The overall goal of this procedure is to reproduce a functional motor unit in vitro. This is accomplished by culturing human primary muscle cells in conjunction with isolated spinal cord explan from rat embryos. The spinal cord implants are then allowed to innervate the human muscle cells with the explan still attached.
The culture is carefully fed medium until the muscle cells fully differentiate. The molecular and or cellular phenotype of the differentiated muscle cells can then be assessed through a selection of molecular tools. The visual demonstration of this method is critical because first, it is difficult to learn how to isolate the spinal cord.
And most of all, you need to be very delicate when added the cultural medium to the innovative muscle cells. Euthanize a pregnant rat with embryos between stages. ED 12.5 and ED 13.5.
Be precise as the stage is crucial to the whole procedure. Dissect out the uterus and collect the whole embryo chain in HBSS medium with 10%FBS under a stereoscope isolate and decapitate each embryo for each embryo. Isolate the spinal cord in one piece and remove surrounding muscle and connective tissue.
To do that first, fix the embryos with micro scissors. Cut a line on each side of the spinal cord and carefully peel up the skin. Then put the embryo on the side and carefully isolate the spinal cord with the dorsal root ganglion or DRG still attached.
Functional innervation will not occur if the DRGs do not remain attached to the spinal cord. The spinal cord is whiter than the surrounding pink connective tissue and muscle. Carefully remove the surrounding tissues without disturbing the DRGs.
Once isolated, use the needle of a micro syringe to slice each spinal cord transversely into cubes with at least two DRGs attached. These eggplants will be around a cubic millimeter and are now ready to be co cultured with a standard human muscle cell culture. This protocol requires human muscle cells to be cultured from tissue biopsies.
Consult the written protocol for details on this procedure. Begin by removing the fusion medium on the muscle cell culture, leaving a fine layer of medium on the monolayer. Next carefully place the x explan on the muscle cell.
Layer five evenly spaced X explants can fit on each 35 millimeter dish. Slowly add fusion medium onto each x explan dropwise. Do not add the fusion medium too quickly or in too much volume.
Otherwise the x explants will float and fail to innervate the muscle cells. Then very carefully put the dishes back into the incubator. Continue adding media slowly.
When handled carefully, x explants will stop floating in the media and adhere to the cells in about one hour. Once adhered, increase the fusion medium volume to two milliliters per 35 millimeter dish. If after waiting overnight, the X explan are still floating in the medium, then either the muscle cell layer was not sufficiently confluent or the medium was added.
Two, roughly. These X explan will never adhere twice a week. Exchange all of the fusion media dropwise very slowly to avoid cell detachment.
The X explan are quite fragile during the first week and can detach easily. This is a very nice technique because after the innovation, just by measuring or collecting the cultural medium, you can measure the ome of the co cultures. And if you carefully choose the molecular tools, you can differentiate what's coming from the muscle and what's coming from the nerve.
Two days after the innervation neurites emerged from each explan and establish contacts with the muscle cells represented by button like structures. When neurites do not emerge, innervation will fail likely due to a lack of DRG attachment. As early as four to six days after the innervation, a few individual fibers started to contract.
The contractions persisted for many months, maintained simply by changing the culture medium twice a week. After about two weeks as expected, the spinal cord explants themselves degenerated, but the innervations were conserved. A fine layer of nerve components stayed at the surface of the muscle cell layer, and this nervous network was sufficient to maintain the functionality of the motor unit.
One of the big advantages of this technique is the possibility to take samples from patients and to measure or compare the phenotype of the co cultures between the control and the patient sample. And following this procedure, you can use very regular classical techniques like real time PCR, Western blood immunohistochemistry, and you can actually study the muscle or the nerve derd phenotype of disco cultures.
