Neural Methods and Materials

• Culturing of hybrid neuroblastoma derived stem cell line (NDC)

The hybrid neuroblastoma derived cell line was a commercially obtained characterized neural progenitor cell line from Canada. The NDCs were supplemented with Dulbecco’s Modified Eagles Medium (DMEM) (GIBCO, Invitrogen) 10% Fetal Calf Serum (FCS) (PAA laboratories, Germany), 1X Penicillin (10,000 U/ml) and Streptomycin (10000 µg /ml) (PEST) (Thermo Scientific) and were maintained in 75 cm² tissue culture treated flask at 37⁰C with 5% CO₂.

• Preparation of PCL and PEDOT coated PCL electrospun fibres

Ø   PCL Fibres

The aligned and random microfibrous PCL mesh of different diameters ( 1 µm and 10µm ) were prepared using the Electrohydrodynamic (EHD) set up. It consists of a syringe pump that pushes the polymer, made of 10 % w/v PCL dissolved in Chloroform/Methanol (3:1), through a stainless steel needle which is supplied with voltage. Each mesh was fabricated using 21 G needle with an inner diameter of 0.4 mm. The materials were spun onto aluminium foil by varying voltage, collector distance and flow rate of the polymer. PCL sheets were made by spin coating 10% PCL in chloroform on glass slides.

Ø   PEDOT coated PCL mesh

The EDOT (2, 3-dihydrothieno (3, 4-b) - 1, 4-dioxin) monomer and 20% Iron (III) tosylate dissolved in butanol provided by Maria Bolin (ITN) were used for the preparation of PEDOT coated meshes. The Fe (III) tosylate was spin coated onto each PCL fiber mesh at 1200 revolutions per minute (RPM) for 2 minutes. The spin coated meshes were then subjected to the vapors of EDOT monomer heated at 60 ⁰C for 6 hours in an enclosed container. Any residual Fe (III) tosylate was washed away with a series of butanol, isoporpanol and water rinsing steps. The preparation of the biomaterials by electrospinning was done by Abeni Wickham (IGEN) for in vitro culture of NDCs for nerve regeneration.

 

• Sterilization of materials

The PCL and PEDOT coated PCL materials (random (1 µm and 10µm), aligned (1 µm and 10µm) and sheets) were placed in a 24 well plate and rinsed in Dulbecco’s Phosphate Buffered Saline (PBS), (GIBCO, Invitrogen). The materials were kept in 3X Antibiotic solutions (Penicillin and Streptomycin) for overnight at 4 ⁰C. Next day, t he materials were washed thrice with PBS at one hour interval and stored at 4 ⁰C for further use.

• Cell seeding on Materials

The hybrid Neuroblastoma cell line (NDC) maintained in DMEM, 10% FCS, 1% PEST were trypsinized using 0.05% Trypsin-EDTA (GIBCO, Invitrogen) at 37⁰C for 2-3 minutes. The cells were then centrifuged (Eppendorf Centrifuge 5804, Hamburg) at 300x g for 5 minutes. The pellet was suspended in 1ml of DMEM, 10% FCS, 1% PEST and then counted using cell counter (Scepter^TM Hanheld Automated Cell counter, Millipore). The NDCs were seeded on materials kept in a 24 well tissue culture treated plate in the ratio 5000 cells/well. Then the cells were supplemented with DMEM containing 10% FCS, 1% PEST and incubated at 37⁰C and 5% CO_2. The NDCs seeded on PCL/ PEDOT materials were subjected to cell viability test and then used for differentiation studies.

• Cell Viability Assay

After 24 hours of cell seeding, the NDCs on materials were subjected to viability test using Live and Dead Stain (Molecular probes, Invitrogen) as per manufacturer’s protocol. The NDCs on materials were given with Live / Dead stain containing Calcein AM and Ethidium homodimer-1 for 30 minutes at room temperature and then checked for viability through fluorescence using confocal laser scanning microscope (Carl Zeiss LSM 700).

• NDC Differentiation to neurons

The NDCs were treated with specific differentiation Medium made of Keratinocyte Serum Free Basal media (GIBCO, Invitrogen) containing 0.1 µM Dexamethasone (Acros Organics), 50 µg/mL Ascorbic acid (Sigma-Aldrich), 1.5 µM DMSO (Sigma-Aldrich), 20 µM cAMP (Sigma-Aldrich), 0.5 µg/mL NGF (GIBCO, Invitrogen) (Hackett et al., 2010) . The cells were maintained for seven days in the neural differentiation medium and observed under Inverted microscope (ULWCD 3.0 Olympus, Japan) daily for neural morphological changes.

• Characterization of differentiated NDCs by Immunocytochemistry (ICC)

The differentiation of the neural progenitors (NDCs) into neurons was confirmed by the expression of neuronal markers using mouse β-Tubulin-III antibody (Millipore, MA), mouse Neurofilament – Heavy chain antibody (GeneTex, CA). The NDCs in the differential medium were fixed with 4% Paraformaldehyde prepared in PBS of pH -7.4 for 15 minutes at room temperature. The cells were permeabilized using 0.25% Triton-X-100 in PBS for 10 minutes for the expression of intracellular targeted antigens. Blocking of unspecific binding of antibodies was carried out by incubating the cells in 1% Bovine Serum Albumin (BSA) in PBST (0.05% Tween 20 in PBS) for 30 minutes. The cells were incubated with the respective primary antibodies for two hours at room temperature under humid conditions and followed by incubation with secondary antibody goat anti-mouse Alexa fluor 488 (Invitrogen, CA) for one hour at room temperature in dark. The cells were counter stained with DAPI ( 4', 6 – diamidino -2-phenylindole, dihydrochloride ) for one minute and mounted using glycerol. The presence of antigens was detected by fluorescence excited through laser light source and the images were photographed using LSM Zeiss 700, confocal laser scanning Microscope.

• Statistical Analysis

Simple unpaired, one tailed T-test was used to compare the cytotoxicity effect of PCL and PEDOT coated PCL at p≤0.05 level of significance. Similarly, the neurite length measurement was compared between different orientation and diameter fibers of PEDOT coated PCL and PCL and between PEDOT coated and PCL fibers altogether using simple unpaired one tailed t-test at p≤0.05 level. To compare the percentage of neurite formation between each sample as above, two samples T-test between percent was carried out at p≤0.05 level.

                                            Cardiac Methods and Materials

• Isolation and Culturing of Cardiac Stem Cells (CSCs).

With ethical approval from the IRB, cardiac stem cells (CSCs) were isolated from the hearts of 3 week old C57BL/6 mice according to the manufacturer’s protocol (Millipore Cardiac Stem cell isolation kit). They were cultured in medium containing DMEM/Hams F12 with 15mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) containing 10% FCS, 1% PEST, 1X ITS (Insulin-Transferrin-Selenium), 0.5% DMSO, 20ng/ml Epidermal Growth Factor (EGF). The primary CSCs were expanded by passaging at confluency by splitting at a ratio of 1:4 or 1:6. 

• Characterization of CSCs by Fluorescent Associated Cell Sorting (FACS) Analysis

The Cultured CSCs were characterized by FACs analysis using different stem cell markers (Sca-1-PE-cy7, c-kit-PE and CD34-FITC), Cardiac specific cell marker (GATA4), stromal markers (CD44- APC -cy7, CD29- APC ) hematopoietic marker (Cocktail lineage- APC ) and endothelial marker, (CD31-PE-cy7) and with corresponding isotype controls.

Briefly, a single cell suspension (0.5 to 1x 10⁶ cells each) of CSCs at either passage 2 or 3, in 100 µl of buffer (phosphate buffered saline, [PBS]; 0.1% sodium azide; 2% FBS), was incubated with saturating concentrations of respective antibodies for 30 minutes in the dark on ice. After three washes, the cells were centrifuged at 200x g for 5 min, resuspended in ice-cold PBS.  Cell fluorescence was evaluated using a Gallios multicolour flow cytometer (Beckman Coulter AB , Sweden) and the data were analyzed using Kluza software (BD, San Jose, CA). An isotype control was included in each experiment and specific staining was measured from the cross point of the isotype with a specific antibody graph. A total of 10,000 events are acquired to determine the positivity of different cell surface markers used.

• Characterization of CSCs by Immunocytochemistry (ICC)

Confirmation of CSC phenotype was done by immunolocalization of cardiac progenitor cell markers using polyclonal antibodies against the cardiac progenitor marker, Isl-1 and early cardiac marker, GATA-4.  CSC’s at passage 2 were seeded into 24 well plates and cultured upto confluency. After confluency, cells were then fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) for 20 minutes and processed for immunocytochemistry. Blocking of unspecific binding of antibodies was carried out by incubating the cells in 1% Bovine Serum Albumin (BSA) in PBST (0.05% Tween 20 in PBS) for 30 minutes. The cells were incubated with the respective primary antibodies for two hours at room temperature under humid conditions and followed by incubation with secondary antibody goat anti-rabbit Alexa fluor 546 (Invitrogen, CA) for one hour at room temperature in dark. The cells were counter stained with DAPI ( 4', 6 – diamidino -2-phenylindole, dihydrochloride ) for one minute and mounted using Glycerol. The stained preparations are screened with a laser scanning confocal microscope (LSM510; Carl Zeiss Inc., Thornwood NY) using a fluorescent light source (excitation wavelength 480 and 540 nm) to detect the expression of markers.

• Differentiation of CSCs into Cardiomyocytes

The passage 2  CSCs of 80% to 90% confluency were induced by 10µM 5'azacytidine along with DMEM/Hams F12 with 15mM HEPES, 1%FCS, 1%PEST, 1X ITS, 0.5% DMSO for 72 hours and medium was changed every day and After 72 hours, the induced CSCs were supplemented with DMEM/HamsF12 media containing 1%FCS, 1%PEST,1X ITS, 0.5% DMSO, 20ng/ml EGF along with TGF - β 1 (10ng/ml) for 21 days and medium was changed every alternate day.

• Characterization of Differentiated cells by ICC

The immunostaining to confirm cardiomyocytes differentiate was done according to the staining protocol from Cardiomyocyte characterization kit (Chemicon International, Inc). The expression of Cardiomyocyte specific markers such as mouse anti-Troponin -1(1/100 dilution), Mouse anti- Actinin (1/200), Rabbit anti-ANP (Atrial Natriuretic Peptide) (1/200) and Rabbit Anti-GATA-4 (1/200) along with the respective dilutions of isotype control mouse IgG and Rabbit IgG were checked. The CSCs in the differential medium were fixed with 4% Paraformaldehyde prepared in PBS of pH -7.4 for 15 minutes at room temperature. The cells were permeabilized and blocked together by using 1% BSA in 0.3% Triton-X-100 in PBS for two hours in room temperature for the expression of intracellular targeted antigens. The cells were incubated with the respective primary antibodies diluted in blocking solution for two hours at room temperature under humid conditions and followed by incubation with secondary antibody goat anti-mouse Alexa fluor 488 and anti-rabbit-546 (Invitrogen, CA) for one hour at room temperature in dark. The cells were counter stained with DAPI ( 4', 6 – diamidino -2-phenylindole, dihydrochloride ) for one minute and mounted using Glycerol. The presence of antigens was detected by the emission of fluorescence through laser light source using LSM Zeiss 700, confocal laser scanning Microscope.