Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

The overall goal of this procedure is to isolate neural stem cells from the paraventricular region of the adult rat or human spinal cord. This is accomplished by first harvesting the spinal cord via laminectomy. The second step is to cut the spinal cord transversely into one centimeter segments and dissect the paraventricular region from each segment of the spinal cord.

Next, the minced tissue is cut into one millimeter pieces and then enzymatically dissociated. The final step is to separate intact cells by centrifugation through a discontinuous density gradient and plate the cell suspension. Ultimately, neuros sphere formation and the growth of neuro stem cells can be evaluated using immunofluorescence microscopy.

Today I am going to demonstrate how to isolate neural stem cells from the paraventricular region of the adult rat spinal cord. These cells can then be used to help answer key questions in the neural stem cell field, such as which exogenous factors promote lineage restriction or how these cells respond to various stimuli or stress. To begin, prepare the culture, media and dissection buffers as described in the accompanying text protocol.

Next, following an overdose of anesthetic, sterilize the rat's skin and use large dissection scissors to remove the dorsal surface, exposing the vertebral column. Hold the dissection scissors perpendicular to the dorsal surface and transversely. Cut the vertebral column above the hind limbs.

Then use smaller scissors to longitudinally cut the dorsal muscle overlying the vertebral column in a rostral direction to expose the spinous processes starting at the exposed coddle end. Insert small blunt bone cutting instrument extraorally into the lateral aspect of the spinal canal in the space between the spinal cord and the vertebral column. Place the blade shallow and parallel to the cord.

Then make small cuts into the lamina and carefully peel away the lamina to expose the spinal cord. This angle prevents damage to the underlying spinal cord. Continue to remove the lamina in the rostral direction.

To expose the thoracic and cervical spinal cord using blunt tissue forceps, gently lift the spinal cord from the vertebral column and sever the roots with micro scissors To release the cord, place the excised spinal cord in a Petri dish containing ice cold, sterile rat dissection buffer. Rinse the tissue with the ice cold dissecting buffer and use scissors to cut the spinal cord transversely into one centimeter segments. For each segment of tissue, use fine forceps to hold the tissue with one hand, and with the other hand use micro scissors to carefully remove the white matter along with most of the gray matter, leaving only the periventricular region of the spinal cord.

Pull the dissected periventricular tissue into a 10 centimeter sterile Petri dish containing ice cold rat dissection buffer. To begin the isolation of rat neural stem cells, use micro scissors to mince the dissected per ventricular tissue into one cubic millimeter pieces. Next, prepare the enzymatic dissociation buffer by first adding five milliliters of Earl's balanced salt solution to a vial of papain from a papain dissociation kit.

Place the vial at 37 degrees Celsius for 10 minutes so that the papain is completely dissolved and the solution appears clear. Next, add 500 microliters of Earl's balanced salt solution to the vial of DNAs from the dissociation kit. And gently mix, add 250 microliters of the DNA solution to the vial containing papain and gently mix.

Next, add the minced tissue approximately 0.2 grams into the vial, and place the vial on a rocker platform at 37 degrees Celsius for 45 minutes to one hour. After incubation tri rate the mixture with a 10 milliliter pipette to dissociate any remaining tissue pieces to yield a cloudy cell suspension. Transfer the cell suspension into a sterile 15 milliliter conical tube and centrifuge at 300 Gs for five minutes.

At room temperature during centrifugation, prepare the OVO moid solution by mixing 2.7 milliliters of Earl's balance salt solution with 300 microliters of the reconstituted albumin OVO MOID inhibitor solution. In a 15 milliliter conical tube, add 150 microliters of the DNA solution previously prepared to the oval moid solution and mix by inverting the tube, discard the supernatant from the pelleted cells, and immediately resus suspend the cell pellet in the diluted DNA albumin inhibitor mixture. Next, prepare a discontinuous density gradient by adding five milliliters of albumin inhibitor solution to a 15 milliliter tube and using a five milliliter pipette gently and slowly layer the cell suspension on top of the albumin inhibitor solution.

Centrifuge the sample at 70 gs for six minutes at room temperature. Once finished, discard the supernatant and resuspend the cell pellet in one milliliter of prewarm rat EFH medium that is prepared as described in the accompanying text protocol. Count live cell density with a hemo cytometer and plate the cells into a T 25 culture flask at a density of 10 cells per microliter in EFH.

Incubate the flasks at 37 degrees Celsius in 5%carbon dioxide and allow the cultures to grow undisturbed for one week to avoid aggregation of spheres. Shown here is a transverse section of rat spinal cord that has been stained with luxal fast blue, as well as hematin and eoin to show the boundaries of white and gray matter. The dotted outline designates the dissected remaining periventricular tissue that was isolated in this protocol from this region.

Neurospheres like the ones shown here, will grow free floating in suspension culture at high magnification. Micro spikes can be seen protruding from the neurospheres neurospheres proliferate as shown by KI 67 positive staining where neurospheres were dissociated and plated on matrigel coated wells. Additionally, they will primarily express nest in which is a marker for neural precursor cells.

Following this procedure, exogenous factors may be added to the cultured cells to promote differentiation. The adult neural stem cells or their progeny can also be transplanted into various animal models to assess their ability for regenerative repair. I hope you find this video useful, and good luck with your experiments.