The overall aim of the protocol is to generate a humanized mouse model with functional human immune system and liver using human hematopoietic stem cells and hepato site. This method can help answer key questions in genetic liver humanized mice. This method provides a powerful tool to study immunopathology caused by HIV as observed in people.
Demonstrating the process will be by Yimin Sun, a supervisor from the pathology and microbiology department, along with Weimin Wang and Edwrd Makarov, research technologist. To begin, transfer umbilical cord blood collected in heparinized tubes to a sterile laminar flow cabinet. Add PBS until the volume is increased to 35 milliliters.
Layer the sample on top of lymphocyte separation medium and centrifuge it 400 times G and at four degrees celsius for 35 minutes with no breaks. Next, carefully remove the top plasma layer and use a transfer pipette to transfer the white buffy coat interface to a new tube. Re-suspend the buffy coat in 30 to 40 milliliters of ice cold buffer.
Using a pipette, combine 20 microliters of the cell suspension with 20 microliters of 0.4%trypan blue. Pipette 10 microliters of this mixture into the outer opening of either of the two chambers of a counting slide, and insert the slide into an automated cell counter to count the cells. Centrifuge the cells at 300 times G and at four degrees celsius for 10 minutes.
Carefully aspirate the supernatant and add 300 microliters of ice cold buffer. Add 100 microliters of human FC receptor blocking reagent and 100 microliters of monoclonal mouse anti-human CD34 antibody conjugated MicroBeads for up to 100 million cells. Incubate for 30 minutes at four degrees celsius.
Add 10 milliliters of ice cold buffer to wash the cells and centrifuge it 300 times G and four degrees celsius for 10 minutes. Carefully remove the supernatant and re-suspend the pellet in 500 microliters of ice cold buffer. After this, place a positive selection LS column in the magnetic activated cell sorting field and pass it through with three milliliters of ice cold buffer.
Load the sample into the LS column that can entrap MicroBeads bound to human CD34 positive cells in samples, and allow it to flow under the influence of gravity into the collection tube. Wash the column three times with ice cold buffer and collect the eluent in the same collection tube. Then, plunge the column with five milliliters of ice cold buffer to allude the CD34 positive cells into a new collection tube.
Repeat the procedure to achieve a purity of over 90%Next, use trypan blue dye and a hemocytometer to count the eluded CD34 positive cells. After counting, centrifuge the cells at 300 times G for five minutes. Discard the supernatant and re-suspend the cells in 125 microliters of PBS for an injection to be used immediately in transplantation.
To check the purity of the CD34 positive eluent, take 50 microliters of the suspension and incubate it with 10 microliters of PE conjugated anti-human CD34 antibody at four degrees celsius for 30 minutes. After this, wash and re-suspend the cells in PBS and proceed to perform flow cytometry. After acquisition, analyze the data as outlined in the text protocol.
First, retrieve and thaw the cryopreserved hepatocytes as outlined in the text protocol. Then, remove the bio cap and pour the thawed hepatocytes into a 50 milliliter conical tube containing warmed thawing medium. Rock the tube by hand for a few seconds to suspend the cells.
Pellet the cells at 100 times G and at room temperature for eight minutes. Wash the pelleted cells in PBS with 0.1%BSA and pool them with either fresh or thawed HSPCs in PBS to a final volume of 80 microliters per mouse. First, attach one end of a sterile extension tube to a 30 gauge needle and the other end to a one milliliter syringe.
Fill the syringe with the suspension of pooled HEPs and HSPCs. Fit the syringe in the notch of a repetitive dispensing pipette and adjust the dispenser to dispense 10 microliters in each press. Shave each mouse as outlined in the text protocol and scrub the left side of the body of each mouse with 10%povidone iodine followed 70%isopropyl alcohol.
Using Vannas type scissors, make an small incision in the skin, muscle and peritoneum at the left of the peritoneal wall to enter the peritoneal cavity approximately 5 millimeters below the lower edge of the rib cage. Locate the spleen and use forceps to pull it slightly to the operating area for easy access and insert the 30 gauge needle into the lower pole of the spleen. Next, unlock the plunger of the dispensing pipette and dispense 10 microliters of the volume at a time, with a limit of 60 to 80 microliters per spleen.
Retract the needle slowly and clip the spleen with ligating clips using a ligation applier. Then, use cotton-tipped applicators wetted with sterile PBS to push the spleen back into the body cavity. Use an interrupted suture pattern to close the muscle layer of the abdominal wall.
Accomplish skin closure with an interrupted suture pattern using non-absorbable sutures. Transfer all needed materials to a designated Biosafety Level Two Plus facility. Wear personal protection equipment including a disposable cover all gown, shoe covers, a face mask and double gloves, including cut resistant gloves, at all times while working with the virus.
Select mice with a reconstitution of more than 15%of human CD45 positive cells and with a presence of human albumin in the serum for HIV-1 infection. Intraperitoneally inject the mice with 1, 000 to 10, 000 tissue culture infectious doses of 50 HIV-1 ADA in a volume of 100 to 200 microliters per mouse. After euthanizing the mice, excise the liver from each mouse as outlined in the text protocol.
Collect and fix the livers in 4%paraformaldehyde overnight. The establishment of a dual humanized mouse model with human liver and immune cells can be easily monitored at each step with very simple ELIZA and flow cytometry, respectively. Flow cytometry is regularly performed to evaluate the development of a functional immune system and to see the effect of HIV infection on immune cells.
In dual humanized mice, the development of functional immune cells can range from 15 to 90%of the lymphocyte gate. For the evaluation of the engraftment of human hepatocytes, ELIZA for human-specific albumin levels is performed monthly on mouse serum. Mice engrafted with both HSPCs and HEPs show human-specific albumin levels ranging from approximately seven micrograms per milliliter to 377 micrograms per milliliter at one month, continuing grow over the time of observation.
The effect of HIV infection on human immune cells in the blood of dual humanized mice is monitored by flow cytometry and on HEPs in the liver by human-specific albumin ELIZA. By five weeks, HIV-1 causes a decrease in human albumin levels in the serum and there is a depletion of human CK18 positive hepatocytes in the liver sections of dual humanized mice. A lower of ratio of CD4 to CD8 is typically observed in the blood and liver of HIV-infected mice, compared to levels noted in the same mouse before infection.
Blood should be layered carefully on top of lymphocyte separation medium for isolations of buffy coat. For the surgery, hold the spleen gently and insert the needle in lower pole of the spleen. Following the isolation of hematopoietic stem cells, it is crucial to check the purity of CD34 positive hematopoietic stem cells to avoid CD3 positive cells and acute injections of hepatocyte from different donor.
As the humanized mice show physiological aspect of human immune system and liver, the developed mice could be utilized to study physiopathogenesis of HIV and physical infections and cirrhosis.
