Synthetic mammalian neuromuscular junction and method of making
A method for forming neuromuscular junctions includes forming functional neuromuscular junctions between motoneurons and muscle cells by co-culturing one or more human motoneurons and one or more human muscle cells in a substantially serum-free medium. A synthetic mammalian neuromuscular junction includes a human motoneuron functionally linked to a human muscle cell in a substantially serum-free medium. An artificial substrate may be used to support the one or more neuromuscular junctions.
1. A method for forming at least one synthetic neuromuscular junction, the method comprising:
co-culturing differentiated human skeletal muscle stem cells adhered to an artificial surface and overlayered with differentiated human spinal cord stem cells in a serum-free medium; and
forming at least one functional neuromuscular junction between a differentiated human skeletal muscle stem cell and a differentiated human spinal cord stem cell.
2. The method of claim 1 , further comprising adding NbActiv4 to the serum-free medium.
3. The method of claim 1 , wherein the artificial surface comprises a silicon based substrate monolayer deposited thereon.
4. The method of claim 3 , wherein the silicon based substrate monolayer comprises DETA.
5. The method of claim 3 , wherein the silicon based substrate monolayer is deposited on the artificial surface in a predetermined pattern.
6. A method of forming synthetic neuromuscular junctions, the method comprising:
adhering differentiated human skeletal muscle stem cells to an artificial surface;
overlayering differentiated human spinal cord stem cells onto the differentiated human skeletal muscle stem cells;
culturing the artificial surface in a serum-free medium, and
monitoring for formation of functional neuromuscular junctions.
7. The method of claim 6 , wherein the artificial surface is coated with DETA.
8. The method of claim 6 , further comprising adding NbActiv4 to the serum-free medium.
9. A co-culture for forming at least one synthetic mammalian neuromuscular junction, comprising: differentiated human skeletal muscle stem cells adhered to an artificial surface and overlayered with differentiated human spinal cord stem cells in a serum-free medium.
10. The co-culture of claim 9 , wherein the medium comprises the components in Table 1.
11. The co-culture of claim 9 , wherein the serum-free medium comprises at least one synaptogenesis promoting component and one or more trophic factors.
12. The co-culture of claim 9 , wherein one or more of the differentiated human spinal cord stem cells is functionally linked to one or more of the differentiated human skeletal muscle stem cells.
13. The co-culture of claim 9 , wherein the artificial surface comprises a silicon based monolayer substrate deposited thereon.
14. The co-culture of claim 13 , wherein the silicon based monolayer substrate is deposited in a predetermined pattern.
15. The co-culture of claim 13 , wherein the silicon based monolayer substrate comprises DETA.
16. The co-culture of claim 9 , wherein the serum-free medium further comprises NbActiv4.