CN113941378A - Multi-cavity electrophysiological micro-nano detection-based neural organoid chip and detection method - Google Patents
Multi-cavity electrophysiological micro-nano detection-based neural organoid chip and detection method Download PDFInfo
- Publication number
- CN113941378A CN113941378A CN202111198208.5A CN202111198208A CN113941378A CN 113941378 A CN113941378 A CN 113941378A CN 202111198208 A CN202111198208 A CN 202111198208A CN 113941378 A CN113941378 A CN 113941378A
- Authority
- CN
- China
- Prior art keywords
- organoid
- chip
- cavity
- organoids
- electrophysiological
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/12—Specific details about manufacturing devices
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
- B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Clinical Laboratory Science (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Sustainable Development (AREA)
- Biomedical Technology (AREA)
- Dispersion Chemistry (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Analytical Chemistry (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- Electromagnetism (AREA)
- Cell Biology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a nerve organoid chip based on multi-cavity electrophysiological micro-nano detection and a detection method, wherein the nerve organoid chip comprises a microelectrode array chip and an organoid culture cavity, and the microelectrode array comprises 6 groups of working electrodes and 4 reference electrodes; the organoid culture cavity comprises a culture cavity body and a multi-cavity part, wherein the culture cavity body is a square structure with a cylinder dug inside and is packaged on the sensor chip; the multi-cavity part main part is the tubbiness, and the bottom has 6 cylindrical through-holes, corresponds 6 working electrode groups of microelectrode array, and both sides are cantilever structure, and collocation screw and nut adjustment multi-cavity part position effectively controls the organoid gathering and distributes in electrode array region, increases organoid and working electrode's contact probability. The invention can realize the monitoring of the electrophysiological activity of the nerve organoid, is particularly suitable for acquiring and recording smell response signals of the olfactory organoid, and has the characteristics of real-time no damage, high sensitivity, high stability, good reproducibility and the like.
Description
Technical Field
The invention relates to the technical field of bionic sensing, in particular to a nerve organoid chip based on multi-cavity electrophysiological micro-nano detection and a detection method.
Background
Organoids are formed by stem cells which differentiate and self-assemble in vitro, a tissue structure with biological functions similar to organs. Organoids are important as a micro in vitro tissue culture model in research such as organ tissue generation, disease pathology, drug research and development. The biosensor is a sensor which takes cells, tissues and the like as sensitive elements, converts morphological and physiological changes of the cells into electric signals, records and outputs the electric signals in real time, and has the characteristics of no need of marking, long-time monitoring and real-time monitoring. The biosensor can monitor the physiological activities of cells in a multi-scale manner, and is an emerging technology which is favored by researchers. Most biosensors mainly monitor cells cultured in two dimensions, still have the limitation of not being close to the in-vivo physiological state, and are produced by combining three-dimensional cells or organoids with a sensor chip and a micro-fluidic system to construct a sensing system which is high in sensitivity, specificity and response and is closer to the in-vivo. The growth condition of the organoid in the chip is quantitatively monitored in real time by using a biosensing technology, so that the unmarked and non-invasive recording is realized, and the method has important application prospects in the fields of drug research and development, tissue generation, disease generation and the like. The invention provides a neural organoid chip and a detection method based on multi-cavity electrophysiological micro-nano detection, and aims to solve the problem that traditional two-dimensional cultured cells are easy to attach to the surface of a sensor chip and are not easy to move, but organoids have three-dimensional structures, so that the positions of the organoids are not easy to fix when the organoids are planted on the surface of the sensor chip, and the probability of contacting electrodes is low.
Disclosure of Invention
The invention aims to provide a neural organoid chip based on multi-cavity electrophysiological micro-nano detection and a corresponding detection method aiming at the defects of the prior art.
The purpose of the invention is realized by the following technical scheme: a nerve organoid chip based on multi-cavity electrophysiological micro-nano detection is composed of a multi-channel microelectrode array chip manufactured by a micro-nano processing technology, a PCB (printed Circuit Board) adapter plate and a multi-cavity organoid culture cavity;
the microelectrode array chip takes square glass as a substrate, the middle area of the chip is a microelectrode array for detection, and comprises 60 round working electrodes and 4 1/4 round-structured reference electrodes, each 10 working electrodes form a group, 6 groups of working electrodes are uniformly distributed on each vertex of a hexagon, the center of the hexagon is the center of the chip, the reference electrodes are distributed at four corners of the chip, gold wire transfer points for transferring with a PCB transfer board are arranged on the periphery of the reference electrodes, and the reference electrodes are connected with the electrodes through leads; the microelectrode array chip is adhered to the PCB adapter plate through UV glue and subjected to gold wire electric welding to form a sensor chip part;
the center of the PCB adapter plate is provided with a circular through hole, corresponds to the middle area of the microelectrode array chip and is used for observing the contact conditions of the microelectrode array and the organoid and the electrode;
the organoid culture cavity comprises a culture cavity body and a multi-cavity part, wherein the culture cavity body is of a square structure with a cylinder dug out inside and is packaged on a sensor chip; the main part of multi-chamber part is the tubbiness structure, and the bottom has 6 cylindrical through-holes, and the position of through-hole corresponds 6 work electrode points on the microelectrode array chip respectively, and both sides are cantilever structure, and collocation screw and nut use for the position of adjustment multi-chamber part, when 6 work electrode points of group on the microelectrode array chip are aimed at to 6 cylindrical through-holes, the nut of screwing makes the multi-chamber part is fixed cultivate on the chamber body, the gathering of effective control organoid distributes in electrode array region, increases organoid and work electrode's contact probability.
Further, cultivate chamber body and multicavity formula part and all adopt ya keli material.
Further, the side length of the microelectrode array chip is 12mm, and the diameter of the working electrode is 30 μm.
Furthermore, the multi-cavity part can be detached at any time, so that the electrode is convenient to clean.
Further, a layer of platinum black particles is electroplated on the exposed electrode surface, and the biocompatibility of the chip is improved by combining a chip surface modification technology, wherein the modified substances comprise gelatin, polylysine and laminin.
The invention also discloses a method for detecting electrophysiological signals based on the neural organoid chip, which comprises the following steps:
(1) fixing the multi-cavity part on a culture cavity body of the sensor chip by using a screw and a nut on the cantilever, observing and adjusting under a microscope to ensure that each group of 10 electrodes is exactly positioned in each through hole circle, and then screwing the screw to prevent the multi-cavity part from displacing;
(2) transferring organoids to a microelectrode array under a stereomicroscope; initially containing the culture solution as little as possible, so that the organoids fall into the cylindrical through holes at the bottom of the multi-cavity part, and the micro-needles are used for ejecting air bubbles in the holes when necessary; standing in an incubator for a period of time, and observing the distribution of the organoids and the contact condition of the organoids and the electrodes under a microscope after the organoids are settled to the bottom and attached to the microelectrode array; then adding a proper amount of organoid culture solution to maintain the survival state of organoids, and carrying out electrophysiological signal recording after culturing for a period of time in a constant-temperature incubator;
(3) organoids were stimulated and electrophysiological recordings were performed: after recording the stable self-emission signals of the organoids as a contrast, the electrophysiological response signals of the organoids to the stimulation can be recorded; and (3) sucking out the organoid culture solution in the culture cavity by using a pipette, adding a fresh culture solution, recording electrophysiological signals after adding different concentrations and different types of stimulators, and cleaning before adding the stimulators each time.
Furthermore, the nerve organoids are olfactory organoids, and the olfactory organoids are stimulated by smell to perform electrophysiological recording.
Further, the olfactory organoid is obtained by inducing the culture and differentiation of primary olfactory progenitors/stem cells in olfactory epithelium of a mouse by using a three-dimensional culture technology, and mature olfactory sensory neurons can be expressed after culturing in matrigel and organoid culture solution for about 2 weeks; dissolving matrigel with cell recovery solution, releasing olfactory organoids, transferring the olfactory organoids to a microelectrode array under a body microscope, and culturing in a constant temperature incubator for 12-24h for signal recording.
Further, after stable self-emission signals of the olfactory organoids are recorded as a contrast, electrophysiological response signals of the olfactory organoids to odor stimulation are recorded for subsequent analysis; the stimulant adopts a mixed solution with concentration gradient of 6 odorous substances, wherein the mixed solution comprises methyl salicylate, acetophenone, citral, isoamyl acetate, carvone and n-valeraldehyde; at the start of signal recording, organoid medium in the culture chamber was aspirated with a pipette, fresh medium was added, and then the odor mixture solution was added for stimulation.
Compared with the prior art, the short plate has the following beneficial effects: the invention provides a multi-cavity electrophysiological micro-nano detection-based neural organoid chip, which is successfully applied to olfactory organoid detection, greatly simulates the neural mechanism and physiological structure of odor perception of mammals, can be popularized and applied to physiological activity monitoring of various neural organoids, and has the characteristics of real-time no damage, high sensitivity, high stability, good reproducibility and the like.
Drawings
FIG. 1 is a schematic diagram of the multi-chamber neural organoid chip of the present invention;
FIG. 2 is a top view of the multi-chamber neural organoid chip of the present invention;
FIG. 3 is a schematic diagram of the electrode distribution of the multi-channel microelectrode array chip of the present invention;
FIG. 4 is a schematic diagram of a multi-chamber configuration of an organoid culture chamber according to the present invention;
FIG. 5 is a photomicrograph of olfactory organoids attached to electrode sites on a chip according to an embodiment of the invention;
FIG. 6 is a diagram of a neural organoid chip of the present invention for detecting the response signal of olfactory organoids to a mixed odorant;
in the figure: 1. the device comprises a PCB (printed circuit board) adapter plate, 2, a microelectrode array chip, 3, a square culture cavity body, 4, multi-cavity parts, 5, a cylindrical through hole, 6, a working electrode, 7, a reference electrode and 8, olfactory organs.
Detailed Description
The invention is described in detail below with reference to the figures and the specific embodiments, but the invention is not limited thereto.
The invention provides a nerve organoid chip based on multi-cavity electrophysiological micro-nano detection, and takes olfactory organoids as an example to collect nerve activity signals so as to verify the feasibility and the practicability of the chip.
As shown in figures 1 and 2, the invention designs and manufactures a multi-cavity electrophysiological micro-nano detection-based neural organoid chip, which mainly comprises a multi-channel microelectrode array chip 2 manufactured by a micro-nano processing technology, a PCB adapter plate 1 and a multi-cavity organoid culture cavity structure, and can monitor physiological activities of organoids in real time, nondestructively and for a long time. For example, the method can be applied to acquisition and recording of smell response signals of olfactory organs, and completes feature extraction and pattern recognition of the signals through a neural decoding algorithm to construct a smell recognition model, thereby realizing high sensitivity, specificity and stability of the smell and quick detection and recognition.
The microelectrode array chip 2 is manufactured by taking square glass with the side length of 12mm as a substrate through the process flows of thin film sputtering, photoetching, wet etching and the like. Before processing, a photoetching mask is required to be designed in AutoCAD software according to actual size, wherein the photoetching mask comprises a metal layer and an insulating layer, and the shape and the spatial arrangement condition of electrodes are required to be drawn on the metal layer. The middle area of the chip is a microelectrode array (MEA) for detection, which comprises 60 working electrodes 6 and 4 reference electrodes 7, the diameter of the circular working electrodes 6 is 30 μm, every 10 working electrodes 6 are in a group, and 6 groups are uniformly distributed on each vertex of the hexagon, the center of the hexagon is the center of the chip, the 1/4 circular reference electrodes 7 are distributed at four corners of the chip, and the periphery is gold wire transfer points which are transferred with the PCB transfer board 1 and are connected with the electrodes by leads with the line width of 10 μm. After the microelectrode array chip 2 is manufactured, the PCB adapter plate 1 is bonded by UV glue, and gold wire electric welding is carried out to form a sensor chip part.
A circular through hole is reserved in the center of the PCB adapter plate 1, corresponds to the middle area of the chip, and is convenient for observing the contact condition of the microelectrode array and the organoid and the electrode by using a microscope and the like. The shape and spatial arrangement of the electrodes are shown in fig. 3, and the design is done in AutoCAD software.
The organoid culture chamber comprises two parts, wherein one part is packaged on a sensor chip, a square culture chamber body 3 with a cylinder dug out is arranged inside the chamber body, an acrylic material can be adopted, and organoid culture solution, stimulating substances and the like can be added in the middle during experiments. The other part is an embedded multi-cavity part 4 with a cantilever structure, can be made of acrylic materials, can be fixed in a culture cavity through screws and nuts, and is designed and finished in SolidWorks software. As shown in fig. 4, the main body of the multi-cavity part 4 is a barrel-shaped structure, the bottom of the multi-cavity part is provided with 6 cylindrical through holes 5, the positions of the through holes correspond to 6 groups of working electrode points on the MEA chip respectively, cantilever structures are designed on two sides of the through holes, and screws and nuts are matched for use and used for adjusting the positions of the multi-cavity part. The multi-cavity part can be detached at any time, so that the electrode is convenient to clean.
In addition, in order to improve the signal-to-noise ratio of the MEA, a layer of platinum black particles is electroplated on the exposed electrode surface, and the electrode impedance is reduced by increasing the specific surface area, so that the noise is reduced and the signal-to-noise ratio is improved. Meanwhile, the biocompatibility of the chip is improved by combining a chip surface modification technology, for example, the surface of the chip is modified by gelatin, polylysine, laminin and the like, so that the signal-to-noise ratio and the stability of the organoid nerve response signal are effectively improved.
The nerve organoid chip based on multi-cavity electrophysiological micro-nano detection provided by the invention is subjected to performance test by taking olfactory organoids as an example. The olfactory organoid is obtained by inducing the culture and differentiation of primary olfactory progenitors/stem cells in olfactory epithelium of a mouse by using a three-dimensional culture technology, and can express mature olfactory sensory neurons after being cultured in matrigel and organoid culture solution for about 2 weeks. Dissolving matrigel with cell recovery solution, releasing olfactory organoids, transferring the olfactory organoids to MEA under a body microscope, and culturing in a constant temperature incubator for 12-24h for signal recording. The method comprises the following specific steps:
(1) organoid culture: after 3 weeks old C57 mice were sacrificed by intraperitoneal injection of excess anesthetic urethane, the heads were broken, the head tissues were stripped off and quickly placed in Ringer's solution mixed with ice and water. The left and right skull were separated along the median suture with an orthopedic scissors, the olfactory epithelial region of the nasal septum was exposed, the olfactory epithelial tissue was cut off with a surgical scissors, and transferred into the pre-cooled Ringer's solution along with the nasal septum. Both stroma and epithelium were separated under a micromanipulator and epithelium was transferred to an appropriate amount of 0.25% Trypsin & 0.02% EDTA in a 15mL centrifuge tube. Digesting in 37 deg.C constant temperature incubator for 30-45min while shaking every 5-10 min. Fetal calf serum was added at 110. mu.L/mL to stop digestion, gently blown for 15min and then filtered through a filter screen while preventing air bubbles. Centrifuging at 200g for 5min, discarding supernatant, adding a certain amount of organoid culture solution, and gently blowing and beating. Mixing with matrigel at a ratio of 1:2, adding 60 μ L per well into 24-well plate, and standing in 37 deg.C incubator for 15 min. After coagulation, about 500. mu.L of organoid culture medium was added to each well and cultured in a 37 ℃ incubator. The culture medium was replaced with fresh medium every 1 day, and the growth state of organoids was observed under a microscope.
(2) Electrophysiological recording of olfactory organoids: after the olfactory organoids express mature olfactory sensory neurons (about 2 weeks of culture), the matrigel is dissolved with cell recovery solution, the olfactory organoids are released, and the olfactory organoids are transferred to MEA under a stereomicroscope. Before this, it is necessary to fix the multi-cavity parts to the culture chamber body of the sensor chip by using screws and nuts on the cantilever, and observe and adjust under a microscope so that each group of 10 working electrodes is exactly positioned in each through hole circle, and then tighten the screws to prevent the parts from being displaced. When transferring olfactory organoids to MEA, initially minimal culture solution is included to allow organoids to fall into the cylindrical through-hole at the bottom of the multi-chamber part, requiring microneedles to expel air bubbles in the hole if necessary. Standing in the incubator for a while until the organoids settle to the bottom and attach to the MEA, and observing the distribution of olfactory organoids 8 and contact with the electrodes under a microscope, as shown in FIG. 5. Then adding a proper amount of organoid culture solution to maintain the survival state of organoids, and culturing in a constant temperature incubator for 12-24h and then recording signals. The neural signal acquisition System used a 64-Channel MEA1060 System of Multi-Channel System, Germany, with a single Channel sampling rate of 40kHz, and the raw signals were stored in a computer and subsequently analyzed off-line using MATLAB software. In the signal acquisition process, in order to simulate the organism environment, a double-channel biological sample temperature controller is introduced into the signal acquisition system to keep the organoid culture environment at 37 ℃.
(3) The smell stimulates olfactory organoids and electrophysiological recording is performed: the olfactory sensory neurons can convert odor chemical signals into nerve activity signals, neuron action potentials can be triggered after specific binding of odors and receptors, and the microelectrode arrays can record the action potentials for researching the influence of odor stimulation on olfactory organs. After recording the stable self-emitted signal of the olfactory organoid as a control, the electrophysiological response signal of the olfactory organoid to the odor stimulus can be recorded for subsequent analysis. The irritant is a concentration gradient mixed solution of 6 odorous substances, including methyl salicylate, acetophenone, citral, isoamyl acetate, carvone and n-valeraldehyde. The stock solution of the odor was diluted to 2M with dimethyl sulfoxide and then diluted with Ringer's solution to obtain a mixture of solutions of different concentration gradients. At the start of signal recording, organoid medium in the culture chamber was aspirated with a pipette and stimulated by adding 900. mu.L of fresh medium followed by 100. mu.L of odor mix. As shown in FIG. 6, after the olfactory organ is subjected to odor stimulation, the distribution frequency of spike is obviously improved, and the same result is obtained by repeated experiments. The results show that the nerve organoid chip based on multi-cavity electrophysiological micro-nano detection can realize the monitoring of the electrophysiological activity of the nerve organoid and has the characteristics of no damage in real time, high sensitivity, high stability, good reproducibility and the like.
The above-described embodiments are intended to illustrate rather than to limit the invention, and any modifications and variations of the present invention are within the spirit of the invention and the scope of the appended claims.
Claims (10)
1. A nerve organoid chip based on multi-cavity electrophysiological micro-nano detection is characterized by consisting of a multi-channel microelectrode array chip manufactured by a micro-nano processing technology, a PCB (printed Circuit Board) adapter plate and a multi-cavity organoid culture cavity;
the microelectrode array chip takes square glass as a substrate, the middle area of the chip is a microelectrode array for detection, and comprises 60 round working electrodes and 4 1/4 round-structured reference electrodes, each 10 working electrodes form a group, 6 groups of working electrodes are uniformly distributed on each vertex of a hexagon, the center of the hexagon is the center of the chip, the reference electrodes are distributed at four corners of the chip, gold wire transfer points for transferring with a PCB transfer board are arranged on the periphery of the reference electrodes, and the reference electrodes are connected with the electrodes through leads; the microelectrode array chip is adhered to the PCB adapter plate through UV glue and subjected to gold wire electric welding to form a sensor chip part;
the center of the PCB adapter plate is provided with a circular through hole, corresponds to the middle area of the microelectrode array chip and is used for observing the contact conditions of the microelectrode array and the organoid and the electrode;
the organoid culture cavity comprises a culture cavity body and a multi-cavity part, wherein the culture cavity body is of a square structure with a cylinder dug out inside and is packaged on a sensor chip; the main part of multi-chamber part is the tubbiness structure, and the bottom has 6 cylindrical through-holes, and the position of through-hole corresponds 6 work electrode points on the microelectrode array chip respectively, and both sides are cantilever structure, and collocation screw and nut use for the position of adjustment multi-chamber part, when 6 work electrode points of group on the microelectrode array chip are aimed at to 6 cylindrical through-holes, the nut of screwing makes the multi-chamber part is fixed cultivate on the chamber body, the gathering of effective control organoid distributes in electrode array region, increases organoid and work electrode's contact probability.
2. The neural organoid chip based on multi-cavity electrophysiological micro-nano detection according to claim 1, wherein the culture cavity body and the multi-cavity parts are made of acrylic materials.
3. The neural organoid chip based on multi-cavity electrophysiological micro-nano detection according to claim 1, wherein the side length of the microelectrode array chip is 12mm, and the diameter of the working electrode is 30 μm.
4. The neural organoid chip based on multi-cavity electrophysiological micro-nano detection according to claim 1, wherein the multi-cavity parts can be disassembled at any time, thereby facilitating electrode cleaning.
5. The neural organoid chip based on multi-cavity electrophysiological micro-nano detection according to claim 1, wherein a layer of platinum black particles is plated on the exposed electrode surface, and the biocompatibility of the chip is improved by combining a chip surface modification technology, wherein the modification substances include gelatin, polylysine and laminin.
6. The neural organoid chip based on multi-cavity electrophysiological micro-nano detection according to claim 1, wherein the chip is applied to acquisition and recording of olfactory organoid odor response signals.
7. A method for detecting electrophysiological signals based on the neural organoid chip of any of claims 1 to 6, comprising the steps of:
(1) fixing the multi-cavity part on a culture cavity body of the sensor chip by using a screw and a nut on the cantilever, observing and adjusting under a microscope to ensure that each group of 10 electrodes is exactly positioned in each through hole circle, and then screwing the screw to prevent the multi-cavity part from displacing;
(2) transferring organoids to a microelectrode array under a stereomicroscope; initially containing the culture solution as little as possible, so that the organoids fall into the cylindrical through holes at the bottom of the multi-cavity part, and the micro-needles are used for ejecting air bubbles in the holes when necessary; standing in an incubator for a period of time, and observing the distribution of the organoids and the contact condition of the organoids and the electrodes under a microscope after the organoids are settled to the bottom and attached to the microelectrode array; then adding a proper amount of organoid culture solution to maintain the survival state of organoids, and carrying out electrophysiological signal recording after culturing for a period of time in a constant-temperature incubator;
(3) organoids were stimulated and electrophysiological recordings were performed: after recording the stable self-emission signals of the organoids as a contrast, the electrophysiological response signals of the organoids to the stimulation can be recorded; and (3) sucking out the organoid culture solution in the culture cavity by using a pipette, adding a fresh culture solution, recording electrophysiological signals after adding different concentrations and different types of stimulators, and cleaning before adding the stimulators each time.
8. The method of claim 7, wherein the neural organoids are olfactory organoids, and the electrophysiological recording is performed by odor stimulation of the olfactory organoids.
9. The method of claim 8, wherein said olfactory organoid is obtained by inducing culture differentiation of primary olfactory progenitors/stem cells in mouse olfactory epithelium by three-dimensional culture technique, and expressing mature olfactory neurons after culturing in matrigel and organoid culture for about 2 weeks; dissolving matrigel with cell recovery solution, releasing olfactory organoids, transferring the olfactory organoids to a microelectrode array under a body microscope, and culturing in a constant temperature incubator for 12-24h for signal recording.
10. The method of claim 8, wherein after recording a stable self-emitted signal of the olfactory organoid as a control, recording an electrophysiological response signal of the olfactory organoid to the odor stimulus for subsequent analysis; the stimulant adopts a mixed solution with concentration gradient of 6 odorous substances, wherein the mixed solution comprises methyl salicylate, acetophenone, citral, isoamyl acetate, carvone and n-valeraldehyde; at the start of signal recording, organoid medium in the culture chamber was aspirated with a pipette, fresh medium was added, and then the odor mixture solution was added for stimulation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111198208.5A CN113941378B (en) | 2021-10-14 | 2021-10-14 | Nerve organoid chip based on multi-cavity electrophysiological micro-nano detection and detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111198208.5A CN113941378B (en) | 2021-10-14 | 2021-10-14 | Nerve organoid chip based on multi-cavity electrophysiological micro-nano detection and detection method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113941378A true CN113941378A (en) | 2022-01-18 |
| CN113941378B CN113941378B (en) | 2023-03-03 |
Family
ID=79330474
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111198208.5A Active CN113941378B (en) | 2021-10-14 | 2021-10-14 | Nerve organoid chip based on multi-cavity electrophysiological micro-nano detection and detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113941378B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114414531A (en) * | 2022-01-30 | 2022-04-29 | 福州大学 | In-situ on-line detection method and device for metabolic molecules of organoid |
| CN115248238A (en) * | 2022-07-09 | 2022-10-28 | 浙江大学杭州国际科创中心 | Nano trap microelectrode array device and controllable preparation method and application thereof |
| CN117143728A (en) * | 2023-08-18 | 2023-12-01 | 柔脉医疗(深圳)有限公司 | Microfluidic electronic chip, organoid model preparation method and protein analysis method |
| WO2024066251A1 (en) * | 2022-09-26 | 2024-04-04 | 中国人民解放军陆军军医大学 | Integrated organ chip and preparation method therefor |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003056321A1 (en) * | 2001-12-17 | 2003-07-10 | Capital Biochip Company, Ltd. | Apparatus for stimulating an animal cell and recording its physiological signal and production and use methods thereof |
| CN101712925A (en) * | 2009-11-23 | 2010-05-26 | 浙江大学 | Multi-scale integrated cell impedance sensor for detecting behavior of single cells and cell groups |
| CN103293293A (en) * | 2013-06-24 | 2013-09-11 | 浙江大学 | Preparation method of electrochemistry immunosensor for unmarked carcinoembryonic antigen detection |
| WO2015192089A1 (en) * | 2014-06-12 | 2015-12-17 | Axion Biosystems, Inc. | Multiwell microelectrode array with optical stimulation |
| CN208911728U (en) * | 2018-09-26 | 2019-05-31 | 海清新(深圳)净化科技有限公司 | A kind of active carbon filter core and air purifier filter device |
| CN211471437U (en) * | 2019-11-27 | 2020-09-11 | 浙江霍德生物工程有限公司 | Culture assembly |
| CN111758028A (en) * | 2018-02-26 | 2020-10-09 | 富士胶片株式会社 | Flow path device |
| WO2021097447A1 (en) * | 2019-11-15 | 2021-05-20 | University Of Central Florida Research Foundation, Inc. | 3d microelectrode array (mea) for optical and electrical probing of electrogenic cells |
| CN113125509A (en) * | 2021-04-15 | 2021-07-16 | 浙江大学 | Human body odor detection and masking effect evaluation method based on biological mixing system |
-
2021
- 2021-10-14 CN CN202111198208.5A patent/CN113941378B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003056321A1 (en) * | 2001-12-17 | 2003-07-10 | Capital Biochip Company, Ltd. | Apparatus for stimulating an animal cell and recording its physiological signal and production and use methods thereof |
| CN101712925A (en) * | 2009-11-23 | 2010-05-26 | 浙江大学 | Multi-scale integrated cell impedance sensor for detecting behavior of single cells and cell groups |
| CN103293293A (en) * | 2013-06-24 | 2013-09-11 | 浙江大学 | Preparation method of electrochemistry immunosensor for unmarked carcinoembryonic antigen detection |
| WO2015192089A1 (en) * | 2014-06-12 | 2015-12-17 | Axion Biosystems, Inc. | Multiwell microelectrode array with optical stimulation |
| CN111758028A (en) * | 2018-02-26 | 2020-10-09 | 富士胶片株式会社 | Flow path device |
| CN208911728U (en) * | 2018-09-26 | 2019-05-31 | 海清新(深圳)净化科技有限公司 | A kind of active carbon filter core and air purifier filter device |
| WO2021097447A1 (en) * | 2019-11-15 | 2021-05-20 | University Of Central Florida Research Foundation, Inc. | 3d microelectrode array (mea) for optical and electrical probing of electrogenic cells |
| CN211471437U (en) * | 2019-11-27 | 2020-09-11 | 浙江霍德生物工程有限公司 | Culture assembly |
| CN113125509A (en) * | 2021-04-15 | 2021-07-16 | 浙江大学 | Human body odor detection and masking effect evaluation method based on biological mixing system |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114414531A (en) * | 2022-01-30 | 2022-04-29 | 福州大学 | In-situ on-line detection method and device for metabolic molecules of organoid |
| CN114414531B (en) * | 2022-01-30 | 2023-07-28 | 福州大学 | In-situ on-line detection method and device for organoid metabolic molecules |
| CN115248238A (en) * | 2022-07-09 | 2022-10-28 | 浙江大学杭州国际科创中心 | Nano trap microelectrode array device and controllable preparation method and application thereof |
| CN115248238B (en) * | 2022-07-09 | 2024-04-02 | 浙江大学杭州国际科创中心 | Nano trap microelectrode array device and controllable preparation method and application thereof |
| WO2024066251A1 (en) * | 2022-09-26 | 2024-04-04 | 中国人民解放军陆军军医大学 | Integrated organ chip and preparation method therefor |
| CN117143728A (en) * | 2023-08-18 | 2023-12-01 | 柔脉医疗(深圳)有限公司 | Microfluidic electronic chip, organoid model preparation method and protein analysis method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113941378B (en) | 2023-03-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113941378B (en) | Nerve organoid chip based on multi-cavity electrophysiological micro-nano detection and detection method | |
| Gross et al. | Recording of spontaneous activity with photoetched microelectrode surfaces from mouse spinal neurons in culture | |
| EP0765384B1 (en) | Device for the study of organotypic cultures and its uses in electrophysiology and biochemistry | |
| Scott et al. | Morphological and electrophysiological characteristics of dissociated chick embryonic spinal ganglion cells in culture | |
| Pine | A history of MEA development | |
| Stoppini et al. | A new extracellular multirecording system for electrophysiological studies: application to hippocampal organotypic cultures | |
| KR100291052B1 (en) | A method for measuring physicochemical properties of a tissue or a cell, a method for inspecting a drug, and a device thereof | |
| KR100466954B1 (en) | Apparatus and method for screening, olfactory mucosa stimulating compound found by the screening method, and therapeutic apparatus and electrode section for measurement | |
| CN110055221A (en) | A kind of class cerebral disease treatment tissue model and the preparation method and application thereof based on cell three-dimensional printing technique | |
| JP3204875B2 (en) | Cell potential measurement device | |
| KR20120121910A (en) | Integrated cultivation and measurement device for label-free detection and classification of cellular alterations, in particular for generation and characterisation of cell-spheroids, components and uses thereof | |
| Li et al. | Stretchable mesh microelectronics for the biointegration and stimulation of human neural organoids | |
| Maher et al. | Microstructures for studies of cultured neural networks | |
| Calvet | Patterns of spontaneous electrical activity in tissue cultures of mammalian cerebral cortex vs. cerebellum | |
| Valero et al. | Head-mounted approaches for targeting single-cells in freely moving animals | |
| CN114636744A (en) | Microelectrode array chip based on nano porous membrane and high-flux intracellular electric signal continuous monitoring system | |
| Schlapfer et al. | Spontaneous bioelectric activity of neurons in cerebellar cultures: evidence for synaptic interactions | |
| Cerina et al. | The potential of in vitro neuronal networks cultured on micro electrode arrays for biomedical research | |
| Yang et al. | Voltage-clamp recordings of postsynaptic currents in substantia gelatinosa neurons in vitro and its applications to assess synaptic transmission | |
| Egert et al. | Heart on a chip—extracellular multielectrode recordings from cardiac myocytes in vitro | |
| DE10142393C1 (en) | Apparatus measuring bioelectrical signals from spheroids, includes commensurately-sized chamber ringed internally with coplanar electrodes | |
| Zhuang et al. | A high sensitive in vivo biosensing detection for odors by multiunit in rat olfactory bulb | |
| WO2005071057A1 (en) | Magnetic or electric field stimulating device and method for promoting, restraining, or obstructing growth and function of living cell or living tissue using the magnetic or electric field stimulating device | |
| R. DeWeese | Whole‐Cell Recording In Vivo | |
| Obien et al. | CMOS-based high-density microelectrode arrays: technology and applications |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |