Xeno-free proliferation of human bone marrow mesenchymal stem cells

Abstract

The proliferation of human bone marrow mesenchymal stem cells (MSCs) employing xeno-free materials not containing fetal calf serum (FCS) and porcine trypsin was investigated for the regenerative medicine of cartilage using MSCs. Four sequential subcultivations of MSCs using a medium containing 10% FCS and recombinant trypsin (TrypLESelect™) resulted in cell growth comparable to that with porcine trypsin. There was no apparent difference in the cell growth and morphology between two kinds of MSC stored in liquid nitrogen using 10% FCS plus DMSO or serum-free TC protector™. MSCs were isolated from human bone marrow cells, stored in liquid nitrogen, and sequentially subcultivated four times employing conventional materials that included FCS, porcine trypsin, and DMSO, or xeno-free materials that included serum-free medium (MesenCult-XF™), TC protector™ and TrypLESelect™. Cells in the culture using the xeno-free materials maintained typical fibroblast-like morphology and grew more rapidly than the cells in the culture using the conventional materials, while the cell surface markers of MSCs (CD90 and CD166) were well maintained in both cultures. Chondrogenic pellet cultures were carried out using these subcultivated cells and a medium containing TGFβ3 and IGF1. The pellet culture using cells grown with the xeno-free materials showed an apparently higher gene expression of aggrecan, a chondrocyte marker, than the pellet culture using cells grown with the conventional materials. Consequently, MSCs that are isolated, stored, and grown using the xeno-free materials including the serum-free medium (MesenCult-XF™), TC protector™, and recombinant trypsin (TrypLESelect™) might be applicable for regenerative medicine of cartilage.

Serum‐free human MSC medium supports consistency in human but not in equine adipose‐derived multipotent mesenchymal stromal cell culture

“…This was not the case when equine bone marrow derived MSCs were cultured in a different serum‐free human MSC medium (StemPro® SFM), after which a widely consistent CD29 and CD90 expression was found . However, other groups reported a reduction of CD90 + MSCs after serum‐free culture of human bone marrow derived samples as well . Furthermore, in the current study, expression of the exclusion marker antigens CD34, CD45, and MHCII was very low in all equine samples cultured in DMEM (< 2%) but not in all samples cultured in MACS/MACS II medium, in some of which small CD34 + , CD45 + or MHCII + subpopulations were found.…”

“…Published studies on serum‐free culture media mainly focus on human MSCs, which were obtained from bone marrow in the majority of studies , but also from adipose tissue or birth‐associated tissues . In contrast, serum‐free culture of large animal MSCs has only been addressed in very few studies so far .…”

Different culture media affect growth characteristics, surface marker distribution and chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells

“…Although several reports have propagated easy and efficient use of serum-free or xeno-free MSC expansion media [17,45,46], none of these media have made it to a wide distribution, somewhat reflecting our own experience that the use of these media is still not as practicable as advertised.…”

“…However, there is an enormous variance in cell isolation techniques and expansion conditions for MSCs [12,17-19]. Basal culture media and supplements have become a considerable market with the growth of mesenchymal stromal cell research.…”

An exploratory clinical study on the safety and efficacy of an autologous fibroblast‐seeded artificial skin cultured with animal product‐free medium in patients with diabetic foot ulcers

“…Considerable efforts have been made to eliminate animal‐derived materials from the culturing process, and several animal‐product‐free media for mesenchymal stem cells have been reported (30). An animal‐product‐free medium for fibroblast cultivation may be available in the near future.…”