Human mesenchymal stem/stromal cells (hMSCs) have generated great interest in regenerative medicine mainly due to their multidifferentiation potential and immunomodulatory role. Although hMSC can be obtained from different tissues, the number of available cells is always low for clinical applications, thus requiring in vitro expansion. Most of the current protocols for hMSC expansion make use of fetal bovine serum (FBS) as a nutrient-rich supplement. However, regulatory guidelines encourage novel xeno-free alternatives to define safer and standardized protocols for hMSC expansion that preserve their intrinsic therapeutic potential. Since hMSCs are adherent cells, the attachment surface and cell-adhesive components also play a crucial role on their successful expansion. This review focuses on the advantages/disadvantages of FBS-free media and surfaces/coatings that avoid the use of animal serum, overcoming ethical issues and improving the expansion of hMSC for clinical applications in a safe and reproducible way.

Human bone marrow-derived mesenchymal stem/stromal cells (hMSCs) are considered promising therapeutic agents in the field of cell therapy and regenerative medicine, mainly due to their relative facility to be isolated, multi-differentiation potential, and immunomodulatory role. However, their application in clinics requires a crucial step of in vitro expansion. Most of the protocols for hMSCs in vitro culture use foetal bovine serum as medium supplement that, being from animal origin, presents several safety concerns and may initiate xenogeneic immune responses after cells transplantation. This work reports the optimization of a pharmaceutical-grade xeno-free strategy for hMSCs in vitro expansion based on the supplementation of basal medium with a pharmaceutical-grade human plasma-derived supplement for cell culture (SCC) and 2 human growth factors (bFGF and TGFβ1), plus a coating of human plasma fibronectin (Fn). After 4 weeks in culture, this strategy improves hMSCs expansion yield about 4.3-fold in comparison with foetal bovine serum supplementation and 4.5-fold compared with a commercially available xeno-free medium. hMSCs expanded in SCC-based formulation maintained their phenotype and differentiation capacity into osteogenic, adipogenic, and chondrogenic lineages, without alterations in cell karyotype. Overall, the SCC-based medium appears to be an excellent alternative for the xeno-free expansion of hMSCs as therapeutic agents for clinical applications.

The use of human Mesenchymal Stem Cells (hMSC) as therapeutic agents for advanced clinical therapies relies on their in vitro expansion. Over the last years, several efforts have been made to optimize hMSC culture protocols, namely by mimicking the cell physiological microenvironment, which strongly relies on signals provided by the extracellular matrix (ECM). ECM glycosaminoglycans, such as heparan-sulfate, sequester adhesive proteins and soluble growth factors at the cell membrane, orchestrating signaling pathways that control cell proliferation. Surfaces exposing the synthetic polypeptide poly(L-lysine, L-leucine) (pKL) have previously been shown to bind heparin from human plasma in a selective and concentration-dependent manner. To evaluate its effect on hMSC expansion, pKL was immobilized onto self-assembled monolayers (SAMs). The pKL-SAMs were able to bind heparin, fibronectin and other serum proteins, as demonstrated by quartz crystal microbalance with dissipation (QCM-D) studies. hMSC adhesion and proliferation were significantly increased in pKL-SAMs compared to controls, most probably related to increased heparin and fibronectin binding to pKL surfaces. This proof-of-concept study highlights the potential of pKL surfaces to improve hMSC in vitro expansion possible through selective heparin/serum protein binding at the cell-material interface.