CN102337211B - cell culture device - Google Patents

Abstract

The invention relates to a cell culture device comprising: a fixed bottom plate, a cell culture system, a light control system and an electrode system arranged on the fixed bottom plate. The cell culture device achieves the culture of nerve cells through the specific structure of microfluidic technology. For example, nerve cells can grow in the cell culture chamber, but axons can only grow in the nerve conduit. The nerve cells in the culture chamber pass through the nerve cells. The axons in the catheter constitute the nerve loop, and the reproducibility and control of the cell growth area are relatively strong; and then the nerve cells are regulated with high spatio-temporal resolution by laser, which has strong selectivity and specificity, and can realize the control of nerve cells. The excitation and inhibition of the neural circuit are assisted by electrode system stimulation, and the behavior and function of the neural circuit are detected in real time, with high spatial and temporal resolution, and reliable regulation and detection results.

Description

cell culture device

【Technical field】

The invention relates to the field of neural engineering, in particular to a cell culture device based on microfluidic technology.

【Background technique】

With the increasing incidence of nervous system diseases, its research is gradually becoming a hot spot. However, due to the complexity of the nervous system, it is very difficult to study some problems of the nervous system in vivo, especially the exploration of neural circuits and neural plasticity. Under such conditions, in vitro culture of nerve cells has become an ideal method. Regular connections between neurons can be established through in vitro culture, and the complex three-dimensional connections between neurons can be reduced to two dimensions, making observation and research easier.

The traditional method of culturing nerve cells in vitro is to regulate the growth of nerve cells on the culture medium through chemically patterned surface, physical microstructure construction (Physical structure) or solution gradients (Solution gradients) to form nerve cells. circuit, and then regulate nerve cells through functional electrical stimulation (Functional electrical stimulation) or local perfusion (Local perfusion).

Among them, the surface chemical treatment is to chemically treat the surface of the cell culture substrate into a biocompatible interface with a certain shape, so as to attract nerve cells to grow in these areas; It is processed into an interface with a certain microstructure to guide the growth of nerve cells in a designated area; the concentration gradient of the solution is to affect the growth of nerve cells in a certain area by controlling the concentration of biologically active substances (such as polypeptides and proteins). Functional electrical stimulation is a regulation method that has been used clinically, and it has played a good role in the treatment of diseases such as essential tremor, Parkinson's disease and dystonia.

However, by regulating the growth of nerve cells to form neural circuits through surface chemical treatment, physical microstructure construction or chemical concentration gradients, the reproducibility and control of the cell growth area is relatively poor, so the reproducibility of the experiment is poor. Although functional electrical stimulation has excellent time resolution, its spatial resolution is poor, and it is not selective and specific, making it difficult to inhibit nerve cells. Although local perfusion has high spatial resolution, the position of perfusion is random, it is difficult to control accurately, and the reproducibility is poor; in addition, the temporal resolution of local perfusion is poor, which cannot meet the electrophysiological requirements. Therefore, there is a traditional lack of a cell culture device that can be used for nerve cell culture, neural circuit construction, and regulation and detection at a higher spatiotemporal resolution.

【Content of invention】

Based on this, it is necessary to provide a cell culture device that can be used for nerve cell culture, neural circuit construction, regulation and detection at a higher spatio-temporal resolution.

A cell culture device includes: a fixed base plate and a cell culture system arranged on the fixed base plate, a light regulation system and an electrode system; The control system is placed above the nerve conduit to regulate the photostimulation of the culture in the nerve conduit; the electrode system communicates with the nerve conduit to assist in the regulation of the culture in the nerve conduit, and to monitor the behavior and function of the neural circuit in real time. detection.

Applied to the above scheme, preferably, the cell culture device also includes a drug perfusion system fixed on the fixed base plate, the drug perfusion system includes a drug perfusion pool, a drug catheter and a waste liquid pool, and the drug perfusion pool is connected to the waste liquid pool through a drug catheter , The drug conduit communicates with the nerve conduit.

Applied to the above scheme and its combination, preferably, the electrode system includes an external contact point, a wire and an electrode point, the external contact point is connected to the electrode point through a wire, and the nerve guide is connected to the electrode point.

Applied to the above scheme and its combination, preferably, the electrode points are electrochemically deposited with conductive materials for reducing the impedance of the electrode points, and the electrode points are modified with enzyme compounds by enzyme immobilization technology for collecting biochemical signals.

Applied to the above solutions and combinations thereof, preferably, the electrode system is formed on the fixed base plate by sputtering or photolithography.

Applied to the above solutions and combinations thereof, preferably, the light stimulation position of the light regulation system corresponds to the position of the electrode points.

Applied to the above scheme and its combination, preferably, the light control system includes a laser light source, an optical fiber interface, an optical fiber, and a waveguide for controlling the guiding and diameter of the laser beam, and the waveguide is connected to the laser light source through the optical fiber and the optical fiber interface.

Applied to the above scheme and its combination, preferably, the electrode system includes a plurality of one-to-one corresponding external contact points, wires and electrode points, and the plurality of external contact points are evenly distributed around the fixed base plate, and the plurality of wires and electrode points form electrodes Correspondingly, the light control system includes a plurality of waveguides with different diameters, and the plurality of waveguides form a waveguide array, and the positions of the waveguides in the waveguide array correspond to the positions of the electrode points in the electrode array; There are microfluidic switches.

Applied to the above solutions and combinations thereof, preferably, the nerve guide has a width of 1-10 μm and a length of 100-1000 μm; the waveguide has a straight diameter of 1-10 μm.

Applied to the above scheme and its combination, preferably, wherein the cell culture system includes two sets of cell culture chambers, each group includes two connected cell culture chambers, the two groups of cell culture chambers are connected through 4 nerve conduits, and the nerve conduits The diameter is 5 μm and the length is 900 μm; the electrode system is provided with 16 square electrode points along the axial direction of each nerve guide, the length and width of the electrode points are both 5 μm, and the distance between the electrode points in the axial direction of each nerve guide is 50 μm; The light regulation system includes 16 waveguides, and the diameter of the waveguide is 2 μm; the drug perfusion system includes a plurality of drug conduits orthogonally connected to the nerve conduits.

Applied to the above solutions and combinations thereof, preferably, the light control system includes a laser light source, an optical fiber interface, a sub-interface and an optical fiber, and the optical fiber is connected to the laser light source through the sub-interface and the optical fiber interface.

Applied to the above scheme and its combination, preferably, the electrode system includes a plurality of one-to-one corresponding external contact points, wires and electrode points, and the plurality of external contact points are evenly distributed around the fixed base plate, and the plurality of wires and electrode points form electrodes Correspondingly, the light control system includes a plurality of optical fibers with different diameters, and a plurality of optical fibers form an optical fiber array, and the positions of the optical fibers in the optical fiber array correspond to the positions of the electrode points in the electrode array; There are microfluidic switches.

Applied to the above solutions and combinations thereof, preferably, the nerve conduit has a width of 1-10 μm and a length of 100-1000 μm; the straight diameter of the optical fiber is 1-10 μm.

Applied to the above scheme and its combination, preferably, wherein the cell culture system includes two sets of cell culture chambers, each group includes two connected cell culture chambers, the two groups of cell culture chambers are connected through 4 nerve conduits, and the nerve conduits The diameter is 5 μm and the length is 900 μm; the electrode system is provided with 16 square electrode points along the axial direction of each nerve guide, the length and width of the electrode points are both 5 μm, and the distance between the electrode points in the axial direction of each nerve guide is 50 μm; The light control system includes 16 optical fibers with a diameter of 2 μm; the drug perfusion system includes a plurality of drug conduits orthogonally connected to the nerve conduits.

Applied to the above scheme and its combination, preferably, the material of the fixed bottom plate is glass, silicon or silicon dioxide; the material of the cell culture chamber and the nerve guide is transparent polydimethoxysiloxane.

The cell culture device achieves the culture of nerve cells through the specific structure of microfluidic technology. For example, nerve cells can grow in the cell culture chamber, but axons can only grow in the nerve conduit. The nerve cells in the culture chamber pass through the nerve cells. The axons in the catheter constitute the nerve loop, and the reproducibility and control of the cell growth area are relatively strong; and then the nerve cells are regulated with high spatio-temporal resolution by laser, which has strong selectivity and specificity, and can realize the control of nerve cells. The excitation and inhibition of the neural circuit are assisted by electrode system stimulation, and the behavior and function of the neural circuit are detected in real time, with high spatial and temporal resolution, and reliable regulation and detection results.

【Description of drawings】

1 is a schematic structural view of a cell culture device according to an embodiment;

Fig. 2 is a schematic structural diagram of the cell culture system in the embodiment of Fig. 1;

Fig. 3 is a schematic diagram of the distribution of the electrode array and the nerve guide in the embodiment of Fig. 1;

Fig. 4 is a schematic diagram of the distribution of the cell culture system and the drug perfusion system in the embodiment of Fig. 1;

Fig. 5 is a schematic diagram of the distribution of nerve guides and drug guides in the embodiment of Fig. 1;

Fig. 6 is a schematic diagram of the distribution of the light regulation system and the cell culture system in the embodiment of Fig. 1;

Fig. 7 is a schematic diagram of the distribution of the light regulation system and the cell culture system according to another embodiment.

【Detailed ways】

In the following, the cell culture device will be further described in detail mainly in conjunction with the accompanying drawings and specific examples.

As shown in FIG. 1 , a cell culture device 100 in a preferred embodiment includes: a fixed base plate 110 , a cell culture system 120 , an electrode system 130 , a drug perfusion system 140 and a light regulation system (not shown). Wherein, the cell culture system 120 , the electrode system 130 , the drug perfusion system 140 and the light control system are fixed on the fixed base plate 110 .

The fixed bottom plate 110 can be made of hard material, and mainly plays the role of supporting and fixing. Materials that can be used include glass, silicon, and silicon dioxide. The fixed bottom plate 110 in this embodiment is a square glass bottom plate. In other implementation manners, the fixed bottom plate 110 can also be made of other materials such as silicon or silicon dioxide, and the shape is not limited to square, and can also be circular.

Referring to FIG. 2 , the cell culture system 120 of this embodiment includes two groups of four cell culture chambers 122 . Each group of cell culture chambers is communicated through a main conduit 124 . The main conduit 124 between the two groups communicates through four nerve conduits 126 provided on the main conduit 124 . The nerve conduit 126 communicates with the two main conduits 124 so that the two sets of cell culture chambers 122 can communicate through the nerve conduit 126 . The cell culture chamber 122 and the nerve guide 126 are mainly used to control and guide the growth of nerve cells, the connection between nerve cells and the formation of nerve circuits. The nerve guide 126 of different specifications can be designed according to the required research model, the width of the nerve guide 126 is 1-10 μm, and the length is 100-1000 μm. Preferably, the width of the nerve guide in this embodiment is 5 μm, and the length is 900 μm. The nerve guide 126 of this design can ensure that nerve cells do not enter the nerve guide 126, and only axons grow in the nerve guide 126, thereby An ordered neural network can be obtained.

The cell culture system 120 is preferably made of flexible, transparent, and air-permeable materials, such as silicone rubber. The cell culture system of this embodiment is made of highly hydrophobic polydimethoxysiloxane (PDMS). In order to allow nerve cells to grow better in the cell culture chamber 122, the inner surfaces of the cell culture chamber 122, the main conduit 124, and the nerve conduit 126 of this embodiment can be processed by techniques such as surface grafting, surface coating, and layer-by-layer self-assembly. Biocompatibility treatment. In addition, before cell culture, the inner surfaces of the cell culture chamber 122, the main conduit 124, and the nerve conduit 126 can be further coated, such as polylysine coating, laminin coating, and the like.

Please refer to FIG. 1 and FIG. 3 , the electrode system 130 includes an external contact 132 , wires 134 and electrode points 136 . The external contacts 132 are connected to the electrode points 136 through wires 134 , and each external contact 132 corresponds to one electrode point 136 . A plurality of external contacts 132 are uniformly arranged around the fixed base 110 , and a plurality of wires 134 and electrode points 136 form an electrode array. Each side of the fixed bottom plate 110 in this embodiment is provided with 16 external contact points 132 . For further convenience, the external contact point 132 is designed so that it can be directly connected to a general-purpose electrophysiological measuring instrument for electrophysiological measurement, such as the electrophysiological measuring instrument of Plexon Company of the United States and MEA Company of Germany. An electrode array formed by 16 wires 134 and square electrode points 136 is sequentially arranged in the axial direction of the nerve guide 126 , the length and width of each electrode point 136 are 5 μm, and the interval between every two electrode points 136 is 50 μm. The nerve guide 126 communicates with the electrode point 136, so that the electrode array can perform electrophysiological measurement and electrical pulse stimulation on the culture in the nerve guide 126 with high spatial and temporal resolution.

Preferably, the electrode system 130 of this embodiment is formed on the surface of the fixed bottom plate 110 by sputtering or photolithography. The material of the wire 134 and the electrode point 136 can be one or more of gold, platinum, iridium oxide, titanium nitride, conductive polymer, and carbon-based material. In actual use, the electrode system 130 can be used as an auxiliary regulation method. The electrode array can perform functional electrical pulse stimulation on nerve cells, induce the growth of nerve cells by changing the electric field strength in the microenvironment, and Ability to study neural circuits and neuroplasticity. In order to reduce the contact impedance of the electrode point 136 and enhance the signal-to-noise ratio of the electrophysiological signal, a modified material such as iridium oxide, conductive polymer or carbon-based material can be deposited on the surface of the electrode point 136 by electrochemical means. For example, after modifying the electrode point 136 made of iridium oxide with a carbon-based material, the impedance of the electrode point 136 can be reduced by 50% to 90% at a measurement frequency of 1KHz, and the background noise of the obtained electrophysiological signal is small, and the signal-to-noise ratio high. Further, enzyme immobilization technology can be used to immobilize and modify the enzyme compound on the electrode point 136 to form a biosensor to detect the microenvironment around the axon. The detected signals include the concentration of neurotransmitters, amino acid concentrations, ion concentrations, and oxygen concentrations. , temperature, pH value, etc., so as to obtain changes in the concentration of neurotransmitters, glutamate, and important ions in the axon during the regulation process, as well as changes in important indicators such as oxygen, temperature, and pH in the environment. For example, after the electrode point 136 can be modified with tyrosinase, a neurotransmitter dopamine sensor can be obtained, and the dopamine sensor can be used to detect the concentration of dopamine released at the position of the axon within a certain spatial range, so that the neural circuit can be studied Function.

Furthermore, by setting microfluidic switches in each electrode array, the corresponding electrode points 136 can be turned on or off in a targeted manner, and the nerve guide 126 can be selectively stimulated with local electric pulses.

Please refer to FIG. 1 , FIG. 4 and FIG. 5 , the drug perfusion system 140 includes a drug perfusion tank 142 , a drug conduit 144 and a waste liquid tank 146 . The drug perfusion pool 142 is connected to the waste liquid pool 146 through a drug conduit 144 . The drug conduit 144 communicates with the nerve conduit 126 and can stimulate the axons in the microenvironment in the nerve conduit 126 with drugs. The drugs used may be neurotrophic factors, neurotransmitters, amino acids or genes (such as light-sensitive protein genes, etc.) and the like. The drug perfusion system 140 of this embodiment includes nine drug conduits 144 . The nine drug conduits 144 are in orthogonal communication with the nerve conduit 126 . Multiple drug conduits 144 can be used to perform multi-point drug stimulation on the axons or to perform high-throughput drug screening, the drug release is uniform, and the experimental results are reliable. Further, each drug conduit 144 can be provided with a microfluidic switch, and the corresponding drug conduit can be turned on or off through the microfluidic switch, so that the axons in the nerve conduit 126 can be specifically stimulated with drugs.

As shown in FIG. 6 , the light regulation system 150 is disposed above the cell culture system 120 . The light control system 150 includes a laser light source (not shown), an optical fiber interface 152, an optical fiber 154, and a waveguide 156 for controlling the direction and diameter of the laser beam. The waveguide 156 is connected to the laser light source through the optical fiber 154 and the optical fiber interface 152 . The waveguide 156 guides the laser light emitted by the laser light source into the nerve guide 126 to perform photostimulation and regulation on the axons and the like. During implementation, the laser light source mainly emits blue (wavelength = 472nm) or yellow (wavelength = 593nm) laser, and the blue or yellow laser is introduced into the nerve guide 126 through the optical fiber 154 and the waveguide 156 for irradiation. Since the blue or yellow laser light only acts on the cells into which the light-sensitive gene is introduced, the light regulation system 150 is cell-specific and can effectively excite or inhibit neurons. Before using the light regulation system 150, it is necessary to transfer and express the light-sensitive gene into the neuron of the relevant circuit through a specific promoter. Wherein, the excitatory channel protein gene is Channelrhodopsin-2 (ChR2), and the inhibitory channel protein gene is Helorhodopsin (NpHR). The light regulation system 150 provides light stimulation to the neurons with the light-sensitive gene transferred in the nerve conduit through the optical fiber 154 and the waveguide 156, thereby causing the excitation or inhibition of the neurons. Stimulating neurons expressing excitatory channelrin genes with blue light will excite such neurons, while stimulating neurons expressing inhibitory channelrin genes with yellow light will inhibit such neurons. Neural circuits can be regulated through excitation or inhibition, so as to conduct related research. Since the light regulation system 150 is cell-specific, this light-sensitive gene neural regulation technology has a relatively high spatiotemporal resolution.

The light modulation system 150 may include a plurality of waveguides 156, and the diameter of the waveguides may be 1-10 μm. A plurality of waveguides 156 form a waveguide array by micromachining techniques. The incident laser light can be divided into laser arrays with a fixed size through the waveguide array. At the same time, microfluidic switches can be added to the waveguide array to selectively perform local photostimulation, with strong purpose and high temporal and spatial resolution. The photo-stimulation site guided by the waveguide 156 corresponds to the position of the electrode point 136, so that the electrode point 136 can collect electrophysiological signals at the photo-stimulation site more accurately and specifically. The light regulation system 150 of this embodiment has 16 waveguides 156 distributed in the axial direction of each nerve guide 126 . The waveguide 156 has a diameter of 2 μm. The 16 waveguides 156 correspond to the 16 electrode points 136 .

In other embodiments, the optical control system can also replace the waveguide by arranging optical fibers with different diameters. As shown in FIG. 7 , the light control system 250 in this embodiment includes a laser light source (not shown in the figure), an optical fiber interface 252 , a branch interface 254 and an optical fiber 256 . The optical fiber 256 is connected to the laser light source through the sub-port 254 and the optical fiber interface 252 . The diameter of the optical fiber 256 may be 1-10 μm. Preferably, the diameter of the optical fiber is 2 μm. The optical fiber 256 directly guides the laser light emitted by the laser light source into the nerve guide 126 . A plurality of optical fibers 256 can form an optical fiber array, and a microfluidic switch can be added to the optical fiber array to selectively perform local light stimulation on the nerve guide 126 .

The cell culture device 100 achieves the culture of nerve cells through the specific structure of microfluidic technology. For example, nerve cells can grow in the cell culture chamber 122, but axons can only grow in the nerve guide 126, and the cell culture chamber 122 Nerve cells form a nerve loop through the axons in the nerve conduit 126, and the reproducibility and control of the cell growth area are relatively strong; and the laser is used to regulate the nerve cells with high spatio-temporal resolution, with strong selectivity and specificity , can realize the excitation and inhibition of nerve cells; through the stimulation of the electrode system 130, the nerve loop can be assisted to regulate, and the behavior and function of the nerve loop can be detected in real time, with high spatial and temporal resolution, and reliable regulation and detection results.

The above-mentioned embodiments only express several implementation modes of the present invention, and the description thereof is relatively specific and detailed, but should not be construed as limiting the patent scope of the present invention. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims (11)

1. A cell culture device, characterized in that, comprising: a fixed base plate and a cell culture system, a light regulation system and an electrode system arranged on the fixed base plate; The nerve conduit for the growth of nerve axons in the culture room; the light regulation system is placed above the nerve conduit for light stimulation regulation of the culture in the nerve conduit; the electrode system communicates with the nerve conduit for Auxiliary regulation of the culture in the nerve conduit, and real-time detection of the behavior and function of the neural circuit; the electrode system includes an external contact point, a wire and an electrode point, and the external contact point is connected to the electrode through the wire The nerve guide is connected to the electrode point; the electrode point is electrochemically deposited with a conductive material for reducing the impedance of the electrode point, and the electrode point is modified with an enzyme compound by an enzyme immobilization technique for collecting biochemical signal; the light stimulation position of the light control system corresponds to the position of the electrode point; the light control system includes a laser light source, an optical fiber interface, an optical fiber and a waveguide for controlling the guiding and diameter of the laser beam, and the waveguide passes through The optical fiber and the optical fiber interface are connected with the laser light source. the 2. The cell culture device according to claim 1, further comprising a drug perfusion system fixed on the fixed base plate, the drug perfusion system comprising a drug perfusion tank, a drug conduit and a waste liquid tank, the The medicine perfusion pool is connected with the waste liquid pool through the medicine conduit, and the medicine conduit is connected with the nerve conduit. the 3. The cell culture device according to claim 1, wherein the electrode system is formed on the fixed bottom plate by sputtering or photolithography. the 4. The cell culture device according to claim 2, wherein the electrode system includes a plurality of one-to-one corresponding external contact points, wires and electrode points, and the plurality of external contact points are evenly distributed around the fixed base plate , a plurality of wires and electrode points form an electrode array, correspondingly, the light control system includes a plurality of waveguides with different diameters, a plurality of waveguides form a waveguide array, and the positions of the waveguides in the waveguide array are the same as those in the electrode array The positions of the electrode points are in one-to-one correspondence; a microfluidic switch is arranged in the electrode array, the waveguide array and the drug catheter. the 5 . The cell culture device according to claim 4 , wherein the nerve guide has a width of 1-10 μm and a length of 100-1000 μm; the waveguide has a diameter of 1-10 μm. the 6. The cell culture device according to claim 5, wherein the cell culture system comprises two groups of cell culture chambers, each group comprises two connected cell culture chambers, and four groups of cell culture chambers are passed between the two groups of cell culture chambers. A nerve conduit is connected, and the diameter of the nerve conduit is 5 μm, and the length is 900 μm; The electrode system is sequentially provided with 16 square electrode points along the axial direction of each nerve guide, the length and width of the electrode points are both 5 μm, and the distance between the electrode points in the axial direction of each nerve guide is 50 μm; The light control system includes 16 waveguides, and the diameter of the waveguides is 2 μm; The drug perfusion system includes a plurality of drug conduits in orthogonal communication with the nerve conduits. the 7 . The cell culture device according to claim 2 , wherein the light control system comprises a laser light source, an optical fiber interface, a branch interface and an optical fiber, and the optical fiber is connected to the laser light source through the branch interface and the optical fiber interface. the 8. The cell culture device according to claim 7, wherein the electrode system includes a plurality of one-to-one corresponding external contact points, wires and electrode points, and the plurality of external contact points are evenly distributed around the fixed base plate , a plurality of wires and electrode points form an electrode array, correspondingly, the light control system includes a plurality of optical fibers with different diameters, a plurality of optical fibers form an optical fiber array, and the position of the optical fiber in the optical fiber array is the same as that in the electrode array The positions of the electrode points are in one-to-one correspondence; a microfluidic switch is arranged in the electrode array, the waveguide array and the drug catheter. the 9 . The cell culture device according to claim 8 , wherein the nerve guide has a width of 1-10 μm and a length of 100-1000 μm; and the optical fiber has a diameter of 1-10 μm. the 10. The cell culture device according to claim 9, wherein the cell culture system comprises two sets of cell culture chambers, each group comprising two connected cell culture chambers, and four sets of cell culture chambers are passed between the two groups of cell culture chambers. The nerve conduit is connected, and the diameter of the nerve conduit is 5 μm and the length is 900 μm; The electrode system is sequentially provided with 16 square electrode points along the axial direction of each nerve guide, the length and width of the electrode points are both 5 μm, and the distance between the electrode points in the axial direction of each nerve guide is 50 μm; The light control system includes 16 optical fibers, and the diameter of the optical fiber is 2 μm; The drug perfusion system includes a plurality of drug conduits in orthogonal communication with the nerve conduits. the 11. The cell culture device according to claim 1, wherein the material of the fixed bottom plate is glass, silicon or silicon dioxide; the material of the cell culture chamber and the nerve guide is transparent polydimethylformaldehyde Oxysiloxane. the

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