Cell Tissue Res (2009) 337:361–369 DOI 10.1007/s00441-009-0836-4
REGULAR ARTICLE
An improved method for isolating Schwann cells from postnatal rat sciatic nerves Yujun Wei & Jianli Zhou & Zhenghuan Zheng & Aijun Wang & Qiang Ao & Yandao Gong & Xiufang Zhang
Received: 10 April 2009 / Accepted: 24 June 2009 / Published online: 29 July 2009 # Springer-Verlag 2009
Abstract The major difficulty in Schwann cell (SC) purification is contamination by fibroblasts, which usually become the predominant cell type during SC enrichment in vitro. Current reported measures are mainly limited by either high cost or complicated procedures with low cell yields or purity. Our objectives have been to develop an efficient, easily applicable, rapid method to obtain highly purified SC from the sciatic nerve of newborn rats. The method involves two rounds of purification to eliminate fibroblasts with the novel combined use of cytosine-Barabinoside hydrochloride (Ara-C) action and differential This work was supported by Tsinghua-Yue-Yuen Medical Sciences Fund, the National Natural Science Foundation of China (contract grant numbers: 30670528, 30700848, 30772443), Beijing Municipal Science & Technology Commission (BMSTC, contract grant number: H060920050430), National Basic Research Program of China (also called the 973 Program, contract grant number: 2005CB623905), and the National Natural Science Foundation of Beijing (contract grant number: 7082090). Y. Wei : Z. Zheng : Y. Gong : X. Zhang (*) Department of Biological Sciences and Biotechnology, State Key Laboratory of Biomembrane and Membrane Biotechnology, Tsinghua University, Beijing 100084, China e-mail:
[email protected] J. Zhou Department of Physics and Mathematics, Kunming Medical Collage, Kunming, Yunnan 650031, China A. Wang Department of Bioengineering, University of California, Berkeley CA 94720, USA Q. Ao Department of Neurosurgery of Yuquan Hospital, Tsinghua University, Beijing 100049, China
cell detachment. Cultured cells were first treated with Ara-C for 24 h. The medium was replaced with the growth medium containing 20 ng/ml human heregulin1-β1 extracellular domain (HRG1-β1 ECD). After another 48 h in culture, the cells were treated with 0.05% trypsin, following which SCs, but not fibroblasts, were easily detached from the dishes. The advantage of this method is that the two steps can eliminate the fibroblasts complementarily. Ara-C eliminates most of the fibroblasts growing among SCs, whereas the differential cell detachment technique removes the remainder growing under or interacting with the SC layer. A purity of more than 99% SCs has been obtained, as confirmed by cell morphology and immunostaining. The purified SCs have a spindle-shaped, bipolar, and sometimes tripolar morphology, align in fascicles, and express S-100. The whole procedure takes about 10 days from primary culture to the purified SCs growing to confluence (only half the time reported previously). This protocol provides an alternative method for investigating peripheral nerve regeneration and potentially could be used to produce enough SCs to construct artificial nerve scaffolds in vitro. Keywords Schwann cells . Isolation . Cytosine arabinoside . Differential cell detachment . Peripheral nerve regeneration . Rat (Sprague Dawley)
Introduction Schwann cell (SCs), the principal supporting cells of the peripheral nervous system, are crucially involved in the functional recovery of injured peripheral nerves (Bunge 1993). After peripheral nerves are transected, Wallerian degeneration takes place. SCs, together with macrophages remove necrotic tissue and myelin debris. Furthermore, SCs
362
proliferate to form Büngner bands, which help the regrowing axons to elongate their growth cones in the direction of denervated targets. The whole regeneration process is completed when the regenerative axons are remyelinated by SCs. The nerve regeneration process is also regulated by surface cell adhesion molecules (Shibuya et al. 1995), cellular matrix protein (Rothblum et al. 2004), and neurotrophic factors (Fawcett and Keynes 1990; Haastert et al. 2005) synthesized directly or indirectly by SCs. One of the promising methods for peripheral nerve repair is to use artificial nerve grafts containing SCs (Mosahebi et al. 2001). Compared with the use of a conduit alone, the bio-hybrid graft has many advantages. It uses biodegradable materials and cells that help raise the recovery of the injured organs and has been applied in clinical settings (Vacanti et al. 2001). Therefore, the production of a large number of viable SCs in a short period, which is essential for the application of tissue engineering, is necessary for clinical use. For this purpose, many efforts have been made during the last few decades to establish an optimal method for the rapid isolation and high enrichment of SCs. The enrichment of a large population of SCs within a short time span is a difficult task, since these cells are easily contaminated by fibroblasts and proliferate poorly. The abundant connective tissues in nerves further complicate their enrichment during the following purification procedure. To date, several methods have been described for obtaining SCs, including antimitotic treatment (Calderon-Martinez et al. 2002), a combination of antimitotic treatment and antibody-mediated cytolysis by employing complements (Brockes et al. 1979), a repeated explantation method (Oda et al. 1989), differential adhesion methods (Pannuzio et al. 2005), immunoselective methods (Manent et al. 2003), in vivo or in vitro predegeneration (Komiyama et al. 2003; Mauritz et al. 2004), the cold jet technique (Jirsová et al. 1997), differential detachment methods (Jin et al. 2008), and a combination of the in vitro predegeneration and cold jet technique (Haastert et al. 2007, 2009). These methods can provide highly purified SCs with various efficiencies. However, they have certain limitations, such as the requirement for special equipment, complicated procedures, or high cost. We have modified the techniques in several previously reported methods (Calderon-Martinez et al. 2002; Jin et al. 2008) and added new strategies to establish an easier but nevertheless fast method to enrich SCs with high purity.
Cell Tissue Res (2009) 337:361–369
mixture (DMEM/F-12) were from Invitrogen. Cytosine-Barabinoside hydrochloride (Ara-C), penicillin, and streptomycin were from Sigma. Human heregulin1-β1 extracellular domain (HRG1-β1 ECD) was from R&D. Fetal bovine serum (FBS) was obtained from Hyclone. All the culture plates were from Corning (Costar brand). All other reagents were local products of analytical grade. The compositions of the optimized culture media employed for the isolation procedures of SCs are presented in Table 1. Primary antibodies employed for immunocytochemistry, including rabbit anti-S-100 protein monoclonal antibody, were obtained from Sigma. Secondary antibodies, including fluorescein isothiocyanate (FITC)-conjugated goat antirabbit IgG (H+L), were purchased from Invitrogen. Postnatal Sprague-Dawley rats (1–3 days old) were obtained from the Beijing Haidian Experimental Animal Center. All animal experiments were carried out in accordance with US National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications no. 80–23) revised 1996 and were approved by the Beijing Administration Committee for Experiment Animals. Primary culture Postnatal Sprague-Dawley rats were killed by decapitation. Both sciatic nerves were dissected out from each pup. Nonnervous tissue was gently striped under a dissecting microscope, and the nerve segments were stored in icecold DMEM/F-12 containing 1% penicillin/streptomycin. The nerve segments were washed in DMEM/F-12 twice, then re-suspended in 0.3% collagenase type II solution (100 μl per segment) and incubated for 30 min at 37°C. After incubation, the enzymatic solution was carefully Table 1 Composition of cell culture media (DMEM/F-12 Dulbecco’s modified Eagle medium/F-12 Ham nutrient mixture, FBS fetal bovine serum, Ara-C cytosine-B-arabinoside hydrochloride, HRG1-β1 ECD human heregulin1-β1 extracellular domain) Culture medium
Composition
SC basal medium
DMEM/F-12 containing: 10% FBS 1% penicillin/streptomycin DMEM/F-12 containing: 10% FBS 10 μM Ara-C 1% penicillin/streptomycin DMEM/F-12 containing: 10% FBS 20 ng/ml HRG1-β1 ECD 1% penicillin/streptomycin
SC purification medium
Materials and methods Materials Collagenase type II was purchased from Merck. Trypsin and Dulbecco’s modified Eagle medium/F-12 Ham nutrient
SC growth medium
Cell Tissue Res (2009) 337:361–369
363
removed, and an equal volume of 0.25% trypsin-EDTA was added. The nerve segments were incubated for another 5 min at 37°C and then mechanically dissociated until they formed a homogeneous suspension. SC basal medium was then added to the suspension at a ratio of 4:1 in order to terminate the activity of the trypsin. The mixture was centrifuged at 800– 1000 rpm for 5 min. The supernatant was discarded, and the cells were re-suspended in SC basal medium. Finally, the cells were plated at a sub-confluent concentration onto 60-mm culture plates and cultivated at 37°C, 5% CO2 for 24 h before starting the isolation of SCs (one 60-mm culture plate per five peripheral nerve segments).
under the phase-contrast microscope until the maximum amount of cells was dissociated (no more than 30 s). A four-fold volume of FBS was added to the cell suspension to terminate the activity of trypsin. The resultant cell suspension was transferred into 15-ml conical tubes and centrifuged at 800–1000 rpm for 5 min. The centrifuged supernatant was removed, and the pellet was re-suspended in the medium (containing half the volume of the above collected supernatant medium and half the volume of fresh SC basal medium) and then plated onto culture dishes (1:3).
First round of SC purification
The purity of the SCs was determined on the basis of cell morphology. Cell morphologies of SCs and fibroblasts were differentiated by phase-contrast microscopy. Cells with a bipolar or tripolar, spindle-shaped morphology were identified as SCs, whereas flat or polygonal cells were considered to be fibroblasts. To quantify SC purity, six fields of vision from each culture plate were randomly selected. The numbers of SCs or fibroblasts were counted. The percentage of SCs was counted relative to the total number of counted cells (SCs per total cells), and means±SD were obtained.
After the cells had been cultured for 24 h, SC basal medium was replaced with SC purification medium to eliminate fibroblasts. This medium was removed 24 h later, and the cell debris was washed away by gently rinsing the monolayer twice with DMEM/F-12. The SC growth medium was then added to the dishes, and the cells were cultured for another 48 h before the second round of the isolating step was started.
SC purity
Second round of SC purification SC proliferation This is the most important step for obtaining the maximum number and purity of viable SCs. The detailed procedure is as follows. After the 48-h culture (see above), SC growth medium was discarded, the cells were washed twice with phosphate-buffered solution (PBS, pH 7.2), and 0.05% trypsin-EDTA was added (1 ml enzymatic solution per 60mm culture plate). Then, the culture plates were continuously shaken vertically for 1 min and subsequently horizontally until most of SCs were detached from the dishes. All the above procedures were examined with a phase-contrast microscope. As soon as the majority of SCs were suspended, a four-fold volume of SC basal medium was added to the cell suspension to terminate the activity of the trypsin. The suspended cells were then collected into a conical tube and centrifuged at 800–1000 rpm for 5 min. After removal of the supernatant, the pellet was resuspended in SC growth medium and plated onto a culture plate at a density of 5×104 cells/ml. Half of the medium of the cells was replaced by an equal volume of fresh SC basal medium twice a week. To determine whether the differential cell detachment was necessary, cells without the second isolating step were maintained as a control. After cultures had grown to confluence (5–6 days), the supernatant medium was collected. The cells were washed twice with PBS (pH 7.2), and 2.5% trypsin-EDTA was added to the culture dishes. During trypsinization, cell dissociation from the dishes was monitored continuously
To assess the proliferation of the purified SCs, cells were initially seeded at 4.3×104 cells/well onto 24-well plates. After the SCs had grown for 1, 3, or 5 days, the proliferation of the cells was determined by MTT assay, which detected mitochondrial dehydrogenase activity of viable cells spectrophotometrically. Briefly, cells were cultured for 1, 3, and 5 days, and 100 μl MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5diphenyltetrasodium bromide, 5 mg/ml) was added to each well and incubated at 37°C for 4 h. Then, the MTT solution was carefully removed, and the insoluble blue formazan crystal was dissolved in 1 ml dimethyl sulfoxide; 100 μl solution was aspirated from each well and poured into a 96well plate. The absorbance of each well was measured by an enzyme-linked immunosorbent assay at 570 nm. In addition, the MTT assay was performed on a directly counted SCs series (0.2×105, 0.4×105, 0.8×105, 1.6×105, 3.2×105, 6.4×105), and the absorbency values were plotted against the counted cell numbers to established a standard calibration curve. Viable cell numbers were then determined from the standard curve based on their MTT absorbency. Cells numbers were represented as means±SD. Immunocytochemistry analysis SCs were characterized immunocytochemically by antibodies directed against S-100 protein, based on the protocol
364
Cell Tissue Res (2009) 337:361–369
previously described by Komiyama et al. (2003). Briefly, cells cultured on coverslips were fixed with 4% paraformaldehyde (PFA) in PBS for 10 min and then permeabilized with 0.05% Triton X-100 for 10 min. Non-specific sites were blocked with 5% goat serum for 1 h. Then, the cells were incubated with rabbit anti-S-100 protein monoclonal antibody (1:200) at room temperature for 1 h, washed three times with PBS, and incubated with the corresponding secondary antibodies (1:200, FITCconjugated goat anti-rabbit IgG) for 2 h at room temperature. After a second rinsing step with PBS, the cells were incubated with 4′, 6′-diamidino-2-phenylindole dihydrochloride (DAPI, 1:1000) to counterstain cell nuclei for 5 min at room temperature. The coverslips were then washed three times in PBS (5 min each), and finally, the cells were mounted in antifade solution. Labeled cells
were examined with fluorescence microscopy. The images were digitally recorded and processed with Image-Pro Plus. To quantify the purity of the finally purified SCs further, six fields of vision from each slide were randomly selected. The numbers of cells positive or negative for S-100 staining were counted. The percentage of SCs was counted relative to the total number of counted cells (SCs per total cells), and means±SD were obtained.
Fig. 1 Phase-contrast photomicrographs of primary cultured cells. a Most suspended cells adhered onto the dishes after 24 h of primary culture and developed with two distinct shapes that represented two types of cells: fibroblasts (white arrows) and Schwann cells (SCs;
black arrows). b Fibroblast (white arrow) growing among SCs. c Fibroblast (white arrow) growing under the layer of SC. d SC (black arrow) with bipolar morphology. e SC (black arrow) with tripolar morphology. f Fibroblast (white arrow). Bars 100 μm (a) 25 μm (b–e)
Statistical analysis Data are expressed as means±SD. For comparison of quantitative measures, the values were subjected to statistical analysis by using Students t-test, with significance at P
