DEVICE FOR THE EXAMINATION OF NEURONS

Abstract

The present invention relates to a device for the examination of neurons and to a method for examining neurons, and more specifically for examining neurons growing in a three-dimensional network.

Claims

1. A device for recording electrical activity of or stimulating neural cells in a three-dimensional neural network, comprising: a first compartment configured to contain neurons and to maintain said neurons in a three-dimensional matrix, and at least one microchannel extending from said first compartment and having dimensions allowing the extension of neurites from said first compartment into said microchannel and preventing the entry of the soma of neurons into said at least one microchannel, wherein said at least one microchannel comprises at least one microelectrode embedded therein and arranged to record electrical signals from or administer electrical pulses to neurites extending along said at least one microchannel.

2. The device of claim 1, further comprising a second compartment, wherein a first connecting region comprises the at least one microchannel leading to said second compartment thereby connecting said first compartment with said second compartment.

3. The device of claim 1 further comprising: a third compartment, a second connecting region connecting said second and third compartments, said second connecting region comprising at least one further microchannel having dimensions allowing the passing-through of neurites from said second to said third compartment and preventing the entrance of the soma of neurons into said microchannel(s), wherein said at least one further microchannel comprises at least one further microelectrode embedded therein and arranged to record electrical signals from or administer electrical pulses to neurites extending along said at least one further microchannel.

4. The device of claim 1, wherein the microelectrode(s) comprise(s) a transducer.

5. The device of claim 1, wherein said at least one microchannel or said at least one further microchannel comprise multiple microelectrodes.

6. The device of claim 1, wherein said first connecting region comprises a plurality of said microchannel or said second connecting region comprises a plurality of said further microchannel.

7. The device of claim 1, wherein said dimensions of said microchannel or said further microchannel at its/their smallest dimension(s) are selected from the group consisting of: <5 m, about 4 m, about 3 m, about 2 m, about 1 m, about 0.5 m, about 0.4 m, about 0.3 m, about 0.2 m, and about 0.1 m.

8. The device of claim 1, wherein at least one of said first and, if applicable, second and third compartments comprises transparent material.

9. A method for examining neurons, comprising the following steps: cultivating of neurons in a first compartment in a three-dimensional matrix such that the neurons are not adhering to the walls of said first compartment, wherein at least one microchannel is extending from said first compartment, said at least one microchannel has dimensions allowing the extension of neurites from said first compartment into said at least one microchannel and preventing the entry of the soma of neurons into said at least one microchannel; letting the neurites of said neurons grow along said at least one microchannel, recording electrical signals from or administering electrical pulses to said neurites growing along said at least one microchannel by at least one recording microelectrode or at least one stimulation microelectrode embedded in said at least one microchannel.

10. The method of claim 9, wherein said first compartment is connected via a first connecting region with a second compartment, said first connecting region comprising said at least one microchannel leading to said second compartment thereby connecting said first compartment with said second compartment, and the neurites of said neurons are allowed to grow through said at least one microchannel towards said second compartment.

11. The method of claim 9, wherein the scaffold comprises a hydrogel or a fibrous matrix.

12. The method of claim 9, wherein in addition to or instead of said neurons non-neuronal cells are cultivated.

13. The method of claim 9, wherein the three-dimensional matrix include neurospheres or minibrains or brain organoids.

14. The method of claim 9, wherein test compounds are added to said first or second compartment.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0085] FIG. 1 shows a plan view on top of an embodiment of a microfluidic device assembly according to the invention;

[0086] FIG. 2 shows a cross section along the line II-II of the embodiment depicted in FIG. 1;

[0087] FIG. 3 shows a cross section along the line III-III of the embodiment depicted in FIG. 1;

[0088] FIG. 4 shows a magnification of the microchannels area of the embodiment depicted in FIG. 3;

[0089] FIG. 5 shows a plan view on the bottom of one of the functional units or microfluidic device of the embodiment shown in FIG. 3;

[0090] FIG. 6 shows a detail view on the bottom side of connecting region and the microchannels with integrated microelectrodes of the embodiment shown in FIG. 5;

[0091] FIG. 7 shows a bird's eye view onto an embodiment of a microfluidic device assembly;

[0092] FIG. 8 illustrates a prototype of the device according to the invention, in which neurons are cultivated within a three-dimensional matrix in a bottom sub-compartment with a separate top sub-compartment for liquid perfusion. Microchannels and microelectrodes (not shown here) according to the invention allow recording and/or stimulation of the neurons in a three-dimensional matrix.

[0093] FIG. 9 shows a scanning electron microscope image of the prototype depicted in FIG. 8.

[0094] FIG. 10 illustrates an embodiment of the device according to the invention from a bird's-eye-view.

[0095] FIG. 11 shows a side sectional view of the embodiment of FIG. 10.

[0096] FIG. 12 shows another embodiment of the device according to the invention.

[0097] FIG. 13 illustrates a further embodiment of the device according to the invention.

[0098] FIG. 14 illustrates an embodiment of the connecting region.

[0099] FIG. 15 illustrates various embodiments of microchannels.

[0100] FIG. 16 shows a confocal 3D image of GFP-labelled primary neurons cultivated in an embodiment of the device assembly according to the invention;

[0101] FIG. 17 shows a confocal 3D image of a single GFP-labelled human iPSC-derived neuron growing in the second compartment of an embodiment of the device assembly according to the invention;

[0102] FIG. 18 shows a high resolution detail of a single neurite of the neuron depicted in FIG. 17;

[0103] FIG. 19 shows (left and middle) a differential interference contrast (DIC) microscopic image of human iPSC-derived neurons grown in 3D on an embodiment of a microfluidic device according to the invention. Right panel shows action potentials recorded by microelectrodes integrated into microchannels.

DESCRIPTION OF PREFERRED EMBODIMENTS

1. The Microfluidic Device

[0104] In FIG. 1 a plan view on top of an embodiment of a device assembly is shown under the reference sign 10. The device assembly 10 may be manufactured by means of photolithography using a polymer such as SU-8 or by means of injection moulding using polymers such as cyclic olefin copolymer (COC) or cyclin olefin polymer (COP) or by means of selective laser etching using glass. In the shown embodiment the device assembly 10 has the dimensions of 32 mm (width)32 mm (length)3 mm (height). The device assembly 10 comprises four corresponding devices or functional units or 12a, 12b, 12c, 12c, separated from each other, which allow the cultivation of four independent neuron cell cultures. Each of the units 12a, 12b, 12c, 12d comprises a first 14, a second 16, and a third compartment 18 each configured for the cultivation of neurons. The first, second, and third compartments 14, 16, 18 each comprise a channel section 14a, 16a, 18a and a terminal reservoir 14b, 16b, 18b which are connected with each other. Each of said terminal reservoirs 14b, 16b, 18b comprises a cell seeding area 14c, 16c, 18c.

[0105] In FIG. 2 a cross section along the line II-II of the second compartment 16 of the functional unit 12c is depicted. It illustrates that the terminal reservoir 16b is positioned onto a base plate 20 which may form an integrated part of the device assembly 10 but may also be a glass MEA affixed to the upper unit forming a microfluidic part of the device in a liquid-tight manner. The cell seeding area 16c is embedded into the base plate 20. The terminal reservoir 16b and the cell seeding area 16c lead to the channel section 16a.

[0106] As it can be inferred from FIG. 2 the channel section 16a and the terminal reservoir 16b are both open on top which allows the addition of culture media and test compounds into the device assembly 10. It is also shown that the channel section 16a comprises a lower sub-compartment 16a and an overlying top sub-compartment 16a adjacent thereto. The lower sub-compartment 16a comprises a diameter or a channel width, respectively, which is wider than the diameter or the channel width, respectively, of said top sub-compartment 16a. Both the overlying top sub-compartment 16a and the lower sub-compartment 16a open into the terminal reservoir 16b. The lower sub-compartment 16a extends to the cell seeding area 16c as a result of a subsidence in the base plate 20.

[0107] In FIG. 3 a cross section along the line III-III of the functional unit 12d as shown in FIG. 1 is depicted. It illustrates the arrangement of the channel section 16a, the lower sub-compartment 16a and the overlying top sub-compartment 16a, the terminal reservoirs 16b and the cell seeding areas 16c whichon that representationare located at the left 16b, 16c and the right termini 16b, 16c of the channel section 16a. It is also again shown that both the overlying top sub-compartment 16a and the lower sub-compartment 16a open into the terminal reservoirs 16b and 16b. The lower sub-compartment 16a extends to the cell seeding areas 16c, 16c as a result of a recess in the base plate 20. Microchannels 22 open into the lower sub-compartment 16a, in particular in that part which is outside of the terminal reservoirs 16b and 16b. The microchannels 22 are provided in a connecting region 24 (not shown here) which connect the second 16 and the neighboring first compartment 14 and extend into the lower sub-compartment 14a of the channel section 14a of the first compartment 14 (not shown here). FIG. 4 shows a magnification of the microchannels 22 which are provided in the connecting region 24 which connects lower sub-compartment 16a with lower sub-compartment 14a.

[0108] FIG. 5 shows a plan view on the bottom of the embodiment of a functional unit 12 of the device assembly 10. Shown in black are the connecting regions 24 which comprise the microchannels 22 which connect the second 16 with the neighboring first 14 and with the neighboring third compartment 18, and extend into the respective lower sub-compartments 14a, 18a (not shown here) of the channel sections 14a, 18a of the first and third compartments 14, 18.

[0109] FIG. 6 shows a detail view on the bottom side of connecting region 24 which connects the second 16 with the neighboring first 14 compartment. In the depicted embodiment each of the microchannels 22 comprises a microelectrode 26 embedded therein. The microelectrodes 26 may be integrated into a glass MEA substrate below each of the microchannels 22 and arranged to record electrical signals from or administer electrical pulses to neurites extending along said microchannels 22. Therefore, the microelectrodes 26 include recording and stimulation microelectrodes.

[0110] In a preferred but non-limiting embodiment of the device assembly 10 each connecting region 24 comprises 32 microelectrodes 26, each functional unit 12, therefore, comprises 64 microelectrodes 26 and the entire device assembly 10 comprises 256 microelectrodes 26.

[0111] FIG. 7 shows a bird's eye view onto an embodiment of a device assembly 10 and a magnification of the terminal reservoir 16b and the cell seeding area 16c of the second compartment 16. In this embodiment the bottom of the microfluidic device assembly 10 is consisting of a glass MEA 28 comprising the microelectrodes 26 (not shown here) affixed to the upper microfluidic part 30 in a liquid-tight manner.

[0112] For cultivation the cells are seeded in the cell seeding areas 16c of the second compartment 16 in a hydrogel scaffold which fills the lower sub-compartments 14a, 16a, 18a up to the border to the top sub-compartments 14a, 16a, 18a. Neurites can extend into the neighboring first 14 and third compartment 18 by growing through the microchannels 22. The microelectrodes 26 integrated below each of the microchannels 22 record action potentials along single neurites or may apply electrical pulses thereon.

[0113] FIG. 8 illustrates a prototype of the device according to the invention where microstructures were fabricated directly on glass substrates by employing epoxy-based negative resists such as SU-8 and a UV lithographic process. Additional thicker layers are then laminated on top by using dry film resists (DFR). It is mainly composed by two microfluidic compartments, one on top of each other, separated by a perforated thin DFR. FIG. 9 shows an SEM image of said perforated membrane fabricated by the inventors using lamination of a dry film resist. Membrane thickness is 20 m. Perforations are 30 m in diameter. The bottom compartment was used for seeding neurons dispersed in a 3D matrix such as hydrogel, while the top compartment was later filled with liquid media. Diffusion through the perforated membrane assure that nutrients, gases and catabolites are continuously exchanged between the liquid and the gel compartments, providing the required conditions for long term cultivation of neurons in 3D and application of test compounds. Microchannels 22 (not shown here) are provided in a connecting region 24 (not shown here) and comprise microelectrodes 26 (not shown here). The microchannels allow extension of neurites from neurons dispersed in 3D and thereby allow recording and/or stimulation of their electrical activity by the microelectrodes.

[0114] In FIG. 10 an embodiment of the device according to the invention is depicted from a bird's-eye-view. In FIG. 11 a side view of this embodiment of the device according to the invention is depicted.

[0115] In FIG. 12 another embodiment of the device according to the invention is depicted. In this embodiment an open compartment is provided with its lower connecting region containing seven closed microchannels with single microelectrodes. Neurons extend neurites into the microchannels.

[0116] In FIG. 13 a further embodiment of the device according to the invention is illustrated. In this embodiment of three compartments separated by two connecting regions, each containing seven microchannels with single microelectrodes. Different neural cell types are cultured in the top and bottom compartments. Their cell bodies are constrained to their respective compartments. The cells communicate by synaptic connections after extending neurites through the channels into the central compartment.

[0117] FIG. 14 illustrates the design of the connecting region. In a bird's-eye-view multiple microchannels in a connecting region are depicted, each microchannel having a single microelectrode.

[0118] In FIG. 15 various embodiments of microchannels are illustrated such as a straight channel with single microelectrode (A), a straight channel with multiple microelectrodes (B), a channel with a long microelectrode (C), a channel with varying width (D), a curved channel (E), split channels with multiple microelectrodes (F, G).

2. Fabrication and Evaluation

[0119] General:

[0120] An MEA having a footprint of 49 mm49 mm and containing 256 integrated recording microelectrodes, arranged in a 464 matrix, is designed and fabricated. Patterning of microelectrodes-aligned microchannels (3 m high, 7.5 m wide, 300 m long) on the upper surface of MEA is achieved by photolithography of SU-8.

[0121] At the same time, a range of different designs, materials and fabrication processes is evaluated for their capacity to consistently produce the microfluidic part of the device assembly (32 mm wide, 32 mm long, 3 mm high) compatible with the goal of growing cells in 3D. In particular, materials like COC and COP, considered industry standards for biopharmaceutical applications, are tested for injection moulding. Materials like glass are tested for selective laser etching. The material is tested for the successive MEA bonding process (by solvent bonding or thermal bonding or chemical bonding) as well as for the purpose of culturing viable 3D neuronal networks.

[0122] After bonding, the new hybrid MEA/microfluidic device is subjected to biological testing and functional characterization.

[0123] Design and Mastering:

[0124] The design of the microfluidic device assembly can be transferred to a design for mass manufacturing. After taking care of typical design rules for injection moulding and adapting the design accordingly, metal inserts can be manufactured by micro-milling. Depending on the complexity a new base mould might be required during the development.

[0125] Injection Moulding:

[0126] After realising the insert for the mould, injection tests can be performed with different materials e.g. different grades of COC or COP. This will enable to test and to compare different properties of the moulded material for the succeeding bonding process as well as in the final application.

[0127] Selective Laser Etching:

[0128] The design of the microfluidic device assembly can be transferred to a design for mass manufacturing. After taking care of typical design rules for selective laser etching and adapting the design accordingly, glass microfluidic parts can be produced by selective laser etching.

[0129] Bonding:

[0130] The MEA can be manufactured such that the upper surface will be micro-patterned by a photolithographic process based on SU8. During the bonding task the moulded part may be permanently attached to this SU8 layer. Bonding of injection moulded parts may be done by solvent bonding or thermal bonding or chemical bonding. A bonding process of COC/COP or glass to SU8 is used which will not destroy the micro-features on SU8 and has a sufficient bonding strength for the application.

[0131] Characterisation and Application Tests:

[0132] Characterisation and application tests are performed in parallel. Characterisation includes: geometrical measurements by scanning electron microscopy (SEM), profilometry and transparency checks. Application tests can start in parallel to the bonding tests. This will give first hints about the bonding strength and how this is affected by the different strategies used for bonding.

[0133] Combining Structural and Functional Readouts:

[0134] Due to the optical clarity of the device, capturing structural data by live microscopy will allow reconstructing 3D neuronal morphologies in detail. A recent study performed by the inventors has shown how by using high resolution confocal microscopy it was possible to identify fine neuronal ultra-structures, such as dendritic spines, in live 3D neurons cultured on matrigel scaffolds. Recording neuronal activity by microelectrodes offers the advantage, over other electrophysiological techniques, of being non-invasive. Therefore, measurements can be taken repeatedly at any time-point during cultivation. In combination to imaging experiments this offers the unique possibility to combine continuous acquisition of structural and functional parameters from 3D cultured neurons into a single live assay.

[0135] Platform Validation:

[0136] This will be carried out in order to verify the capacity of the device according to the invention to identify and predict a range of interactions between specific classes of compounds and neuronal pathways. Electrophysiological and structural read-outs can be both employed to monitor the responses to a small ad selected group of reference compounds. This will ensure that the most relevant molecules will be selected, according to a list of criteria such as: i) chemical structure, ii) applications routes (nervous system drugs, other-organ drugs, pesticides) and iii) range of engaged cellular pathways. For testing purposes, the compounds can be divided into 3 calibration sets, containing 5 compounds each (3 positive and 2 negative controls), for the purpose of identifying subtypes of response patterns: [0137] 1. Efficacy Set: neuropharmacologically active drugs. [0138] 2. Safety Set: molecules with demonstrated seizurogenic activity. [0139] 3. Toxicity Set: environmental pollutants and pesticides with a defined mechanism of action.

[0140] Throughput:

[0141] Each microfluidic device assembly, with a footprint of only 49 mm49 mm, may contain a total of 256 microelectrodes, arranged in a matrix of up to 1221 microelectrodes. This will ensure multiple independent experiments on each functional unit, each having enough microelectrodes to sample a large number of neurons. A range of compound concentrations plus control solutions could be run on each chip, providing enough through-put to support rapid screening campaigns.

[0142] Cost-Efficiency:

[0143] Due to the compact size of the microfluidic device assembly, running each single assay will only require a few thousand cells and less than 100 l of test compounds. This means, for example, that one vial of human iPS cells, containing 1-2 million cells on average, will be sufficient for up to 60 independent measurements. This, considering the very high prices of iPS cells, will contribute to keep costs at an acceptable level for this type of assay.

[0144] Bio-Compatibility:

[0145] Most microfluidic devices currently used to create organs-on-chip are made in PDMS. Although this material offers several advantages, such as easy fabrication, optical clarity and gas permeability, on the other hand PDMS can variably absorb small hydrophobic compounds, which may lead to changes in bioavailability for some compounds. For this reason alternative materials such as COC or COP or glass, gold standards in biopharmaceutical testing, can be used to fabricate the microfluidic chip. Although this will require a more complex process for production of the device and thermal or solvent or adhesive bonding to attach it to the MEA substrate, the use of such materials is considered advantageous considering its intended screening purposes.

[0146] Long-Term 3D Cultivation:

[0147] Achieving long-term cell survival in hydrogel scaffolds requires a controlled flow of culture media through the microfluidic chip. The media is in fact used to exchange gases between air and the gel, to provide nutrients and remove metabolites, to apply compounds during drug testing. These features will be implemented in the microfluidic device assembly design, based on a channel containing a hydrogel lane (400 m wide, 150 m high) at the bottom of the device (lower sub-compartment) and a liquid lane (300 m wide, up to 2 mm high) on top of the gel lane (top sub-compartment). In this way media/solutions/drugs can be easily applied/replaced at the top liquid lane via reservoirs and from here they can rapidly diffuse to the cells contained in the bottom gel lane.

[0148] Handling:

[0149] Liquid handling and drug applications will be done as described above, using an open system that does not require any specific equipment, as all liquids in the top lane will move between the reservoirs passing through open channels having low resistance. This provides the option to add robotic control of all liquid handling steps, as eventually required for screening purposes.

[0150] Cross-Platform Functionalities:

[0151] With a footprint of only 49 mm49 mm and an optically-clear glass bottom, the microfluidic device according to the invention will be available for capturing simultaneously electrophysiological and imaging data using standard MEA recording apparatus and confocal/scanning disk microscopy. Microelectrode arrays can be designed compatible to the format of recording hardware/software produced by commercial providers.

3. Cultivation and Examination of Nerve Cells

[0152] The inventors have successfully tested the microfluidic device assembly for its capability to cultivate and examine neurons in the context of 3D neuronal networks.

[0153] FIG. 16 shows a live confocal 3D imaging of GFP-labelled primary neurons in a microfluidic device according to the invention. From a central compartment containing the soma, several neurites extend through the microfluidic channels at the bottom of the device, until they reach the lateral compartments and continue growing in 3D.

[0154] Emulating 3D Brain Networks:

[0155] To partially reconstruct the complex multi-cellular structure of the human brain, commercially available neuronal (excitatory and inhibitory) and glial (astrocytes) cells derived from human iPSCs are grown in 3D inside the microfluidic device assembly, using a range of biomimetic hydrogel scaffolds (bio-derived or synthetic). In order to evaluate cell viability, neuronal outgrowth and 3D architectures obtained using different microfluidic designs, materials and cell types, live imaging experiments are carried out by labelling cells with a range of genetically-encoded fluorescent proteins. Using this approach the inventors obtained data with various prototype devices showing GFP-labelled neurons growing extensively in 3D within few days from seeding. FIG. 17 shows a live confocal 3D imaging of GFP-labelled human iPSC-derived neurons growing in the central compartment of a microfluidic device assembly according to the invention. Neurites can be seen growing extensively in all directions.

[0156] FIG. 18 shows a higher resolution digital reconstruction of previous image shown in FIG. 17. A single neurite (less than 1 m thick) is observed in 3D from different angles, with several dendritic spines being clearly visible.

[0157] Recording Neuronal Activity from 3D Networks:

[0158] As neurites elongate through the dedicated microchannels at the bottom of the microfluidic device assembly according to the invention, microelectrodes integrated below each microchannel will allow measuring action potential propagation and synaptic transmission between neurons connected in 3D. Different microelectrodes sizes and positions within the microchannels are evaluated to identify the ideal dimensions to obtain the best signal-to-noise ratio. The inventors obtained proof-of-concept results showing that iPSC-derived neurons cultured in 3D (FIG. 19, left) are capable to grow neurites through the microchannels (FIG. 19, middle), where integrated microelectrodes could clearly measure from single neurites individual action potentials originating from spontaneously active neurons (FIG. 19, right).

[0159] As can be seen from FIG. 19 which shows differential interference contrast (DIC) live imaging of human iPSC-derived neurons grown in 3D on a microfluidic device according to the invention, cells can be observed at different positions along the z axis. In left panel cell bodies are mainly visible, while in middle panel the neurites can be observed entering the microchannels at the bottom of the device. Right trace shows action potentials recorded from microelectrodes (not visible here) integrated at the bottom of the microchannels.