Isolation and Culture of Rat Mesenchymal Stem Cells

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.