Technique for Isolation and Culture of Rat Jaw Bone Marrow Mesenchymal Stem Cells

This study provides an effective and stable method for obtaining enough high-quality jaw bone marrow, mesenchymal stem cells with strong differentiation ability in a short time. The niche-based method can be applied in the isolation of rat bone marrow mesenchymal stem cells. Our study successfully isolates high-purity jaw bone marrow mesenchymal stem cells, offering a substantial cell source for jaw bone tissue engineering, particularly in the context of bone defect repair.

These advancements hold significant promise for the field. To begin, extract a rat mandible from a euthanized rat. Position rongeurs beak at the junction of an extracted rat mandible's incisors and the first molar.

Cut off the connection between the lower incisors and the mandible, then extract the lower incisors completely. Remove all the molars, followed by the mandibular ramus. Now separate the central part of the mandible along the distal edge of the last molar, and expose the marrow cavity.

Next, fill a one-milliliter disposable syringe with alpha MEM complete medium. Insert the needle into the bone marrow cavity. Flush the bone marrow repeatedly into a culture dish containing alpha MEM complete medium until the bone turns white.

Transfer the flushed out solution into a 15-milliliter centrifuge tube. Centrifuge the solution at 800 G for three minutes at room temperature. When centrifugation is complete, pipette out the supernatant, then add 10 milliliters of alpha MEM complete medium to the tube.

Resuspend the pellet in the medium, and transfer the suspension into a new 10-centimeter wide culture dish. Now use a bone rongeur to divide the flushed mandible into bone slices. Transfer the bone slices into a tube containing three milliliters of 0.1%type II collagenase solution.

Place the bone slice mixture in a shaker for 90 minutes at 37 degrees Celsius and 200 RPM. When incubation is complete, centrifuge the digested mandible at 800 G for three minutes at room temperature. Pipette out the supernatant to discard it, then transfer the harvested cells and the digested bone slices into well plates containing 10 milliliters of alpha MEM complete medium.

Incubate the cultures at 37 degrees Celsius in a humidified incubator under 5%carbon dioxide supplementation. After 72 hours, replace half of the culture medium with fresh medium. When the adherent cells are 80 to 90%confluent, passage them at a ratio of one to two.

Remove the bone slices during the second subculture. After 72 hours of culture, most cells were suspended and round, with a few adhered to the wall. Adherent colonies that were spindles or fibroblast-like appeared after five days of culture.

The adherent cells reached 90%confluency by day seven, and formed a fish school shape. The passaged cells showed homogeneity, were predominantly spindle shaped, and in a vortex-like pattern after confluency.