The overall goal of this procedure is to effectively isolate and expand the population of neural precursor cells that reside in the adult canine hippocampus. This method can be used to obtain and study adult neural precursor cells, which then may help answer key questions in the field of adult neurogenesis. The main advantage of this technique is that it grants researchers access to an alternative higher mammal animal model, which may have closer parallels to the human brain than traditional animal models.
In this procedure, begin extraction within six hours post mortem by sterilizing the dorsal surface of the animals head using povidone iodine. Using a scalpel, make a longitudinal incision along the sagittal midline, across the entire dorsal surface of the cranium to expose the underlying masseter muscles. At the rostral and caudal borders of the cranium make additional five to 10 centimeter perpendicular cuts on both sides, passing behind the eyes and ears respectively to allow the skin to be fully reflected.
Separate the masseter muscles from their attachment to the sagittal crest of the skull and scrape any remaining tissue from the cranium. Using an osculating bone saw, cut the cap of the skull in a circumferential line. Than using a probe or a scalpel, sever any remaining attachments with the dera and carefully remove the skull cap to expose the dorsal surface of the brain.
After that, sever the spinal cord by inserting a scalpel between the upper cervical vertebrae. Gently lift the brain and use a scalpel to carefully free the brain from the cranial fossa. Sever the connective nerves and blood vessels located on it's ventral side.
Then, gently transfer the brain to a container of DPBS. To dissect the hippocampus, rinse the brain in DPBS to remove any blood. Next, place it dorsal side down onto a 150 millimeter Petri dish.
Slowly bisect the brain longitudinally through the midsagittal plane using a wedge brain knife cutting with a single slice that utilizes the full length of the blade. Do not apply additional downward pressure or use a sawing motion as this may damage the tissue. Place both hemispheres medial surface up.
Dissect at the hippocampi using a scalpel and fine forceps and transfer them to a 35 millimeter tissue culture dish. Mince the tissue samples into approximately one cubic millimeter pieces using a scalpel blade. Next, transfer the tissue into a 15 milliliter tube.
Add two millimeters of 0.1%trypsin EDTA and incubate it for seven minutes in a water bath at 37 degrees Celsius. Afterward, halt the enzyme reaction by adding four milliliters of serumedia to the tube. Pellet the suspension by centrifugation at 100 times g for seven minutes and then remove the supernatant.
Re-suspend the pellet in 300 microliters of DPBS and mechanically dissociate it by gently pipetting up and down until a smooth homogenate has formed. Subsequently add 14 milliliters of serumedia to the tube and pass the cell suspension through a 40 micron cell strainer. Next, pellet the suspension by centrifugation at 100 times g for seven minutes.
Remove the supernatant and re-suspend it in complete growth medium. From the cell suspension obtained at isolation, count the number of viable cells using trypan blue dye exclusion and a hemacytometer. Dilute the cell suspension in complete growth medium and seat the cells in the uncoated wells of a six well plate.
Incubate the cells at 37 degrees Celsius for 14 to 28 days and replace 50%of the growth media every seven days. Measure the diameter of the neuro spheres regularly and allow the cells to grow to no more than 100 micrometers in diameter. To passage as a floating culture, when the neuro spheres reach approximately 100 micrometers in diameter, combine all the neuro sphere containing media into a single tube.
Pellet the cells by centrifugation at 100 times g for seven minutes and remove the supernatant. Next, re-suspend the pellet in one milliliter of 0.1%trypsin EDTA and incubate it for seven minutes in the water bath at 37 degrees Celsius. Afterward, gently pipet up and down 100 times using a 200 microliter pipet tip to ensure dissociation of the neuro spheres.
Halt the enzyme reaction by adding five milliliters of serumedia to the tube and centrifuge it at 100 times g for seven minutes. After seven minutes remove the supernatant, re-suspend the cell pellet in 1 milliliter of complete growth medium and count the number of viable cells. Subsequently, dilute the cell suspension in complete growth medium and seat the cells in the uncoated wells of a six well plate.
Incubate the cells at 37 degrees Celsius for 14 to 28 days and replace 50%of the growth medium every seven days. In this procedure, add a sufficient amount of 0.1%gelatin solution to cover the tissue culture dish surface and cure it in an incubator at 37 degrees Celsius for one hour prior to processing the neuro spheres for adherent culture. Next, combine all the media from the floating neuro sphere culture into a single tube.
Pellet the neuro spheres by centrifugation at 100 times g for seven minutes and then remove the supernatant. Re-suspend the pellet in one milliliter of 0.1%trypsin EDTA and incubate it for seven minutes in a water bath at 37 degrees Celsius. Gently pipet up and down 100 times using a 200 microliter pipet tip.
After that, add five milliliters of seramedia to halt the enzyme reaction and centrifuge it at 100 times g for seven minutes. Discard the supernatant and re-suspend the cell pellet in 1 milliliter of complete growth medium. Then count the number of viable cells using trypan blue dye exclusion and a hemacytometer.
Next, dilute the cell suspension in complete growth medium remove the gelatin from the tissue culture flasks and seed the cells. Afterward, incubate the cells at 37 degrees Celsius. Replace the media with fresh, warmed complete growth media every three days until the culture reaches approximately 80%confluence after approximately seven days.
Neuro precursor cells were able to proliferate as floating neuro spheres or when seeded at one times tenth to the fourth cells per square centimeter onto 0.1%gelatin coated culture flasks as an adherent monolayer. No morphological differences were observed between dorsal and ventral hippocampal neuro precursor cells and both adherent cultures were able to undergo under 10 population doublings without any observed slow down in passage time. Nestin and Sox2 neural stem cell gene expressions in adherent culture were not significantly different across the hippocampal dorsal axis.
When cultured in BDNF for 28 days, adult canine neuro precursors from both the dorsal and ventral hippocampus differentiate into mature neuronal encyclical cell types. While attempting this procedure, it's important to remember work quickly and maintain sterile working conditions. After watching this video, you should have a good understanding of how to isolate and expand neuro precursor cells from the adult canine hippocampus.
