Schwann cell-like dADCs viability, VEGF secretion and proliferation

<b>Culture of cells</b> Adipose-derived stem cells were isolated from adult Sprague Dawley rats and differentiated into a Schwann cell-like phenotype (dADSCs) as previously described [1], and were maintained in Minimum Essential Media (MEM- with GlutaMAX; Gibco) containing 10% (v/v) foetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin solution, supplemented with 14M forskolin (Sigma), 10ng/mL basic fibroblast growth factor (bFGF; Pepro Tech Ltd., UK), and 252ng/ml neuregulin NR G1 (R&amp;D Systems, UK). The cells were kept at sub-confluent levels in a 37°C incubator with 5%CO₂ and passaged with trypsin/EDTA (Invitrogen, UK) approximately every 72 h. <br> [1] Kingham, P. J. <i>et al. </i>Adipose-derived stem cells differentiate into a Schwann cell phenotype and promote neurite outgrowth in vitro. <i>Experimental Neurology </i>207, 267–74 (2007). <br> <b>Fabrication of cell-seeded collagen gels</b> To prepare the gels, 8 volumes of type I rat tail collagen (2mg/ml in 0.6% acetic acid; First Link, UK) were mixed with 1 volume of 10x MEM (Sigma) and the mixture neutralised using sodium hydroxide before mixing with 1 volume of cell suspension. 240µL of cellular collagen suspension was pipetted into individual wells of a 96-well plates then incubated at 37°C for 15 min for the gels to set, before being stabilised by plastic compression for 15 min (RAFT, Lonza). The number of cells in the starting suspensions was varied to give final cell densities of 39, 77, 154, 231 and 385 million cells/ml stabilised gel. The resulting gels were immersed in 200µL media (MEM with GlutaMAX, Gibco), apart from the highest seeded density gels, which were transferred to a 24-well plate with 1mL media. Samples were incubated at 37C in a humidified incubator with 5%CO₂ for 24h and 5 days respectively. In both cases the oxygen level inside the incubator was controlled and maintained (using Biospherix ProOx 110) at each of the following concentrations: 1%, 3%, 5%, 10%, 16%. <br> <b>Metabolic viability assay</b> The medium from each well was aspirated and frozen for further analysis, then viable cell density of each gel after incubation was determined using CellTiter-Glo 3D Cell Viability Assay (Promega). Gels were transferred to 100µL fresh medium (MEM- with GlutaMAX) in a white opaque 96-well plate and 100µL reagent was added, mixed for 3 min at 175 rpm and left at room temperature for a further 25 min before luminescence was measured. Analysis involved subtracting baseline luminescence (from cell-free controls) and determining equivalent viable cell density using a standard curve generated from comparator gels tested immediately after the plastic compression step. <br> <b>Proliferation assay (Ki67 Staining)</b> Gels for immunocytochemistry were fixed using 4% paraformaldehyde (PFA) overnight at 4°C. All storage washes and dilutions were performed using PBS. Cells were permeabilised in 0.5% TritonX-100 (Sigma) for 30 min. Following 3 x 5 min washes, non-specific binding was blocked with 5% normal goat serum (Dako, Ely, UK) for 30 min. After another wash step, a primary antibody used to detect Ki67 (rabbit IgG; 1:250 (AB15580 Abcam)) was incubated overnight at 4 °C. Following 3 x 10 min washes, secondary antibody (anti-rabbit dylight 488; 1:300 (Vector Laboratories)) was added for 90 min. Hoechst 33258 (1 mg/ml) was also added in the secondary antibody solution. The mean number of proliferating cells and cells/field was determined using fluorescence microscopy (Zeiss Axio Lab.A1) via quantification of Ki67 and Hoechst staining in 3 pre-determined areas per gel. <br> <b>VEGF-A Enzyme-linked immunosorbent assay (ELISA)</b> Secreted VEGF-A protein levels were determined by ELISA. The cell culture supernatant from the wells was collected and analysed with a VEGF-A sandwich ELISA kit (RayBiotech, GA, USA) according to the manufacturer’s protocols. In brief, samples were diluted 10-200 fold to fit the standard curve (0-80 pg/ml). All samples were analysed in duplicate and the end-absorbance was measured at 450nm (BioTek Synergy microplate reader).<br>