Simple method to isolate mesenchymal stem cells from bone marrow using xeno-free material: A preliminary study

Attempts to look for FBS substitute showed that platelet rich plasma (PRP) was able to replace fetal bovine serum (FBS), and in some studies even showed better performance than FBS. However, most studies that compared FBS to xeno-free materials such as platelet rich plasma, allogeneic human AB serum, or autologous plasma to culture bone marrow derived stem cells did not conduct the study on primary cultures, as they used passaged cells. Therefore, in this study we used PRP supplemented medium in primary cultures to isolate MSCs from bone marrow without mononuclear cell enrichment step. Processing of bone marrow was done by simple centrifugation at 400g to separate the pellet from supernatant, followed by centrifugation of supernatant to get the remaining stem cells. Both first and second pellets were cultured in 10% PRP containing alpha minimum essential medium (αMEM). The time needed for the cells to attach in the supernatant derived primary culture, time to become confluent and harvested, cell yields in primary culture harvest (pellet and supernatant) were noted. After second centrifugation, supernatant yielded a pellet, and both pellets from first and second centrifugation contained mesenchymal stem cells (MSCs) that can be isolated due to their plastic adherent property. Supernatant derived pellet culture became confluent faster compared to those from first centrifugation. In conclusion: PRP supplemented α MEM can be used to isolate MSCs from bone marrow using simple centrifugation method.