Ex vivo expansion of human mesenchymal stem cells in defined serum-free media

Abstract

Human mesenchymal stem cells (hMSCs) are presently being evaluated for their therapeutic potential in clinical studies to treat various diseases, disorders, and injuries. To date, early-phase studies have indicated that the use of both autologous and allogeneic hMSCs appear to be safe; however, efficacy has not been demonstrated in recent late-stage clinical trials. Optimized cell bioprocessing protocols may enhance the efficacy as well as safety of hMSC therapeutics. Classical media used for generating hMSCs are typically supplemented with ill-defined supplements such as fetal bovine serum (FBS) or human-sourced alternatives. Ideally, culture media are desired to have well-defined serum-free formulations that support the efficient production of hMSCs while maintaining their therapeutic and differentiation capacity. Towards this objective, we review here current cell culture media for hMSCs and discuss medium development strategies.

Figure 1

Cultivation of primary human BM MNCs using different media including three commercial media. Cells were inoculated at 150,000 BM MNCs/cm² into fibronectin-coated T-25 flasks, each containing 8 mL of Mesencult-XF (a), StemPro MSC SFM Xeno-Free (b), MSCGM-CD (c), and PPRF-msc6 (d), and a classical FBS medium (10% FBS DMEM) (e). After 60 h, nonadherent cells in each medium were removed, and fresh medium was added to the adherent cells (100% medium change). The adherent cells were allowed to grow for additional 10 days with 50% medium change every other day, and then stained with crystal violet to visualize colonies generated.

Figure 2

Cultivation of nonadherent cell fractions removed from the culture of primary BM MNCs in different media. Nonadherent cells and spent medium removed from each of the flasks, demonstrated in Figure 1, were replated into a new T-25 flask coated with gelatin containing 4 mL of the fresh medium—that is, Mesencult-XF (a), StemPro MSC SFM Xeno-Free (b), MSCGM-CD (c), PPRF-msc6 (d), and 10% FBS DMEM (e). After 60 h, nonadherent cells and medium in each flask were discarded, and fresh medium was added to the adherent cells (8 mL per flask). The adherent cells were allowed to grow for additional 8 days with 50% medium change every other day and then stained with crystal violet to visualize colonies generated.

Figure 3

Idealized growth response curve versus nutrient concentration illustrating the procedure for determining the “optimum” concentration of a nutrient. The range of concentrations that support optimum growth (referred to as a “plateau”) is determined, and its midpoint on the plot is selected as the concentration to be used in future media (Adapted from [92]).

Figure 4

An overview demonstrating a process for the development of a defined serum-free medium for hMSCs. (a) To replace ill-defined serum with defined supplements, a variety of medium constituents, including basal media, additional nutrients (e.g., lipids and vitamins), binding proteins, physiochemical reagents (e.g., buffer), hormones, growth factors, and attachment factors were selected. (b) A sequential strategy was designed for screening effectively the selected basal media and medium additives to develop a defined serum-free condition that supports the isolation and expansion of hMSCs [76].