Establishment of three-dimensional culture of neural stem/progenitor cells in collagen Type-1 Gel

Purpose: Recent studies demonstrated that transplanting neural stem/precursor cells (NSPCs) into an injured spinal cord of adult rat promotes functional recovery. The functional recovery from spinal cord injury might be enhanced by transplanting NSPCs with a scaffold that fills the cavity, entraps the NSPCs in the cavity, and acts as an attachment for neurite extension. We recently focused on collagen type-1 as a scaffold for NSPC transplantation into the injured spinal cord. In the present study, we determined the optimal conditions for culturing NSPCs in 3D collagen type-1 gel with respect to cell survival and cell migration. We, then, evaluated the ability of NSPCs to differentiate under the optimal condition. Methods: NSPCs were derived from the striatum of rat embryos. To determine the optimal cell density and collagen concentration for 3D collagen gel culture for NSPC, we performed viability assay and migration assay. Then, we examined the proportion of phenotypes differentiated from NSPC in that optimal condition. Results: In viability assay, the viability rate increased as the NSPC density increased, and peaked at 1 × 10⁷ to 5 × 10⁷ cell/ml. For collagen concentration, the viability rate increased as the collagen concentration decreased. In migration assay, cell migration was most extensive at collagen concentrations between 0.3 and 0.75 mg/ml. Migration distances gradually declined as collagen concentrations increased. In the optimal condition, NSPCs differentiated into neurons (40.1%), astrocytes (53.1%), and oligodendrocytes (5.3%) in 3D collagen gel culture. Conclusion: The optimal conditions for NSPC culture in 3D collagen gel was a cell density between 1 × 10⁷ and 5 × 10⁷ cells/ml and a collagen concentration between 0.5 and 0.75 mg/ml. Under the condition, NSPCs could differentiate into neurons, astrocytes, and oligodendrocytes.