Speaking of significance, generating NK cells from an expandable source is critical for applications such as cancer immunotherapy. The advantage of this technology is that expanded potential stem cells possessing broader differentiation capacity can be used instead of regular IP cells. For predifferentiation of hEPSCs, remove the hEPSC medium in which cells were maintained and add one milliliter of DFK medium.
Incubate for two to three days at 37 degrees Celsius and 5%carbon dioxide. To check the formation of embryoid bodies, after removing the spent DFK medium, wash the cells once with one milliliter of PBS. Add 500 microliters of 0.05%trypsin and incubate the plate at 37 degrees Celsius and 5%carbon dioxide for three to seven minutes, based on the morphology.
Once the trypsin is removed, and two milliliters of DFK medium to harvest the cells. Spin down the cells at 300 G for three minutes at room temperature. After removing the supernatant, add one milliliter of DFK medium and one microliter of Y-27632 and resuspend the cells by flicking.
Count the cells using a hemocytometer and dilute the cell suspension with DFK medium and Y-27632 to obtain 4, 000 cells per 25 microliters of medium. Place 30 to 40 drops of cell suspension on the cap of a 10-centimeter Petri dish and pour some PBS into the lower dish to prevent the evaporation of the droplets. Gently invert the cap to cover the dish and incubate at 37 degrees Celsius and 5%carbon dioxide for three days.
For collecting the embryoid bodies from the droplet, using a one-milliliter pipette containing PBS, pump a little PBS into the droplet and aspirate out the medium, including the embryoid bodies. Then, transfer these embryoid bodies into a 15-milliliter tube and spin them down at 100 G for one minute at room temperature. After removing the supernatant, add one milliliter of medium A.Transfer the collected embryoid bodies to a non-adherent 24-well plate and consider it day zero.
To perform mesoderm patterning of the embryoid bodies, incubate the plate for three days at 37 degrees Celsius with 5%carbon dioxide. On day two, replace 500 microliters of spent medium A with fresh medium A of the same volume. To perform hematoendothelial specification of the embryoid body, remove 700 microliters of medium A from the well and add the same volume of medium B.Culture for seven days, changing the half-media every two days.
To transfer the patterned embryoid bodies onto an air/liquid interface, slightly tilt the plate so the embryoid bodies aggregate at the bottom, and using a one-milliliter pipette, remove as much of medium B as possible. Now pick up the embryo bodies with a pipette by aspirating the remaining medium and transfer them to a trans-well in a 24-well plate, To perform lymphoid progenitor expansion, add 500 microliters of NK-1 medium to the lower compartment of the trans-well, culture for 14 days, and perform a medium change every other day. To coat the plate, dilute the coating material in PBS, add one milliliter of diluted coating solution to each well of a 12-well plate, and cover the opening of the plate with wrapping film.
Incubate the plate at four degrees Celsius overnight. Add 200 microliters of PBS into the trans-well and slowly pipette up and down around five to six times to harvest the cells released from the organoids. Transfer the harvested cell suspension to a two-milliliter tube and spin down the cells at 500 G for five minutes at room temperature.
After removing the supernatant, resuspend the cells in one milliliter of NK-1 medium. Remove all the coating solution from the well and wash each well of a 12-well plate with one milliliter of PBS twice. Next, split the harvested cells at a ratio of 1:2 and transfer the cell suspension onto a coated 12-well plate.
Add one milliliter of NK-1 medium so each well has 1.5 milliliters of medium. For changing the medium, allow the cells to sink to the bottom for one to two minutes. Then, carefully aspirate out 750 microliters of medium and spin it down as demonstrated previously.
After discarding the supernatant, resuspend the obtained pellet in 750 microliters of NK-1 medium by pipetting five to six times and transfer it into the original well. In the present study, the hEPSC-derived products at the endpoint were analyzed. Approximately 15%of the cells dissociated from 3D culture system from two batches induced at different time points were CD3-negative, CD56-positive, and 16%of the cells were CD45-positive, CD-56-positive, which is in line with the percentages of CD3-negative, CD56-positive cells seen in earlier trials.
The percentages of CD3-negative, CD56-positive cells in the cells harvested from the 2D culture system ranged from 30 to 6%The hEPSC-derived products during day 18 culture from 2D system showed expression of both ectopic CD56 and CD107a in 12%of cells after two hours of antibody stimulation, and approximately 28%of the harvested cells were CD94-positive, CD159a-positive. The in vitro differentiation products were tested for cytotoxicity against human erythroleukemia cells K562. When co-cultured with tumor targets for three hours, the differentiated products display mild cytotoxicity compared to an interleukin 2-independent permanent cell line NK92mi cells.
Following this method, cells such as T cells, B cells, and erythrocytes can be generated.
