Isolation, In Vitro Expansion, and Characterization of Mesenchymal Stem Cells from Mouse Epididymal Adipose Tissue

My research focus on adipose-derived mesenchymal stem cells as a new approach treatment, associated or not associated with other therapies, to improve inflammation from several diseases. We demonstrated that using adipose mesenchymal stem cells in conjunction with praziquantel has significantly controlled the granulomatous reaction in Schistosoma mansoni infection, highlighting the benefits of this combined treatment approach. Our protocol address the research gap in the meticulous collection of epididymal adipose tissue under aseptic conditions, the detailed physical processing involving vigorous shaking, and identifies the optimal time for utilizing adipose-derived stem cells.

Our protocol offers significant advantages, notably the abundant availability and the potential straightforward isolation of mesenchymal stem cells. Additionally, mesenchymal stem cells possess potent therapeutic properties including anti-inflammatory and tissue generation capabilities. To begin, place a euthanized mouse on a corkboard with the ventral side facing up.

Using tweezers, lift the shaved skin in the center of the abdomen and make an incision with a pair of sterile scissors. Cut the skin open to detach it from the peritoneum. Then, lift the peritoneal layer and cut it.

With a new set of sterile tweezers and scissors, gently grip, lift, and cut the entire epidermal adipose tissue located above the testes. Place the excised fat into a 50 milliliter sterile conical tube containing basal medium on ice. Next, wash the epididymal tissue with PBS.

With sterile tweezers, transfer the epididymal adipose tissue into a tube containing 15 to 20 milliliters of digest solution. Cut the tissue into fragments with sterile scissors. Then, incubate the tissue fragments at 37 degrees Celsius for one hour.

Dilute the sample in an equal volume of basal medium to inactivate the collagenase. Centrifuge the suspension at 250G for 10 minutes at four degrees Celsius. After discarding the supernatant, resuspend the pellet in one milliliter of basal medium.

Next, seed a well of a six-well plate containing three milliliters basal medium with isolated cells. Incubate the cells overnight at 37 degrees Celsius with 5%carbon dioxide. The next day, add three milliliters of basal medium to the PBS-washed cells.

Cells extracted from the adipose tissue displayed mesenchymal stem cell morphology matches. To perform the functional characterization of mouse adipose-tissue-derived stem cells by osteogenic differentiation, first detach the cells using trypsin EDTA. Collect the trypsinized cells into a conical tube.

Next, seed the cells into the wells of a six-well plate with basal medium. Incubate the plates at 37 degrees Celsius under 5%carbon dioxide supplementation. After 24 hours, replace the medium with osteogenic medium.

Then, stain the cells with von Kossa stain to assess the mineralized nodules. Wash the cells two times with PBS after aspirating the medium. Fix the cells in two milliliters of 70%ethanol overnight at room temperature.

Next, add two milliliters of 5%silver nitrate solution into the wells. After washing the cells with distilled water, incubate the cells in two milliliters of 5%sodium thiosulfate for five minutes. Counterstain the washed cells with two milliliters of eosin for 40 seconds.

After washing away the stain, place the plate under an optical microscope to view the stained cells. The adipose-tissue-derived stem cells showed multipotency to differentiate into osteoblasts, adipoblasts, and chondroblasts. Von Kossa staining showed the presence of mineralized nodules, characteristic of osteoblasts.

The adipogenic cells showed the presence of lipid vacuoles in the cytoplasm of the stem cells, while Alcian Blue staining revealed the presence of glycosaminoglycan in the extracellular matrix.