The present study was approved by the ethics committee of Shanghai Jiao Tong University School of Medicine (Shanghai, China). All surgical interventions, treatments and postoperative animal care procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals. Three adult Rhesus monkeys (4-year-old males, weighing 5.88–8.24 kg) were purchased from Ping'an Animal Reproduction Center of Chengdu (reproduction license no. SCXK 2008–013; Chengdu, China). All monkeys were individually housed at the Department of Laboratory Animal Sciences at Shanghai Jiao Tong University School of Medicine, at a temperature of 21°C with 55% humidity under a 12-h light/dark cycle with free access to food and water.

Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Hyclone (GE Healthcare Life Sciences, Little Chalfont, UK). Collagenase NB4 was obtained from Serva Electrophoresis GmbH (Heidelberg, Germany). Neutral protease Dispase II was from Roche Applied Science (Madison, WI, USA). Heregulin-β1 and b-FGF were sourced from PeproTech, Inc. (Rocky Hill, NJ, USA). Forskolin was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Cytosine-B-arabinoside hydrochloride (Ara-C), penicillin-streptomycin and 0.25% trypsin-EDTA were obtained from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). All the culture plates were BD Falcon; BD Biosciences (Franklin Lakes, NJ, USA). The compositions of the culture media used for SC isolation are presented in Table I. The following primary antibodies were used for immunofluorescence and flow cytometry: Rabbit anti-S100 calcium binding protein B (S100β; cat no. Z0311) polyclonal antibody (Dako; Agilent Technologies, Santa Clara, CA, USA), anti-glial fibrillary acidic protein (GFAP; cat no. ab7260) polyclonal antibody and anti-nerve growth factor receptor (P75NTR; cat no. ab8874) polyclonal antibody (Abcam, Cambridge, UK). The Alexa Fluor 488-conjugated goat anti-rabbit-IgG secondary antibody (cat no. R37116) was purchased from Invitrogen (Thermo Fisher Scientific, Inc.).

All surgical procedures were performed under sterile surgical conditions and general anesthesia. The animals were anesthetized by intramuscular injection of a mixture of ketamine (20 mg/kg) and xylazine (2 mg/kg). Briefly, the bilateral common peroneal nerve was exposed with an incision that extended from the lower interface along the biceps femoris distally towards the popliteal fossa. Two 7-cm segments of the common peroneal nerves were then excised. The nerves were immediately washed three times with ice-cold phosphate-buffered saline (PBS, pH 7.2) supplemented with 2% FBS. The epineurium was gently stripped off using fine-pointed forceps and microscissors under an anatomical microscope. The common peroneal nerve segments were cut into short pieces (2–3 mm in length), placed into one 6-well plate and cultured first in basal medium (BM) for 3 h and then switched to Schwann cell culture medium (SCCM). The cells were kept in a humidified atmosphere of 5% CO₂ at 37°C. The nerve pieces were monitored for evidence of in vitro pre-degeneration on the 2nd, 5th and 7th day of culture. The finalized process of SC isolation and purification is summarized and presented in Fig. 1.

For dissociation of the nerve pieces, the pre-degeneration medium was discarded, and the nerve fragments were transferred to a 15-ml conical tube containing 0.2% (0.27 U/ml) collagenase NB4 in DMEM and incubated at 37°C for 45–60 min until the cells were dispersed into a homogeneous suspension. Following centrifugation of the suspension at 600 × g for 5 min at room temperature, the supernatant was removed and discarded, and the cells were re-suspended in SCCM. The cells were subsequently seeded onto one laminin-coated six-well plate and cultivated at 37°C in 5% CO₂ for 48 h prior to isolation of the SCs.

Differential detachment methods were applied as previously described (10). Following cultivation in the six-well plate for 48 h, the SCCM was replaced with 0.1 ml/cm² of Dispase II (1.25 U/ml) diluted in DMEM. The cells were then incubated at 37°C for 10–15 min and subsequently shaken horizontally for 1 min to release rounded-up cells. The suspended cells were transferred to a 15-ml conical tube and centrifuged at 600 × g for 5 min at room temperature. The supernatant was discarded, and the cells were re-suspended in purification medium (PM) to reduce the fibroblast contamination, and cultured for 24 h. After another 24-h cultivation period, the cells were subjected once more to the aforementioned purification steps (Dispase II treatment) and re-suspended in SCCM (Fig. 1).

In order to characterize the isolated cells, S100β, P75NTR and GFAP immunostaining was performed. The cells were fixed to slides with 4% paraformaldehyde (PFA) for 15 min at room temperature, washed three times with PBS, treated with 0.3% Triton X-100 to permeabilize the membranes, washed again in PBS and blocked with 10% bovine serum album (Sigma-Aldrich; Merck KGaA) at 37°C for 30 min. The slides were subsequently incubated with rabbit anti-S100 polyclonal antibody (1:200), rabbit anti-P75NTR polyclonal antibody (1:500) or rabbit anti-GFAP polyclonal antibody (1:500) at 37°C for 1 h. The slides were washed three times with PBS and incubated with a FITC-conjugated goat anti-rabbit IgG secondary antibody (1:500; cat no. F2765) for 1 h at 37°C, and finally incubated with 4′, 6′-diamidino-2-phenylindole dihydrochloride (DAPI; 1:500; Invitrogen; Thermo Fisher Scientific, Inc.) for 2 min at room temperature to stain the cell nuclei. Images were captured under a fluorescence microscope (Olympus Corporation, Tokyo, Japan) and processed using Image-Pro Plus version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). Cells in three randomly selected fields were counted to calculate the SC purity.

To further analyze SC purity using specific marker molecules, P0 (before purification) cells and P2 (after purification) cells were collected after 1 min incubation with 0.25% trypsin-EDTA at 37°C, washed with and suspended in blocking buffer (2% FBS in PBS) and incubated with rabbit anti-P75NTR antibody (1:500) for 30 min at 4°C. The cells were subsequently washed twice with blocking buffer and incubated with an Alexa Fluor 488-conjugated goat anti-rabbit-IgG secondary antibody (1:1,000) for 15 min at 4°C. Cells were analyzed using an EPICS Altra flow cytometer (Beckman Coulter, Inc., Fullerton, CA, USA). Cells without primary antibody treatment were used as a blank control.

SCs were morphologically identified from fibroblasts under a phase-contrast microscope and counted in three randomly selected fields to obtain an average number. Cells with a bipolar or tripolar shape were identified as SCs and flat or polygonal cells were identified as fibroblasts. Following trypsinization, all cells (SCs and fibroblasts) were counted with a hemocytometer to determine the total yield. Purity was defined as the percentage of SCs relative to the total number of counted cells. Cell yields were expressed as the mean of 10⁶ cells per biopsy. Cell number was presented as the mean ± standard deviation (SD).

Data were presented as the mean values ± SD. For quantitative comparison and analysis, an unpaired Student's t-test and one way analysis of variance followed by Dunnett's post hoc test were used to compare differences between groups. All statistical analyses were performed using SPSS version 18.0 (SPSS, Inc., Chicago, IL, USA). P-values <0.05 were considered statistically significant.

Following 2 days of pre-degeneration treatment, several cells began to migrate out of the nerve explants and attach to the culture dish. The number of cells that migrated out of the nerve explants increased with increased treatment time. The cells migrating into the BM were predominantly fibroblasts and rarely SCs (Fig. 2A). Following 2, 5 and 7 days of pre-degeneration, the nerve explants were subjected to enzymatic digestion and examined to determine SC yield prior to seeding the cells onto laminin-coated six-well plates. The cell yield (Fig. 2B) was 50±8.2×10⁴ cells/well after 2 days of pre-degeneration in SCCM, and 44±7.2×10⁴ cells/well after 2 days of pre-degeneration in BM. The cell yield in SCCM (120±21×10⁴ cells/well) was 1.25-fold higher compared with the BM media (96±1.5×10⁴ cells/well) after 7 days of pre-degeneration, and was 2.84 times higher compared with the immediate culture cells that had not undergone pre-degeneration (43±1.5×10⁴ cells/well). In addition, the purity of the SCs was slightly higher after 7 days of pre-degeneration and culture in SCCM (63.17±7.1%) compared with 7 days of pre-degeneration and culture in BM (58.33±6.2%) (Fig. 2C). These results demonstrated that SC purity and yield were greater than the fibroblast purity and yield following in vitro pre-degeneration in SCCM.

The majority of cells suspended in SCCM adhered to the laminin-coated flasks within 24 h and exhibited two distinct morphologies: SCs and fibroblasts (Fig. 3A). Fibroblasts were characterized by a regular polygonal shape and ovoid nucleus (Fig. 3B). The SCs were characterized as small, elongated bipolar (Fig. 3C), tripolar (Fig. 3D) or multipolar (Fig. 3E) cells with a bright nucleus. The fibroblasts were tightly attached to the cell plate and adhered under the SCs (Fig. 3F), which facilitated the removal of the SCs.

Following the first dispase treatment, the SCs appeared to proliferate faster than the fibroblasts (Fig. 4A); however, the fibroblasts subsequently became the predominant cell type. Therefore, Ara-C was used to inhibit fibroblast growth during the 48–72 h period. Following the first round of SC purification on day 9 and the use of Ara-C (Fig. 4B), SC purity markedly increased to 85.1±7.4%, determined by cell counting based on morphological differences. However, the remaining fibroblasts continued to proliferate rapidly and an additional round of purification was performed on day 10. Following the second round of purification and subsequent 48 h culture, SC purity reached 96.3±3.9% (Fig. 4C and D). A significant difference in SC purity was observed between days 9 and 12. To determine the final cell yield, the enriched SCs were identified by cell counting using a hematocytometer on days 9, 10 and 12. The cell yield increased and reached 2.1±0.2×10⁶ cells/well following the second round of purification (Fig. 4E). These results demonstrated that cell yield and SC purity were significantly increased following two rounds of purification.

Immunostaining for S100β protein (predominantly expressed in SC nuclei), P75NTR (primarily expressed on the cell surface and may be used as a marker in flow cytometric analysis) and GFAP (expressed in cells lacking fibronectin) were used to differentiate SCs from fibroblasts. Purified SCs exhibited typical bipolar or tripolar morphology and oval nuclei and unlike fibroblasts, were immunopositive for S100β,P75NTR and GFAP (Fig. 5).

To provide further quantitative evidence of SC purity, the cells were analyzed before and after purification by flow cytometry with a P75NTR-specific rabbit antibody and tagged with an anti-rabbit IgG antibody (Alexa Fluor 488). Following pre-degeneration, cell suspensions obtained from collagenase digestion of the nerve explants were incubated for 48 h, immunostained for P75NTR, and analyzed by flow cytometry. The percentage of P75NTR-positive SCs was 65.2±1.3% at 48 h and 98.4±1.5% following one purification cycle. There was a significant difference in cell percentages (Fig. 6).