Culture of human mesenchymal stem cells using a candidate pharmaceutical grade xeno-free cell culture supplement derived from industrial human plasma pools

Abstract

IntroductionFetal bovine serum (FBS) is an animal product used as a medium supplement. The animal origin of FBS is a concern if cultured stem cells are to be utilized for human cell therapy. Therefore, a substitute for FBS is desirable. In this study, an industrial, xeno-free, pharmaceutical-grade supplement for cell culture (SCC) under development at Grifols was tested for growth of human mesenchymal stem cells (hMSCs), cell characterization, and differentiation capacity.MethodsSCC is a freeze-dried product obtained through cold-ethanol fractionation of industrial human plasma pools from healthy donors. Bone marrow-derived hMSC cell lines were obtained from two commercial suppliers. Cell growth was evaluated by culturing hMSCs with commercial media or media supplemented with SCC or FBS. Cell viability and cell yield were assessed with an automated cell counter. Cell surface markers were studied by indirect immunofluorescence assay. Cells were cultured then differentiated into adipocytes, chondrocytes, osteoblasts, and neurons, as assessed by specific staining and microscopy observation.ResultsSCC supported the growth of commercial hMSCs. Starting from the same number of seeded cells in two consecutive passages of culture with medium supplemented with SCC, hMSC yield and cell population doubling time were equivalent to the values obtained with the commercial medium and was consistent among lots. The viability of hMSCs was higher than 90%, while maintaining the characteristic phenotype of undifferentiated hMSCs (positive for CD29, CD44, CD90, CD105, CD146, CD166 and Stro-1; negative for CD14 and CD19). Cultured hMSCs maintained the potential for differentiation into adipocytes, chondrocytes, osteoblasts, and neurons.ConclusionsThe tested human plasma-derived SCC sustains the adequate growth of hMSCs, while preserving their differentiation capacity. SCC can be a potential candidate for cell culture supplement in advanced cell therapies.

Single-cell high-content imaging parameters predict functional phenotype of cultured human bone marrow stromal stem cells

“…Also, highly proliferative hBM‐MSCs exhibit better engraftment in preclinical mice models . The hBM‐MSCs in the current study had a similar PDT (76.5 ± 34 hours) as observed in previous studies . We further observed that hBM‐MSC cultures enriched in small cell and nuclei exhibited greater proliferative capacity.…”

“…The AUC of cell proliferation curves was generally used as a good metric to summarize the growth curve . Nevertheless, we also calculated the average value of PDT for all donor cells (76.5 ± 34 hours), which was similar to previously reported PDT for primary hBM‐MSCs (between 72 and 120 hours) . Furthermore, we found a strong negative correlation between AUC and PDT values for analyzed cell populations ( r s = −0.73, P < .0001, n = 53, Figure S2A).…”

Optimization of the use of a pharmaceutical grade xeno‐free medium for in vitro expansion of human mesenchymal stem/stromal cells

“…Human plasma‐derived supplement for cell culture medium (SCC), produced by the pharmaceutical company Grifols (Parets del Valles, Spain), was recently described as a potential candidate for xeno‐free supplementation of hMSCs expansion media (Diez, Bauman, Gajardo, & Jorquera, ). Besides being a pharmaceutical‐grade product, SCC has the advantages of (a) providing uniformity between batches, because it is obtained from human plasma pools of over 1,000 donors and (b) be safe, which is assured by every individual donation testing for viral markers and pathogenic agents and by steps with viral clearance capacity during its production process (Diez et al, ; Nieto JIJ & Diez, ). Moreover, SCC is a GMP‐compliant product, being every step of its development well‐defined and validated under standard operating procedures.…”

“…This work aimed to optimize the use of a pharmaceutical grade xeno‐free strategy, based on SCC‐supplemented medium (Diez et al, ) to improve the expansion of hMSCs for clinical applications.…”

Human Bone Marrow Mesenchymal Stem/Stromal Cells Preserve Their Immunomodulatory and Chemotactic Properties When Expanded in a Human Plasma Derived Xeno-Free Medium

“…In Díez et al [50], commercial bone marrow-derived hMSC were expanded in the same medium used here but with additional platelet lysate and fibroblast growth factor. Platelet lysate supplementation is controversial, and its effect on immunomodulatory MSC properties is not clear, with some studies showing that it reduces hMSC immunosuppressive properties [40, 41, 61, 66].…”