Optimizing Isolation and Purification of Murine Glomerular Mesangial Cells

One of our research focuses is on multiple myeloma-related renal impairment. To support our research, we've developed and now optimized protocols for primary culture of murine mesangial cells. The necessary equipment for the procedures is easily accessible, and the procedural steps are straightforward.

The acquisition of target cells requires only two to three weeks. This technique facilitates kidney research and supports studies investigating mesangial cell function in disease models, therapeutic development, or signal transduction research. To begin isolation of murine mesangial cells, or MCs, from the properly euthanized animal, using scissors and tweezers, aseptically remove the kidneys from the mouse and place them into a Petri dish.

Rinse the kidneys with Earle's balanced salt solution, then use tweezers to carefully remove the connective tissue and kidney capsule. Vertically halve the kidney using a scalpel. Add three to four milliliters of collagenase I solution to the Petri dish and grind the tissue thoroughly with a plastic cell pestle.

Pipette all tissue and solution from the Petri dish into a 15-milliliter centrifuge tube. Rinse the Petri dish with one to two milliliters of collagenase I solution to ensure complete tissue transfer. Place the tube in a shaker at 37 degrees Celsius, shaking at 120 revolutions per minute for 30 minutes.

Then, add an equal volume of stop solution or culture media one to the mixture. Pipette the solution from the centrifuge tube and pass it through a 100-micron cell strainer. Collect the filtrate in a 50-milliliter centrifuge tube.

Pass the filtrate through a 40-micron cell strainer, retaining only the cells on the strainer. Rinse the strainer repeatedly with culture media one to maximize cell collection. Transfer the collected cells from the strainer into a new 50-milliliter centrifuge tube.

Centrifuge the suspension at 600g for five minutes at room temperature. After centrifugation, resuspend the cells in a cell culture dish according to the desired cell density. Place the dishes in a 37-degree Celsius cell incubator with 95%air and 5%carbon dioxide.

Add one to two milliliters of PBS along the inner edge of the culture dish to rinse the cells. Use a pipette to remove all the PBS from the bottom of the dish, then add 10 milliliters of culture media one along the inner edge of the dish. Once the cells reach 80%confluence, usually within seven to 10 days, wash the cells twice with sterile PBS.

Add 0.25%trypsin solution to the dish to trypsinize the cells. Place the dish in a 37-degree Celsius incubator. When most cells exhibit a rounded morphology and detach from the surface, terminate the trypsinization by adding an equal volume of media two.

Centrifuge the cells at 600g for five minutes at room temperature, then resuspend them in media two. The MCs isolated in this study exhibited significantly high expression of alpha-SMA, fibronectin, and vimentin. Immunofluorescent staining of purified and cultured MCs performed after 10 days of culture in media two showed that more than 95%of the cells expressed both alpha-SMA and vimentin.