Collagen microsphere fabrication
The collagen microspheres were fabricated as reported . In brief, Type I collagen (extracted from bovine Achilles tendon) solution (5 mg/ml) emulsified in liquid paraffin oil (Sigma-Aldrich, St. Louis, MO, USA) containing surfactant (Span 20, Sigma-Aldrich) (0.3% (v/v)) and stirred using a ceramic-top stirring plate (Fisher Scientific, Pittsburg, PA, USA) for five minutes. 1-ethyl-3-(3-dimethylaminopropryl) carbodiimide (EDC) (Sigma-Aldrich) (50% in water (v/v)) was added to the emulsified mixture, and was stirred for 1 h to form collagen microspheres. Ethanol (50% (v/v) in water) was then added into the mixture and stirred for five minutes to separate the collagen microspheres from the oil phase. The mixture was then centrifuged at 3,500 rpm for five minutes. This step was repeated three times to remove the supernatant. The collagen microspheres were washed with sterile phosphate-buffered saline (PBS) and centrifuged again (3,500 rpm five minutes) to remove the supernatant. Sterile PBS was added to the centrifuge tube to re-suspend the microspheres and the tube with the microsphere suspension was stored at 4°C before the cells were seeded. In the studies of cell viability assay and the co-culture of microspheres delivered OPCs and DRGs, 20% of microspheres generated by 1 ml collagen solution were placed in each well of a 48-well plate. The microspheres fully covered the bottom of the well and cells were seeded on top of the microspheres. To study the OPCs’ growth and differentiation on collagen microspheres with immunocytochemistry, 5% of the microspheres generated by 1 ml collagen solution were placed in each well of a 48-well plate. The immunolabeled cells on microspheres and on a cell culture plate can be easily visualized with the low density of the microspheres in the cell culture wells.
OPCs isolation and culture
Procedures used in animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) and completed at the Wichita State University, Wichita, KS, USA. OPC cultures were established as reported previously . In brief, postnatal day P1 to 2 rats were sacrificed and cerebral cortexes were isolated from the brain. The cortex tissues were each triturated gently and sequentially twice through needles with a 1-cc syringe. The tissue suspension was passed through a 70 mm nylon cell strainer (BD Falcon™, Durham, NC, USA) which was placed on a 50 ml conical tube and the flow-through was collected. The isolated cells were cultured for about seven days. The OPCs were then isolated from the mixed cell culture layer by mechanically shaking the cell culture flasks. The collected OPCs were cultured in cell culture dishes coated with Poly-D,L-ornithine (5 mg/ml in PBS, Sigma-Aldrich). The OPCs were fed with OPC growth medium containing DMEM (Life Technologies™, Grand Island, NY, USA), Sato media (DMEM, 100 μg/ml transferrin, 100 μg/ml BSA, 0.2 μM progesterone, 16 μg/ml putrescine, 40 ng/ml sodium selenite (Sigma-Aldrich)), 1% penicillin-streptomycin, 2 mM L-glutamine (Life Technologies™), 5 μg/ml insulin, 10 nM D-biotin, 1 mM sodium pyruvate, 5 μg/ml N-acetyl cysteine (Sigma-Aldrich), Trace Elements B (1x, Mediatech, Inc., Manassas, VA, USA) and 10 ng/ml PDGF and 10 ng/ml bFGF (Peprotech, Rocky Hill, NJ, USA). Then the OPCs were passaged or grown on collagen microspheres.
Cell viability assays
The viability and proliferation of OPCs, B104 cells and PC12 cells, which were seeded on collagen microspheres, were studied by monitoring their metabolic activity using alamarBlue assay (Pierce Biotechnology, Rockford, IL, USA). To perform the alamarBlue assay, the sterile PBS containing collagen microspheres was centrifuged and the microspheres were resuspended in the cell culture medium. The collagen microspheres were placed into the 48-well plates and the bottom of the cell culture wells were fully covered by the microspheres. After the microspheres were settled down at the bottom of the cell culture wells, the cells (15,000 cells/well) were added into the cell culture medium and the cells grew on the microspheres. In the control study, the cells (15,000 cells/well) were directly seeded in 48-well plates coated with poly-D,L-ornithine (5 mg/ml in PBS). After culturing for four days, the medium was gently removed from the wells and the cells were then incubated with culture medium containing 10% (v/v) alamarBlue reagent for two hours. Then the cell culture medium containing alamarBlue reagent was transferred to a 96-well plate (100 μl/well) and absorbance was measured at a wavelength of 570 nm and 600 nm in a microplate reader (Synergy Mx Monochromator-Based Multi-Mode Microplate Reader, Winooski, VT, USA). The cell viability of the 3T3 cells and PC12 cells growing on the collagen microspheres was analyzed by fluorescence-based LIVE/DEAD® assays (Life Technologies™). The Calcein and EthD-1 labeled cells were counted and quantified to show the rate of live cells on the collagen microspheres.
DRG isolation and culture
DRGs from 15-day-old Sprague Dawley rat embryos were isolated and cultured in the collagen-coated 24-well-plate wells . The whole DRGs were cultured in Neurobasal media supplemented with B27, 1% penicillin/streptomycine, 2 mM L-glutamine (Life Technologies™), 20 ng/ml 2.5S nerve growth factor (NGF, Alomone Labs, Jerusalem, Israel), 8.625 μg/ml uridine and 3.75 μg/ml of 5-fluoro-2- deoxyuridine (Sigma-Aldrich). The DRGs were cultured for 10 days before they were co-cultured with OPCs.
OPCs and DRG co-culture
OPCs (15,000 cells/well) were seeded on the collagen microspheres and cultured with OPCs growth medium for two days. The collagen microspheres with pre-seeded OPCs were then transferred into the wells with the growth of DRGs . In the control group, OPCs (15,000 cells/well) were added into the DRG culture wells. The co-culture of the DRG and OPCs were maintained for eight days in the DMEM with Sato media, 1% penicillin-streptomycin, 2 mM L-glutamine, 5 μg/ml insulin, 10 nM D-biotin, 1 mM sodium pyruvate, 5 μg/ml N-acetyl cysteine, Trace Elements B and Triiodothyronine (Sigma-Aldrich). Negative control was performed to treat the cultured DRG with the co-culture medium without adding OPCs in the cell culture.
OPCs and DRGs were fixed with 4% paraformaldehyde in PBS solution. The phenotype of the cultured OPCs was determined with anti-A2B5 antibody (a gift from Dr Qing Lu, University of Texas Southwestern Medical Center). The differentiated OPCs were labeled with anti-O4 antibody (a gift from Dr Qing Lu, University of Texas Southwestern Medical Center) and anti-Myelin Basic Protein (MBP) antibody (Millipore, Billerica, MA, USA). Dual labeling of DRG neuron axons and oligodendrocyte were performed to study the myelination of the DRG axons using anti-IIIβ-tubulin antibody (Millipore) and anti-MEB antibody, respectively. The images were taken using Zeiss Axio Observer microscope (Carl Zeiss Microscopy, LLC, Thornwood, NY, USA).
Statistical analysis was conducted using a two-tailed Student’s t-test. A P-value of 0.05 was considered to be statistically significant. Data are expressed as means ± standard deviation.