Human mesenchymal stem cell culture: rapid and efficient isolation and expansion in a defined serum-free medium

Human mesenchymal stem cells (hMSCs) are typically obtained for research or therapeutic applications by isolating and subculturing adherent cells from bone marrow on tissue-culture substrates using growth media. The purity and properties of the resulting populations can be affected greatly by the conditions under which they are cultured. Fetal bovine serum (FBS), although ill-defined, has been widely used as a critical requirement for conventional hMSC culture. However, a defined serum-free medium would greatly facilitate the development of robust, clinically acceptable bioprocesses for reproducibly generating quality-assured cells. The present study provides evidence demonstrating that a defined serum-free medium (PPRF-msc6) shows several beneficial features over a conventional FBS-containing medium for the production of hMSCs. When compared to control FBS-based cultures, PPRF-msc6 medium supported the derivation of hMSCs from primary cultures of bone marrow cells in a more rapid and consistent manner. Furthermore, hMSCs cultured in PPRF-msc6 exhibited: (a) a greater colony-forming capacity in primary as well as passaged cultures; (b) negligible lag phase and explicit exponential growth; (c) lower population doubling times (21-26 h vs 35-38 h between passage levels 1 and 10); (d) a greater number of population doublings (62 ± 4 vs 43 ± 2; over a 2 month period); and (e) a higher degree of homogeneity in size. Our data demonstrate that PPRF-msc6 is an important development which opens the door for the rapid, efficient and reproducible production of hMSCs in clinical settings.