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 co-culture for forming at least one synthetic mammalian neuromuscular junction, comprising: fetal mammalian skeletal muscle cells adhered to an artificial surface and overlayered with fetal mammalian spinal motoneurons in a serum-free medium, wherein the serum-free medium comprises a mixture of the components of Table 3 at the concentrations listed in Table 3, the components of Table 4 at the concentrations listed in Table 4, the components of Table 5 at the concentrations listed in Table 5, creatine, estrogen, and cholesterol.
2. The co-culture of claim 1 , wherein one or more of the fetal mammalian spinal motoneurons is functionally linked to one or more of the fetal mammalian skeletal muscle cells.
3. The co-culture of claim 1 , wherein the artificial surface comprises a silicon based monolayer substrate deposited thereon.
4. The co-culture of claim 3 , wherein the silicon based monolayer substrate is deposited in a predetermined pattern.
5. The co-culture of claim 3 , wherein the silicon based monolayer substrate comprises trimethoxysilylpropyl diethylenetriamine (DETA).
6. The co-culture of claim 1 , wherein the fetal mammalian skeletal muscle cells have a density of about from 700 to about 1000 cells/mm 2 .
7. The co-culture of claim 1 , wherein the fetal mammalian spinal motoneurons have a density of about 100 cells/mm 2 .