Hi, I'm Ryan O'Mara, a PhD student from the University of Ottawa and Ottawa Hospital Research Institute. This protocol will describe methods on how to produce enriched populations of oligodendrocyte precursor cells from mice, which may be differentiated alone or in combination with mouse dorsal root ganglion neurons to produce myelinating cold Cultures. Let's begin to give A brief overview of the procedure.
Neonatal mouse courtes are dissociated and seated into T 25 flasks. After a culture period of nine days, an astrocyte monolayer forms on the base of the flask on top of which oligodendrocyte precursor cells are proliferating. Oligodendrocyte precursor cells are then obtained and purified and can be subsequently differentiated into mature oligodendrocytes in an enriched culture.
If myelinating co cultures are desired. Doce root ganglion neurons from P five to P 10 mice are cultured alongside the mixed glial cultures. Over a period of nine days, these neurons form a dense neurite bed on which oligodendrocyte precursor cells may be seeded.
This allows for oligodendrocyte mediated wrapping of the neurites, thus resulting in a myelinating cold culture. First Whole brains from P zero to P two mice are placed in ice cold MEM and transferred to a dissection microscope using a scalpel cut through the meningeal layer of each cortex in a longitudinal manner, Then pry off the meninges from the underlying cortex. Next, remove the olfactory bulbs With the brain ventral side up.
Make deep longitudinal incisions where the cortices meet the ventral cephalon With the brain dorsal side up pry off the cortices, then dice the cordis into about four pieces each. Transfer The cortices into a 15 milliliter conical tube containing 350 microliters of ice cold. MEM per brain.
Brains of the same genotype are pooled in the same tube, so that four brains would require 1400 microliters of MEM. Once the dissection is completed, move the pooled brains to a 37 degree water bath for three minutes. Next, in a sterile tissue culture hood, pass the brain pieces through a P 1000 pipette tip to break them up into smaller pieces.
Add 75 microliters of prewarm OPC Pap pain per brain to the conical tube. Then Incubate in a 37 degree water bath for 20 minutes. Inverting the tube every two minutes to prevent tissue aggregation.
Once the incubation is completed, add two milliliters of mixed glial culture. Media per brain to the digest, allowed to stand for 10 minutes. After 10 minutes, aliquot the tissue suspension into five milliliter plastic tubes.
The number of tubes should match the number of brains in the preparation using a flame Polish glass pasture pipette tate the contents of each tube slowly at first, Then with increasing intensity as pieces begin to dissociate. Stop tri rating once there are no visible chunks of tissue left in the suspension. Pool the contents of each tube into a 50 milliliter conical tube containing four milliliters of mixed glial culture.
Media per brain after gently inverting the tube several times, aliquot the cell suspension into 15 milliliter conical tubes. The number of tubes should match the number of brains in the preparation centrifuge the tubes at 300 times gravity for five minutes and resus suspend the pellets in one milliliter of mixed glial culture media. Add the resuspended cells from each conical tube to poly L lysine coated T 25 flasks that were pre equilibrated with five milliliters of mixed glial culture media.
The number of flasks should equal the number of brains Dissected, Incubate the flasks, and at 37 degree incubator at 8.5%CO2. After three to four hours, the cells should be adhered to the substrate. However, there will be much debris persisting in the media, which may adversely affect cell viability.
To avoid this, replace the media after three to four hours with fresh mixed glial culture media as you can. This media change removes much of the cellular debris. The next day.
Many cells should be evident on the base of the flask. On the third day, an astrocyte monolayer should be forming on the base of the flask. At this point, perform a two thirds media change by removing four milliliters of media and adding back four milliliters of fresh media.
This step should be repeated every three days. On the sixth day, an astrocyte monolayer should be clearly visible on top of which oligodendrocyte precursor cells are proliferating. At this point, supplement the culture media with the insulin at a final concentration of five micrograms per milliliter on the ninth day, the oligodendrocyte precursor cells are ready to be separated from the astrocyte monolayer.
To do this, place the flasks on an orbital shaker and a 5%CO2 incubator. Allow the flask to equate to this incubator for one hour. Then shake the cells at 50 RPM for 45 minutes.
This step removes many loosely attached contaminating cells. Perform a full media change by removing all of the media and adding back four milliliters of fresh culture media. Replace the flask in a 5%CO2 incubator and allow to equate for three hours.
At the three hour mark, check the coloration of the culture media. The culture media should be red indicating a balanced pH. If the color is towards orange or purple, adjust the incubator CO2 level accordingly.
Maintenance of a balanced pH is important for the survival of the oligodendrocyte precursor cells during the overnight shake. Once a balanced pH has been achieved, shake the flask at 220 RPM for approximately 16 hours. The next morning, remove The flask from the shaker and transfer the media from the flasks to 10 centimeter tissue culture dishes.
These dishes will serve as means to further purify the oligodendrocyte precursor cells by differential adhesion. Each dish should receive media from two flasks, incubate the dishes at 5%CO2 for 30 minutes. At the 15 minute mark, give each dish a gentle nudge to prevent oligodendrocyte precursor cells from adhering to the plastic.
After the 30 minute incubation, examine the dishes under phase contrast. Oligodendrocyte precursor cells are evident as small clumps of cells. Some of these cells may have adhered to the dish.
Loosen the adhered oligodendrocyte precursor cells from the dish by gentle agitation. Be careful. Too much agitation may result in loosening of the contaminating cell types.
Transfer the cell suspension to a 15 Milliliter conical tube and centrifuge at 300 Times gravity for five minutes. Resuspend the pellet gently with a P 1000 pipette tip. Followed by a P 200 pipette tip count the cells with a hemo cytometer purified oligodendrocytes may then be seated onto laminate coated cover slips for differentiation into enriched oligodendrocyte cultures at day one.
Post seeding the enriched oligodendrocyte precursor cells have relatively simple morphology extending only a few processes. By day three, differentiating oligodendrocytes have extended numerous processes reminiscent of immature oligodendrocytes. By day six, oligodendrocytes have extended myelin like membranous sheets at any point during this time.
Course cover slips can be fixed and processed for immuno cyto chemistry. Rather than seeding the purified Oligodendrocyte precursor cells on laminin coated cover slips, they may be seeded onto dorsal root ganglion neurite beds to produce myelinating co cultures. This next section will describe methods on how to produce These cultures.
To begin isolate whole spines from P five to P 10 mice and transfer them to a dissection microscope with dissection scissors. Cut through the ventral medial region of the spine in a longitudinal fashion. Carefully remove the dorsa root ganglia or DRGs with fine tipped four steps.
Transfer the ganglia to Hank's buffered salt solution in a Petri dish. Trim any DRGs of their excessively long roots. Using a P 1000 wide boar pipette tip.
Transfer the ganglia to a 1.5 milliliter centrifuge tube. Keep the tube on ice until all mice have been processed. When DRG isolation is completed, centrifuge the tube at approximately 300 times gravity.
Transfer the tube to a sterile tissue culture hood and remove the supernat. Add 500 microliters of prewarm DRG papine to the tube and incubate in a 37 degree water bath for 10 minutes. When the pepane digestion is complete centrifuge, then replace the Papine solution.
With 500 microliters of Prewarm collagenase a solution incubate at 37 degrees for 10 minutes. Repeat the centrifugation step and replace the collagenase A solution with one milliliter of DRGN culture media. After our final centrifugation step, add one milliliter of DRGN media and with a flame polish glass pasture pipette tri.
Rate the ganglia slowly at first, then with increasing intensity as the tissue begins to dissociate. Once no more tissue chunks are visible, transfer the cell suspension to a Petri dish containing seven milliliters of DRGN media. Incubate the dish and a 37 degree incubator at 8.5%CO2 for one and a quarter hours.
After this incubation, observe the Petri dish. Under phase contrast, DRG neurons are evidenced as large bodied cells. Many contaminating cells should be firmly adhered to the Petri dish.
Transfer the media to a 15 milliliter conical tube and rinse the dish with four milliliters of DRGN media to collect any remaining neurons. Centrifuge the conical tube At 300 times gravity for five minutes. Re suspend the resulting pellet and 500 microliters of DRGN media.
Count the cells using a HEO cytometer. Be sure to only count the DRG neurons seed 30 to 50, 000 neurons per 12 millimeter laminate coated cover slip in one milliliter of DRGN media culture. The cells in an 8.5%CO2 tissue culture incubator.
The next morning perform a full media change by replacing the DRGN media with ol media supplemented with 10 micromolar FUD R every second day. Subsequent to this, perform a three quarters media change with FUD r supplemented OL media When Examined under the phase contrast microscope at day one, neurons have begun to extend a few processes. At day three, the neuronal processes have lengthened.
At day six, a neurite bed has developed composed of many intermingled processes. At day nine, this neurite bed has increased in density and is ready for the reception of oligodendrocytes by growing mixed brain cultures. Alongside the neuron cultures, oligodendrocyte precursor cells may be purified at day nine of the neuron cultures.
They can then be seated onto the neurite beds. By adding 50, 000 cells to each well of a 24 well dish. At day six of co-culture cells, reminiscent of oligodendrocytes can be seen interacting with the DRG neurons.
These cultures can now be fixed and processed for immuno cyto Chemistry. When Enriched oligodendrocyte cultures are labeled with antibodies against the oligodendrocyte markers, NG two mag and MVP immunologically positive cells are evidenced displaying stage specific morphologies. These cultures originate as a population of NG two positive oligodendrocyte precursors, and over the six days of differentiation, produce mag positive mature oligodendrocytes.
When SDS page is performed on lysates obtained from oligodendrocyte enriched cultures, it can be seen that oligodendrocytes express CNPP and MVP over the six day time course. When looking at DRG neurons prior to the addition of oligodendrocytes dense Taj one and neurofilament positive neurite beds are evidenced at day nine. At day six of Oligodendrocyte DRG neuron co-culture, many MBP positive oligodendrocytes can be seen interacting with the neuron neurites at higher magnification.
Oligodendrocytes are seen to wrap these neurites with MBP positive membrane reminiscent of myelination, a digitally magnified view shows the extent of overlap of the MVP positive membrane on the neurofilament positive process. Hi, I'm Scott Ryan, a postdoctoral fellow at the Ottawa Hospital Research Institute. These techniques provide a means of generating a liga, endocyte and DRG neuron co-culture systems from mice.
This technique is valuable in studying both experimentally induced and transgenic models of myelinating disease. It's also effective for screening novel therapeutic compounds.
