In vitro pain model and pain measurement method using the same

Abstract

This invention provides an in vitro pain model and a pain measurement method using the same. [Solution] A culture measurement device is provided, comprising one first chamber, one second chamber, and one channel connecting the first chamber and the second chamber; and a nerve organoid chip comprising a plurality of nerve cells, one nerve spheroid (a group of nerve cell bodies), and at least one axon bundle extending from the nerve spheroid and its terminal.

Description

Technical Field

[0001] The present invention relates to an in vitro pain model and a pain measurement method using the same.

Background Art

[0002] Measuring the pain experienced by a subject is difficult and is currently greatly limited to the observation of the subject's behavior and self-report. However, since both observation and self-report are subjective, it is difficult to objectively measure pain.

[0003] In recent years, culture devices capable of rapidly growing axon bundles extending from nerve cells in vitro, artificial axon bundles, bundles of artificial axon bundles, artificial nerves, and nerve grafts using the same have been developed (Patent Document 1). However, an in vitro pain model and a pain measurement method using the same that enable objective measurement of pain are not known.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] The present invention provides an in vitro pain model and a pain measurement method using the same.

Means for Solving the Problems

[0006] As a result of intensive research, the inventors have developed a nerve organoid chip including a culture measurement device having an electrode and a nerve organoid. The present invention includes the following [1] to [B2]. [1] It includes one first chamber, one second chamber, and one flow path connecting the first chamber and the second chamber, Culture measurement device; and It consists of multiple nerve cells and has one nerve spheroid (a group of nerve cell bodies), and at least one axon bundle extending from the nerve spheroid and its terminal. Nerve organoids A neural organoid chip that includes The nerve spheroid is located in the first chamber, and the at least one axon bundle is located in the channel. Here, (1) At least one electrode (α) is placed in the first chamber and the nerve spheroid is in contact with the at least one electrode (α); and / or (2) At least one electrode (β) is installed in the flow path, and at least one axon bundle is in contact with the at least one electrode (β), By measuring the potential of one or more of the at least one electrode (α) and / or the at least one electrode (β), it is possible to detect electrical signals from the nerve organoid. Nerve organoid chip. [2] The nerve organoid chip according to [1], wherein the nerve cells are nerve cells derived from stem cells or primary cultured nerve cells. [3] The nerve organoid chip according to [1], wherein the nerve organoid is a nerve organoid of a sensory nerve. [4] The nerve organoid chip according to [1], wherein cancer cells are present in the second chamber and are in contact with at least one axon bundle or its terminal. [5] The nerve organoid chip according to [1], wherein the cancer cells are cancer cells derived from pancreatic cancer.

[0007] [A1] A method of using a neural organoid chip according to any one of [1] to [5] as an in vitro pain model, comprising: adding any test object into the measurement device and measuring one or more potentials in the at least one electrode (α) and / or the at least one electrode (β) to detect an electrical signal from the neural organoid. [A2] The method according to [A1], further comprising calculating at least one value selected from the group consisting of firing frequency, firing intensity, and conduction velocity from the electrical signal.

[0008] [B1] A method for manufacturing a neural organoid chip, comprising: (i) comprising one first chamber, one second chamber, and one flow path connecting the first chamber and the second chamber, where at least one electrode (α) is installed in the first chamber; and / or at least one electrode (β) is installed in the flow path, preparing a culture measurement device; (ii) seeding neural cell spheroids in the first chamber of the measurement device; and (iii) culturing the neural cell spheroids to grow axon bundles therefrom and extend them into the flow path. The manufacturing method comprising the above steps. [B2] The manufacturing method according to [B1], further comprising, after step (iii), seeding and culturing cancer cells in the second chamber and contacting them with the at least one axon bundle or its end.

Advantages of the Invention

[0009] The present invention can provide an in vitro pain model and a pain measurement method using the same.

Brief Description of the Drawings

[0010] [Figure 1](A) A schematic diagram (plan view, elevation view, cross-sectional view) of a six-well culture measurement device, which is an embodiment of the present invention. (B) A schematic diagram of a neural organoid chip, which is an embodiment of the present invention. A: The first chamber (also referred to as the cell body culture chamber); a: A small well-shaped (concave) structure in the first chamber; B: The second chamber (also referred to as the terminal side chamber); C: The flow path (also referred to as the axon growth flow path); C’: A flow path different from the flow path C. α1-4: At least one electrode (α) (also referred to as the cell body measurement electrode); β1-4: At least one electrode (β) (also referred to as the axon measurement electrode). By placing the six-well culture measurement device on the head stage, potential measurement becomes possible. [Figure 2] A photograph of a neural organoid chip (± cancer cells) in the example. [Figure 3] (A) In the example, the measured potential changes at each electrode before and after adding morphine (1 mg / ml) are shown. (B) In the example, the effects of co-culture with cancer cells (Capan-1) and opioid analgesics (1 mg / ml; oxyfast and morphine) are shown. [Figure 4] (A) In the example, the effect of morphine on the firing intensity is shown. (B) In the example, the concentration-dependent effect of morphine on the firing frequency is shown. (C) In the example, the effect of co-culture with cancer cells on the firing intensity is shown. (D) In the example, the concentration-dependent effect of morphine on the firing frequency under co-culture with cancer cells is shown.

Mode for Carrying Out the Invention

[0011] Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, but are not necessarily limited thereto. The object, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description herein, and those skilled in the art will be able to easily reproduce the present invention from the description herein. The embodiments and specific examples of the invention described below are examples of preferred embodiments of the present invention and are provided for illustrative or explanatory purposes only; the present invention is not limited thereto. It will be apparent to those skilled in the art that various modifications and modifications can be made based on the description herein, within the intent and scope of the present invention as disclosed herein.

[0012] One embodiment of the present invention is a nerve organoid comprising a plurality of nerve cells, having one nerve spheroid (i.e., a group of nerve cell bodies), and at least one axonal bundle extending from the nerve spheroid and its terminal. A nerve cell is a cell that makes up each part of the nervous system, either the central nervous system or the peripheral nervous system. In this invention, the term "neuron" is used synonymously with "neuron" to refer to the entire cell. Nerve cells are mainly divided into three parts: the cell body containing the cell nucleus, the dendrites that receive input from other cells, and the axon and its terminal that output signals to other cells. The cell body (soma) is the part of a nerve cell where organelles such as the cell nucleus are concentrated, and where the dendrites and axon meet. The axon is a projection-like structure extending from the cell body, and is responsible for outputting signals in nerve cells. The length of the axon varies greatly within nerve cells; in humans, it ranges from a few micrometers long that connects to adjacent cells to over one meter long that extends into the spinal cord. Nerve cells include motor nerve cells, sensory nerve cells, and autonomic nerve cells. An axon bundle is a bundle of (single) axons extending from multiple nerve cells. While not specifically limited, it can consist of two or more, ten or more, or even one hundred or more axons.

[0013] Stem cells are cells that possess the ability to self-renew by dividing and creating identical cells (self-renewal) and the ability to differentiate into other types of cells, allowing them to proliferate indefinitely. Examples of pluripotent stem cells include embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells). Neural stem cells are another type of multipotency stem cell. These can be induced to differentiate into nerve cells. Nerve cells derived from stem cells are nerve cells that have been differentiated from stem cells. By using nerve cells derived from stem cells, contamination by pathogens and other pathogens can be avoided.

[0014] Primary cultured nerve cells refer to cell cultures that directly utilize animal tissue. Primary cultured cells are thought to maintain a state close to that of living organisms and are therefore used in various research applications. Regarding the cells that make up nerve tissue, almost all nerve system cells, including neurons (nerve cells) and glial cells (neuroglial cells) such as astrocytes, oligodendrocytes, Schwann cells, and microglia, can be primary cultured.

[0015] The length of the axon bundle is not particularly limited as long as it fits within the following channel (C), but may be 1 mm or more, 5 mm or more, 10 mm or more, 20 mm or more, 30 mm or more, 40 mm or more, 50 mm or more, or 100 mm or more. The diameter of the axon bundle is not particularly limited as long as it fits within the following channel (C), but may be 10 μm or larger, 25 μm or larger, 50 μm or larger, or 100 μm or larger. The length and diameter of the axon bundle can be measured, for example, by analyzing images of the axon bundle taken using an optical microscope or digital camera, as the length of the axon bundle in the image. Similarly, the diameter of the axon bundle can be measured as the minimum thickness of the axon bundle in the image by analyzing images of the axon bundle taken using an optical microscope or electron microscope.

[0016] One embodiment of the present invention is, It includes one first chamber, one second chamber, and one flow path connecting the first chamber and the second chamber, (1) At least one electrode (α) is installed in the first chamber, and / or (2) At least one electrode (β) is installed in the flow path, This is a culture measurement device.

[0017] As illustrated in Figure 1, it is preferable that the first chamber (A) and the second chamber (B) have well-shaped (concave) structures capable of holding liquids such as culture media. Although not particularly limited, the first chamber (A) may have an even smaller well-shaped (concave) structure (a) at its bottom. The lower ends of the side walls of the first chamber (A) and the second chamber (B) are open, and the openings are connected via a flow path (C). If a smaller well-shaped (or concave) structure (a) is provided at the bottom of the first chamber (A), an opening may exist in the well-shaped (or concave) structure (a), and this opening may be connected to the opening at the lower end of the side wall of the second chamber (B) via a flow path (C). Preferably, the flow path (C) does not open to any part other than the connection between the opening of the first chamber (A) and the opening of the second chamber (B). Although not particularly limited, as shown in the cross-sectional view of Figure 1, openings may also exist at the upper end of the side wall of the first chamber (A) and the upper end of the side wall of the second chamber (B), allowing for liquid exchange between the first chamber (A) and the second chamber (B) via a different flow path (C') than the flow path (C). The bottom shape of the first chamber (A) (or small well-shaped (concave) structure (a)) and the second chamber (B) is not particularly limited, but is preferably circular. The bottom area of ​​the first chamber (A) and the second chamber (B) is not particularly limited, but is preferably 7.0 mm². 2 ~28mm 2 Preferably, the bottom area of ​​the small well-shaped (concave) structure (a) in the first chamber (A) is not particularly limited, but is 0.4 mm 2 ~7.0mm 2 Preferred The bottom surface of the first chamber (A) (or the bottom surface of the small well-like (concave) structure (a) is preferably made of a material to which nerve cell bodies can adhere (or is coated with a cell adhesion factor such as adhesive collagen). The width and height of channel (C) are not particularly limited as long as the liquid can move through channel (C) and the axon bundle can extend, but may be 80 to 150 μm, and its height may be 80 to 200 μm. The length of channel (C) is not particularly limited as long as the axon bundle can exist, but may be 0.5 mm or more, 1 mm or more, 5 mm or more, 10 mm or more, 20 mm or more, 30 mm or more, 40 mm or more, 50 mm or more, or 100 mm or more.

[0018] At least one electrode (α) may be installed at the bottom of the first chamber (A), on the well side. If a smaller well-shaped (concave) structure (a) is provided at the bottom of the first chamber (A), it is preferable that at least one electrode (α) is installed at the bottom of the smaller well-shaped (concave) structure (a), on the well side. The number of electrodes (α) is not particularly limited as long as the electrodes (α) do not come into contact with each other, but it is preferable that 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 electrodes are present in the first chamber (A). At least one electrode (β) may be installed on the inside of the flow path (C). The electrode (β) is preferably installed perpendicular to the flow path (C) and is preferably installed at the connection with the first chamber (A) (or the small well-shaped (concave) structure (a) within (A)) or at the connection with the second chamber (B). If there are multiple electrodes (β), they are preferably installed at equal intervals with respect to the length of the flow path. The number of electrodes (β) is not particularly limited as long as the electrodes (β) do not come into contact with each other, but it is preferable that 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 electrodes are present in the flow path (C). The size of electrodes (α) and (β) is not particularly limited as long as the electrodes do not come into contact with each other within the first chamber (A) or flow path (C), but a size of approximately 10 μm to 1 mm x 10 μm to 1 mm is preferred. The material of the electrodes is not particularly limited. Electrodes (α) and (β) can be connected directly or via wiring to an external potential measuring device.

[0019] One embodiment of the present invention is a neural organoid chip containing the above-mentioned culture measurement device and the above-mentioned neural organoid. In one embodiment, as illustrated in Figure 1, the nerve organoid chip has a nerve spheroid located in a first chamber (A) (or in a well-shaped (concave) structure (a)) and in contact with at least one electrode (α), and at least one axon bundle located in the channel (C) and in contact with at least one electrode (β). As a result, it becomes possible to detect electrical signals from the nerve organoid by measuring the potential of electrode (α) and / or electrode (β). The electrical signals from the nerve organoid mimic the electrical signals from nerves in the body, and the nerve organoid chip can be used as an in vitro model of in vivo sensation (sight, hearing, touch, taste, smell). In particular, nerve organoid chips using nerve organoids with high positive rates for thermal pain nerve markers such as TrkA and CGRP enable objective and quantitative evaluation of pain, including touch. The nerve organoid chip may contain a liquid such as a culture medium, as long as it does not interfere with the detection of electrical signals from the nerve organoid. Using a reference electrode in contact with the liquid such as a culture medium, electrical signals (also called action potentials) from nerve cells in contact with electrodes (α) and (β) can be measured.

[0020] In one embodiment of the present invention, the nerve organoid chip may further contain other eukaryotic or prokaryotic cells. Examples include, but are not limited to, cells and tissue aggregates from any normal biological tissue (liver, kidney, heart, lung, muscle, etc.) that can be cultured from mammals (human, mouse, rat, etc.), cells from benign tumors, cells from malignant tumors (including sarcomas) that have the ability to invade surrounding tissues and / or metastasize to distant sites (also known as cancer cells), and microbial cells (cells of beneficial microorganisms such as lactic acid bacteria; cells of pathogenic microorganisms such as streptococci, etc.). A nerve organoid chip containing these cells can mimic the effects of these cells' invasion on nerves. While not particularly limited, it is preferable that such cells be added to the second chamber (B) and brought into contact with the axon bundle or its terminals. The cancer cells may be primary cancer cells derived from the patient or cancer cells from established cancer lines. While not particularly limited, cancer cells that cause cancer pain (including neuropathic pain) are preferred. Cancer cells known to cause cancer pain include those derived from pancreatic cancer, tongue cancer, pharyngeal cancer, esophageal cancer, stomach cancer, pancreatic cancer, liver cancer, colorectal cancer, etc.

[0021] One embodiment of the present invention is a method for using the above-mentioned neural organoid chip as an in vitro sensory model, The method includes the step of detecting an electrical signal from the nerve organoid by adding an arbitrary test substance to the measuring device and measuring the potential of at least one electrode (α) and / or at least one electrode (β). The type of sensory model used depends on the type of nerve cells forming the nerve organoid, but in the case of a nerve organoid chip using nerve organoids with a high positive rate for temperature and pain nerve markers, it can be used as a model for pain, including touch.

[0022] The test substance is added to the culture measurement device and brought into contact with the nerve organoid. Although not particularly limited, if the device is filled with a liquid such as a culture medium, the liquid containing the test substance may be added to the culture measurement device. The addition location may be the first chamber (A), the second chamber (B), or a different channel (C') from the channel (C). The test subjects are not particularly limited, but those that may affect the activity of nerve organoids are preferred. In addition to the eukaryotic or prokaryotic cells mentioned above, the test subjects include, but are not limited to, compounds, extracts, etc. Compounds, extracts, etc. include neurotransmitters (serotonin, norepinephrine, dopamine, etc.), psychotropic drugs, opioid analgesics (morphine, oxycodone, etc.) or non-opioid analgesics, viruses, and chemicals that cause chemical sensitivity. After adding the test substance, the potential from the nerve organoid is measured. Changes in the potential of electrode (α) and / or electrode (β) are detected using a reference electrode in contact with a liquid such as culture medium, by connecting electrode (α) and / or electrode (β) directly or via wiring to a potential measuring instrument. At this time, the potentials of multiple electrodes (α) or multiple electrodes (β) may be measured. Such measurements can be used to normalize the changes in the potentials of electrode (α) and electrode (β). By comparing the potential of the test substance with that of nerve organoids before its addition, the effect of the test substance on sensory perception can be estimated.

[0023] To quantify the effect on the sensory perception of the test object, the ignition frequency, ignition intensity, conduction velocity, etc., may be calculated from the obtained electrical signal data (also called ignition data). The ignition intensity may also be calculated by detecting the maximum and minimum voltage values ​​after removing abnormal values ​​from the obtained electrical signal data, and then using the formula: Ignition Intensity = Maximum Value - Minimum Value. The firing frequency may be calculated by determining the number of times a certain threshold is crossed from the obtained electrical signal data, and then averaging the frequency (number of times / second) over all data. The conduction velocity may be determined by measuring the waveform of the electrical change at different electrodes (α) and (β), and defining conduction velocity as the velocity of waveform movement (for example, by comparing the waveform data of electrode (β1) measuring the axon bundle 0.1 mm away from the cell body with electrode (β2) measuring the axon bundle 0.2 mm away, the time lag between the waveform detected by electrode (β1) and the waveform detected by electrode (β2) can be defined as the velocity of waveform movement).

[0024] One embodiment of the present invention is a method for manufacturing the above-mentioned nerve organoid chip. Such manufacturing methods (i) Steps to prepare the culture measurement device described above; (ii) The step of seeding nerve cell spheroids into the first chamber of the measuring device; and (iii) A step of culturing nerve cell spheroids and thereby growing axonal bundles from the spheroids and extending them into the channel. Includes. Step (i) may include filling the culture measurement device with culture medium. The culture medium is filled into at least the first chamber (A), the second chamber (B), and the channel (C). Preferably, the culture medium is added until a different channel (C') is filled. Any conventionally known culture medium suitable for culturing nerve cells can be used as the culture medium. For example, DMEM (Dulbecc's modified Eagle medium) or Neurobasal medium can be used as the base medium, with supplements such as N2 or B27, or neurotrophic factors such as BDNF (Brain-derived neurotrophic factor) added. In step (ii), nerve cells are seeded inside the first chamber (A) or a small well-shaped (concave) structure (a). The number of seeded cells and the cell density can be set as appropriate. There are no particular limitations, but the number of seeded cells is 1 × 10⁻⁶. 3 ~1 × 10 5 It is preferable. It is also possible to seed nerve cell spheroids. It is preferable that at least a portion of the nerve cell body is in contact with electrode (α). In step (iii), the axons of multiple nerve cells extend and form axon bundles that contact the electrode (β) within the channel (C). It is preferable to culture until the ends of the axon bundles reach the second chamber (B). During this time, the culture medium may be changed as appropriate.

[0025] In the above method for manufacturing a neural organoid chip After step (iii), the process may further include seeding and culturing cancer cells in the second chamber and bringing them into contact with at least one axon bundle or its terminal. [Examples]

[0026] Example: Evaluation of the effects of opioid analgesics on cancer cell co-culture using nerve organoid chips. 1. Manufacturing of neural organoid chips Sensory nerve cell bodies (spheroids) differentiated from human iPS cells were seeded inside a small well-like (concave) structure (a) in the first chamber (A) shown in Figure 1, and cultured for 19 days in an incubator at 37°C / 100%RH / 5% by volume of carbon dioxide, with culture medium changes. The culture medium was prepared by adding the following components to Neurobasal plus medium (Thermo Fisher Scientific), the basal medium: GDNF 20 ng / ml, BDNF 20 ng / ml, β-NGF 20 ng / ml, NT-3 10 ng / ml, B27Plus supplement (1x), and Penicillin-Streptomycin (Thermo Fisher Scientific) (1 / 100).

[0027] 2. Co-culture of cancer cells and addition of opioid analgesics After measuring the electrical potential (indicated as "before co-culture" in the figure), pancreatic cancer-derived Capan-1 cells (1x10⁶) were placed in the second chamber (B) of some of the neural organoid chips. 3 ~1x10 5Capan-1 cells were seeded. Co-culture was then carried out for 9 or 10 days (Figure 2). Other neural organoid chips were cultured without seeding Capan-1 cells. In all cultures, the culture medium was changed to a 10-20% serum medium suitable for cancer cell culture at the start of co-culture. After measuring the potential (indicated as "Capan-1 co-culture" in the figure), opioid analgesics such as morphine or Oxyfast (oxycodone) were added to the culture medium at concentrations of 1 mg / ml (indicated as "1 / 10" in the figure), 100 μg / ml (indicated as "1 / 100" in the figure), 10 μg / ml (indicated as "1 / 1000" in the figure), or 1 μg / ml (indicated as "1 / 10000" in the figure). After culturing for 3 days following the addition of the opioid, the potential of the nerve organoids was measured. The measurements were performed using the MCS MEA2100 system (headstage, interface board, temperature controller (37°C environment)) and Multi Channel Experimenter software (measurement frequency 20 kHz). Data analysis was performed using Multi Channel Analyzer software.

[0028] 3. result The results are shown in Figures 3 and 4. The addition of morphine significantly reduced the firing intensity and firing frequency of nerve organoids (Figures 3A and 4A,B). This indicates that the present invention can mimic the analgesic effect of morphine in living organisms (which is due to the suppression of nerve action potentials). When cancer cells were co-cultured, the activity of nerve organoids was significantly activated, increasing their firing intensity (Figure 4C), which was then suppressed by opioid analgesics (Figure 3B). This indicates that the present invention can serve as an in vitro model for cancer pain. Furthermore, the electrical signals (firing frequency) of nerve organoids activated by co-culture were suppressed in a concentration-dependent manner by opioid analgesics (Figure 4D). This indicates that the present invention allows for the in vitro quantification of the effects of analgesics. [Industrial applicability]

[0029] This invention allows for the objective evaluation of pain. It is said that approximately 10-30% of cancer pain is intractable pain accompanied by nerve compression and infiltration that does not respond to opioid analgesics. Using this invention, it becomes possible to quantitatively screen for analgesics that are even more effective than strong opioid analgesics such as morphine.

Claims

1. It includes one first chamber, one second chamber, and one flow path connecting the first chamber and the second chamber. Culture measurement device; and It consists of multiple nerve cells and has one nerve spheroid (a group of nerve cell bodies), and at least one axon bundle extending from the nerve spheroid and its terminal. Nerve organoids A neural organoid chip including, The nerve spheroid is located in the first chamber, and at least one axon bundle is located in the channel. Here (1) At least one electrode (α) is placed in the first chamber and the nerve spheroid is in contact with the at least one electrode (α); and / or (2) At least one electrode (β) is installed in the flow path, and at least one axon bundle is in contact with the at least one electrode (β), By measuring the potential of one or more of the at least one electrode (α) and / or the at least one electrode (β), it is possible to detect electrical signals from the nerve organoid. Nerve organoid chip.

2. The nerve organoid chip according to claim 1, wherein the nerve cells are nerve cells derived from stem cells or primary cultured nerve cells.

3. The nerve organoid chip according to claim 1, wherein the nerve organoid is a nerve organoid of a sensory nerve.

4. The nerve organoid chip according to claim 1, wherein cancer cells are present in the second chamber and are in contact with at least one axon bundle or its terminal.

5. The nerve organoid chip according to claim 4, wherein the cancer cells are cancer cells derived from pancreatic cancer.

6. A method for using a neural organoid chip as an in vitro sensory model according to any one of claims 1 to 5, A method comprising the step of detecting an electrical signal from a nerve organoid by adding an arbitrary test substance to the measuring device and measuring the potential of one or more of the at least one electrode (α) and / or the at least one electrode (β).

7. The method according to claim 6, further comprising the step of calculating at least one value selected from the group consisting of ignition frequency, ignition intensity, and conduction velocity from the electrical signal.

8. A method for manufacturing nerve organoid chips, (i) comprising one first chamber, one second chamber, and one flow path connecting the first chamber and the second chamber, At least one electrode (α) is installed in the first chamber; and / or at least one electrode (β) is installed in the flow path. The process of preparing a culture measurement device; (ii) A step of seeding nerve cell spheroids in the first chamber of the measuring device; and (iii) A step of culturing the nerve cell spheroids and thereby growing axonal bundles from the spheroids and extending them into the channel. A manufacturing method that includes this.

9. The manufacturing method according to claim 8, further comprising the step of seeding and culturing cancer cells in the second chamber after step (iii) and bringing them into contact with at least one axon bundle or its terminal.

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