The overall goal of this procedure is to derive glial restricted precursors or GRP cells from fetal spinal cords. This is accomplished by extracting the spinal cords from E 13 fetuses and transferring them to culture plates with GRP Selective Media. Next immuno panning is used to enrich the GRP population and eliminate the non GRP lineage.
The purified GRP cells can be frozen and put into long-term storage. The purified GRP cells can be thawed and used for future experiments, such as showing the migration of transplant GRP cells through a single cell wide passage. Visual demonstration of this method is critical as the surgical steps can be difficult to learn, especially to the inexperienced eye.
To demonstrate today are Roche De Silva and Joel Marx. Who are students in our lab Obtain pregnant mouse dams on embryonic day E 12 or E 13 of fetal development from a commercial source or prepared in-house day. E one is defined by the presence of a vaginally located mucus plug.
The day after a fend female is housed with a male having sanitized all of the instruments and tissue culture hood. Prepare a clean Petri dish with 20 milliliters of cold dissection medium. Next, prepare the mouse with an anesthetizing injection of chloral hydrate.
Once the mouse fails to show a pain reflex from a toe pinch, for instance, euthanize it via cervical dislocation or decapitation, We prefer not to use CO2 to anesthetize the animal because of the possible downstream effect on the viability of the fetuses and ultimately the derived cells. After disinfecting the abdominal area, open the abdomen using large scissors and forceps to remove the uterus. Depending on the strain the uterus will contain eight to 14 fetuses.
Place the uterus in fresh cold dissection, medium in a clean Petri dish to isolate the fetuses. First, wash away blood and excess tissues. Then extract each fetus from the uterine horn and separate them from the embryonic sac and placenta.
Using small scissors, transfer the fetuses to a dish of fresh, cool dissection.Medium. The cool medium will lower the metabolic activity and preserve the viability of the derived cells Under a dissection microscope. Use forceps and spring scissors to cut the skin along the spinal cord, peel it back and expose the spinal cord.
Transect the spinal cord at around C one and at the beginning of the tail. Then carefully remove the spinal cord with all its bone and cartilage transfer and collect each prepared spinal cord in a single dish. With fresh dissecting medium, We harvest the entire spinal cord, even though there's a greater population of GRP cells located.
Eventually, however, the GRP medium selects for the GRP phenotype and that population is further maximized by H two B five immuno To prevent overgrowth of meningeal tissue. In subsequent cell cultures, remove as much of the meninges as possible from the spinal cord due to the nascent protruding peripheral nerves. This is more difficult than removing meningeal tissue from extracted cerebral.
Once as much of the meninges has been removed as possible, transfer the spinal cords to a 20 millimeter Petri dish and proceed with dissociating. The cells in preparation. Prewarm an aliquot of trypsin for at least half an hour in a 37 degree Celsius water bath.
The following steps should be done quickly to enhance the viability of the derived spinal cord tissue. Transfer the spinal cords to a 50 milliliter centrifuge tube containing trypsin and DNAs. Then briefly tritrate the mixture with a pipeter.
Incubate the mixture for 10 minutes at 37 degrees Celsius tri. Rate it again and then continue the incubation for another 10 minutes. Then add five milliliters of GRP medium in centrifuge for five minutes.
At 1000 RPM aspirate the supernatant and resus. Suspend the pellet in 10 milliliters of GRP medium with DNAs one. Now incubate for another 10 minutes and pellet the cells at 1000 RPM for five minutes.
Aspirate the supernatant and resuspend the pellet. In fresh GRP medium. Pass the cells through a 40 to 70 micron cell strainer and plate them on PLL laminin coated T 25 flasks.
Incubate the cells and change 100%of the media the next day and then every other day or more often if nutrients are pleated. At this point, GRPs have attached and have started extending processes and they can be optimized using immuno panning. Continue growing the cells until they're 85 to 90%confluent or a weak has passed.
Then harvest cells using a rubber policeman or trypsin. Resus resuspend the cells in fresh media and expand them in three T 25 flasks, changing the media at least every 48 hours When the flasks have reached con fluency, harvest them as before into five milliliters of GRP media. Tri rate the cell suspension to break up clumps and transfer them to A two B five coated bacteriological.
Petri dishes incubate the cells on the A two B plates for one hour at room temperature without shaking. Then aspirate the media and wash the plates four to eight times with PBS to maintain high stringency for non-specific binding of cells to the plate. Using a rubber policeman.
Collect the washed cells into two milliliters of GRP media and then transfer the suspension to PLL lamina coated plates or flasks with GRP medium. Once the cells have reached 85 to 90%confluence, harvest the musing trypsin, then dilute and inactivate the trypsin with eight milliliters of GRP stock, medium, and collect the cells as a pellet. Resuspend the volume of cells from a T 25 flask in one milliliter of GRP freezing medium or cells from a T 75 flask in two milliliters of GRP freezing medium.
Adjust the cell suspension concentration if needed, and transfer the cells to cryogenic vials slowly freeze the cells overnight in a cryogenic container containing isopropanol at minus 80 degrees Celsius the following day. Transfer the cells to long-term storage. The cells will be 60 to 80%viable after thawing six months to one year later.
Viability is largely dependent on the quality of the PLL laminin coating. After two days in culture, plated spinal cord cells of different morphologies were observed to maximally enrich. The GRP population cells are immuno pan to select for the A two B five population.
If there is a problem with neuro epithelial cell contamination, double immuno panning to first eliminate the NCA positive population followed by A two B five selection can be employed. A T three free B 27 supplement was added to reduce the contamination of mature oligodendrocytes. Purified GRP cells were identifiable by their small soma and two or three short processes.
Often there was also a small persistent population of A two B five negative cells that were neuro epithelial and capable of becoming either GRP or N rp. Fortunately, the longer the cells were maintained on GRP medium, the more likely they adopted the GRP phenotype. The basic techniques of this procedure can be used to derive other cell types, which can answer additional questions such as which cell is better suited for cell replacement therapy or can cortex derive neurons be good models for in vitro myelination.
