The overall goal of this cell culture technique is to enrich for bone marrow neural progenitors to facilitate their differentiation into functional Schwann cells. This method addresses a key issue in stem cell and regenerative medicine:how to use a limited source of neuroprogenitors to generate glia to assist in post-traumatic axonal regrowth and remyelination. An advantage of this technique is the use of efficient culture conditioning to facilitate neural cell expansion from bone marrow stromal cells for their use in autologous cell therapy.
After two days in culture, remove 75%of the spent medium and the non-adherent cells and rinse the adherent cells with three 10 millimeter PBS washes. After the third wash, replace the PBS with 10 milliliters of marrow stomal cells, or MSC, growth medium and return the culture to the incubator. Visible colonies should appear by day six to seven.
On day 10, wash the culture in fresh PBS and detach the cells with 1.5 milliliters of recombinant enzymatic cell dissociation reagent. After five minutes at 37 degrees Celsius, add three milliliters of MSC growth medium to neutralize the reaction and collect the detached cells by centrifugation. Resuspend the pellet in five milliliters of fresh MSC growth medium and seed the cells at a four times 10 to the fourth cells per square centimeter density in a new 10 centimeter tissue culture plate.
The cells should reach 80 to 90%confluency within two days of passaging. For hypoxic preconditioning, first release the ring clamp and clean the exposed parts with 70%ethanol. Further disassemble the hypoxia chamber into components and place them into a laminar flow hood for sterilization under UV light for 15 minutes.
Next, wash an 80 to 90%confluent MSC culture with 10 milliliters of PBS as just demonstrated and treat the cultures with fresh MSC growth medium supplemented with 25 millimolar hepes. Place the treated culture into the reassembled hypoxia chamber and tighten the ring clamp. Seal the connecting ends of the chamber to ensure that there is no gas leakage and flush a gas mixture of 99%nitrogen and 1%oxygen into the chamber at a flow rate of 10 liters per minute.
After five minutes, place the chamber into the cell culture incubator for 16 hours. The next morning, detach the cells as just demonstrated and collect them by centrifugation. Resuspend the pellet in neural progenitor medium and seed six times 10 to the third cells per square centimeter onto low-attachment 6 well plates for their culture at 37 degrees Celsius and 5%CO2 for 12 days.
Sizable non-adherent spheres of cells should be observed by day six to seven. With more neurospheres apparent in hypoxia conditioned cultures than those under normoxia by days 10 to 12. On day 12, use a 10 milliliters pipet to transfer the neurospheres into a 15 milliliters conical tube and collect the neurospheres by centrifugation.
Resuspend the spheres in DMEM/F12 and plate five to 10 neurospheres per square centimeter onto poly-D-lysine laminin coated 6 well plates in 1.5 milliliters of glial induction medium per well. Maintain the sphere cell culture for at least seven days, refreshing the medium every three days. To harvest the dorsal root ganglia, transfer the first embryo into a 10 centimeter culture dish containing room temperature PBS under a dissecting microscope in the prone position and insert micro dissecting forceps along either side of the spinal cord.
Using blunt dissection, separate the spinal cord from the surrounding soft tissue using forceps to excise the spinal cord along the neck opening and tail stub. Remove the residual soft tissue from the freed spinal cord until only the spinal cord, nerve roots, and attached dorsal root ganglion remain. Detach the individual dorsal root ganglia from their connecting nerve roots.
Then, use a pipet pen equipped with a one milliliters pipet tip to transfer up to 100 dorsal root ganglia into 1.5 milliliters microcentrifuge tube of PBS. Collect the ganglia by centrifugation and resuspend the pellets in 200 microliters of recombinant enzymatic cell dissociation reagent per tube. After 10 minutes at 37 degrees Celsius, centrifuge the dorsal root ganglia again using a 200 microliter pipet tip to gently triturate the pellets in dorsal root ganglion neuron maintenance medium.
Seed the dorsal root ganglion cells at five times 10 to the third cells per square centimeter onto poly-D-lysine laminin coated 6 well plates in 1.5 milliliters of dorsal root ganglion neuron maintenance medium per well. After two days in culture at 37 degrees Celsius and 5%CO2, wash the cells and return the neuronal cell cultures to the incubator in fresh dorsal root ganglion neuron purification medium for another two days. After three to four maintenance and purification cycles, the dorsal root ganglion cell cultures should test positive for the neuronal marker Tuj-1 and negative for the glial cell marker S100 beta.
On day seven of the Schwann cell-like cell culture, rinse the cells in PBS followed by dissociation with 0.5 milliliters of recombinant enzymatic cell dissociation reagent per well at 37 degrees Celsius for five minutes. Resuspend the cell pellet in co-culture medium and seed the Schwann cell-like cells onto the purified dorsal root ganglion neuron culture at one times 10 to the third cells per square centimeter for 14 days of co-culture at 37 degrees Celsius and 5%CO2. Healthy MSCs exhibit a tapered morphology in contrast to the quadrangular appearance of MSCs that have been maintained for a high number of passages and have lost their multipotency.
Expanded MSC colonies should demonstrate the expression of MSC markers, the absence of hematopoietic stem cell markers, and the capacity for differentiation into adipogenic, osteogenic, and chondrogenic lineages. Healthy MSC culture that have been subjected to hypoxic preconditioning, demonstrate neurospheres that are larger in both number and size. The purity of the dorsal root ganglion neuron network can be confirmed by the expression of the neuronal marker Tuj-1 and the concomitant absence of the Schwann cell marker S100.
After 14 days in co-culture with dorsal root ganglion neurons, Schwann cell-like cells acquire the features and functional properties of mature fate-committed Schwann cells. Once mastered, the neural progenitor subpopulation can be expanded in either human or rat BMSCs in 10 days if the cell expansion technique is performed properly. While attempting this procedure, it's important to remember to confirm that the BMSC sample is endowed with nestin-positive neuroprogenitors and the CD45 positive hematopoietic progenitors have all been removed.
After watching this video, you should have a good understanding of how to source BMSCs, to enrich for the neural progenitor subpopulation, and to direct neural progenitor differentiation into fate-committed Schwann cells for the use in enhancing post-traumatic axonal regrowth and remyelination. Following this procedure, myelinating glia, such as mature Schwann cells, can be derived for their use in the development of therapies for demyelinating injuries and diseases of both the peripheral and central nervous system.
