Ficoll paque premium gradient solution

The SD rats were euthanized by intra-peritoneal injection of an overdose of ketamine (100 mg/ml) and xylazine (100 mg/ml) from Ilium Troy Laboratory, Australia. Femoral and tibiae bones were then dissected aseptically. The central canal of the bone was injected with 5 ml of Dulbecco’s Modified Eagle’s Medium (DMEM, catalog # 11885–084, Gibco, Life Technologies, USA) containing 20% (v/v) fetal bovine serum (FBS, catalog # 10270098, Gibco, Life Technologies, USA), 1% MEM Non-essential amino acid (Catalog # M7145, Sigma-Aldrich, USA) and 1% Antibiotic-antimycotic (Catalog # 15240062, Gibco, Life Technologies, USA) to extrude the marrow tissue. Then the tissue was gently dissociated into single cells mixture and layered onto a Ficoll-Paque PREMIUM gradient solution (Catalog # 17544202, GE Healthcare Bio-sciences, Sweden) and centrifuged at 2000 rpm for 20 min. Mononuclear cells were extracted and plated on T25 cm² tissue culture flasks at a density of 1 × 10⁷ marrow cells and incubated in a humidified chamber at 37°C with a 5% CO₂ supply. After 24 hours the non-adherent cells were removed by total media replacement, and the attached cells were grown. When the MSCs became 80% confluent, they were detached with 0.25% trypsin/EDTA and then subcultured. BM MSCs were characterised by flow cytometry, and neuronal induction was conducted at passage 4.

For this study, Sprague Dawley rats were purchased from the animal facility of Universiti Sains Malaysia. MSCs were extracted from bone marrow tissues of three SD rats using a previously described method [19 (link)]. Briefly, three SD rats (4 weeks old) were euthanised using an overdose mixture of ketamine-xylazine (Ilium Troy Laboratory, Blacktown, Australia) via intraperitoneal injection. Femoral and tibial bones were then aseptically dissected, and 5 mL of 20% DMEM was injected into the central canal of the bones to extrude the marrow tissue. Next, the cell mixture was separated using Ficoll-Paque PREMIUM gradient solution (GE Healthcare Bioscience, Uppsala, Sweden), and mononuclear cells were extracted. The collected cells from three SD rats were plated at a density of 1 × 10⁶ marrow cells and incubated in a humidified chamber at 37 °C with 5% CO₂. After 24 h, floating cells were removed using total media replacement. At 80% confluence, MSCs were detached with TrypLE™ Express stable trypsin replacement enzyme without phenol red (Life Technologies, Carlsbad, CA, USA) and subcultured until passage 3 for neural induction.