The overall aim of this procedure is to obtain pure astrocyte cultures by isolating and culturing mixed cortical cells from P one to P four mouse pups. This is accomplished by first harvesting brain cortices of mouse pups and removing the meninges. The second step is to cut the cortex into small pieces, dissociate the cortex pieces by trypsin and tation to obtain single cells and to plate the single cells on poly de lycine coated tissue culture flasks.
Next, after seven to eight days, astrocytes, microglia, and oligodendrocytes will have formed different layers and the separation of astrocytes from microglia and oligodendrocytes is achieved by shaking the tissue culture flask on an orbital shaker. The final step is the ionization and harvest of the remaining astrocytes, which are then replated in a tissue culture flask. Ultimately 12 to 14 days after the first cell split, astrocytes are plated at the appropriate concentration for experiments and cell purity can be examined by immuno cyto chemistry using astrocyte specific markers.
The method outlined here is based on the astrocyte culture preparation from rodent neonatal brains originally described by McCarthy and develops in 1980. This method can help answer key questions in the neuroscience field, such as questions regarding the interplay between o osteocytes and neurons, as well as the role of as osteocytes in certain S disease, where as osteocytes become activated and contribute to inhibitory scar formation. Visual demonstration of this method is useful for young scientists to point out the critical steps of the isolation method and to visualize the astrocytic morphology during the different steps of the isolation procedure to ensure pure and healthy astrocyte cultures.
Demonstration of this procedure will be done by two graduate students of my laboratory, Sebastian Hilker and Khristian Bora Before beginning this procedure ensure that all tissue culture reagents and dissection reagents and materials are prepared in advance according to the instructions in the written protocol. After sacrificing a P one to P four mouse pup, perform a midline incision posterior to anterior along the scalp to reveal the skull, carefully cut the cranium from the neck to the nose, then make a cut anterior to the olfactory bulb and another inferior to the cerebellum. To disconnect the cranium from the skull base, use flat tip forceps to gently flip the cranial flaps to one side.
Next, cut away the olfactory bulbs and the cerebellum, and then lift out the brain. Deposit the brain into a dish of chilled HBSS and place on ice. Now repeat the procedure to harvest the brains from an additional three animals.
Four brains will provide enough astrocytes to seed a T 75 tissue culture flask at the proper density. Once all of the brains have been harvested, transfer the dish containing the brains to a stereo microscope. Next to isolate the cortices, grab the posterior end of each brain with fine forceps and perform a midline incision between the hemispheres.
Then insert a second set of forceps to the created groove and peel away the plate like structure of the cortex from the rest of the brain. Once all cortices have been isolated, carefully peel the meninges away from the cortex by pulling with the fine forceps. This step avoids contamination of the final astrocyte culture by meningeal cells and fibroblasts.
Transfer the prepared cortical hemispheres into a second dish filled with chilled HBSS and place on ice. Continue accordingly with all four cortices. Finally, cut each hemisphere into four to eight small pieces using sharp blades under sterile conditions.
Transfer the pieces of cortex into one 50 milliliter falcon tube and add HBSS to a final volume of 22.5 milliliters. Then add 2.5 milliliters of 2.5%trips in. Replace the cap mix and incubate the tissue in the water bath at 37 degrees Celsius for 30 minutes.
Mix by shaking every 10 minutes. Then after centrifusion for five minutes at 300 times G to pellet. The cortex tissue pieces carefully decant the S supernatant.
Add 10 milliliters of astrocyte plating medium to the pellet, and use a 10 milliliter pipette to vigorously pipette the medium containing the tissue up and down 20 to 30 times until a single cell suspension is obtained. Next, add astrocyte plating medium to a final volume of 20 milliliters and count the cells using a hemo or automated cell counter. According to standard procedures, one preparation of four mouse pup cortices should yield 10 to 15 times 10 to the six dissociated single cells.
Finally aspirate the poly de lycine from a previously prepared flask and transfer the dissociated cell suspension to the flask. Incubate the culture at 37 degrees Celsius in the tissue culture incubator for two days, and then change the media. The media should then be changed every three days.
Thereafter, the astrocytes should appear complement with an overlying layer of microglia after seven to eight days in culture. At this point, set the T 75 flask on an orbital shaker set at 180 rotations per minute for 30 minutes to remove microglia, discard the supinate containing microglia, or spin it down and plate for culture. Then add 20 milliliters of fresh astrocyte culture medium, and continue by shaking the flask at 240 RPM for six hours to remove oligodendrocyte precursor cells.
Since some OPCs will not completely detach from the astrocyte layer, continue to shake vigorously by hand for one minute. In order to prevent OPC contamination, again, discard the supinate or spin it down on plate to culture your OPCs. Rinse the remaining confluent astrocyte layer twice with PBS, aspirate the PBS and add five milliliters of trips in EDTA in the tissue culture incubator at 37 degrees Celsius.
Check for detachment of astrocytes every five minutes and enforce the detachment of astrocytes by hitting the flask against the palm of your hand two to three times. Once the astrocytes have detached from the flask, add five milliliters of astrocyte culture medium. Spin the cells at 180 times G for five minutes, aspirate the supinate and add 40 milliliters of fresh astrocyte plating.Medium.
One T 75 flask of mixed cortical cells should yield around one time cent of the six cells enriched for astrocytes after the first passage plate the cells in two T 75 culture flasks and incubate 37 degrees Celsius in the tissue culture incubator. Change the medium every two to three days, 12 to 14 days after the first massage plate, the astrocytes at the appropriate cell concentration 24 to 48 hours before performing the experiment. One T 75 tissue culture flask should yield around 1.5 to two times 10 to the six cells after the second split.
This image shows mixed cortical cells one day after plating. The black arrows indicate astrocytes attached to the bottom of the flask. Dying neurons can be seen floating in the supinate three days after plating of mixed cortical cells.
The astrocyte layer is forming as indicated by the black arrows and neurons are almost absent. The same mixed cortical culture is seen here five days after plating. The first microglia and OPCs on top of an astrocyte layer are apparent as indicated by the black arrows as seen here.
The astrocyte layer is completely confluent seven days after plating mixed cortical cells. This image shows the enriched astrocyte culture. Two days after splitting.
The attached cells show astrocyte morphology with low density. Here the areas indicate single cells. Two weeks later, the astrocyte layer has reached high density.
The scale bar represents 10 microns. This image demonstrates the purity of the astrocyte cultures. Each panel shows cells that have been immunostain for the astrocyte markers, G-F-A-P-G-L-A-S-T-S 100 B Aquaporin four A LDH, one L one, and BLBP shown in green.
The nuclei are revealed by a DAPI counter stain. After watching this video, you should have a good understanding of how to isolate and culture cortical astrocytes. The isolation and culture of cortical astrocytes described in this protocol provides a powerful tool for investigating astrocyte biology.
Since their manageability in diverse applications can greatly complete their investigation in vivo.
