Mammalian Cell In Vitro Topological Neuron Network

Abstract

A method of creating a three-dimensional surface topography network for the containment and growth of mammalian neuron cells for high throughput screening of potential biologically active drug compounds is provided. In the present disclosure a nn array of wells is created on a carrier substrate that contains both wells and interconnected channels between wells to facilitate, in one embodiment, axon growth between neuron cells and subsequently the creation of a living interactive neuron network in vitro that emulates in vivo neuron behavior.

Claims

1. A biocompatible substrate comprising a network of wells for culturing cells in vitro and channels connecting the wells, comprising: two or more wells including media and connected by channels configured form a network between the two or more wells and to facilitate intercellular connections, wherein at least one of the wells has neurons, or stem or progenitor cells capable of differentiating into neural cells.

2. The substrate of claim 1 wherein the network is a ring, mesh, star, line, tree or bus topology.

3. The substrate of claim 1 wherein the substrate comprises glass or a synthetic polymer.

4. The substrate of claim 1 wherein the substrate comprises an electrode array.

5. The substrate of claim 1 wherein at least one well includes neuromuscular, cardiac, liver, kidney, pancreas, or skin cells.

6. The substrate of claim 1 wherein at least one well includes motor neurons.

7. The substrate of claim 1 wherein at least one well includes non-motor neurons.

8. The substrate of claim 7 wherein the non-motor neurons comprise cortical neurons, hippocampal neurons or dorsal root neurons.

9. The substrate of claim 1 wherein adjacent wells have different cell types.

10. The substrate of claim 1 wherein at least one channel has a diameter of about 5 to 25 microns.

11. The substrate of claim 1 wherein at least one channel has a length of about 5 to 25 microns.

12. A method of making a biocompatible substrate comprising a network of wells for culturing cells in vitro and channels connecting the wells, comprising: providing a multi-well plate, wherein the diameter of the wells in the plate is about 10 microns to about 25000 microns; and fabricating one or more channels between one or more of the wells, wherein the width of the channels is about 2 microns to about 250 microns, thereby forming a network of interconnected wells.

13. The method of claim 12 wherein the channels have a width of about 5 to about 25 microns.

14. The method of claim 12 wherein the channels have a length of about 5 to about 25 microns.

15. The method of claim 12 wherein the network is a ring, mesh, star, line, tree or bus topology.

16. The method of claim 12 which is formed by liquid casting, injecting molding, thermal and/or UV micro embossing, micro machining, thermoforming, and/or high pressure stamping.

17. A method to monitor cellular activity, comprising: providing the substrate of claim 1; contacting the cells in the wells with one or more compounds; and monitoring the activity of the cells after contact.

18. The method of claim 17 wherein electrical activity is monitored.

19. The method of claim 17 wherein the non-motor neurons comprise cortical neurons, hippocampal neurons or dorsal root neurons.

20. The method of claim 17 wherein one or more of the channels comprise axons.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 shows the various network configurations that can be fabricated in the present disclosure on a carrier substrate.

[0021] FIG. 2 shows a 3 by 3 array of wells and well interconnection channels to facilitate the growth of axons between neurons using the present disclosure.

DETAILED DESCRIPTION

[0022] Referring to FIG. 2, this shows a simple 3 by 3 array useful in the present disclosure. It is comprised of wells 10 and channels 20 that interconnect the wells to form a simple mesh network. The array can be fabricated on a number of substrates such as glass, polymer films, molded micro plates, and plates with electrode arrays. The array of the can be a simple nn with n=1, or where each n can range from 1 to about 10,000, e.g., n is 1 to about 100. The well diameter to contain the neurons can range from about 10 microns to about 25.4 millimeters. The well can be in any number of geometric shapes, such as a circle, square, or polygon. The wells 10 can also contain a volume of liquid to nourish the cells and promote growth and generally can contain micro liters to milliliters of appropriate growth media.

[0023] The substrate 30 can be fabricated by a number of techniques well known in the art such as liquid casting, injecting molding, thermal and/or UV micro embossing, micro machining, thermoforming, and/or high pressure stamping. In one embodiment, injection molding and/or micro embossing are employed. In one embodiment, the substrate may be transparent if subsequent optical analysis is to be conducted on the topological network. However, if electrophysiology is desired to observe the behavior of the network during exposure to a biologically active compound, the substrate need not be transparent.

[0024] The channels 20 that connect the wells 10 can vary in width, length and depth depending on how complex and what type of topological network is being fabricated. In one embodiment, the channels are generally about 2 microns to about 100 microns in width, e.g., about 5 microns to about 15 microns. The channel depth is approximately equal to the depth of the well 10 walls. The channels can be fabricated by micro molding, micro embossing, micro machining, or laser ablation. The channel allows axons that are generated by the neurons to communicate to adjacent or distant wells 10. The wells 10 can be populated with neurons only or other cells types as well such as neuromuscular, cardiac, liver, kidney, pancreas, and skin to create complex neuron organ in vitro models.

[0025] In one example using UV micro embossing on a polymer film, the process formed a neuron topological substrate. In this example the wells 10 and channels 20 were created in one step by the use of an embossing tool that creates the microstructure geometries of interest in a high speed roll to roll process. The wells had a diameter of approximately 1 millimeter and the channels that interconnect the wells had a width of about 10 microns.

[0026] In another example a 384 well plate well known in the art was converted into a neuron topological substrate by laser engraving or ablating 10 micron channels between the wells to form a mesh topological network on the plate. In yet another example a 96 well electrode plate was converted into a topological neuron network by micro machining 10 micron channels between the wells.

[0027] In all the aforementioned examples the topological arrays with the appropriate mammalian cells, growth media and incubation conditions are subsequently exposed (the wells) to active biological compounds. The topological neuron arrays can then be either optically or electrically monitored to observe the physical and logical behavior of the axon cabling between the cells of the mesh network. In one embodiment, a neuron in the array is multipolar. In one embodiment, a neuron in the array is pseudo-unipolar. In one embodiment, a neuron in the array is bipolar. In one embodiment, a neuron in the array is a Purkinje cell. In one embodiment, a neuron in the array is a granule cell. In one embodiment, a neuron in the array is a pyramidal cell. In one embodiment, a neuron in the array is a chandelier cell. In one embodiment, a neuron in the array is a spindle neuron. In one embodiment, a neuron in the array is a stellate cell.

[0028] In one embodiment, the wells in the array have the same growth medium. For example, for neurons, an exemplary medium is BrainPhys (Stem Cell Technologies, Vancouver, Canada), and for heart cells, an exemplary medium is medium for iPS cells. In one embodiment, certain wells having neurons, e.g., hippocampal, cortical or dorsal root ganglion neurons, are adjacent to wells having motor neurons and those cells are maintained in the same medium.

[0029] In one embodiment, the length of a channel is about 5 microns to about 1 millimeter. In one embodiment, the length of a channel is about 5 microns to about 30 microns. In one embodiment, the length of a channel is about 10 microns to about 20 microns.

[0030] In one embodiment, the diameter (width) of a channel is about 5 microns to about 250 microns. In one embodiment, the width of a channel is about 5 microns to about 30 microns. In one embodiment, the width of a channel is about 10 microns to about 20 microns.

[0031] In one embodiment, when cells in different wells in the array have different growth medium, the channels that link wells may be of a size that limits media diffusion between adjacent wells. For example, in an array with brain, liver and heart cells, the channels may be of a diameter of about 5 to about 100 microns, e.g., from about 15 to about 25 microns. In one embodiment, the diameter of the channel minimizes diffusion into adjacent wells by the medium due to surface tension.

[0032] In one embodiment, to allow for axon generation, the channels are from about 15 to about 25 microns in diameter. In one embodiment, the base of the channel and the base of the well are at the same level. In one embodiment, the channel is U-shaped. In one embodiment, the shape of the channel is a cylinder. In one embodiment, the channel is angular in shape, e.g., a rectangular prism (cuboid), a triangular prism or a hexagonal prism.

[0033] The subject matter herein is described by example and different ways of practicing the subject matter have been described. However the subject matter covered by this application is not limited to any one specific embodiment or use or their equivalents. While particular embodiments of the method for fabricating a substrate having cell micro arrays with subsequent drug dosing have been described it will be appreciated by those skilled in the art that changes and modifications may be made thereto without departing from the invention and as set forth in the following claims.