MICROELECTRONIC HUMAN BLOOD BRAIN BARRIER

Abstract

The present disclosure provides a planar microelectronic human blood brain barrier stack used to model drug effects and transport across the brain capillary endothelial barrier to neurons. In one embodiment the stack is comprised of a carrier substrate, electrode arrays, astrocytes, extracellular matrix and brain capillary endothelial cells.

Claims

1. A microelectronic planar blood brain barrier device, comprising: a planar substrate; one or more electrodes in contact with the planar substrate; a first layer comprising a plurality of mammalian neurons in contact with the one or more electrodes and also optionally the planar substrate; a second layer comprising one or more agents that are biocompatible and optionally adhere to at least some of the plurality of neurons; and a third layer comprising a plurality of endothelial cells in contact with the one or more agents.

2. The device of claim 1 wherein the substrate further comprises one or more cell binding molecules.

3. The device of claim 2 wherein the molecules comprise a peptide or a polypeptide.

4. The device of claim 3 wherein the peptide or polypeptide includes fibronectin, laminin, Arg-Glu-Asp-Val-Tyr (REDV) or Lys-Arg-Glu-Asp-Val-Try (KREDVY).

5. The device of claim 1 wherein the substrate comprises glass, silicon, standard printed circuit board (PCB), or flexible polymeric film.

6. The device of claim 5 wherein the film comprises Kapton, polycarbonate, or polyester (PET).

7. The device of claim 1 wherein the thickness of the substrate is from about 1 micron to about 2 millimeters or about 25 to 250 microns.

8. (canceled)

9. The device of claim 1 wherein the one or more electrodes comprise copper, silver, gold, nickel, aluminum, indium tin oxide, graphene, carbon nanotubes, carbon nanobuds, or silver nanowires.

10. The device of claim 1 wherein the electrodes have an electrical resistivity of less than 100 ohms per square.

11. The device of claim 1 wherein the electrodes have an electrical resistivity of less than 10 ohms per square.

12. The device of claim 1 wherein the mammalian neurons are astrocytes.

13. The device of claim 12 wherein the astrocytes are human astrocytes.

14. The device of claim 1 wherein the one or more agents in the second layer include one or more of gelatin, collagen, hyaluronic acid, cellulose, chemically modified cellulose, silicone, chitosan, vegetable protein, agar, polyacrylamide, polyvinylalcohol, polyols, fibronectin, vitronectin, laminin, matrigel, polylysine, or polyvinylprylidone.

15. The device of claim 1 wherein the thickness of the second layer is from about 10 nanometers to 250 microns or 0.5 to 5 microns.

16. (canceled)

17. The device of claim 1 wherein the endothelial cells comprise capillary endothelial cells.

18. The device of claim 17 wherein the endothelial cells comprise brain capillary endothelial cells.

19. (canceled)

20. The device of claim 1 wherein the one or more electrodes comprise gold plated copper and the one or more agents in the second layer include extracellular matrix.

21. The device of claim 2 wherein the one or more cell binding molecules comprise KREDVY.

22. A method of using a device, comprising: providing the device of claim 1; contacting the endothelial cells in the device with one or more test compounds; and detecting whether the one or more compounds alter the activity of the neurons in the device.

23. The method of claim 22 wherein the activity detected is action potential, impedance or conduction velocity.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0015] FIG. 1 shows a cross sectional view of one embodiment of the device.

DETAILED DESCRIPTION

[0016] The following detailed description is directed towards the various embodiments of the invention. Although one or more of these embodiments may be preferred, the embodiments disclosed should not be interpreted, or otherwise used, as a limiting the scope of the disclosure, including the claims. In addition one skilled in the art will understand that the following description has broad application, and the discussion of any embodiment is meant only to be exemplary of that embodiment, and not intended to intimate that the scope of the disclosure, including the claims, is limited to that embodiment.

[0017] In one embodiment, multielectrode arrays (MEAs) in combination with multilayer cellular stacks of cells that are layered on top of the MEAs are used to measure electrophysiological changes in the neuron layer in contact with the MEA electrodes, as a result of potential therapeutic compounds crossing the capillary endothelial cells forming a barrier above the neuron cell layer.

[0018] In one embodiment, a microelectronic planar blood brain barrier device is provided. The device may include a planar substrate; one or more electrodes disposed on the planar substrate; a first layer comprising a plurality of mammalian neurons disposed on the one or more electrodes and also optionally the planar substrate; a second layer comprising one or more agents that are biocompatible and are disposed on at least some of the plurality of isolated neurons; and a third layer comprising a plurality of isolated endothelial cells disposed on the one or more agents. In one embodiment, the substrate, electrodes, or both, further include one or more cell binding molecules disposed thereon. In one embodiment, the cell binding molecules include a peptide or a polypeptide. In one embodiment, the substrate is formed of glass, silicon, standard printed circuit board (PCB), or flexible polymeric film. In one embodiment, the film is formed of Kapton, polycarbonate, or polyester (PET). In one embodiment, the thickness of the substrate is from about 1 micron to about 2 millimeters. In one embodiment, the thickness of the substrate is about 25 to 250 microns. In one embodiment, the thickness of the substrate is about 100 to 500 microns. In one embodiment, the one or more electrodes include copper, silver, gold, nickel, aluminum, indium tin oxide, graphene, carbon nanotubes, carbon nanobuds, or silver nanowires. In one embodiment, the electrodes have an electrical resistivity of less than 100 ohms per square. In one embodiment, the electrodes have an electrical resistivity of less than 50 ohms per square. In one embodiment, the electrodes have an electrical resistivity of less than 10 ohms per square. In one embodiment, the electrodes have an electrical resistivity of less than 5 ohms per square. In one embodiment, the mammalian neurons are astrocytes, e.g., human astrocytes. In one embodiment, the layer having the mammalian neurons is a single cell layer. In one embodiment, the layer having the mammalian neurons comprises 2 to 10 cell layers. In one embodiment, the one or more agents in the second layer include one or more of gelatin, collagen, hyaluronic acid, cellulose, chemically modified cellulose, silicone, chitosan, vegetable protein, agar, polyacrylamide, polyvinylalcohol, polyols, fibronectin, vitronectin, laminin, matrigel, polylysine, or polyvinylprylidone. In one embodiment, the thickness of the second layer is from about 10 nanometers to 250 microns. In one embodiment, the thickness of the second layer is 0.5 to 5 microns. In one embodiment, the endothelial cells comprise capillary endothelial cells, e.g., human capillary endothelial cells. In one embodiment, the endothelial cells comprise brain capillary endothelial cells. In one embodiment, the layer having the mammalian endothelial cells is a single cell layer. In one embodiment, the layer having the mammalian endothelial cells comprises 2 to 10 cell layers. The device may be employed to screen compounds for their ability to cross the endothelial layer and alter the activity of the neurons in the device.

[0019] FIG. 1 shows a cross section of one embodiment. The multilayer stack 60 is comprised of an electrode support 10, conductive electrodes 20, neurons 30, extracellular matrix 40, and capillary endothelial cells 50.

[0020] The electrode support 10 can be formed of materials including but not limited to glass, silicon, standard printed circuit board (PCB), or flexible polymeric film such as Kapton, polycarbonate, or polyester (PET) film. The thickness of the support 10 may range from about 1 micron to about 2 millimeters, e.g., about 25 to 250 microns. The support 10 may be opaque or transparent and in one embodiment comprises transparent PET.

[0021] The conductive electrodes 20 may be formed of materials including but not limited to a conductor such as copper, silver, gold, nickel, aluminum, indium tin oxide, graphene, carbon nanotubes, carbon nanobuds, or silver nanowires. The electrodes 20 may have an electrical resistivity of less than 100 ohms per square, e.g., less than 10 ohms per square. The electrodes may be patterned in any geometric shape or size width lines, e.g., interdigitated conductive lines. The width of the lines may vary from about 1 to about 300 microns, e.g., about 50 to 100 microns. In one embodiment, copper electrodes 10 that have been flash plated with gold make the surface more biologically compatible for cell attachment and viability.

[0022] Once the multielectrode array 20 has been fabricated on a support material 10 the next step is to attach neurons to the electrodes, e.g., gold plated electrodes. Good cell adhesion and attachment allows for enhanced cell functioning, viability and measurement of the electrophysiology of the neurons during therapeutic drug exposures of the stack. In one embodiment, gold-coated copper electrodes 20 may be plasma cleaned to remove any surface contamination and then reacted with a 20 mM solution of alkanethiols of 11-mercaptoundecanoic acid (MUA) for 5 to 10 minutes. This results in a self assembled monolayer (SAM) or MUA on the surface. The electrodes may then be immersed into a 150 mM solution of 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDAC) and 30 mM N-hydroxysuccinimide (NHS) for 30 minutes to attach the NHS group to the terminus COOH of the SAM layer. The finished activated electrode structure may then be sterilized with 70% ethanol for 15 minutes and exposed to various proteins that have binding sites for cells. For example, binding protein or polypeptides that may be used include but are not limited to fibronectin, laminin, Arg-Glu-Asp-Val-Tyr (REDV) or Lys-Arg-Glu-Asp-Val-Try (KREDVY). In one embodiment, KREDVY is employed to enhance cell binding and viability after cell attachment.

[0023] Neurons 30 are subsequently cultured by techniques well known in the art onto the protein-activated electrodes 20. There are many types of human neurons that can be used such as those derived from primary cells, or those derived from induced pluripotent stem cells (iPScs). There are about 10,000 specific types of neurons in the human brain but generally speaking they can be classified as motor neurons, sensory neurons, and interneurons. In one embodiment, astrocytes are employed as they play a role as the first layer of neurons adjacent to the brain capillary endothelial barrier (EB). Astrocytes process and modulate molecules that are transported through the EB before entering the brain. In one embodiment, iPSc derived astrocytes are employed as the neuron 30 layer.

[0024] Brain capillary endothelial cells (BCECs) 50 grow on extracellular matrix 40 in order to form very tight cell-to-cell contacts or junctions. A layer of extracellular matrix (ECM) 40 may be added between the neurons 30 and the BCECs. This is accomplished by applying a dilute solution 0.001 to 5% by weight in solution of the matrix into wells or chambers defined by the MEAs. Typical ECM components or synthetic polymers that can be used include but are not limited to gelatin, collagen, hyaluronic acid, cellulose, chemically modified cellulose, silicone, chitosan, vegetable protein, agar, polyacrylamide, polyvinylalcohol, polyols, fibronectin, vitronectin, laminin, matrigel, polylysine, polyvinylprylidone, or other polypeptides, or any combination of the aforementioned materials with or without crosslinking. The ECM layer may also contain adsorbed or absorbed polypeptides such as REDV and KREDVY to further enhance cell adhesion to the ECM or synthetic polymer containing layer. In one embodiment, gelatin and/or hyaluronic acid are the ECM components used in the ECM layer. The ECM may be deposited onto the astrocyte surface 30 and allowed to equilibrate for 12 to 24 hours before adding the last layer of the stack, the BCECs. The thickness of the ECM layer can range from 10 nanometers to 250 microns, e.g., 0.5 to 5 microns.

[0025] In one embodiment, the cells used for the BCEC layer are the hCMEC/D3 BBB cell line, which was derived from human temporal lobe microvessels and immortalized with hTERT and SV40 large T antigen. They are a model of human blood-brain barrier (BBB) function. The cell line is available from EMD Millipore Corporation in Temecula, Calif., is well characterized and easily cultured and grown. This BCEC layer 50 may be used to study pathological and drug transport mechanisms with relevance to the central nervous system.

[0026] Once the microelectronic planar BBB stack 60 is fabricated it may be used to study drug transport and effects on the astrocytes 30 that are bound to the MEA electrodes on the opposite planar surface to the BCEC layer. Electrophysiology properties of the astrocytes can be monitored and measured such as action potential, impedance, and conduction velocity. If drug or drug candidates are added to the BCEC side of the planar stack and if they pass through the BCEC layer their affect or lack thereof can be easily monitored electronically by the MEA array. Both drug efficacy and toxicity to both the BCEC and astrocyte layers may be measured.

[0027] In one embodiment, the in vitro BBB cell stack is in one or more wells of a plate, e.g., a multi-well plate, each having one or more electrodes on the bottom surface of the wells in contact with neurons in the cell stacks. The cell stack may be cultured in media or any physiologically compatible solution, or reside in a gel. One or more test compounds may be added to individual wells with cell stacks using, for example, micropipettes or an automated pipetting device.

[0028] In one embodiment, a substrate has a plurality of BBB cell stacks in a microarray having a plurality of electrodes, at least one of the electrodes in contact with neurons in the cell stacks. The substrate may be placed in a receptacle so that the cell stacks on the substrate may be cultured in media or any physiologically compatible solution.

[0029] The above discussion is meant to be illustrative of the principle and various embodiments of the present invention. Numerous variations and modifications will become apparent to those skilled in the art once the above disclosure id fully appreciated. It is intended that the following claims be interpreted to embrace all such variations and modifications.