WP1 - MSC Isolation and Characterization

Workpackage 1: MSC Isolation and Characterization Adult human bone marrow derived MSCs hold great promise as clinical treatments for several pathologies due to their multipotency. Given the low percentage of MSCs present in adult bone marrow, approximately 1 in 100,000 (Pittenger et al 1999), compounded by the need for significant numbers of MSCs for clinical treatments, in vitro culturing of MSCs is necessary for clinical application. MSCs can indeed be isolated and expanded with high efficiency, and induced to differentiate to multiple lineages under defined culture conditions.

Currently there is no standardized approach for culturing adult human MSCs. Various combinations of basal medium, fetal bovine serum, glucose concentration, plating density, passaging density and plastic surface quality affect the phenotype of the cultured MSCs (Sotiropoulou et al 2006). MSC isolation and expansion from adult bone marrow using direct plating is the generally accepted method for cell isolation (Bruder et al 1997; Pittenger et al 1999; Digirolamo et al 1999). However, variations on MSC isolation methods include density gradients (Shahdadfar et al 2005), gravity sedimentation (Carranico et al 2008), antibody technology (Guo et al 2006) and DNA binding (Logeart-Avramoglou et al 2005). Previous studies also show isolation of specific MSCs populations by selection of CD164+ cells (Sorrentino et al 2008) or CD271+ cells (Logeart-Avrmoglu et al 2005). Variations such as these in the isolation and subsequent culture conditions of this already heterogenous cell population leads to great diversity in MSC characteristics and potential between research laboratories and clinics.

The most common technique for isolating MSCs, direct plating, involves subjecting the whole marrow sample to fractionation on a density gradient solution such as Percoll, after which the cells are plated at densities ranging from 1 × 104 cells/cm2 to 0.4 × 106 cells/cm2 (Lodie et al 2002; McBride et al 2003; Phinney et al 1999). Cells are generally cultured in basal medium such as Dulbecco's modified Eagle's medium (high glucose) in the presence of pre-selected 10% fetal bovine serum(FBS) (Phinney et al 1999; Pittenger et al 1999; Digirolamo et al 1999). Primary cultures are usually maintained for 12-16 days, during which time the nonadherent haematopoietic cell fraction is depleted and the MSC population of interest is adherent to the plastic substrata. The addition of growth factor supplements such as fibroblast growth factor-2 (FGF-2) to primary cultures of human MSCs was reported to lead to an enhanced osteogenic potential (Martin et al 1997). As MSCs are expanded in large-scale culture for human applications it will be important to identify defined growth media, without or with reduced FBS, to ensure more reproducible culture techniques and enhanced safety. Significant advances have been made regarding cell culture low serum conditions (Jiang et al 2002; Pytlik et al 2009), as discussed in WP4 below.

Figure 1: In vitro differentiation assays, such as osteogenic culture pictured here, are used as a primary method to characterize isolated MSCs by nearly any method of isolation.

Each laboratory has its own procedures for MSC culturing, making it nearly impossible to compare published data between laboratories. Combined, this variability results in inconsistency both in vitro as well as in clinical trials. There is therefore an obvious need for standardized best practice culture methods in laboratories across Europe, including both serum containing and serum free culture medium.

The objective of WP1 is to determine the optimal combination of basal medium, fetal bovine serum, glucose concentration, plating density, passaging density and plastic ware for the propagation of human MSCs. Additionally, the use of growth factors to enhance proliferation or differentiation will be examined. The culture of MSCs using these methods will be compared across participating laboratories to ensure comparability of phenotype, population doubling and differentiation. A common SOP will be developed, validated and adhered to for future experiments as well as published for adoption in the research community.