Mesenchymal stem cells for regenerative therapy: optimization of cell preparation protocols

Abstract

Administration of bone marrow-derived mesenchymal stem cells (MSCs) is an innovative approach for the treatment of a range of diseases that are not curable by current therapies including heart failure. A number of clinical trials have been completed and many others are ongoing; more than 2,000 patients worldwide have been administered with culture-expanded allogeneic or autologous MSCs for the treatment of various diseases, showing feasibility and safety (and some efficacy) of this approach. However, protocols for isolation and expansion of donor MSCs vary widely between these trials, which could affect the efficacy of the therapy. It is therefore important to develop international standards of MSC production, which should be evidence-based, regulatory authority-compliant, of good medical practice grade, cost-effective, and clinically practical, so that this innovative approach becomes an established widely adopted treatment. This review article summarizes protocols to isolate and expand bone marrow-derived MSCs in 47 recent clinical trials of MSC-based therapy, which were published after 2007 onwards and provided sufficient methodological information. Identified issues and possible solutions associated with the MSC production methods, including materials and protocols for isolation and expansion, are discussed with reference to relevant experimental evidence with aim of future clinical success of MSC-based therapy.

Figure 1

Background of the clinical trials of MSC-therapy reviewed in this article. A total of 47 clinical trials that have reported sufficient information on the MSC preparation were selected to review in this article. (a) A wide range of diseases were targeted by MSC therapy. (b) Both autologous and allogeneic MSCs were used for MSC therapy. (c) The number of MSCs administered was 1 × 10⁶/kg body weight or more in the majority of clinical trials. Some trials repeated the injection. See Supplementary Table 1 as well.

Figure 2

Protocols and materials used for MSC isolation and expansion. Different methods and materials were used in the recent MSC-therapy clinical trials, in terms of method for bone marrow (BM) preparation for MSC isolation (a), culture flask used for MSC isolation (b), culture medium used for MSCs expansion (c), and serum used for MSC expansion (d). PL: platelet lysate; FBS: fetal bovine serum.

Figure 3

Plating density for MSC isolation. Plating cell densities of BM mononuclear cells for MSC isolation used in 26 clinical trial reports are presented. 1.5-1.6 × 10⁵ cells/cm² was most frequently used.

Figure 4

Plating density for MSC expansion. Plating cell densities for MSC expansion used in 25 clinical trial reports are presented. 3,000–6,000 cells/cm² was commonly used.

Figure 5

Preparations of MSCs. (a) Over 70% clinical trials used MSCs that received 1–5 passages. (b) Doses of trypsin used for passaging were widely varied. (c) Cryopreserved MSCs were used in 35% of clinical trials reviewed. In 47 clinical trials studied, two trials used both fresh cells and cryopreserved cells, thus the total number of reports shown in the graph is 49 (see Supplementary Table 1). (d) It was common to use saline as injection vehicle.