MSCs Isolation and Culture Ex Vivo

Abstract

Mesenchymal stem cells (MSCs) can almost be found in any adult organ. They can be isolated and expanded within several weeks up to hundreds of millions of cells. The cell isolation based on the surface antigen expression may significantly enrich for the desired cell population and reduce the time required for cell expansion. This chapter aims to introduce the isolation and ex vivo culture method of MSCs widely used in China, including direct adherence, density gradient centrifugation and magnetic microbead or flow cytometry. To keep cell lines and store cells for later use, this chapter also introduces MSCs cryopreservation and thawing procedures.

Mesenchymal stem cells (MSCs), also named as mesenchymal progenitor cells, are self-renewable and multipotent stem cells that can differentiate into a variety of cell types. It has been demonstrated that MSCs can differentiate in vitro or in vivo into some lineage cells (Pittenger et al, 1999), including osteoblasts (Sila-Asna et al, 2007; Friedman et al, 2006), chondrocytes (Bernardo et al, 2007), myocytes (Toma et al, 2002), adipocytes (Sanchez-Ramos et al, 2000) and neurocytes (Sanchez-Ramos et al, 2000). Classically, MSCs have been obtained from the bone marrow sometimes referred to as marrow stromal cells (Campagnoli et al, 2001; Erices et al, 2000; Lee et al, 2004; Suva et al, 2004). While the terms, mesenchymal stem cell and stromal cell have been used interchangeably, they are now increasingly recognized as separate entities. Stromal cells are a highly heterogenous cell population consisting of multiple cell types with differing potential for proliferation and differentiation. In contrast, MSCs represent a more homogenous sub-population of mononuclear progenitor cells possessing stem cell features and specific cell surface markers. McCulloch and Till first revealed the clonal nature of marrow cells in the 1960s (Becker et al, 1963; Siminovitch et al, 1963). An ex vivo assay for examining the clonogenic potential of multipotent marrow cells was later reported in the 1970s by Friedenstein and colleagues (Friedenstein, 1966, 1974). In this assay system, stromal cells were referred to as colony-forming unit-fibroblasts (CFU-f).

MSCs can be derived from other non-marrow tissues, such as the liver and adipose, as well as amniotic fluid and umbilical cord blood (Campagnoli et al, 2001; Erices et al, 2000; Lee et al, 2004). MSCs comprise 0.001%≈0.1% of the total population of marrow nucleated cells, and can be expanded in vitro extensively without loss of function or phenotype (Pittenger et al, 1999; Gerson, 1999). Neither hematopoietic surface molecules (CD34, CD45, and CD14) nor endothelial markers (CD34, CD31, and vWF) are detectable on the cellular membrane of MSCs (Prockop, 1997; Majumdar et al, 1998). MSCs are recognized as expressing a large number of polyglucoproteins, such as intercellular adhesion molecules (CD44, CD29, CD90), stromal cell markers (SH-2, SH-3, SH-4) and cytokine receptors [interleukin-1 (IL-1) receptor, tumor necrosis factor-alpha (TNF-α) receptor] (Majumdar et al, 1998). Techniques are now available to isolate bone-marrow-derived mononuclear cells and expand the purified MSCs ex vivo in some conditions without change of phenotype or loss of function.