallis test, n = 4 independent experiments per group) nor in terms of morphological complexity (Figures 4A,B) (p = 0.75, one-way ANOVA, two-sided, n = 4 independent experiments per group). Thus, we concluded that cryopreserved OPCs could adequately develop into mature OLs.

After confirmation of successful OL differentiation, the last function to be evaluated was myelination, the OL characteristic directly related to saltatory conduction. It has been demonstrated in previous studies that myelination can be achieved in vitro by culturing primary OLs on aligned nanofibers of appropriate caliber, mimicking axons in the white matter of the brain (Bechler, 2019). For in vitro myelination, OPCs were seeded onto inserts with aligned polycaprolactone (PCL) nanofibers of ~700 nm diameter, stabilized for 2 days, and subsequently differentiated for 4 days. Upon immunofluorescent staining with MBP, instances of MBP⁺ myelin processes enwrapping the fibers could be observed in both control and cryopreserved groups, indicating that cryopreserved OLs were able to form myelin sheath-like structures around axon-mimicking structures (Figure 5). In summary, cryopreserved OPCs could recapitulate in vitro OL functions in a similar manner as non-frozen controls.