Method of co-culturing mammalian muscle cells and motoneurons
View Patent ↗The invention provides a method of co-culturing mammalian muscle cells and mammalian motoneurons. The method comprises preparing one or more carriers coated with a covalently bonded monolayer of trimethoxysilylpropyl diethylenetriamine (DETA); suspending isolated fetal mammalian skeletal muscle cells in serum-free medium according to medium composition 1; suspending isolated fetal mammalian spinal motoneurons in serum-free medium according to medium composition 1; plating the suspended muscle cells onto the one or more carriers at a predetermined density and allowing the muscle cells to attach; plating the suspended motoneurons at a predetermined density onto the one or more carriers and allowing the motoneurons to attach; covering the one or more carriers with a mixture of medium composition 1 and medium composition 2; and incubating the carriers covered in the media mixture.
1. A method of co-culturing mammalian muscle cells and motoneurons, the method comprising:
suspending fetal muscle cells and fetal spinal motoneurons in a serum-free medium according to composition 1 of Table 1;
placing the suspended fetal muscle cells and fetal spinal motoneurons onto a monolayer of covalently bonded trimethoxysilylpropyl-diethylenetriamine supported on an underlying carrier surface; covering the carrier comprising muscle cells and motoneurons in a mixture of serum-free medium composition 1 of Table 1 and serum-free medium composition 2 of Table 2; and
incubating the covered carrier comprising muscle cells and motoneurons.
2. The method of claim 1 , wherein the fetal muscle cells and motoneurons originate from fetal rats.
3. The method of claim 1 , wherein the underlying carrier surface comprises a glass cover slip.
4. The method of claim 1 , wherein incubating is under mammalian physiological conditions.
5. The method of claim 1 , wherein incubating is at approximately 37° C. in an atmosphere of air with about 5% CO 2 and 85% humidity.
6. The method of claim 1 , wherein the mixture of medium composition 1 of Table 1 and medium composition 2 of Table 2 comprises approximately equal volumes of each composition.
7. The method of claim 1 , further comprising changing the covering medium to a Neurobasal/B27/Glutamax-based medium comprising creatine, estrogen, and cholesterol without growth factors as incubating proceeds.
8. The method of claim 1 , further comprising changing the covering medium to a Neurobasal/B27/Glutamax-based medium comprising creatine, estrogen, and cholesterol without growth factors during the first week of incubation.
9. The method of claim 8 , further comprising periodically changing the covering medium after the first week with fresh Neurobasal/B27/Glutamax-based medium comprising creatine, estrogen, and cholesterol without growth factors.
10. The method of claim 1 , further comprising, during the incubating, monitoring the carrier for formation of myotubes by the incubated muscle cells.
11. The method of claim 1 , further comprising, during the incubating, monitoring the carrier for formation of neuromuscular junctions between the incubated motoneurons and muscle cells.