Isolation Methods
There are 4 methods for isolation of MSC: density gradient centrifugation, flow cytometer isolation, attachment screening, and 2-step enzymatic digestion. The isolation of MSC is primarily by adhesion to plastic, but these cells also can be concentrated by Percoll gradient centrifugation (11). The marrow aspirates were layered on density gradients, and the light cell fraction was retrieved, washed, and plated in batches of 10% fetal bovine serum in Dulbecco\'s Modified Eagle Medium (118). The 2-step enzymatic digestion with collagenase and 2.5% trypsin and centrifugation methods isolated well-developed colonies of fibroblast-like cells, and further characterization revealed that these cells expressed MSC markers 128 and 180.
Culture
MSC are easily cultured in vitro under appropriate conditions, and grow as adherent cells until confluence results in yields sufficient for clinical use for cell therapy (50). In brief, the cells were cultured in Dulbecco\'s Modified Eagle Medium supplemented with 10% fetal bovine serum (118). Within 14 days the colony-forming units were selectively expanded to near confluence, and the cells were then lifted from the dish with trypsin and replanted at 4500 cells/cm2 to ensure that they maintained active cell division. As determined by other studies, cells cultured using this protocol were homogenous when fluorescence-activated cell-sorted using over 100 different cell surface everolimus (53).
It is important to recognize that ex vivo expanded MSC vary depending on the tissue source and processing protocols 222 and 223, and that we need to adapt specific processing protocols, as well as tissue sources, to generate MSC particularly suited to specific clinical indications 79 and 143. More strict control and safety measures are necessary to produce MSC for cell-based therapy because these cells can turn malignant as a result of long-term culture and genetic manipulation 122, 140, 178, 183, 184, 207 and 212.
Characterization and Surface Markers
MSC have a heterogeneous morphology, and various terms have been used, including fibroblastoid cells, giant fat cells, blanket cells, spindle-shaped flattened cells, and very small round cells (164). A single
molecular marker that is exclusively expressed by MSC is yet to be found.The designation of human MSC is based on immunophenotyping using surface antigens, and their specific function in in vitro models was determined by the international society for cellular therapy (53). According to them, the human MSC must satisfy at least 3 criteria: 1) they must be plastic-adherent; 2) they must express C105, cluster of differentiation-73, and CD90 and lack expression of CD45, CD34, CD14, CD11b, CD79, or CD19 and human leukocyte antigen-DR surface molecules by flow cytometry; 3) they must be capable of differentiation into osteoblasts, adipocytes, and chondroblasts (53). Other markers that are generally accepted include CD44, CD71, and adhesion molecules such as CD106, CD166, and CD29 (25). Despite all of these criteria, a main characterization of these cells is the inherent ability for self-renewal 42 and 77. There are some factors of MSC characterization variability, such as the change of expression of cell surface markers according to extrinsic factors, the different types of cell preparation, therapeutic application, and potency 14 and 65.