The overall goal of this procedure is to generate genetically modified chimeric antigen receptor murine T cells and evaluate their efficacy in the context of standard of care. For high grade glioma, this is accomplished by first beginning standard of care, whole brain radiation and temozolomide administration to mice. Next, a retrovirus that expresses the car vector is produced, then murine T cells are transduced with the car retrovirus.
Finally, CAR T cells are delivered to mice treated with standard of care, ultimately delivery of CAR T cells in conjunction with whole brain irradiation and temozolomide. Administration is used to test whether host conditioning by standard of care enhances the efficacy of an adoptive T-cell platform. This method can help answer key questions in the brain tumor immunotherapy field, such as the anti-tumor efficacy of novel immunotherapies in the context of standard of care for high grade gliomas.
Though this method can provide insight into the effectiveness of gene modified mi and T-cells against high grade gliomas. It can also be applied to other systems such as the evaluation of T-cell therapy against melanoma, lung, and other brain metastasis tumor models. Generally speaking, individuals new to this method may struggle because this procedure requires several steps to be performed in parallel.
So planning and organizing tasks is critical. Demonstrating the procedure will be Catherine Uni and Carter s Deva, both talented grad student from my laboratory. After preparing three milligrams per milliliter of temozolomide or TMZ in DMSO, place the solution in a beaker of water over a hot plate at 65 degrees Celsius for 10 to 15 minutes.
Once the TMZ is fully dissolved, the solution should become pale yellow in color. Fully load 15 one milliliter syringes with TMZ solution. TMZ should be administered within 90 minutes of preparation to ensure that no TMZ precipitates out of solution to carry out whole brain irradiation power on the x-ray irradiator and allow it to warm up, ensure that the appropriate filter is in place and input the proper voltage and current settings.
Once mice have been sedated, according to the text protocol, place sedated mice on the grid with their heads positioned in the area that receives the highest x-ray intensity. Use lead tubing to shield the bodies from the neck down to block systemic x-ray delivery. Place the mice inside the x-ray machine when x-ray delivery is complete.
Remove the animals from the irradiator. When the animals regain sufficient consciousness and maintain sternal recumbent, place them back in their appropriate cages. After preparing D 10 medium and harvesting HEC 2 93 T cells according to the text protocol, add one milliliter of cell suspension at 7.5 times 10 to the six cells per milliliter into each of 1610 centimeter poly L lysine coated plates containing 10 milliliters of D 10 medium incubate overnight the following day.
And after preparing the car plasmid and transfection reagent as outlined in the text protocol, add the lipid DNA complex dropwise to the HEC 2 93 T-cell incubate for six to eight hours. Then replace the medium with 12 milliliters of fresh T-cell medium or TCM. Next, pour one spleen over a 70 micrometer mesh cell strainer and disaggregate by using the blunt end of the inside of a five milliliter syringe to generate a single cell suspension.
After preparing fresh TCM pool, the disaggregated spleen in a 50 milliliter conical tube in TCM and pellet, the cells eliminate red blood cells by using five milliliters of one x lysis buffer per spleen to resuspend the cells mixing well by gently pipetting up and down. Place the cell suspension in a water bath at 37 degrees Celsius for five minutes. Remove the lysis reaction from the water bath and add TCM at a one-to-one ratio with lysis buffer to neutralize the reaction wash by spinning at 300 Gs for 10 minutes.
After aspirating the supernatant use two milliliters per spleen of TCM to resuspend the pellet To count the cells add 10 microliters of cell suspension to 190 microliters of trian blue. Once the total number of cells has been calculated, use TCM supplemented with two micrograms per milliliter of conna A and 50 international units per milliliter of recombinant human interleukin two to dilute the cells to a concentration of two times 10 to the sixth cells per milliliter. Add two milliliters or four times 10 to the sixth cells to each well of a 24 well treated tissue culture plate and incubate overnight at 37 degrees Celsius and 5%carbon dioxide on the day after the splenectomy.
Prepare PBS containing 25 micrograms per milliliter. Recombinant human fibronectin fragment or RHFF and coat non tissue culture treated 24 well plates by adding 0.5 milliliters of the solution to each well the following day. Remove the solution from the wells and add one milliliter per well of 2%BSA in PBS incubate at room temperature for 30 minutes.
Then remove the BSA by firmly upending the plate before adding two milliliters of PBS to wash. After collecting the viral supernatant from the HEC 2 93 T cells, add fresh TCM to a final volume in milliliters equal to the number of RHFF coated wells, plus an additional three milliliters. Remove the cultured spleen cytes from the incubator and resus suspend by gently pipetting up and down two to three times in each.
Well transfer the cell suspensions to a 50 milliliter conical tube. Next, resuspend the cytes in viral supernatant to one times 10 to the six cells per milliliter. Then add one milliliter per well of the plete suspension to the wells of the RHFF coated plates.
Now spin the plates for 90 minutes at 32 degrees Celsius and 770 GS with an acceleration of four and a brake setting of zero during the spin. Prepare 50 international units per milliliter of recombinant human IL two in TCM. After the spin, add one milliliter of the medium to each well of the plates and incubate overnight.
If T cells reach greater than 80%confluence, split the cells by gently pipetting up and down in each well two to three times. Then transfer one milliliter from each well into new wells of a fresh 24 well tissue culture treated plate. Add one milliliter of fresh TCM with 50 international units per milliliter of IL two to each well, if the cells do not reach greater than 80%confluence change half the medium by slowly pipetting off one milliliter of medium from the top of each well and replacing it with one milliliter of fresh TCM containing 50 international units per milliliter of recombinant human IL two resuspend CAR T cells by gently pipetting up and down three times in each well and transfer to a 250 milliliter centrifuge tube.
Spin the cells at 300 GS for 10 minutes. Next, completely aspirate the supernatant without disturbing the pellet. Use PBS to wash the cells, then count them before washing a second time.
A typical CAR T-cell yield is approximately one times 10 to the sixth cells per well with PBS Resus. Suspend the cells at a concentration of five times 10 to the seventh per milliliter for an injection of one times 10 to the seventh in a volume of 200 microliters to inject into tumor bearing mice use a 27 to 31 gauge needle to load 500 to 1000 microliters into an insulin syringe ensuring that all bubbles are expelled. CAR T cells were generated by transduction with the E GFR V three CAR retroviral vector, which contains the rine stem cell virus or MSCV long terminal repeats.
The extended gag region and envelope splice site and a viral packaging signal. The EG FFR V three car containing the human anti EEG FRV three single chain variable fragment. 1 39 was cloned into the retroviral vector in tandem with murine CD eight, tm CD 28 4 1 BBB and CD three zeta intracellular regions Following transduction, EG FFR V three CAR T cells can be quantified and phenotyped by flow cytometry with a two color panel comprised of a STRT bico rine conjugated biotinylated, EG FFR V three, derived multier and anti CD three fit C.Using the protocol described in this video, car expression is routinely observed in 55 to 70%of CD three positive murine T cells.
CAR T cells can be further phenotyped by the addition of other fluorescently labeled antibodies to the two color car panel as seen in this example, staining for CD eight and CD four shows that 70%of transduced cars are CD eight positive T cells while 20%are CD four positive T cells Once mastered. This technique can be done in 12 hours over a one week period if it is performed properly Following this procedure. Other methods like Elisa, Ellie spot and or intracellular cytokine staining may be performed in order to answer additional questions.
For example, are your chimeric antigen receptor transduced T cells functionally responsive to antigen recognition and is their functionality antigen specific? Don't forget that working with retroviruses, temozolomide and x-ray radiation can be extremely hazardous and percussions such as operating under bsl. Two conditions, wearing personal protective equipment, performing experiments in the designated areas, and obtaining proper training for animal handling.
And the use of x-ray equipment should always be taken while performing these procedures.
