From: Esmaeli, A., Moshrefi, M., Shamsara, A., Eftekhar-vaghefi, S. H., & Nematollahi-mahani, S. N. (2016). Xeno-free culture condition for human bone marrow and umbilical cord matrix-derived mesenchymal stem/stromal cells using human umbilical cord blood serum. International Journal of Reproductive BioMedicine, 14(9), 567.

In this study hBM-MSCs and hUC-MSCs were cultured in media supplemented with either hUCS or FBS.

Culture of human BM-MSCs
The complete culture medium consisted of Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% FBS (Gibco, Grand Island, NY, USA), 100 IU/ml penicillin, 60 μg/ml streptomycin sulfate, and 50 μg/ml amphotericin B. Culture flasks were incubated at 37^oC in the humidified air with 5% CO₂. After 24 hr, the floating cells were removed, while the attached cells were washed three times with pre-warmed PBS. The flasks were then filled with 15 ml of complete medium supplemented with 1:100 insulin-transferrin-selenium solutions and incubated at the same conditions. The medium was refreshed every 4-6 days according to the rate of cell propagation. After reaching >90% confluence, the cells were detached from the substratum by 3 ml 0.25 g/l trypsin and 0.2 g/l EDTA.

Culture of human UC-MSCs
hUC-MSCs were harvested using a previously reported method. Briefly, umbilical cords of neonates that were delivered by Cesarean section were transported in PBS to laboratory. Umbilical cord vessels and amniotic membrane were separated under sterile conditions. The remaining matrix was divided into small pieces (>1 mm) and loaded onto the bottom of 6 cm petri dishes. 1 ml complete medium was added to each petri dish and the dishes were incubated at 37^oC humid atmosphere with 5% CO₂ in the air for 24 hr. 7 ml of complete medium was then added to each petri dish and after 1 wk,
matrix pieces were removed. We used cells at passage 2-7 in our experiments. The mitotic index was calculated as the proportion of the number of cells undergoing mitosis to total number of cells present in the microscopic field.