Neuronal cells cultured on microparticles and methods of using the neuronal cells
View Patent ↗The present invention provides methods for culturing neuronal cells for transplantation into a subject. The methods include culturing neuronal cells with microparticles to provide a microparticle and neuronal cell culture composition, wherein the microparticles are coated with a compound that provides for attachment of neuronal cells. The present invention also provides methods of screening the cultured neuronal cells as well as kits and systems for the method of screening.
1. A method for forming a three dimensional culture of neuronal cells, said method comprising:
depositing a first population of microparticles on a solid substrate, wherein said depositing forms a single first two-dimensional layer of said microparticles, wherein the solid substrate comprises a layer of neuronal cells and wherein said first population of microparticles is coated with cAMP that attracts axons of the neuronal cells;
culturing the neuronal cells for a period of time sufficient for growth of the axons of the neuronal cells to the first population of microparticles; and
depositing a second population of microparticles comprising neuronal cells on the surface of said first two-dimensional layer, wherein said depositing forms a single second two-dimensional layer of said microparticles, thereby forming the three dimensional culture of the neuronal cells.
2. The method of claim 1 , wherein said neuronal cells are assayed for neuronal activity.
3. The method of claim 2 , wherein said assaying for neuronal activity comprises staining the neuronal cells with an antibody that binds to a neuronal cell process.
4. The method of claim 3 , wherein said neuronal cell process is a dendrite or an axon.
5. The method of claim 1 , wherein said neuronal cells are transfected with a gene of interest encoding a detectable molecule; and
wherein said neuronal cells are assayed for neuronal activity by detecting said detectable molecule, wherein the detectable molecule is a fluorescent protein and wherein the neuronal activity comprises growth of a neuronal cell process or synaptic activity.
6. The method of claim 5 , wherein said neuronal activity comprises growth of a neuronal cell process.
7. The method of claim 6 , wherein said neuronal cell process is a dendrite or an axon.
8. The method of claim 5 , wherein said fluorescent protein is Green Fluorescent Protein or Tandem-Dimer-Tomato.
9. The method of claim 5 , wherein said fluorescent protein is under the control of a synapsin promoter.
10. The method of claim 5 , wherein said detectable molecule is a light-gated glutamate receptor.
11. The method of claim 1 , wherein said neuronal cells are cultured in a medium that contains a mitotic inhibitor.
12. The method of claim 11 , wherein said mitotic inhibitor is araC.
13. The method of claim 1 , wherein said microparticles are silica beads, latex beads, agarose beads, collage beads, Polyacrylamide beads, PLL-coated beads or PLGA beads.
14. The method of claim 1 , wherein said compound that provides for attachment of neuronal cells is poly-L-lysine.