Giemsa Staining for Colony Formation Assay: An In Vitro Staining Procedure to Assess Proliferative Capability of Mesenchymal Stem Cells

1. Seeding for Expansion Cell Culture and CFU Plates

1. Resuspend the cells (consisting of erythrocytes and mononuclear cells) in 50 ml of basal medium: Alpha-MEM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 mg/ml streptomycin and 2.5 mg/ml amphotericin B. Count the cells diluted 1:10 in Trypan Blue solution using a Neubauer chamber.
2. Plate the cells in cell culture flasks at the density of 5 x 10⁷ cells/cm² and incubate in a humidified incubator at 37 °C. If available, use hypoxic (5% oxygen) conditions. For the CFU assay control, plate 10⁹ cells in a cell culture dish of 10 cm diameter.
3. After 3 days of culture, mesenchymal stem cells are attached to the cell culture dish while other cells remain in suspension. Change the media with basal medium supplemented with 5 ng/ml basic fibroblast growth factor (bFGF). For the CFU assay control, treat the cell culture dish likewise.
4. Continue cell culture for a total of 2 weeks, exchanging media three times a week. If cells exceed 80% confluency, split by trypsinization. For the CFU assay control, exchange the media likewise, but do not split the cells for the course of the two weeks.

2. Giemsa Stain for CFU Assay

1. Prepare 10 ml of Giemsa-solution per dish: dilute the stock solution (7.6 g/l Giemsa in glycerol:methanol) 1:10 in sterile water (always prepare a fresh working dilution).
2. Remove the media from the cell culture dish and wash the cells with PBS. Be very precautious when applying liquid to the petri dish and avoid washing off the cells.
3. Fix cells in pure Methanol for 5 min at RT. Discard the Methanol. Add the Giemsa-solution and incubate for 60 min in a humidified incubator at 37 °C.
4. Wash two times with PBS. Air dry the plate head first on a paper towel. Count the colonies manually. This is best achieved using a marker pen on the back of the plate.