Robust differentiation and potent immunomodulation of human mesenchymal stromal cells cultured with a xeno-free GMP protein supplement

Abstract

Background/Aims

Human mesenchymal stromal cells (hMSC) are multipotent adult cells commonly used in regenerative medicine as advanced therapy medicinal products. The expansion of these cells in xeno-free supplements is highly encouraged by regulatory agencies due to safety concerns. However, the number of supplements with robust performance and consistency for hMSC expansion are limited. Here, we evaluate a xeno-free human plasma-derived protein supplement (Plastem, Grifols) for the expansion and functional evaluation of hMSCs.

Methods

hMSC from bone marrow, adipose tissue and umbilical cord were obtained from two suppliers and cultured in Dulbecco's modified Eagle's medium (DMEM/F-12) supplemented with fetal bovine serum 10% (FBS), human platelet lysate 5% (hPL) or Plastem 10%+ hPL0.5%. Cell proliferation was evaluated after culturing hMSC for 13 days with trypan blue exclusion. hMSC immunophenotype was assessed by flow cytometry of surface markers expression. Multipotentiality assay determined the ability of hMSC to differentiate into osteogenic, chondrogenic and adipogenic lineages after 21 days, by using specific staining. Immunomodulatory properties of hMSC were analyzed by measuring suppression of human peripheral blood mononuclear cell (PBMC) proliferation in co-culture with hMSC.

Results

Plastem 10% + hPL 0.5% supported robust and sustained hMSC growth with a similar efficiency to the reference supplement FBS 10%. hMSC cultured with the xeno-free supplement presented a similar morphology comparable to FBS-supplemented cells and maintained typical expression of markers: positive (>95%) for CD90, CD73 and CD105; and negative (<5%) for CD45, CD14, CD19, CD34 and HLA-DR. Likewise, hMSC showed potent, in vitro differentiation potential into osteogenic, chondrogenic and adipogenic lineages, outperforming the results obtained with traditional reference supplements in several instances. They retained their immunomodulatory properties, inhibiting the proliferation of phytohemagglutinin (PHA)-stimulated PBMCs with a notable enhancement of the immunomodulatory capacity of hMSCs compared to conventional reference supplements.

Conclusions

Plastem allowed hMSC expansion while preserving phenotype and showed remarkable differentiation and immunomodulatory properties, supporting its use for cell therapy manufacturing processes as a robust, xeno-free alternative to FBS and hPL. Moreover, Plastem can be manufactured at an industrial level, making it a scalable solution for widespread application.