Dmem lg medium

Four week-old SD rats were sacrificed and the primary BMSCs were isolated from the femurs and tibias under a sterile condition. Following flushing the bone marrow cavity with DMEM-LG medium (Invitrogen Life Technologies, Grand Island, NY, USA) and removing large tissues with a 200-mesh nylon filter, the isolated bone marrow cells were cultured in DMEM-LG medium supplemented with 10% fetal bovine serum (FBS; Invitrogen Life Technologies), 1% penicillin and streptomycin (Invitrogen Life Technologies) at 37°C in an environment with 5% CO₂. The medium was refreshed every 3 days and cells were harvested by multiple digestions and passaged when cell confluence reached >80%. Cells of the fifth passage were collected and subjected to flow cytometry analysis. These cells were maintained in adipogenic, osteogenic and chondrogenic differentiation medium (Cyagen Biosciences, Sunnyvale, CA, USA) followed by Oil Red O staining, Alizarin Red S staining and Toluidine Blue staining, respectively. The surface markers were detected using a BD FACSC™ II flow cytometry system (BD Biosciences, San Jose, CA, USA). The antibodies utilized in this analysis included anti-CD44-PE, anti-CD73-APC, anti-CD90-FITC, anti-CD105-PerCP-Cy5.5, anti-CD11b-PE, anti-CD19-PE, anti-CD34-PE and anti-CD45-PE (BD Biosciences). Isotype-matched antibodies were used as controls. Cells were harvested between 3–5 passages.

Cryopreserved BM-MSC, AD-MSC and DP-MSC were used for the study. Informed consent was obtained from the patients or legal representatives at the time of bone marrow, adipose tissue or extracted tooth/teeth collection for previous research projects. Age of patients ranged from 24 to 40 years for both BM-MSC and AD-MSC and 20–28 years for DP-MSC. Cells of 5 healthy donors of each MSC type, which were cryopreserved at first passage, were revived in DMEM-LG medium (Gibco, USA) with 10% fetal bovine serum (FBS) (Hyclone, USA) and penicillin (100 U/ml) streptomycin (100 μg/ml) (Gibco, USA) (Expansion medium), expanded and characterized. Viability of the revived cells was measured by trypan blue staining (Invitrogen, Life Technologies, USA). Followed by characterization (by surface marker profiling using flow cytometry and trilineage differentiation potential), cells from 3^rd to 5^th passage were used for all the further experiments.
Growth and proliferation rate was assessed by MTT assay and population doubling time was also studied (Supplementary material).

Cryopreserved BM-MSC, AD-MSC and DP-MSC (n = 5 each) were revived in DMEM-LG medium with 10% FBS (pre-heated to 37 °C). The cells were allowed to adhere to the culture dish by keeping them undisturbed for 24 h and were expanded thereafter. Before cryopreservation, BM-MSCs were obtained by direct plating of bone marrow on to the culture dish and AD-MSC and DP-MSCs were obtained by explant culture. No enzymes were used for the cell extraction.
After expansion of the hMSCs, the cells were characterized by flow cytometric enumeration (Supplementary Materials). Followed by characterization by surface marker profiling using flow cytometry and trilineage differentiation potential of hMSCs, cells from 3rd to 5th passage were used for all further experiments [13 (link)]. Briefly, hMSCs were revived in DMEM-LG medium (Gibco, Gaithersburg, MD, USA) with 10% FBS. Upon attaining confluence, cells were characterized by differentiating them into adiopocytes, chondrocytes and osteocytes (trilineage differentiation potential) and were enumerated by flow cytometry for the expression of surface markers like CD105, CD73, CD90, CD29, HLA class 1 & class II and CD34/45 (10,000 cells were acquired for enumeration) [14 (link),15 (link),16 (link)].