Design
An in vitro cytological study.
Time and setting
The experiment was conducted between May 2010 and May 2011 at the Experiment Center of Shanghai First People’s Hospital, Shanghai Jiao Tong University School of Medicine, China.
Materials
Two Sprague-Dawley rats on embryonic day 14, of clean grade, female, weighing 350 g, were provided by Shanghai Laboratory Animal Center, China (License No. SYXK (Hu) 2009-0086). All experiments were carried out in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, issued by the Ministry of Science and Technology of China[25].
Methods
Isolation and culture of rat neural stem cells
Neural stem cells were isolated and cultured as previously described[15]. Brie?y, the neocortices of rat embryos on embryonic day 14 were dissected, cut into small pieces and mechanically triturated in cold PBS. The dissociated cells were collected by centrifugation for 10 minutes at 1 000 r/min, and re-suspended in serum-free medium containing Dulbecco’s Modi?ed Eagle’s Medium /F12 medium (Gibco, New York, NY, USA) supplemented with 2% B27 (Gibco), 20 ng/mL epidermal growth factor and 20 ng/mL basic fibroblast growth factor (Peprotech, London, UK). The number of viable cells was counted by trypan blue exclusion assay in a hemocytometer (Corning, Steuben County, NY, USA). Cells were plated on untreated Petri dishes in the culture medium and incubated with 95% air/5% CO2 (Thermo Electron Corporation, USA) at 37°C. The culture medium was changed every 3–4 days. After 7 days, mechanically dissociated neural stem cells and undissociated neurospheres were replated in a new culture ?ask at a density of 1 × 105 cells/mL with fresh culture medium.
Neural stem cell immobilization in the collagen gel
Under sterile conditions, collagen type I collagen (BD Biosciences, San Jose, CA, USA) was dissolved in 0.001 M acetic acid to formulate a 0.3% collagen solution. To prepare the collagen gel, the collagen solution was diluted with 2 × Dulbecco’s Modi?ed Eagle’s Medium/F12 and sterile H2O to achieve a ?nal concentration of 1.0 mg/mL (maintaining physiological osmolarity, 250–300 mOsM). After adjusting the pH of the collagen solution to 7.4 by the addition of 1 M NaOH, the solution was chilled in an icebath to prevent gel formation. Cells were added at a concentration of 1 × 105/mL to obtain a ?nal concentration of 0.5 mg/mL collagen. The cell-collagen solution was allowed to warm at room temperature and after approximately 10 minutes, 0.2 mL aliquots of the collagen-cell suspension were placed into wells of 48-well tissue culture plates. The gels were placed in an incubator (37°C, 5% CO2, 20% O2, 99% Rh) for 1–2 hours to allow gel formation. After neural stem cells were immobilized in the three-dimensional collagen gel, 0.4 mL of cell media was added to the top of the gels and the cell-gel culture was returned to the incubator. The cells were cultured in gels, with an exchange of culture media every 3–4 days.
Passaging of neural stem cells cultured in three-dimensional collagen gels
After neural stem cells were cultured in three-dimensional collagen gels for 6–7 days, the neurospheres were released from the cell-collagen constructs using collagenase type I (Sigma) treatment for 10 minutes at 37°C. The resulting suspension was centrifuged at 1 500 r/min for 10 minutes at room temperature. The supernatant was discarded and the cell pellet was treated with accutase for 10 minutes. Then, single cell suspension was reseeded in the three-dimensional collagen gel and suspension culture system at 1 × 105 cells/mL.
Quantification of process outgrowth of neural stem cells
At indicated experimental time points, images were taken from random fields of neurospheres, which were cultured on poly-L-lysine-coated coverslips or collagen gels. The length of the 20 longest processes on each neurosphere was measured from the edge of the neurospheres to the tip of the processes. The lengths of the processes were measured with DP71 Image software (Olympus, Tokyo, Japan). The length of the processes of 20 independent neurospheres was calculated for each group and the means and standard deviation were also determined.
Differentiation of neural stem cells cultured in the three-dimensional collagen gel
To evaluate the pattern of neural stem cell differentiation in the three-dimensional culture system, we measured the proportion of each phenotype that differentiated from neural stem cells in the three-dimensional cell-gel culture compared with the suspension culture. When the neural stem cells were cultured in a three-dimensional collagen gel and suspension for 4, 7 and 14 days, the neural stem cells from both were transferred onto poly-L-lysine-coated coverslips and incubated in culture medium containing 1% fetal bovine serum for 7 days. Cells were ?xed in ice-cold 4% paraformaldehyde for 20 minutes at room temperature and washed twice in PBS. After incubation for 10 minutes with 0.3% Triton X-100, the cells were further incubated with 10% goat serum for another 15 minutes, followed by primary antibody incubation in PBS overnight at 4°C. The primary antibodies in this study were rabbit anti-nestin polyclonal antibody (1:500; Chemicon, Temecula, CA, USA), mouse anti-βIII-tubulin monoclonal antibody (1:100; Chemicon), rabbit anti-glial fibrillary acidic protein polyclonal antibody (1:200; Chemicon) and mouse anti-2',3'-cyclic nucleotide 3'-phosphodiesterase monoclonal antibody (1:200). Following three washing processes with PBS, AlexaFluor 488 goat anti-rabbit monoclonal antibody (green, 1:200; Invitrogen, Paisley, UK) and AlexaFluor 594 goat anti-mouse monoclonal antibody (red, 1:200; Invitrogen) were added at room temperature for an hour. After another three washes with PBS, all nuclei were stained with glial fibrillary acidic protein for 5 minutes at room temperature and examined under a fluorescence microscope (BX-51; Olympus). Twenty high power fields were randomly chosen from each of the three groups, then the total number of cells (glial fibrillary acidic protein-immunopositive cells) and the cells which were βIII-tubulin-, glial fibrillary acidic protein- and 2',3'-cyclic nucleotide 3'-phosphodiesterase- immunopositive were counted and the proportions of each phenotype were calculated.
Statistical analysis
All quantitative data are expressed as mean ± SD. SPSS 11.5 statistical software (SPSS, Chicago, IL, USA) was applied to process statistical analysis. Multi-group comparisons were performed by one-way analysis of variance and intergroup comparisons were performed by two-sample t-test. Differences were considered statistically significant at P < 0.05.
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