This technique is intended to help us better understand the biology of normal hematopoiesis and disease in the bone marrow by safely injecting and transplanting valuable patient samples into the mice intraorally, followed by aspiration of the bone marrow. It has long been known that it is more efficient to transplant small amounts of valuable samples directly into the bone marrow of mice for experiments done by intravenous injection. However, there are no actual technical videos, so it was generally considered a high hurdle technique.
Our protocol enables any valuable sample to be easily and safely engrafted in the mouse with a small number of cells. There is a high demand for this technology, and with the help of this video, anyone can quickly learn it. We hope that everyone can master this technology and significantly contribute to the advancement of science.
To begin prepare cell suspensions with up to 3 million sample cells using 20 microliters of FACS buffer or thaw medium. Keep these on ice before injection. Disinfect the leg containing the femur to be injected with three sets of alternating scrubs using either a povidone iodine or chlorhexidine scrub and 70%ethanol soaked gauze sponges.
Stabilize the leg by pinching the femur with the thumb and index fingers. Push the tibia with either the ring or the fifth finger to keep the tibia bent from the femur. Ensure proper positioning for successful injection.
Use the edge of an empty sterile 27G needle to peck the patellar tendon on top of the femur. To ensure the best insertion point, insert the 27G needle with an attached syringe just under the patellar tendon. Lodging it securely between the two condyles of the femur, swivel the needle outward and upward to align it parallel with the shaft of the femur.
Rotate the needle in circles while advancing it into the femoral marrow cavity. Insert the needle until there is a noticeable reduction in resistance. Confirm correct needle positioning by lateral movement.
Create negative pressure by pulling the needle plunger back while moving the needle within the bone marrow cavity. Check for blood or marrow in the syringe to ensure the needle is in the bone marrow cavity. After ensuring correct needle placement, remove it slowly and insert the cell containing syringe through the same root.
It is important to remember the root and angle of the first injection when switching to a new needle. Once the cells are successfully injected into the femur, carefully remove the needle and syringe, maintaining pressure on the syringe. Then remove the mouse from the nose cone and place it on a clean paper towel to prevent aspiration of bedding.
For recovery, keep the mouse on a heating pad or other controlled surface. Ensure recovery from anesthesia before placing them back on the mouse rack. Observe mice for signs of distress or infection over the next 24 hours.
Begin by preparing for the procedure before aspirating the bone marrow, wet a 0.5 milliliter 27G syringe with CSM by filling and expelling CSM two to three times. Then gently aspirate the bone marrow from the anesthetized mouse, yielding 20 to 50 microliters of bone marrow. Then fully remove the needle from the femur and suspend the aspirated sample in 500 microliters of CSM.
Remove the mouse from the nose cone and place it on a clean paper towel to prevent aspiration of bedding during recovery. Monitor all mice to verify recovery from anesthesia prior to placing them on the back of the mouse rack. Pellet cells from the bone marrow with a five minute spin at 300G at four degrees Celsius.
Aspirate the supernatant and add ACK lysis buffer to each tube. Vortex the cells for resuspending them and place them on ice for 5 to 10 minutes. Filter the cell suspension through nylon mesh into a new tube.
Rinse the original tube with FACS buffer and pass it through the nylon mesh, pellet cells again by spinning for five minutes at 300G at four degrees Celsius. Aspirate the supernatant from the pelleted cells and add the staining solution containing the desired antibodies. Keep the tubes on ice for 20 minutes.
Wash the cells and proceed to analyze them as per the experimental setup. Transplantation techniques can be easily evaluated with minimal mouse invasion using a bioluminescence image. Here transplantation of a human derived K562 cell line with Acalook was observed in a xenograft mouse model on the side of the injected femur.
Transplantation of CD34 positive HSPCs resulted in more than 10 to 20%of human cell engraftment at eight weeks post transplantation. Similarly, transplanting cord blood derived HSCs resulted in up to 10%human engraftment at eight weeks post transplantation. Further successful transplantation of adult bone marrow derived HSPCs, leukemia stem cells, CRISPR Cas9 edited cord blood derived HSPCs, and patient derived AML iPSCs was achieved using this procedure.
