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This protocol genetically modifies T-cells to introduce chimeric antigen receptors specific for tumor antigens, and then propagates clinically significant numbers on designer artificial antigen presenting cells. Begin with electro transfer of the sleeping beauty. Transpose on and transpose A DNA plasmids into quiescent mononuclear cells isolated from peripheral or umbilical cord blood.

Then measure the transfection efficiency using flow cytometry. Next co-culture, the Transfected T cells over seven days on K 5 62 derived artificial antigen presenting cells with the addition of exogenous cytokines in four week stimulation. Cycles of proliferation proceed to analyze cryopreserve and release the electroporated and propagated car positive T cells for human application.

The sleeping beauty transpose on transpose system offers an alternative to viral based gene transfer approaches to stably introducing a gene into the cell of choice. One of the major advantages of the sleeping beauty system is that one can integrate genes at really at a reduced cost compared to DNA plasmids that integrate through illegitimate non-homologous recombination. Thus, the advantage is that the sleeping beauty system can be used to express a gene at low cost and high efficiency.

Generally, individuals new to this method will initially have to overcome the efficiency of electro T cells in selective propagation of car positive T cells on artificial antigen presenting cells. Both steps are challenging to master because of the need to co-culture. Genetically modified T cells on artificial antigen presenting cells.

This method is useful in the field of adoptive immunotherapy, particularly in effective gene delivery and the manufacturer of clinically significant number of T cells. The broader application extends towards therapy of cancer because T cells from immune system can be modified to be tumor specific. Demonstrating the procedure will be Margaret Dawson and Matthew Fiola.

Technicians from my laboratory First dilute the peripheral blood with an equal volume of P-B-S-E-D-T-A and umbilical cord blood with four volumes of P-B-S-E-D-T-A slowly layer 25 milliliters of diluted blood onto 12 milliliters of fial in a 50 milliliter centrifuge tube. Centrifuge the fial gradient at 400 Gs for 30 to 40 minutes with no break. Now transfer the mononuclear cell fraction to a fresh 50 milliliter centrifuge tube with P-B-S-E-D-T-A.

Bring up the volume to 50 milliliters and harvest the cells by centrifugation. Gently wash the cells once in 50 milliliters of complete culture, media and centrifuge at 400 Gs for 10 minutes. Then pool the cell pellets in CCM perform a cell count.

Using the triam blue exclusion method. The mononuclear cells can now be used for electroporation or cryopreserved for future use. If using cryopreserved MNC quickly thaw two times 10 to the eighth cells for a full scale electroporation, gently transfer the cells to an appropriately sized centrifuge tube containing prewarm.

Complete phenol free RPMI culture media and pellet the cells by centrifugation resuspend the mononuclear cells In P-F-R-P-M-I perform a cell count and transfer the cells to an appropriately sized cell culture vessel at a concentration of 10 of the six cells per milliliter. Equilibrate the cells in a humidified incubator for two hours. Harvest the MNC by centrifugation, then gently resuspend and combine the cell pellets in P-F-R-P-M-I and enumerate the cells Now transfer two times 10 to the eighth MNC to a sterile 50 milliliter centrifuge tube and centrifuge at 200 Gs for 10 minutes without breaks.

Aspirate the supernatant so that no residual media remains around the cell pellet. Prepare a sterile 12 well plate with 10 wells containing four milliliters of warm P-F-R-P-M-I and equilibrate the plate in a humidified incubator at 37 degrees Celsius with 5%carbon dioxide. Then reconstitute the Lonzo Nucleo effector solution from the human T-cell kit as per manufacturer's instructions for each reaction.

Q Vet prepare 100 microliters of DNA master mix containing supplemented NU nuclear effector solution. 15 micrograms of the transpose on plasmid and five micrograms of the transpose plasmid. Next, disperse the pellet of T cells and resuspend a NU nuclear affection solution DNA master mix at a final cell concentration of two times 10th of the seventh cells per 100 microliters.

Carefully transfer 100 microliters of the cell suspension to each of 10 lonza nucleo affection cuvettes being careful to avoid bubbles. Now tap the qve once and electro using the program U 0 1 4 for unstimulated T-cells. Transfer the cuvettes and the Equilibrating 12 well plate to the culture hood using an amox of fine tip transfer pipette at 500 microliters of the prewarm culture medium from the corresponding well to each vete.

Transfer the electroporated cells to the plate and allow the cells to recover in the cell culture incubator for two hours. Then transfer the cells from all wells to a sterile centrifuge tube and pellet the cells after one wash, disperse the cell pellet gently and resus suspend in CCM to achieve a single cell suspension. After performing a cell count, adjust the cell concentration to tend to the six cells per milliliter in CCM.

Then transfer the cell suspension to a culture flask and place in the incubator overnight to generate EGFP transfected cells. Repeat the electroporation protocol using five times 10 of the six cells per vete with five micrograms of amox control EGFP plasmid. Harvest the electroporated cells, then perform a cell count using the trien blue exclusion method.

Then stain one to two times 10 of the six cells with antibody specific for CD three, CD four, CD eight, and human IgG FC gamma as a measurement of car expression. Acquire the cells on fax caliber. Analyze the data using FCS express software to determine expression of car.

Then calculate the car positive cells in culture. The A PC clone number four would derive from K 5 62 cells to co-expressed desired T-cell co-stimulatory molecules Thaw an aliquot of frozen irradiated, A A PC in a 37 degree Celsius water bath. Then wash the cells and CCM enumerate the viable cells by trip blue exclusion and calculate the number of viable A A PC required for stimulation.

Mix the electroporated cells expressing car and the gamma irradiated A A PC in a sterile container at a ratio of one to two. Adjust the A PC ratio for the expression of car based on flow cytometry the day after electroporation Add 30 nanograms per milliliter of IL 21 to the cell suspension. Then Eloqua tended the six cells per milliliter in T 75 flasks or view life culture bags and return to the culture incubator, the electro transfer of DNA plasmids and propagation of T cells on gamma irradiated.

A A PC can be used to generate clinically appealing numbers of T cells derived from peripheral blood and umbilical cord blood for human applications. In this characterization of genetically modified T-cells from peripheral blood expression of EGFP at day zero of first stimulation cycle controls for the efficiency of gene transfer expression of CD 19 specific car is assessed by flow cytometry on CD three positive, CD eight positive and CD four positive T cells at approximately 24 hours after electroporation. And then 28 days after co-culture on A A PC kinetics of car expression on T cells grown in a functionally closed system.

The propagation of peripheral blood derived car positive T cells over time can generate clinically appealing numbers of T cells that stably express the car. After four weeks of co-culture on A A PC, the average fold expansion of CD three positive T cells is about 19, 800 with 90%car expression. After watching this video, you should have a good understanding of how to elect parade and propagate T cells that express a desired car.

Remember to monitor for the overgrowth of NK cells. The electroporation and propagation of T cells is typically accomplished over four seven day stimulation cycles. Further investigations such as cytokine release and cytotoxicity assays can be performed to evaluate the redirected specificity of the genetically modified T cells In the field of adoptive immunotherapy.

This technique helps us to understand the therapeutic potential of T cells that are infused into humans.