Mouse Wound Models and Preparation of Single-Cell Suspensions

Our research mainly focused on the impact of umbilical cords of MSC-derived extracellular vehicles on wound healing at the single cell level. And the primary objective with this part of the study is to establish a stable, acute, full thickness injury mass model, which is a crucial foundation for our experiments. The wound healing process is extremely complex, involving the synchronized interplay of various cells in time and space.

Single cell and sequencing has become the latest method of studying cellular ingenuity and predicting cell differentiation and developmental trajectories during bone healing. And obtaining the high quality single cell suspension from distorted tissues is the key to this research. Our protocol creates a more stable, full thickness in injury mice model, avoiding hair cycle and side tension issues, and prevents mouse scratching and natural activity from affecting results.

And additionally, we use the mixed enzymes for efficient tissue dissociation, yielding high quality single cell suspensions. To begin, randomly assign mice to control and experimental groups and house them in a specific pathogen free animal facility. Provide mice with unrestricted access to standard food and autoclave tap water.

Gently secure the mouse in a prone position and clean the abdomen with alcohol cotton swabs. After anesthetizing the mouse, apply depilatory cream to the back and wait for two minutes. Gently wipe away the cream with moist gauze to avoid damaging the skin.

Then wipe the skin with dried sterile gauze. Arrange the autoclave skin biopsy punch, scissors, forceps, and fresh surgical cloth on a sterile platform. Place the anesthetized mouse in a prone position and disinfect the dorsal skin with three alternating swabs of Betadine and 70%alcohol.

Dip the stamp in ink and make a mark on the upper middle part of the mouse's back. Using surgical scissors carefully excise the entire skin layer of the back along the circular mark, exposing the muscle layer completely. Leave the wound open without dressing, allowing it to naturally form a scab.

Administer 0.1 milliliters of 10%buprenorphine hydrochloride subcutaneously near the wound. Place the mouse back into its individual cage in a prone position. Following anesthesia, observe and photograph the mouse wound every two days to measure wound size.

Subcutaneously, inject 100 micrograms of human umbilical cord mesenchymal stem cell derived exosome around the incision site of an anesthetized mouse from the treatment group. Inject 200 microliters of PBS around the incision site of the control group mouse. Placing wounds on both sides of a mouse resulted in noticeable differences in wound area due to mouse skin elasticity.

The human umbilical cord mesenchymal stem cell derived exosome treatment group exhibited faster healing compared to the control group. Complete healing occurred more rapidly in the stamp model than in the sutured metal ring group.