Marrow stromal cells (MSCs), also known as mesenchymal stem cells or colony-forming units (CFU), are nonhematopoietic multipotent stem-like cells that adhere to culture dishes. They are capable of clonal expansion in culture, support hematopoietic stem cell proliferation, and demonstrate extensive differentiation capacities. MSCs share characteristics with other multipotent stem cells (e.g., neural stem cells, hematopoietic stem cells, side population cells, and liver stem cells) as they possess the ability to self-renew and give rise to differentiated progeny.
Rat MSCs (mMSCs) were harvested from the bone marrow of the femurs and tibias of 6- to 12-month-old Lewis rats by inserting a 21-gauge needle into the shaft of the bone and flushing it with 30 ml of complete α-modified Eagle’s medium (αMEM) containing fetal bovine serum (FBS, 20%), L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), and Amphotericin B (25 ng/ml). Cells were filtered through a 70-µm nylon filter and the cells from one rat were plated into one 75 cm2 flask. The cells were grown in complete αMEM for 3 days, the medium was replaced with fresh medium, and the adherent cells were grown to 90% confluency to obtain samples, defined herein as passage zero (P0) cells. rMSCs at P0 were washed with phosphate-buffered saline (PBS) and detached by incubation with trypsin (0.25%) and EDTA (0.1%) for 5-10 minutes at 37ºC. Complete medium was added to inactivate the trypsin. The cells were centrifuged at 450 × g for 10 minutes, the medium was aspirated, and the cells were resuspended in 1-10 ml complete medium. The cells were then counted in duplicates, using a hemacytometer, and then plated as P1 in 58 cm2 plates, at densities ranging from 0.5 cells/cm2 (low-density) to 5,000 cells/cm2 (high-density). Complete medium was replaced (refeeding) every 3-4 days over the 12- to 14-day period. All cells used for the experiments were P5 or earlier.