1. Techniques for isolation and propagation ofMesenchymal stem cells and their characteristic features:MesenchymalStem cells (MSCs) are multipotent cells capable of self-renewal and candifferentiate into various specialised cells 1.
Mesenchymal stemcells can be isolated from various sources and are characterised by differentsurface markers depending on the source 2. 1.1.Isolation of Mesenchymal stem cells:MSCsare found associated with Marrow and non-marrow sources and can be found in:· Adipose Tissue· Amniotic fluid· Bone Marrow· Dental tissues · Synovial Fluid· Umbilical cord (blood)· Wharton’s Jelly· Peripheral blood and blood vesselsThereare various isolation techniques for obtaining MSCs and the techniques widelyused are listed below:A) Density gradient centrifugation:Density gradient centrifugation is a most widely used technique for isolationof MSCs from biological fluids such as blood- Bone marrow aspirate andumbilical cord blood, Amniotic fluid, Synovial fluid etc., 1,3. Reagents such asFicoll 3, Hyaluronic acidhydrogel 4, etc., are usedto mixed with the blood samples and are centrifuged to obtain layers of cellsseparated by the density gradient and the Mesenchymal cells are isolated.
B) Enzymatic degradation:Enzymatic degradation plays an important role in the isolation of MSCs and usedwidely to obtain MSCs from Umbilical cord, Adipose tissue, Wharton’s jelly,Skin and dental tissues 1,2. Enzymes likeCollagenase I, Trypsin-EDTA, Collagenase V, Dispase etc., are used fordigestion and separation of the cells from the extracellular matrix (ECM) 5.
1.2. Propagation of MSCs:MSCsare plastic adherent cells and can be easily propagated if sourced from bloodsamples such as bone marrow aspirate and umbilical cord blood. The samples fromvarious sources can be grown in culture flasks and MSCs can be selected usingfollowing techniques 1,5,6:1) Plastic non-adherent, floating cells canbe washed off after culturing for 24-48 hrs.
However, high specificity cannotbe guaranteed.2) Positive or negative selection can beperformed to exclude nonspecific cells,3) Magnetic activated cell sorting (MACS)where immunomagnetic beads which have specific antibodies bind to MSCs and aresubjected to magnetic field and unwanted cells are washed off.4) Flowcytometric separation like Fluorescence activated cell sorting (FACS) 7.Theobtained MSCs are then grown in Dulbecco’s modified eagle media (DMEM) with10-30% foetal bovine serum (FBS), with or without the use of antibiotics in a5% CO2 incubator at 37o C 5.
1.3. Characteristics of MSCs fromvarious sources:MorphologicallyMSCs appear to be small cell bodies with thin, long cellular processesresembling fibroblasts and contains a large, round nucleus 8.
Irrespective ofsource the MSCs express three surface markers namely, CD73, CD90, CD105, thatare useful in detection of the cells. However, lack surface markers namely,CD11b, CD14, CD19, CD34, CD45, CD79a and Human Leukocyte Antigen – antigen D related(HLA-DR) 9. Depending on thesource of the MSCs, the differentiation potential varies. Evidently, BoneMarrow derived MSCs (bMSCs) have high osteogenic and chondrogenic potentialcompared to Adipose derived MSCs (aMSCs). While Adipogenesis is similar both inbMSCs and aMSCs 1.