The aim at this procedure is to prepare antigen protected functional red blood cells. This is accomplished by first preparing the HPG polymer in the second step. The HPG is functionalized through the addition of succinyl succinate groups.
Next whole blood from healthy donors is washed with saline to separate out the red blood cells. In the final step, the isolated red blood cells are treated with the functionalized HPG. Ultimately, the protection of the surface antigens on the modified cells by the functionalized HPG is assayed by flow cytometry.
The main advantage of our technique compared to existing methods is that HPG conjugations yield functional red blood cells that circulate normally. The implication of this technique extend toward chronic blood transfusion. As HPG modified red blood cells have reduced antigenicity and normal circulation in vivo.
Demonstrating the procedure will be Dr.Ian, a post doctor, fellow and constant school, a technician in my laboratory Three days before collecting whole blood from consented healthy donors dry lyophilized HPG in a round bottom flask overnight, and a vacuum at 90 degrees Celsius. The next morning, remove the flask from the oil bath and allow it to reach room temperature and then dissolve the dried HPG in two milliliters of anhydrous pyridine. Next, add a catalytic amount of dimethyl amino paridine to the HPG solution to functionalize approximately eight of the hydroxyl groups on the HPG with carboxyl groups.
Over the next 10 minutes, add Sica hydride dissolved in 0.5 milliliters, perine dropwise to the solution, and then stir the mixture overnight at room temperature under Argonne the next day precipitate the HPG mixture in 40 milliliters of four degrees Celsius. Acetone in a 50 milliliter to centrifuge tube for 15 minutes at 27, 000 times G at four degrees Celsius. Then decant the supinate and flush the precipitate with Argonne at room temperature to remove any residual acetone.
To activate the carboxyl groups with Saxon and Mial succinate groups. Groups dissolve the carboxyl functionalized HPG in two milliliters of anhydrous DMF. Then add NHS and Diop profile CARBIMIDE to the HPG solution and stir the mixture overnight at room temperature under Argonne.
The next morning proceed with purifying the S-S-H-P-G by precipitation in cold acetone. Afterwards flushing with argonne to remove any residual acetone. Then determine the purity of the modified HPG and the degree of the carboxyl and succinyl succinate functionalization by proton NMR analysis.
After obtaining informed consent, collect the blood from the healthy donor while the purity of the S-S-H-P-G is being evaluated. Centrifuge should the donor whole blood for four minutes at 1000 times G and room temperature. Use a paster pipette to remove the supinate and the buffy coat and then transfer the packed red blood cells to a 15 milliliter conical centrifuge tube.
Add PBS to the cells and then mix by inversion to obtain a uniform cell distribution. Then centrifuge the cells in PB S3 times removing the SUP natant. After each wash, transfer the pact RBC pellet to a 1.6 milliliter eend orph tube and add 300 microliters of PBS to bring the volume up to 400 microliters immediately after confirming the purity of the modified HPG transfer 150 milligrams of the S-S-H-P-G into a one Dr.Glass vial.
After weighing the polymer, add 300 microliters of PBS to the S-S-H-P-G and vortex it to dissolve the polymer. Then add the S-S-H-P-G polymer solution to the 20%hemid washed red blood cells. Next, gently vortex the suspension and then place the red blood cell HPG mixture onto an orbital shaker at room temperature.
After an hour, wash down the solution four times with PBS and saline removing the supinate each time. Then add 300 microliters of saline to the final 100 microliter palt of packed red blood cells to a final 20%hematocrit concentration. Now allow eight milliliters of donor whole blood cells to clot for 30 minutes at room temperature.
Then after spinning down the cells, collect about three milliliters of the serum transfer one milliliter of the serum into a 60 degree Celsius water bath. To prepare heat inactivated serum. After 30 minutes, add 60 microliters of fresh serum and 60 microliters of heat inactivated serum into separate eph tubes.
Each containing 60 microliters of 20%hematocrit HPG modified red blood cells. Then add 60 microliters of the same into tubes containing the same volume of unmodified red blood cells, and incubate all the red blood samples for one hour at 37 degrees Celsius. To quantify the amount of hemoglobin in the cell suspensions first place 5.9 microliters of each of the samples in triplicate into a 96 well plate.
Then mix each sample with 294 microliters of drab kins reagent and read the absorbance of the hemoglobin sano derivative with a spectrophotometer at 540 nanometers to quantify the amount of hemoglobin in the supinate centrifuge, the rest of the cell samples, and then plate 50 microliters of each of the supinate in triplicate in a 96 well plate. After mixing the cells with 250 microliters of drab kins reagent, measure the absorbance of the hemoglobin ano derivative as just demonstrated for this step. Begin by centrifuging 50 microliters of HPG modified RBC with 10%hematocrit in an eend orph tube.
After discarding the S supernatant at 110 microliters of MTS diluent to the cell pellet and pipette the solution up and down a few times to homogenize the cell suspension. Now load 11 microliters of the cell suspension into each column of a mini gel. Then place MTS cards into a centrifuge card holder and spin the cards for six minutes at 156 times G.The protection of the surface antigens can then be determined from the location of the red blood cells in the mini gel columns according to the manufacturer's description.
For this step, after setting up a dextran PEG two-phase separation system, spin the dextran PEG solution to obtain the two-phase system. Then carefully aliquot one milliliter of the upper PEG phase into a labeled tube with a concave bottom. Add 20 microliters of 20 to 30%hemog crypt HPG modified RBC to the tube mixing the cell suspension by gentle flicking.
Next, add 0.5 milliliters of the cell containing upper phase to 0.5 milliliters of the lower dextran face, and then mix the system again with more gentle flicking. Now rest the tube on bench to allow the system to separate after two minutes. The red blood cells should localize in the upper PEG phase in the lower dextra phase or in both red blood cells.
Derivatized with high levels of HPG are located in the upper PEG phase to evaluate the protection of red blood cell surface antigens by flow cytometric analysis. First, add 20 microliters of saline to three facts tubes for each sample. Then add five microliters each of control unmodified and HPG modified red blood cells in triplicate to the tubes.
Next, add 25 microliters of fit C monoclonal anti reus D to each tube, and incubate the samples at 37 degrees Celsius in a water bath. After 30 minutes, wash the samples in 1.5 milliliter saline, two times removing the supinate each time after the second wash, Reese bend the red blood cells in one milliliter of saline. Finally, acquire at least 5, 000 events on the red blood cell control gate to analyze the protection of surface antigens.
In this histogram, the normal expression of the Resus e-Antigen represented by the green peak on unmodified red blood cells from healthy human donors is shown when the red blood cells are grafted to HPG, the intensity of the Reese's e-Antigen signal decreases as evidenced by the shift in expression to the left denoted by the gray peak. The modification of the red blood cells by HPG also camouflages the expression of CD 47, as demonstrated by the left shift of the gray peak observed in this histogram as well, indicating that masking of the surface proteins occurs in the presence of HPG. Following this procedure, HPG modifier, blood cells can have application in blood transfusion and cell-mediated delivery of therapeutics.
After watching this video, you will have a better understanding of how polymers can be conjugated to red blood cells to modify the antigenicity and to construct the base for drug delivery.
