To begin, culture MSCs In a six well plate with the human MSC medium containing 1%penicillin and streptomycin. Then proceed with labeling ARPE19 Mito-RFP cells with cell trace violet in the cytoplasmic membrane. To do so, add 20 microliters of dimethyl sulfoxide to a vial of cell trace violet reagent, and mix well immediately prior to use.
Grow the ARPE19 Mito-RFP cells to the desired density of 80 to 90%To prepare the loading solution, dilute the cell trace violet stock solution in prewarmed DPBS. Then remove the culture medium from the cells and replace it with one milliliter of loading solution. Incubate the cells for 10 minutes at 37 degrees Celsius.
At the end of the incubation, remove the loading solution. After washing the cells two times with DPBS, add two milliliters of fresh, complete medium. Then incubate the cells for at least 10 minutes at 37 degrees Celsius to allow the cell trace violet to undergo acetate hydrolysis.
Next, prepare a 24 well plate by adding 50 microliters of DPBS into each well. Insert the cover glass into the plate and allow it to settle for one minute. Remove the DPBS from the wells using negative pressure to ensure the cover slide is close to the bottom of the dish.
Once the cell density reaches 80 to 90%remove the original medium and wash the cells once with DPBS, add one milliliter of the trypsin EDTA DPBS mixture and incubate for three to four minutes at 37 degrees Celsius. Then gently tap the six well plate to detach the adhered cells. Add one milliliter of fresh, complete medium to terminate the digestion.
Afterward gently resuspend all the cells with a pipette gun and transfer all collected cells into a 15 milliliter centrifuge tube. Now centrifuge the cells at 200 G for five minutes. Aspirate the supernatant and resuspend the cells in one milliliter of fresh medium.
Place 10 microliters of cell suspension on a counting plate for enumeration. After seeding the MSCs and ARPE19 cells in the 24 well plate, observe the cells under a microscope. Shake the plate until the cells are evenly distributed.
Incubate them in the culture for 24 hours. For indirect co-culture, see 10, 000 ARPE19 cells in 500 microliters of medium per well of a 24 well plate. Place the insert into the well where the cells have been seeded.
Seed 10, 000 mesenchymal stem cells in 100 microliters of medium in the upper cell chamber per well.
