Thymus transplantation is an important method for investigating thymus epithelial cell function and the T-cell maturation in vivo. This simple and efficient protocol can be used to isolate and culture mouse 18.5 thymus for subsequent transplantation into a renal capsule. The protocol for the 18.5 thymus transplantation can be modified for thymic transplantation at other developmental stages or for other tissues of a similar size.
Also, this procedure is very simple. The details for some of the case stamps should be reviewed carefully when performing this technique for the first time. This procedure contains detailed manipulations that can be better illustrated with visualization.
After sacrifice, wipe the embryonic day 18.5 pregnant mouse with 70%ethanol and use autoclaved sterilized instruments to make a v-shaped cut on the abdomen from the bladder up to each horn of the uterus. Use scissors to cut the mesometrium and cervical and vaginal areas and harvest the uterus into a Petri dish containing cold PBS on ice. Cut the anterior uterine wall from one uterine horn to the other to expose the embryos within the envelop to decidua and use fine tweezers to peel back the decidual tissues.
Cut the umbilical cord to release the embryos for transfer into a new Petri dish on ice. To harvest the first thymus, wipe one embryo with 70%alcohol and transfer the embryo into a new Petri dish. After decapitation, fix the embryo in the dish in the supine position and use sterile scissors to cut the lateral chest wall horizontally along the axillary front.
Cut the diaphragm to open the chest. The thymus should be visible as two white lobes located in front of the trachea and adjacent to the heart. Carefully insert bent tip forceps behind the thymus and gently pull up to harvest the tissue.
Check the integrity of the thymus to confirm that it contains two jointed lobes. Then wash the thymus with PBS before using a stereo microscope to trim away the connective tissues and blood vessels. When all of the thymi have been collected, transfer each clean tissue into individual wells of a 24 well plate containing 500 microliters of culture medium per well.
And add 2'deoxygranosine to a final concentration of 1.25 millimolar per well. Then place the isolated thymi in the cell culture incubator for eight days. On the last day of culture, use scissors to cut a scalp vein needle on the infusion tube part at a 45 degree angle and confirm a lack of response to toe pinch in a nude recipient animal.
Place the mouse on the operating table in a right lateral position and use a 0.5%povidone iodine swab to swab the skin in the surgical area two times from the inside to the outside of the body. Use the scalpel to make a five to nine millimeter skin incision parallel to the spine in the left renal area and cut through the subcutaneous tissue and muscle to open the abdominal cavity. Using a pair of tweezers with one hand, lift the muscle and fat tissue from the spine side incision edge of the exposed kidney and use the other hand to gently squeeze out the kidney.
To keep the renal capsule moist during the surgery, wet the surface of the kidney with saline and use the scalp needle to gently scratch the renal capsule on the lower right side about 1/2 to 2/3 the width of the kidney. Slide the infusion tube into the nick of the capsule and gently detach the renal capsule from the kidney long the long side of the kidney about three to four millimeters into the kidney capsule. Then retract the infusion tube.
To implant the thymus into the created subcapsular space, wash one culture thymus two times in fresh saline and connect the clipped infusion tube to a syringe. Slowly aspirate the prepared thymus into the modified infusion tube. And gently insert the infusion tube into the renal capsule until it reaches the superior pole.
To deliver the thymus into the renal capsule, retract the tube slowly while simultaneously gently depressing the plunger. Using an alcohol lamp, slightly heat the scalp needle and after confirming that the whole thymus is inside the subcapsular space, use the heated needle to cauterize the nick. Restore the cauterized kidney to the abdominal cavity and close the peritoneum and muscle with sutures.
Using a modified interrupted vertical mattress suture, close the skin incision with at least three knots and use a povidone iodine swab to disinfect the incision. Then provide a subcutaneous injection of antibiotic and analgesia and place the mouse under an infrared lamp with monitoring until full recovery. Here, an isolate embryonic day 18.5 thymus containing two complete lobes can be observed.
Immediately after implantation, the thymus can be visualized under the renal capsule at the tip of the recipient kidney. After eight weeks of growth within the nude mouse recipient animal, the thymus remains under the capsule, but increases robustly in size. T-cells are produced in both the transplanted thymus and the peripheral blood of nude mouse recipients.
But as expected, no T-cells are detected in the peripheral of non-transplanted nude animals. Further, in this representative experiment, no LacZ gene was detected in the peripheral T-cells harvested from the peripheral blood of nude mouse recipients transplanted with thymus lobes containing the LacZ allele, indicating that peripheral T-cells were generated from the recipient animals. Be gentle when sliding the tube into a renal capsule.
Avoid breaking the capsule. And make sure you have enough space in the for the thymus delivery. Thymic or peripheral T-cell populations can be analyzed using flow cytometer.
Where, immune responses in recipient's peripheral tissues can be analyzed by I2d and the immune histochemical analysis.
