Described in this video is a protocol for creating and using dry macroporous alginate scaffolds known as Drydux that efficiently promote transduction of activated T-cells. Drydux is designed to be used in place of gold standard spinoculation of RetroNectin-coated plates seeded with virus and simplifies the process for transducing cells. Drydux has a transduction efficiency comparable to spinoculation on RetroNectin-coated plates, and serves as a cheaper and more convenient alternative to the conventional transduction method.
First, prepare a 0.4%solution of calcium-D-gluconate. Add sterile filtered DI water to calcium-D-gluconate in a beaker. Stir on medium speed until the calcium-D-gluconate has dissolved, which takes approximately 1 1/2 hours.
Once dissolved, sterile filter the calcium-D-gluconate solution. This solution can be stored at room temperature. Next, prepare a 2%alginate solution.
This can be accomplished using a screw cap vial, or a beaker. Both will be shown here. Carefully add the ultrapure alginate to the vessel.
Add the appropriate amount of DI water to the alginate. Choose the largest stir bar possible that will not impede mixing and add it to the vessel. Stir the solution on high until the alginate dissolves, which takes approximately one hour.
If there are any large alginate clumps on the side of the vessel, tilt the vessel sideways to dislodge them. Small clumps will dissolve over the hour, and are not cause for concern. Once the 2%alginate solution dissolves, reduce the stirring speed.
Slowly add an equal volume of the calcium-D-gluconate solution to the alginate solution. Adding it slowly will help prevent cross-linking clumps from forming. When the calcium-gluconate solution has been added, increase the stirring speed, and stir vigorously for 15 minutes.
Tilt the vessel sideways to remove any clumps that may have formed. After stirring vigorously for 15 minutes, ensure there are no clumps in the solution. Pipette the solution into a 48, or 24-well plate.
For a 48-well plate, pipette 300 microliters into each well. Ensure to cast slowly and change tips often, as the solution will be viscous, and may stick to the tip. For a 24-well plate, pipette one milliliter into each well.
Again, cast slowly, and change pipette tips often. Cover the well plates with a lid, and freeze at negative 20 degrees Celsius overnight. Prepare the plates for the lyophilizer by removing the lids and securing the top with wipes and rubber bands.
Place the plates in the lyophilizer tube, and put on the lyophilizer for 72 hours. After 72 hours, remove the plate from the lyophilizer. The scaffolds are now ready to be used for transduction.
If not using the scaffolds immediately, seal the plate with Parafilm and store at four degrees Celsius until needed. For long-term storage, it is recommended to vacuum seal the plate in addition to sealing with Parafilm. Concentrate viral supernatant by centrifuging two milliliters of viral solution through a centrifugal filter at 1, 500 g for 10 minutes.
100 to 200 microliters of concentrated virus is needed per scaffold. Spin for a few additional minutes if the concentrated virus solution volume is greater than 200 microliters. For each scaffold, make an aliquot of 1 million activated T-cells suspended in 50 microliters of complete cell culture media.
Add the concentrated virus to the cell suspension. The total volume of the cell virus suspension should not exceed 200 microliters for a 48-well scaffold, or 350 microliters for a 24-well scaffolded. Add the cell virus mixture drop-wise to the top of the dry scaffold.
For negative control experiments, use complete cell culture media in place of concentrated virus. Add this cell suspension to the top of the dry scaffold. Incubate the scaffolds in the cell culture incubator at 37 degrees Celsius for 45 to 60 minutes.
Remove the scaffolds from the incubator. The scaffolds should fully absorb the solution during this time. To induce proliferation, add complete cell culture media supplemented with IL-7 and IL-15 to each well.
IL-2 can also be used. For a 24-well scaffold, add one milliliter. For a 48-well scaffold, add 500 microliters.
Incubate at 37 degrees Celsius for 72 hours. The cells will proliferate over the 72 hours, which may turn the media orange. Remove excess media from each well.
To isolate cells from the scaffold, add 0.25 molar EDTA solution to each scaffold. Add one milliliter for 24-well scaffolds, or 300 microliters for 48-well scaffolds. Let the plate sit, or gently agitate for three to four minutes.
Once mostly dissolved, pipette in and out gently inside the well. A few in and out pipetting steps might be necessary to fully dissolve the scaffold. Transfer the solution to a 15-milliliter centrifuge tube.
Wash the cells twice with PBS. Add 12 milliliters of PBS to each centrifuge tube. Centrifuge at 400 g for five minutes.
A cell pellet will form at the bottom of the tube. Aspirate the supernatant solution, and repeat the wash. Be careful to fully remove supernatant with every wash as it is crucial to get rid of all EDTA.
After washing with PBS a second time, the cell pellet is ready for analysis. Flow cytometry was used to determine the transduction efficiency of each group. Non-transduced cells were used as a negative control, and showed no transduction.
Cells seeded on scaffolds without GFP retrovirus also showed no transduction. The Drydux group, which had activated cells seeded onto the dry macroporous alginate scaffolds, showed 85%transduction, just lower than the RetroNectin group. These results demonstrate that Drydux scaffolds effectively transduced cells by retroviral gene transfer without the need for spinoculation of RetroNectin-coated plates.
Drydux scaffolds exhibit high transduction efficiency comparable to RetroNectin, providing an alternative method for transducing T-cells. Due to the low cost of manufacturing the Drydux scaffolds, this process has the potential to significantly lower the cost of CAR T-cell therapies.
