To begin, take a tube of hBM-MSCs and pellet them by centrifugation. Discard the supernatant and resuspend the pellet in an appropriate volume of basal medium. Then, prepare 50 milliliter conical centrifuge tubes containing the required volume of basal medium supplemented with the respective growth factors.
Finally, add the hBM-MSCs from the stock solution at a dilution of one to 66.67 to achieve a concentration of 1.5 times 10 to the fifth cells per milliliter. Remove the polypropylene adhesive film covering the 96 well hydrogel plate to seed the plate. Carefully aspirate the storage buffer covering the hydrogels by positioning the aspirator tip against the wall of the well and slowly moving toward the edge of the inner well.
While seeding, mix the cell suspension periodically to keep the mixture homogenous and add 200 microliters of the cell suspension to each well of the plate. Then, maintain the cultures at 37 degrees Celsius and 5%carbon dioxide in a humidified atmosphere. Alternatively, use an automated plate washer with the aspiration nozzles set at least 3.8 millimeters from the plate carrier and toward the edge of the well and aspirate the storage buffer.
Seed the plate by mixing the cells with a serological pipette and dispensing equal numbers of cells to the well of the plate. Monitor the development of the cultures by Bright-field microscopy with a five times objective and acquire reference images approximately 30 minutes after seeding to evaluate the number of cells added.
