Formation of neuromuscular junctions in a co-culture comprising rat muscle cells overlayered with differentiated human spinal cord stem cells in a serum free medium
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 rat muscle cells in a substantially serum-free medium. A synthetic mammalian neuromuscular junction includes a human motoneuron functionally linked to a rat muscle cell in a substantially serum-free medium. An artificial substrate may be used to support the one or more neuromuscular junctions.
1. A co-culture for forming at least one synthetic mammalian neuromuscular junction, comprising: rat skeletal muscle cells adhered to an artificial surface and overlayered with differentiated human spinal cord stem cells in a serum-free medium comprising retinoic acid (RA), cyclic AMP (cAMP), creatine, estrogen, and cholesterol, wherein the artificial surface comprises a silicon based monolayer substrate deposited thereon.
2. The co-culture of claim 1 , wherein one or more of the differentiated human spinal cord stem cells is functionally linked to one or more of the rat skeletal muscle cells.
3. The co-culture of claim 1 , wherein the silicon based monolayer substrate is deposited in a predetermined pattern.
4. The co-culture of claim 1 , wherein the serum-free medium comprises the components in Table 1.
5. The co-culture of claim 1 , wherein the serum-free medium comprises at least one synaptogenesis promoting component and one or more trophic factors.
6. The co-culture of claim 1 , wherein the serum-free medium further comprises the components listed in Table 2, the components listed in Table 3, and the components listed in Table 4.
7. The co-culture of claim 1 , wherein the silicon based monolayer substrate comprises trimethoxysilylpropyldiethylenetri-amine (“DETA”).
8. A method for forming at least one synthetic neuromuscular junction, the method comprising:
co-culturing rat skeletal muscle 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 rat skeletal muscle cell and a differentiated human spinal cord stem cell,
wherein the serum-free medium comprises retinoic acid (RA), cyclic AMP (cAMP), creatine, estrogen, and cholesterol, and
wherein the artificial surface comprises a silicon based monolayer substrate deposited thereon.
9. The method of claim 8 , wherein the serum-free medium comprises at least one synaptogenesis promoting component and one or more trophic factors.
10. The method of claim 8 , wherein the silicon based substrate monolayer comprises DETA.
11. The method of claim 8 , wherein the silicon based substrate monolayer is deposited on the artificial surface in a predetermined pattern.
12. A method of forming synthetic neuromuscular junctions, the method comprising:
adhering rat skeletal muscle cells onto an artificial surface;
overlayering differentiated human spinal cord stem cells onto the rat skeletal muscle cells;
culturing the artificial surface in a serum-free medium, and
monitoring for formation of functional neuromuscular junctions,
wherein the serum-free medium comprises retinoic acid (RA), cyclic AMP (cAMP), creatine, estrogen, and cholesterol, and
wherein the artificial surface comprises a silicon based monolayer substrate deposited thereon.
13. The method of claim 12 , wherein the artificial surface is coated with DETA.
14. The method of claim 12 , wherein the serum-free medium comprises the components in Table 1.