Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

We outline techniques necessary for the human xenograft skin transplant model, an important tool for the manipulation of immune and non-immune pathways in human tissues in vivo. This technique better recapitulates human skin physiology than in vitro methods, allowing for preclinical experimental manipulation in vivo. Xenograft skin transplants can be used for preclinical testing of compounds targeting human skin inflammation in vivo.

Bandaging mice can be particularly challenging, so we would advise practicing the bandaging process on healthy anesthetized mice prior to performing surgeries. To begin, prepare to dermatome the human skin sample in a sterilized negative pressure tissue culture hood on a sterilized dissection board. Place the skin sample with epidermis side up on the dissection board and wipe the epidermis with a sterile alcohol prep pad followed by PBS.

Now use a 1.5 inch dissecting T pin to fix the closer edge of the skin. Then dermatome the skin specimen at 400 micrometer thickness and apply steady pressure while cutting forward at an angle of 30 to 45 degrees. Place a sterile gauze soaked in sterile PBS at the bottom of the Petri dish and place the skin with epidermis side up onto the wet gauze.

Seal and cover the plate edges with a semi-transparent sealing film to ensure the sample is not contaminated and store the sample at four degrees Celsius before grafting. Prepare the sterile instruments and surgical station for grafting and use autoclaved paper towels as sterile surfaces for instrument and mouse placement. After anesthesia induction, transfer the mouse to a heating pad or another heat source and administer the ophthalmic ointment by dabbing a small drop of ointment on the eye with a gloved finger.

Pinch the skin and subcutaneously inject analgesics at an angle parallel to the body. After lifting the mouse by the tail, expose the abdomen and inject 100 micrograms per 100 microliters of anti-Gr1 intraperitoneally at a 30 degree angle using a one milliliter insulin syringe. Then use animal safe electric clippers to shave the middle and upper portions of the dorsal side of the mouse.

After clearing all the hairs, apply a generous amount of hair removal ointment onto the shaved skin. Wait for 30 seconds to one minute, then wipe the hair removal ointment completely with a paper towel and PBS. After transferring the mouse to the surgical station, sterilize the surgical area and place a sterile plastic wrap over the mouse.

Cut a window in the plastic slightly larger than the size of the area to be grafted. Hold the donor skin firmly in place with the forceps backside and cut a rectangle shaped 10 by 10 millimeter portion of donor skin to be grafted alongside the forceps with a scalpel. Pull the skin away from the body to avoid cutting deeply into the fascia.

To create a graft bed, snip a rectangular area of mouse skin matching the size of the donor skin piece. Then place the donor skin piece with epidermis side up onto the prepared graft bed. Using the back of the forceps, manipulate the skin sliding back and forth until the donor skin lies completely flat against the graft bed.

Add surgical glue tissue adhesive drops where the donor skin meets the mouse skin and hold the mouse and donor skin together with forceps for one to two seconds so that the glue adheres to the tissues. Completely seal the graft's edge and allow the glue to dry thoroughly. Next, cut petrolatum gauze large enough to cover the graft area completely.

Cover the graft with the petrolatum gauze and lightly press the gauze against the skin using forceps. Cut a strip of a transparent film dressing lengthwise so that the width is large enough to cover the mouse's wound. Firmly press the transparent film dressing with the adhesive side down over the gauze.

Quickly roll the mouse to wrap the dressing completely around the torso, ensuring it fits tightly without impeding respiration and all limbs are free for movement. After placing the mouse in a recovery cage, monitor it until it is alert and moving around. Provide a heat source on the part of the cage for at least 15 minutes following recovery.

Analysis of grafts 10 to 21 days post-transplant showed that the graft remained adherent around the edges where the donor's skin meets the mouse's skin and the graft remained thicker and more inflamed than healthy human skin. Analysis by flow cytometry revealed the sustained presence of human immune cells within most grafts, but counts varied between xenografts. The successful graft maintained the human immune cells.

However, despite continued adherence and survival of donor tissue, grafts which failed to maintain the human immune cells are deemed unsuccessful. Analysis of hematoxylin and eosin stained sections of the graft after 35 days showed intact epidermis and the dermis is moderately cellular composed of plump oval fibroblasts with pale syncytial cytoplasm and scattered lymphocytes. Analysis of the graft after 50 days showed similar results with intact epidermis and the dermis containing oval fibroblasts and slightly enhanced extracellular matrix deposition.

Successful skin xenografts rely upon appropriate preparation of skin specimens, suppression of mouse neutrophilic immune responses by anti-Gr1 administration, adherence to aseptic rodent surgery methods, and secure bandaging of mice. The human xenograft skin transplant model allows for researchers to study human immune responses in the intact organ in vivo, providing a new avenue for translational research.