Neurodegenerative diseases and central nervous system (CNS) injuries pose significant challenges due to the limited regenerative capacity of CNS tissues. Understanding and optimizing culture conditions for neural and supporting cells is crucial for advancing regenerative medicine. This thesis aimed to optimize culture conditions for fibroblasts and neural stem cells (NSCs) derived from the cortex of postnatal day (P) 4 opossums. Cortical cells from P4 opossums were thawed and cultured in growth medium (GM) supplemented with fetal bovine serum (FBS) to support fibroblast proliferation. Transition to neuron culture medium (NCM) aimed to promote NSC proliferation and differentiation. Various substrates and coatings were explored to assess their impact on cell morphology and adhesion. Plastic and polymer substrates were found to promote fibroblast adhesion and spreading, with gelatin coating proving superior for fibroblast proliferation compared to PO and laminin. Notably, we successfully established a culture of P4 opossum cortical fibroblasts. Immunofluorescence analysis revealed distinct vimentin distribution patterns between fibroblasts and NSCs, with unexpected SOX2 positivity in both cell types. A subset of larger, round fibroblasts exhibited features indicative of senescence. These cells displayed distinct morphological characteristics such as increased cell and nuclear sizes, which are hallmark features of senescent cells. This thesis enhances our understanding of the optimal conditions for cultivating fibroblasts and NSCs from P4 opossum cortex, providing valuable insights into improving cell proliferation and viability. By systematically addressing substrate, coating, and medium composition, our research contributes to refining cell culture methodologies.