2.1. Electrospinning of PCL nanofibers
The electrospinning setup and the collector used for fabricating and collecting aligned nanofibers are similar to those used in our previous studies [15
]. It consists of five components: syringe pump, syringe, needle, high voltage generator, and collector. The polymer solution used for electrospinning contained 20% PCL (w/v) in a mixed solvent of dichloromethane (DCM) and dimethylformaldehyde (DMF) with a volume ratio of 80:20. Two different collectors, a glass cover slip and a stainless steel frame with an air gap, were employed to collect randomly and uniaxially aligned fibers, respectively. Following electrospinning, the aligned fiber samples were transferred to glass cover slips and then fixed using Silastic Type A Medical Adhesive (Dow Corning Co, Midland, MI, USA). The PCL fibers were sputter-coated with gold before imaging with scanning electron microscope (Nova 200 NanoLab, FEI, Oregon, USA) at an accelerating voltage of 15 kV. Samples prepared for use in cell culture were inserted into a 24-well TCPS culture plate and sterilized via ethylene oxide gas sterilization.
2.2. ES cell culture and embryoid body formation
The protocol for ES cell culture and embryoid bodies (EBs) formation was identical to that described previously [10
]. CE3 and RW4 mouse ES cells were obtained from Dr. Gottlieb and cultured in T25 culture flasks coated with a 0.1% gelatin solution (Sigma-Aldrich, St. Louis, MO) in the presence of 1000 U/mL leukemia inhibitory factor (LIF; Chemicon, Temecula, CA) and 10-4
M β-mercaptoethanol (BME; Invitrogen, Grand Island, NY) to maintain their undifferentiated state. Cells were cultured in complete media consisting of Dubecco's modified eagle media (Invitrogen) supplemented with 10% new born calf serum, 10% fetal bovine serum (Invitrogen), and 0.3 mM of each of the following nucleosides: adenosine, guanosine, cytosine, thymidine, and uridine (Sigma-Aldrich), and passaged at a ratio of 1:4 every two days.
Undifferentiated ES cells were induced to form EBs containing neural progenitor cells using the 4-/4+ retinoic acid treatment protocol [18
]. ES cells were cultured in 100 mm Petri dishes coated with a 0.1% agar solution (Sigma-Aldrich) in complete media in the absence of LIF and BME for 4 days. Retinoic acid (Sigma-Alrdich) at 500 nM was then added to the complete media for the final 4 days of culture. Media was changed every other day during the eight day process.
2.3. Seeding of EBs and ES cells on PCL fibers scaffolds
One mL of neural basal media containing B27 supplement (Invitrogen) was diluted by 1:50 and added to each well of a 24-well plate (Corning, Corning, NY) containing nanofiber samples. Three EBs were seeded onto the surface of PCL fibers in each well. The media was not changed for the rest of the experiment. In order to investigate the differentiation of ES cells on the surface of PCL fibers without forming EBs, ~105 ES cells were transferred from culture flask into each well of 24-well plate and cultured using the 4-/4+ retinoic acid treatment protocol described as above.
Immunohistochemical analysis performed to visualize the spatial distribution of cells and neurites. After 14 days of EB culture, each well was washed with 1 mL of phosphate buffer saline (PBS; Invitrogen) and then fixed for 30 min with 400 μL of 3.7% formaldehyde. When necessary, cells were then permeabilized using 400 μL of 0.1% triton-X in PBS for 30 min. Cells were blocked with 400 μL of 5% normal goat serum (NGS; Invitrogen) in PBS for 1 hour and incubated with primary antibody overnight at 4 °C. The following primary antibody dilutions were used to characterize various mature cells found in cultures: Tuj1 (1:500), O4 (1:100), and GFAP (1:40). Following incubation, each well was washed 3 times with PBS for 5 min each. Appropriate secondary antibodies (1:200 dilution) were applied for 1 hour at room temperature. Each well was then washed with PBS and fluorescent images were taken using a QICAM Fast Cooled Mono 12-bit camera (Q Imaging, Burnaby, BC, Canada) attached to an Olympus microsocope with OCapture 2.90.1 (Olympus, Tokyo, Japan).
2.5. Quantitative analysis of neurite extension from cultured EBs
Behavior of neurite populations extending from EBs cultured on PCL nanofiber samples was examined via quantitative analysis of neurite field morphology. A custom-designed computer program, created using MATLAB (MathWorks Inc., Novi, MI, USA), was utilized to analyze fluorescent micrographs of Tuj1-labeled EBs cultured for 14 days on either random or aligned nanofiber samples. Neurite field eccentricity (a measure of directed neurite growth along a given axis) and maximum length of neurite extension (a measure of the rate of neurite growth) were calculated for each EB/sample. Calculations were accomplished by separately fitting the leading edge of the neurite field and the perimeter of the EB cell mass to standard elliptical equation (1)
at point (h, k) of the form:
where a and b are the ellipse's semimajor and semiminor axes.
Eccentricity of the neurite field was then calculated using the equation (2)
Where values of a and b were obtained from the elliptical equation fit to the leading edge of the neurite field. Maximum length of neurite extension was then calculated as the greatest distance between the elliptical curve marking leading edge of the neurite field and the elliptical curve marking the boarder of the EB cell mass along a line oriented radially from the center of the EB cell mass.
2.6. Analysis of cell phenotype by flow cytometry
After 14 days of EB culture, the medium was aspirated from each well followed by washing with 1 mL of PBS. Then, 250 μL of trypsin-EDTA (0.25%) (Invitrogen) was added to each well and incubated at 37 °C for 15 min, and 250 μL of complete medium was used to cease the reaction. The cells were removed from each well, centrifuged, and resuspended in PBS.
Extracellular antigen labeling was accomplished using O4 marker (1:100; Chemicon) for oligodendrocytes and SSEA-1 marker (1:25; Chemicon) for undifferentiated ES cells. Cells were incubated in PBS containing 5% NGS for 30 min, followed by applying primary antibody diluted with PBS containing 2% NGS for 1 hour. Cells were then washed twice with PBS prior to addition of the secondary antibody (AlexaFluor 488 goat anti-mouse IgM (1:200; Invitrogen)) diluted with PBS containing 2% NGS, which was applied for 1 hour.
Intracellular antigen labeling was accomplished by fixing cells with PBS containing 1% formaldehyde (Sigma) for 30 min. Cells were permeabilized using 0.1% Triton x-100 (Invitrogen) in PBS for 20 min, followed by blocking in PBS containing 5% NGS for 30 min. Primary antibodies (nestin marker (1:100; Chemicon) for neural precursor, β-tubulin III (Tuj 1) marker (1:1000; Covance, Berkeley, CA) for early neuron, and glia fibrillary acidic protein (GFAP) marker (1:40; Immuostar, Hudson, WI) for astrocytes were diluted with PBS containing 2% NGS and applied to the cells for 1 hour. Cells were washed three times with PBS containing 2% FBS. The secondary antibodies, AlexaFluor 488 goat anti-mouse IgG (1:200; Invitrogen) for detecting nestin and Tuj 1 markers and AlexaFluor 488 goat anti-rabbit IgG (1:200; Invitrogen) for detecting GFAP marker, were diluted with PBS and applied to the cells for 1 hour. After incubation, the cells were washed three times and sorted.
Cellular fluorescence was detected using a FACSCalibur flow cytometry (Becton, and Dickinson Company, Franklin, Lakes, NJ) equipped with an argon laser emission of 488 nm. As a control, cells stained with only the same secondary antibody were used to eliminate nonspecific background staining.
2.7. Cell viability analysis
A live/dead viability/cytotoxicity kit (Invitrogen), consisting of calcein AM and ethidium homodimer-1 (Ethd-1), was used to qualitatively assess cell viability. The intracellular esterase present in live cells converts calcein AM, a cell permeable dye, to calcein, resulting in a bright green fluorescence. EthD-1 can penetrate damaged membranes of dead cells where it binds to nucleic acids, producing intense red fluorescence. Media was removed from each well and samples were washed with PBS. The EBs were incubated for 30 min with 400 μL solution containing 2 μM calcein AM and 4 μM EthD-1 dissolved in PBS, respectively, prior to analysis via fluorescent microscopy.
2.8. Statistical analysis
Mean values and standard deviation were reported. Statistical analysis of eccentricity of neurite field and maximum neurite length was performed using unpaired Student t-Test at a 95% confidence level. Comparative analyses between each phenotype were performed using the Scheffe's F post hoc test by analysis of variance at a 95% confidence level. Three sets of 24-well plates containing nanofiber scaffolds with EB cultures were used to provide enough cells (5000 per sort) for cell phenotype analysis and statistical analysis on the results.