The overall goal of this procedure is to create humanized mouse modified with chimeric antigen receptor. This method can help answer key questions in the HIV and immunology fields in regards to testing the efficacy of stem-cell based engineered immunity against HIV infection and other chronic infections and malignancies. Such as a mechanism of immune failure and ways to enhance the immune system and develop a therapy to create better anti-viral or anti-tumor immunity.
The main advantage of this technique is that it is capable of a large amount of experimental manipulations. The humanized BLT mice model mimics human immunity and in this case allows for pre-clinical testings for cell based gene therapies against HIV infection. Demonstrating the procedure will be Valerie Rezek and Brianna Lam, they are technicians form my laboratory.
Begin by using scalpels to cut the thymus into small pieces of about one millimeter cubed in a dish containing supplemented RPMI medium. Then used curved blunt forceps to place each single thymus piece into a single well on a 96 well plate. Add 100-200 microliters of media to all of the wells so that the tissue does not dry.
Visualize the tissue under the microscope to eliminate any tissue that may not be thymus or that has connective tissue attached. Next, remove the confirmed pieces of thymus, combine them into a T25 cell culture flask. Add seven milliliters of antibiotic supplemented RPMI media to the flask and gently rock to mix.
Then place the flask in the tissue culture incubator. This step prevents bacterial contamination of the tissue. Begin the isolation of CD34 positive HSCs from fetal liver by using two scalpels to cut the liver into small pieces of about three millimeters cubed in a dish containing IMDM.
Remove and discard any white connective tissue. Then use a 10 milliliter syringe fitted with a 16 gauge blunt needle to take out the liver pieces and media and transfer to a 50 milliliter conical tube. Gently take up and expel five to seven more times to completely homogenize the tissue.
After preparing 10 milliliters of IMDM media, supplement it with collagenase, hyaluronidase, antibiotics, Dnase, and anti fungal. Pass the media through a 0.22 micron filter. Then add the filtered media to the liver suspension.
After capping the 50 milliliter conical tube containing the liver suspension, seal tightly with self sealing film such as parafilm to prevent leaks. Rotate in a tube rotator in the 37 degrees celsius incubator for 90 minutes. Following the incubation, filter the digested cell suspension through a 100 micron cell strainer into a fresh 50 milliliter tube.
Add PBS to the suspension to bring the total volume up to 50 milliliters. Split this into two 50 milliliter tubes, each containing 25 milliliters of cell suspension. Slowing and gently underlay the cells in each tube with 10 milliliters of a density centrifugation media, such as Ficoll.
Spin at 1200 times g for 20 minutes without break. Following the centrifugation, carefully remove the interface from each tube and transfer it into two separate 50 milliliter tubes. Bring the volume of each tube of interface up to 50 milliliters with PBS.
And then centrifuge at 300 times g for seven to 10 minutes. Combine and wash the cell pellets three times with 50 milliliters of PBS containing two percent FBS, spinning at 300 times g for seven to 10 minutes each time. Following the final spin, re-suspend the pellet in 50 milliliters of RPMI media plus 10 percent FBS.
Count the cells using a hemocytometer. Next, sort CD34 positive cells immediately using a CD34 sorting kit according to the manufacturer's protocol, and save both CD34 positive and CD34 negative fraction. Pour the thymus pieces and medium from the flask into a 60 millimeter dish.
Chill some positive displacement pipette tips by putting them in open 1.5 milliliter sterile screw-cap tubes on ice. Also, place the cell pellets and a volume of gelatinous protein mixture such as matrigel on ice. Anesthetize the recipient mice with an injectible anesthetic according to an approved animal protocol.
Check the anesthesia level of the mouse by squeezing a paw. The absence of a reaction to the pinch indicates a surgical plane of anesthesia. Shave the left side of each mouse from hip to shoulder between the center of the back and the stomach.
Subcutaneously inject six milligrams per kilogram of diluted Carprofen into the animal's shoulder or inguinal triangle. Flush the cannula of the trocar with PBS in a 60 millimeter dish. Using a pair of blunt curved forceps, place a piece of thymus from the 60 millimeter dish just inside the opening of the cannula, then pull back on the trocar to aspirate the tissue into the cannula.
Next, use a positive displacement pipette and a chilled tip to add five microliters of cold gelatinous protein mixture into the tube with the cell pellet. And gently stir to generate the cell suspension. Pipette the cell suspension into the opening of the cannula and slowly pull back on the trocar to load the needle.
Swab the shaved area of the mouse with povidone iodine followed by isopropenyl three times. Determine the darkest spot under the skin which indicates the location of the spleen. The kidney is approximately five millimeters dorsal to the spleen.
After ensuring a suitable level of anesthesia by the absence of a reaction of the paw pinch, use curved forceps to lift the skin and use surgical scissors to make a 15 millimeter incision in the skin parallel to the spleen. Then make a similar cut in the peritoneum layer below. In males, the kidney is easily visible, thus extruded simply by pressing on the abdomen.
Support the kidney with a hemostat or a pair of curved blunt forceps. In females, the ovaries tend to block the kidney from easy extraction. Using a hemostat, pick up the ovary and carefully expose the kidney.
Use needle-tipped forceps to pluck a tiny hole at the posterior end of the kidney capsule. Slide the trocar into this hole and along the kidney until the opening of the cannula is completely covered by the kidney capsule. Gently extrude the tissue under the kidney capsule and pull the trocar back out.
Use the curved forceps to make sure the thymus piece does not come back out with the needle. Next, lift up the peritoneum with the forceps and gently use the hemostat to push the kidney back into place. Tie one double knotted stitch with surgical suture in the peritoneum.
Also place two Autoclips Wound clips to close the skin. Now mix the transduced CD34 positive cells and aspirate 0.5 times 10 to the six cells in a 100 microliter volume into an insulin syringe. Inject these cells into the mouse through retro orbital vein injection.
After injection, swab a small drop of eye lubricant onto each eye, and lay the mouse on its side back in a cage. After implanting all the mice, confirm that the animals have regained conscientiousness and are ambulatory before returning them to the housing room. Mice were sacrificed 10 weeks after surgery and blood, spleen, thymus and bone marrow were harvested.
The resulting cells were stained with anti-human CD45 and anti-human CD4 antibodies and analyzed by flow cytometry. As seen here, CD4 chimeric antigen receptor modified cells can be detected in multiple lymphoid tissues by flow cytometry. The CD4 chimeric antigen receptor modified cells can differentiate into multiple lineages.
Here, splenocytes from CD4 chimeric antigen receptor modified mice were harvested and stained for markers of T and B cells. Cells negative for T and B cell markers were positive for markers of monocytes and macrophages and for natural killer cells. Splenocytes from HIV infected CD4 chimeric antigen receptor mice were stimulated with either HIV infected or uninfected T one cells and their intracellular production of cytokine is shown.
After watching this video, you should have a good understanding of how to construct humanized BLT mice modified with chimeric antigen receptors. Once mastered, this technique can be done in 48 hours if it is performed properly. While attempting this procedure, it is important to remember to work at a steady pace and pay close attention to the state of the mouse being operated on.
For beginners it is important to practice surgery and eye injections before working with tissues and stem cells that are more precious. After its development, this technique paved the way for researchers in the field of HIV and other infectious diseases to explore disease pathogenesis and immune based therapies in the humanized mouse model.
