Method of myelinating isolated motoneurons
View Patent ↗The present invention provides a method of inducing myelination of isolated motoneurons by preparing a non-biological substrate having thereon a covalently attached monolayer of DETA; depositing isolated motoneurons on the substrate in a defined serum-free medium; plating isolated Schwann cells cultured in the defined serum-free medium onto the motoneurons, thereby initiating a co-culture; and passaging the co-culture as necessary into fresh, defined serum-free medium supplemented with L-ascorbic acid at least until the motoneurons form Nodes of Ranvier indicative of myelination. The invention also includes a method of testing for new drugs effective in demyelinating diseases. Additionally, cellular products provided by the invention include an isolated motoneurons myelinated or remyelinated in vitro according to the methods disclosed.
1. A method of inducing myelination of isolated motoneurons, the method comprising:
preparing a non-biological surface having thereon a covalently attached monolayer of N-1[3 (trimethoxysilyl)propyl]diethylenetriamine;
depositing isolated motoneurons on the surface in a serum-free medium of Table 1;
initiating a co-culture by plating isolated Schwann cells cultured in the serum-free medium of Table 1 onto the motoneurons; and
passaging the co-culture as necessary into fresh serum-free medium of Table 1 supplemented with L-ascorbic acid at least until the motoneurons form Nodes of Ranvier indicative of myelination.
2. A method of making myelinated motoneurons in vitro, the method comprising:
co-culturing isolated motoneurons and Schwann cells in a serum-free medium of Table 1 on a surface having thereon a covalently attached monolayer of N-1[3(trimethoxysilyl)propyl]diethylenetriamine; and
passaging the co-culture as necessary into fresh medium of Table 1 supplemented with L-ascorbic acid until the motoneurons are myelinated and Nodes of Ranvier are formed thereon.
3. The method of claim 2 , wherein the surface comprises glass.
4. The method of claim 2 , wherein the covalently attached monolayer of N-1[3(trimethoxysilyl)propyl]diethylenetriamine is patterned.
5. The method of claim 4 , wherein the covalently attached monolayer of N-1[3(trimethoxysilyl)propyl]diethylenetriamine is patterned by photolithography.
6. An isolated motoneuron myelinated in vitro by a method comprising co-culturing isolated motoneurons and Schwann cells in a serum-free medium of Table 1 on a surface having thereon a covalently attached monolayer of N-1[3(trimethoxysilyl)propyl]diethylenetriamine; and passaging the co-culture as necessary into fresh serum-free medium of Table 1 supplemented with L-ascorbic acid until the motoneurons are myelinated and Nodes of Ranvier are formed thereon.
7. The isolated motoneuron of claim 6 , wherein the covalently attached monolayer of N-1[3(trimethoxysilyl)propyl]diethylenetriamine is patterned.
8. The isolated motoneuron of claim 6 , wherein the surface is glass.
9. A culture of motoneurons myelinated in vitro by a method comprising co-culturing isolated motoneurons and Schwann cells in a serum-free medium of Table 1 on a surface having thereon a covalently attached monolayer of N-1[3(trimethoxysilyl)propyl]diethylenetriamine; and passaging the co-culture as necessary into fresh serum-free medium of Table 1 supplemented with L-ascorbic acid until the motoneurons are myelinated and Nodes of Ranvier are formed thereon.
10. The culture of motoneurons of claim 9 , wherein the covalently attached monolayer of N-1[3(trimethoxysilyl)propyl]diethylenetriamine is patterned.
11. The culture of motoneurons of claim 9 , wherein the surface is glass.
12. A mixed culture of isolated Schwann cells and motoneurons in a serum-free medium of Table 1, wherein the motoneurons are myelinated.
13. An isolated dysfunctional motoneuron remyelinated by a method-comprising co-culturing isolated motoneurons and Schwann cells in a serum-free medium of Table 1 on a surface having thereon a covalently attached monolayer of N-1[3(trimethoxysilyl)propyl]diethylenetriamine; and passaging the co-culture as necessary into fresh serum-free medium of Table 1 supplemented with L-ascorbic acid until the motoneurons are myelinated and Nodes of Ranvier are formed thereon.
14. The isolated dysfunctional motoneuron of claim 13 , wherein the covalently attached monolayer of N-1[3(trimethoxysilyl)propyl]diethylenetriamine is patterned.
15. The isolated dysfunctional motoneuron of claim 13 , wherein the surface is glass.