Identification of growth and attachment factors for the serum-free isolation and expansion of human mesenchymal stromal cells

Ex vivo propagation of sparse populations of human mesenchymal stromal cells (hMSC) is critical for generating numbers sufficient for therapeutic applications. hMSC culture media have typically been supplemented with animal serum and, recently, human-sourced materials. However, these supplements are ill-defined and, thus, undesirable for clinical and research applications. Previously reported efforts to develop defined media for hMSC culture only resulted in slow or limited proliferation, and were unsuccessful in expanding these cells from primary cultures. Therefore a major step forward would be the identification of defined, serum-free culture conditions capable of supporting both the isolation and rapid expansion of hMSC.Using classical approaches of medium development, we were able to identify a set of growth and attachment factors that allowed the serum-free isolation and expansion of hMSC from bone marrow.Heparin, selenium and platelet-derived growth factor (PDGF)-BB were found to be inhibitory for the growth of hMSC, whereas basic fibroblast growth factor (bFGF) was critical and worked synergistically with transforming growth factor (TGF)-beta1 to allow significant cell expansion. Ascorbic acid, hydrocortisone and fetuin were also found to be important growth and attachment factors that, in conjunction with substrate-coating proteins, allowed the isolation of hMSC from primary culture and their subsequent expansion.We report a defined medium formulation (PPRF-msc6), consisting of key recombinant and serum-derived components, for the rapid isolation and expansion of hMSC in the absence of serum. This work represents an important step forward for achieving an ideal, completely defined synthetic medium composition for the safe use of hMSC in clinical settings.