Mesenchymal Stem Cells (MSCs) Identification

Summary

MSCs in culture are best assessed by clone formation assays and trilineage differentiation capacity toward osteoblasts, adipocytes, and chondrocytes. However, cell surface marker analysis is beneficial in analyzing the degree of contamination of hematopoietic cells. Source: Human Stem Cell Culture

Operation method

Determination of self-renewing colony-forming units in MSCs

Materials and Instruments

MSCs
Sterile CCM PBSA 0.85% Taipan blue salt solution Trypsin EDTA
Polypropylene centrifuge tubes 15 ml or 50 ml plastic tissue culture dishes 15 cm diameter Pipette tips Non-sterile crystal violet solution Distilled water Modified Neubauer blood cell counter Pipettes

Move

(a) MSCs collection and passaging was performed. The remaining cells in the suspension were recounted to ensure accuracy.

(b) MSCs suspension was prepared with CCM at a concentration of 1000 cells/ml.

(d Prepare three 15 cm Petri dishes by adding 25 ml of preheated fresh CCM to each.

(d) Add 100 μL of suspension (100 cells) to each dish, shake the dish to distribute the cells evenly, and incubate at 37°C with 5% _CO2 for 3 weeks.

(e) After 3 weeks, the medium was aspirated from the CFU culture and the dishes were rinsed with PBSA for 3 times.

(f) Add 10 ml of crystal violet solution to each petri dish and incubate for 5~10 min at room temperature.

(g) Aspirate the staining solution and rinse with distilled water until the background was removed.

(h) Count the number of colonies in each petri dish, take the average and calculate the affixation rate or "CFU potential" (percentage of CFUs formed relative to the number of inocula). Well-cultured MSCs-typically have a CFU potential greater than 40%.

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