Establishing a Three-Dimensional Culture of Rat Brain-Derived Glial Cells
Transcription
24 hours before cell encapsulation, aspirate the medium from two-week primary cultures, and add 1 milliliter of fresh medium to each well. On the day of cell encapsulation, prepare 12.5 milliliters of dilute trypsin and 25 milliliters of medium for each 12-well plate, and warm to 37 degrees Celsius in a water bath. Then, use a 10-milliliter serological pipette to add around 1 milliliter of diluted trypsin to each well of the 12-well plate.
Incubate for 20 to 30 minutes at 37 degrees Celsius and 5% carbon dioxide until the confluent cell layer detaches from the plate. After the incubation, use a 1-milliliter pipette to recover the detached cells, which should appear as a single floating piece, and transfer to a 15-milliliter conical centrifuge tube. Dilute with an equivalent volume of medium and centrifuge at 200 times g for 2 minutes at room temperature.
Following centrifugation, discard the supernatant and resuspend the cell pellet in 10 milliliters of warm medium. Triturate the cells five times with the pipette, then, centrifuge again at 200 times g for two minutes. After the spin, decant the supernatant, resuspend the cells in 5 milliliters of warm medium, and transfer the cells to a 50-milliliter conical centrifuge tube.
Then, using a 10-milliliter syringe with an 18-gauge needle, triturate the cell solution three times and filter the suspension through a 40-micron cell sieve into a new conical centrifuge tube. Next, collect 10 microliters of cell suspension, and dilute 1-to-100 in warm medium, and count the cells with an automated cell counter.
Incubate the remaining cells in a 37 degrees Celsius water bath until the encapsulation step. Warm 12.5 milliliters of naive medium and 12.5 milliliters of conditioned medium for each plant 12-well plate of 3D hydrogels. Then, weigh 7 milligrams of HAMA per plant 12-well plate, and add to sterile filtered for a concentration of 2% weight for volume. Sonicate the solution for 60 minutes at 20 kilohertz until the HAMA is fully dissolved.
While sonicating, prepare individual 10% solutions of both TEA and NVP. Also prepare a 1-millimolar solution of eosin in 1-milliliter aliquots of sterile PBS. Then, for each 12-well plate, make a final 1.4 1.4-milliliter mixture of 1 x 107 cells, 0.5% HAMA, 20% basal lamina mixture in PBS, 0.1% TEA, 0.1% NVP, and 0.01 millimolar eosin Y.
After gently mixing the solution, use a pipette fitted with a 1-milliliter tip to dispense 100 microliters into each PDMS mold. Then, expose the samples to a high-intensity green LED light in an enclosed box for 5 minutes at room temperature.
The five-minute green LED exposure is essential to form the 3D gel. The red color introduced by the eosin Y will become transparent and indicate the termination of the reaction.
Next, add 1 milliliter of warm, conditioned media to each well of a 12-well plate; grasp each coverslip between thumb and forefinger and use curved tweezers to slowly peel off the PDMS mold without displacing the gel. Place the mold into a well with conditioned medium.
Removing the mold is the most challenging step. It should be done with utmost care to prevent dislodging the gel and fracturing the coverslip. This may take practice.
After all molds have been transferred to the wells of the plate, add an additional 1 milliliter of warm DMEM/F12 medium to each well. Incubate the plates at 37 degrees Celsius with 5% carbon dioxide for two weeks before imaging.
