The microbubble activation and expansion protocol is significant because it addresses a major unmet need in cell therapy research and manufacturing, improving the persistence and efficacy of T-cell therapies. Akadeum's buoyancy-based positive selection, activation, and expansion technique limits overstimulation and subsequent T-cell exhaustion during activation and expansion workflows. To begin, incubate 3 times 10 to the 8th commercially obtained PBMCs in 2.5 milliliters of separation buffer, with biotinylated anti-CD3 antibody.
Gently mix the components by pipetting, and incubate them at room temperature for 10 minutes. Add stripped avidin microbubbles to the cells at a ratio of 0.5 to 1, according to the manufacturer's instructions. And mix using a commercial end-over-end rotator at 20 RPM for 10 to 15 minutes at room temperature.
Centrifuge at 400g for five minutes at room temperature. After centrifugation, the positively selected cells will be at the top of the suspension with the stripped avidin microbubbles, and the remaining non-selected cells will be in the cell pellet at the bottom of the tube. Using a nine-inch glass pipette, insert the tip below the bubble cell layer to the bottom of the tube.
Aspirate the cell pellet and subnatant with an electronic pipette, and transfer them to a new tube. Resuspend the bubble cell layer left in the original tube in one milliliter of complete T-cell medium. Centrifuge the subnatant at 400g for five minutes at room temperature, and use it for indirect measurement of purity and recovery.
After centrifugation of the subnatant and unselected cells, aspirate the supernatant, and resuspend the pellet in one milliliter of media before counting. Using an automated cell counter, count the cells in the subnatant with bright field microscopy and determine the number of cells captured in the bubble cell layer, as described in the text manuscript. To create the conjugated anti-CD28 stripped avidin and microbubbles, add the biotinylated anti-CD28 antibody to the commercial micro bubbles, and mix using end-over-end rotation for a minimum of two hours.
Add the anti-CD28 conjugated strep avidin micro bubbles to the prepared bubble cell suspension at a ratio of 1.5 to 1. Mix the components using end-over-end rotation for 15 minutes. Then adjust the total volume to 2 million cells per milliliter with complete T-cell medium, or another desired medium according to the cell number obtained.
Distribute one milliliter of activated cells in a 24 well plate, and incubate in a humidified 5%carbon dioxide incubator at 37 degrees Celsius. After 24 hours, add IL-2 and soluble anti-CD3 to encourage further expansion, as calculated using the initial number of cells plated on day zero. Place the cell plate back into the humidified carbon dioxide incubator, and incubate overnight at 37 degrees Celsius.
Remove half of the medium from the midnatant every two days. Replace it with fresh, complete T-cell medium, and add IL-2 at a concentration of 50 units per milliliter. Count the T-cells twice weekly to assess cell density.
When the cell density exceeds 2 times 10 to the 6th, or 2.5 times 10 to the 6th cells per milliliter, transfer them into a bigger vessel, diluting them to 5 times 10 to the 5th cells per milliliter. Gently mix the contents of each well by pipetting up and down. Remove the entire contents of the well, including the microbubbles, and transfer them into a 1.5 milliliter tube.
Wash each well with 400 microliters of calcium-free and magnesium-free DPBS, and transfer the solution into a 1.5 milliliter tube. Then centrifuge the tube at 400g for five minutes at room temperature. Aspirate the supernatant, and resuspend the cell pellet in 50 microliters of separation buffer.
Next, split 50 microliters of cell suspension into 25 microliters each for activation and exhaustion. Stain the cells with an activation and exhaustion antibody staining cocktail, and incubate them for 10 minutes at room temperature in the dark. Add one milliliter of separation buffer, mix gently, and centrifuge to wash out excess antibodies.
Aspirate the supernatant completely. Resuspend the cell pellet in one milliliter of separation buffer, and transfer it to an appropriate vessel for the flow cytometry analysis. Increases in viable T-cell numbers and transgene positive T-cells were observed between the control sample and the cells that received microbubble co-stimulation.
Increased effector cell populations were also observed in the micro bubble samples. The viable activated T-cells express increased early activation marker CD69, and middle to late activation marker CD25. The total number and percentage of exhaustion markers PD1 positive cells were also significantly higher in cell samples that received microbubble co-stimulation.
Proper mixing and dispensing of microbubbles is essential to ensure accurate dosing, as the microbubbles float to the surface rapidly without agitation. We fully expect it to do so by enabling rapid growth of a large population of T-cells with higher activity than those more exhausted T-cells frequently seen in other methods.
