JP7151966B2 - Apparatus for culturing nerve cells, method for culturing nerve cells, method for quantifying morphological degeneration of axonal bundles by orientation analysis, method for analyzing and identifying proteins in nerve tissue and axonal bundles, and use of nerve cells Method - Google Patents

Description

The present disclosure includes an apparatus for culturing nerve cells, a method for culturing nerve cells, a method for quantifying morphological degeneration of axonal bundles by orientation analysis, a method for analyzing and identifying proteins in nerve tissue and axonal bundles, and It relates to the use of nerve cells.

In vitro (extracorporeal) culture of neurons and axons is necessary for the rapid development of drugs effective against neurological diseases. Conventionally, several devices for culturing nerve cells and axons have been proposed (see, for example, Non-Patent Document 1 and Patent Document 1). These devices are microfluidic platforms with multiple compartments that allow axons to grow and separate.

Taylor, Anne M et al. “A Microfluidic Culture Platform for CNS Axonal Injury, Regeneration and Transport ” Nature methods 2.8 (2005):599-605. PMC. Web. 8 Mar. 2016.

U.S. Patent Application Publication No. 2004/0106192 (A1)

However, the conventional techniques described above are not sufficient. In order to evaluate nerve tissue in drug screening, etc., it is necessary to evaluate cell bodies, axons, and junctions between cell bodies and axons, respectively. cannot be divided and evaluated.

One of the advantages of the present disclosure is the ability to provide an apparatus for culturing neurons that rapidly grows axonal bundles extending from the neurons in vitro.

In a method, a method of culturing neurons with axons, comprising: culturing in one first chamber , one second chamber , and one channel connecting the first and second chambers. supplying fluid, wherein the first chamber, the second chamber and the channel are contained in a module arranged in a culture plate; seeding each neuronal spheroid; culturing the neuronal cells such that an axonal bundle grows and extends within each channel, the spheroids being connected to each other by the axonal bundle. .

A method for quantifying morphological degeneration of axonal bundles by orientation analysis comprises the steps of: applying stress or a drug to an axonal bundle; and measuring the orientation of the axonal bundle to which the stress or drug is applied. A method for quantifying morphological degeneration of an axonal bundle by orientation analysis, wherein the axonal bundle comprises one first chamber capable of receiving a cell body of a nerve cell, and a cell of a nerve cell axons received in and extending into a channel connecting a second chamber capable of receiving a body and connecting spheroids each seeded within both the first and second chambers; In a bundle, the orientation is the direction in which the channels extend.

According to the present disclosure, axon bundles extending from nerve cells can be rapidly grown in vitro.

It is a schematic diagram which shows the background in this Embodiment. It is a figure which shows the concept in this Embodiment. FIG. 2 is a diagram showing a culture plate according to the present embodiment; FIG. FIG. 4 is a diagram showing culture modules in a culture plate in the present embodiment; FIG. 4 shows a culture module with seals according to the present embodiment; 4 is a photograph showing axons grown in channels of a culture plate in the present embodiment. FIG. 10 is another photograph showing axons grown in the channels of the culture plate in the present embodiment. FIG. 4 is a photograph showing an increase in the number of axons grown in the channels of the culture plate in this embodiment. FIG. 4 is a diagram for explaining extension velocity measurement of an axon in a channel of a culture plate according to the present embodiment; 4 is a photograph showing axons extracted from a culture plate in this embodiment. FIG. 10 shows an experiment in which axons of 409B2 cell line-derived motor neurons and skeletal muscles of laboratory mice are spliced together in the present embodiment. FIG. 4 shows a long-term experiment of splicing 409B2 cell line-derived motor neuron axons with skeletal muscle of laboratory mice in this embodiment. FIG. 2 shows an experiment in which axons of motor neurons derived from the 409B2 cell line and skeletal muscle derived from the C2C12 cell line are spliced together according to the present embodiment. 4 is a photograph of an axonal bundle used for protein observation in the present embodiment. 4 is a photograph of an axon bundle used for stretching in the present embodiment. FIG. 10 is a diagram illustrating the results of a second experiment in which axons are stretched according to the present embodiment; 4 is a photograph of motor neurons used for calcium imaging in the present embodiment. FIG. 4 is a diagram explaining the results of an experiment for measuring action potential changes in the present embodiment; FIG. 4 is a schematic diagram showing various examples of a culture module according to the present embodiment; It is a figure which shows the process of isolate|separating an axon from a cell body in this Embodiment. It is a schematic diagram showing the cause of ALS. It is a photograph showing the result of a stress test in the present embodiment. FIG. 10 is a diagram showing axonal orientation evaluation results of a stress test in the present embodiment; FIG. 10 is a photograph showing the results of co-culturing with glial cells in the present embodiment. FIG. FIG. 10 is a photograph of axon bundles extending from cell body spheroids with and without glial cells in the present embodiment. FIG. FIG. 4 is a diagram showing the results of observation of axonal bundles grown in the channels of the culture plate in the present embodiment. 4 is a photograph showing a cross-section of nerve cells containing axonal bundles grown in channels of a culture plate according to the present embodiment. Fig. 10 is another photograph showing a cross section of nerve cells containing axonal bundles grown in the channels of the culture plate according to the present embodiment. 4 is a photograph showing the result of observation with a scanning electron microscope of the surface of an axonal bundle grown in the channels of the culture plate in the present embodiment. 4 is a photograph showing axon bundles when stress is applied in the channels of the culture plate in the present embodiment. 4 is a graph showing experimental results of morphological changes in axon bundles when stress is applied in the channels of the culture plate in the present embodiment. FIG. 3 is a diagram showing myelination of axons in a culture plate module according to the present embodiment. FIG. 2 is a schematic diagram showing applicable fields of the apparatus according to the present embodiment; FIG. 3 is a schematic diagram showing another applicable field of the device according to the present embodiment;

Hereinafter, this embodiment will be described in detail with reference to the drawings.

FIG. 1 is a schematic diagram showing the background of this embodiment, and FIG. 2 is a diagram showing the concept of this embodiment. In FIG. 2, (a) is a schematic diagram of nerve cells, (b) is a diagram showing the results of conventional culture, and (c) is a diagram showing the concept of this embodiment.

This embodiment proposes a new process for culturing neurons with axons, a new apparatus suitable for culturing and a cluster of neurons with cultured axonal bundles. In order to rapidly search for drugs effective against neurological diseases, it is important to culture nerve cells in an environment similar to that in the body. As shown in FIG. 2, a neuron comprises a cell body and an axon with an axon. As shown in FIG. 1, in the body, the axons of motor nerves form bundles, and the axon terminals are joined to a plurality of skeletal muscle cells. Thus, in vitro culture results in cells with bundled axons, with cell bodies, axons and axon terminals spatially separated from each other, as shown in Figure 2(c). It is desirable to generate body masses, ie, spheroids of cell bodies. In order to accurately confirm the effect of a drug, it is necessary to apply the drug to each site, such as the cell body, axon bundle, and axon terminal, and evaluate the effect of the drug at each site. is. However, as shown in FIG. 2(b), conventional culture methods could only provide cell bodies with axons in a random state in which cell bodies and axons are spatially mixed.

Next, the configuration of the device used in this embodiment will be described.

FIG. 3 is a diagram showing a culture plate in this embodiment, FIG. 4 is a diagram showing a culture module in the culture plate in this embodiment, and FIG. 5 is a diagram showing a culture module provided with a seal in this embodiment. be. 3, (a) is a plan view, (b) is an enlarged side cross-sectional view of the culture module in (a), and in FIG. 4, (a) is a photograph of the culture plate, and (b) is the culture module. , and each of (c) to (e) is a schematic plan view of the culture module. In FIG. 5, (a) is a schematic side cross-sectional view of the culture module provided with the seal, (c) is a schematic side cross-sectional view of the culture module with seals and cells, (d) is a schematic side cross-sectional view of the culture module with seals and cell body spheroids, (e) [Fig. 3] is a schematic side cross-sectional view of a culture module without a seal.

In this embodiment, a culture plate 10 is used. As shown in FIGS. 3(a) and 4(a), the culture plate 10 is a rectangular plate-like member and includes a plurality of modules 11 arranged side by side in row and column directions. The culture plate 10 includes a substrate 15 made of a cover slip, which is a transparent glass plate, and a top plate 16 attached to the surface of the substrate 15 . A plurality of modules 11 are formed on the top plate 16 . As shown in FIGS. 3 and 4, each module 11 has a dumbbell-like shape in plan view, and has a U-shaped cross-sectional shape as a whole. It includes two chambers 12b and a channel 13 connecting the bottoms of the first and second chambers 12a, 12b.

The top plate 16 may be a PDMS (poly-dimethyl-siloxane) sheet and can be fabricated using known photolithographic techniques (see, for example, Non-Patent Document 1 and Patent Document 1). It should be noted that the top plate 16 may be, for example, a plate-like member made of other polymers such as Pyrex (registered trademark) or glass, and is made using methods such as hot embossing, drilling, and the like. It can be anything.

Each of the first and second chambers 12a and 12b is a cylindrical recess, and is formed as a well-shaped depression whose bottom is closed by the substrate 15 and whose top is open. One end of the channel 13 opens to the lower end of the side wall of the first chamber 12a, and the other end opens to the lower end of the side wall of the second chamber 12b. The width of the channel 13 is preferably 100 to 150 [μm] and the height is preferably 100 to 200 [μm], but these dimensions are not limited to such numerical values. Can be adjusted as needed. The insides of the first and second chambers 12a and 12b and the channel 13 are filled with the culture medium 18. As shown in FIG. Since the structure of the module 11 is simple, the culture medium 18 can smoothly flow into the module 11 . In addition, since the size of the channel 13 is larger than that in conventional devices (see, for example, Non-Patent Document 1 and Patent Document 1), the culture solution 18 in the first and second chambers 12a, 12b naturally can be mixed with The planar shape of each of the first and second chambers 12a and 12b is desirably circular as shown in FIG. be able to. In the examples shown in FIGS. 3(a), 4(c) and 4(d), since the inlet 13a of the channel 13 has a tapered planar shape, the planar shape of the first chamber 12a is perfect. Although it is not circular but deformed, the planar shape of the second chamber 12b is almost perfectly circular because the inlet 13a of the channel 13 is linear. The axon is effectively guided into the channel 13 when the entrance portion 13a has a tapered planar shape. The axons can form bundles within the channel 13 even if the inlet 13a is linear.

The interior of one of the first and second chambers 12a, 12b is placed or seeded with neuronal cell bodies. Either of the first and second chambers 12a and 12b can be selected as the chamber in which the cell bodies are seeded. It is assumed that the chamber is In the example shown in FIG. 4(c), multiple neuronal cell bodies are seeded separately, ie in a dissociated cell state. Also, in the examples shown in FIGS. 4(d) and (e), multiple neuronal cell bodies are densely seeded, ie, in the form of spheroids. After culturing for a while, the axons of the nerve cells extend through the channel 13 to the second chamber 12b and form a bundle within the channel 13 . The inner diameter of channel 13 is desirably large enough to accommodate an axonal bundle of nerve cells.

As shown in FIGS. 5(a) and (b), a sealing member 17 is placed over the second chamber 12b to close the open end of the second chamber 12b, which is not seeded with nerve cell bodies therein. It is desirable to place The sealing member 17 is preferably made of a PCR (Polymerase Chain Reaction) sealing plate or sealing film, but is not necessarily limited to this, and may be made of any material suitable for sealing. It may be composed. As shown in FIGS. 5(c) and 5(d), when the open end of the second chamber 12b is closed by the sealing member 17, the culture medium is prevented from flowing into the second chamber 12b. It becomes easier to seed and keep cell bodies in the first chamber 12a. As shown in FIG. 5(e), if the open end of the second chamber 12b is left open, the neuronal cell bodies placed in the first chamber 12a will flow into the second chamber 12b together with the culture medium. prone to influx. If nerve cell bodies are seeded in the first chamber 12a in the form of spheroids, the sealing member 17 can be omitted because the cell body spheroids are too large to flow through the channel 13. FIG.

Next, the results of an axon growth experiment conducted by the inventor using the culture plate 10 according to the present embodiment will be described.

FIG. 6 is a photograph showing axons grown in the channels of the culture plate in this embodiment, FIG. 6A is another photograph showing axons grown in the channels of the culture plate in this embodiment, and FIG. Photographs showing an increase in the number of axons grown in the channels of the culture plate in the embodiment, FIG. 4 is a photograph showing axons extracted from a culture plate in this embodiment. In FIG. 6, (a) is a photograph of part of the culture plate, (b) is a photograph of one axon of the modules shown in (a), and in FIG. 6A, (a) is an axon FIG. 6B is a photograph showing an example in which the length of the axon is 4.5 [mm], (b) is a photograph showing an example in which the length of the axon is 9 [mm], and (a) to (f) in FIG. 6B is a photograph showing axons cultured for 0 to 5 days, and in FIG. (c) is a graph showing the measurement results of the axon extension speed. In FIG. 7, (a) is an enlarged photograph of nerve tissue, and (b) is a fluorescent photograph of cell nuclei (marker Hoechst), (c) is a fluorescence photograph of the whole nerve (using marker Tuj 1 ).

In this experiment, motor neurons derived from human iPS cells (409B2 cell line) were used. Motor neuron cell body spheroids were seeded and cultured in the first chamber 12a. Motor neurons were obtained from human iPS cells using well plates or dishes. Cell body spheroids were then obtained using non-adhesive culture plates. The culture was carried out in an environment of 37[°C], _O2 : 20[%], _CO2 : 5[%]. Then, as shown in FIG. 6, the axon grew inside the channel 13 and extended to the second chamber 12b. After culturing, the motor neuron aggregates were extracted, ie removed, from the module 11 of the culture plate 10 . The assembly of motor neurons extracted from the module 11 comprises large axonal bundles extending from the assembly of cell bodies, as shown in FIG. 7(a). As shown in FIG. 7(b), cell nuclei present in the cell bodies and stained with the marker Hoechst were observed in aggregates of motor neurons. In addition, aggregates of motor neurons stained with the marker Tuj 1 were observed, as shown in FIG. 7(c). Axonal bundles were clearly recognized as extending from aggregates of cell bodies. By changing the length and inner diameter of channel 13, axon bundles of various sizes can be obtained. For example, in the example shown in FIG. 6A(a), the axon length is 4.5 [mm], and in the example shown in FIG. 6A(b), the axon length is 9 [mm]. be. For use in observations and experiments, the length of the axon is desirably 1 [mm] or more. Also, the width of the axon bundle can be set to 100 [μm], for example. In this case, it can be seen that about 5500 axons are included when observing a cross-sectional TEM image of the axon bundle as shown in FIG. 20(c) described later.

Further, when the cell body spheroids were seeded in the first chamber 12a and the culture was continued, the process of axon bundles in the channel 13 could be observed. Specifically, as shown in FIG. 6B, the axons in the channel 13 were photographed every culture day, and the number was counted. As shown in FIGS. 6B (a) to (f), the number of axons is 3 when the number of days of culture is 0, the number of axons is 12 when the number of days of culture is 1, and the number of days of culture is 2. The number of axons is 15 when cultured for 3 days, the number of axons is 24 when cultured for 3 days, the number of axons is 28 when cultured for 4 days, and the axons when cultured for 5 days. A bundle was formed with 30 or more pieces.

Furthermore, after seeding the cell body spheroids in the first chamber 12a, the migration speed of the axon terminal in the X-axis direction (longitudinal direction of the channel 13), that is, the extension speed of the axon in the channel 13 was measured. could be measured. In this measurement, a module 11 was used in which graduations 14 including major graduations 14a and minor graduations 14b were provided on both sides of the channel 13, as shown in FIGS. 6C(a) and (b). Then, the position of the axon terminal is measured for each culture day, and the relationship between the number of culture days and the distance to the axon terminal as shown in FIG. was able to establish a relationship with In FIG. 6C(c), the horizontal axis indicates the number of culture days, and the vertical axis indicates the distance to the axon terminal. From the relationship shown in FIG. 6C(c), it is possible to calculate the moving speed of the axon terminal in the channel 13, that is, the extension speed of the axon.

As described above, according to the present embodiment, axon bundles can be obtained by culturing in each module 11 of culture plate 10 . Therefore, by applying drugs to the axonal bundles in each module 11, drug screening can be performed quickly. Furthermore, since the axonal bundles are separated from the cell bodies in each module 11, it is possible to screen drugs that are effective against neurological diseases by precisely applying drugs to the axonal bundles in each module 11. Also, it is possible to ascertain which part of the axonal bundle, for example the distal end or the proximal end, is affected by the drug.

Next, the results of an experiment conducted by the inventor using the culture plate 10 according to the present embodiment to connect axons and skeletal muscles will be described.

FIG. 8 is a diagram showing an experiment in which the axon of the 409B2 cell line-derived motor nerve cell and the skeletal muscle of a laboratory mouse in this embodiment are joined together. FIG. 9 shows the 409B2 cell line-derived motor nerve in this embodiment Fig. 10 shows a long-term experiment of joining cell axons and skeletal muscle of laboratory mice. It is a figure which shows the experiment which joins. 8 and 9, (a) is a photograph of a laboratory mouse myotube, (b) is a schematic plan view of a module, (c) is an enlarged photograph of an axon without myotube, and (d). 10A and 10B are enlarged photographs of an axon with myotubes. In FIG. 10, (a) is a schematic plan view of a module, and (b) is an enlarged photograph of a junction.

In the first experiment, human iPS cell (409B2 cell line)-derived motor neurons were used as neurons, and myotubes from laboratory mice (shown in FIG. 8(a)) were used as skeletal muscles. Motor neuron and cell body spheroids were obtained in the same manner as described above. In addition, their culture was also performed by the same method as described above. As shown in FIG. 8(b), nerve cells were seeded in the first chamber 12a and skeletal muscle was seeded in the second chamber 12b to co-culture nerve cells and skeletal muscle. It was found that when skeletal muscle was seeded in the second chamber 12b, the axonal growth rate was higher and the axonal bundles grew and thickened faster than otherwise. FIGS. 8(c) and (d) show the inside of the channel 13 after 28 days of culture when the myotubes were not seeded in the second chamber 12b and when the myotubes were seeded in the second chamber 12b, respectively. axonal growth state. In the second experiment, for a longer period (43 days) than in the first experiment (28 days), but similar to the first experiment, as shown in Figures 9(a) and (b). module 11 and similar motor neurons and myotubes were similarly used and co-cultured. It has been found that if myotubes are not seeded in the second chamber 12b, the morphology of the axon bundles is degenerated. Figures 9(c) and (d) show the inside of the channel 13 after 43 days of culture when the myotubes were not seeded in the second chamber 12b and when the myotubes were seeded in the second chamber 12b, respectively. axonal growth state.

In the third experiment, motor neurons derived from human iPS cells (409B2 cell line) were used as neurons, and striated muscles derived from mouse myogenic cells (C2C12 cell line) were used as skeletal muscles. As shown in FIG. 10(a), nerve cells were seeded in the first chamber 12a and skeletal muscle was seeded in the second chamber 12b to co-culture nerve cells and skeletal muscle. Then, the junction between the axon and skeletal muscle was stained with α-Bungarotoxin, and the axon was stained with Tuj 1. As shown in FIG. 10(b), junctions between axons and skeletal muscle were observed.

As described above, according to this embodiment, the axon terminal can be attached to the skeletal muscle in the second chamber 12b away from the first chamber 12a where the cell body resides. In other words, this embodiment can provide a situation similar to inside the body. Therefore, by precisely applying the drug to the junction between the axonal bundle and the skeletal muscle in each module 11, it is possible to screen for drugs that are effective against neurological diseases.

Next, the results of an experiment conducted by the inventor using the culture plate 10 according to the present embodiment to grow axons, slice the grown axons, and observe the proteins inside will be described. .

FIG. 11 is a photograph of an axonal bundle used for protein observation in this embodiment. In the figure, (a) is a photograph of a cell body spheroid with axonal bundles extracted outside the culture plate, and (b) is a photograph of an axon in the spheroid shown in (a) (with marker Tau 1 ). (c) is a fluorescence photograph of cell nuclei in the spheroid shown in (b) (using the marker Hoechst), and (d) is a slice of the axonal bundle shown in (a). (e) is a fluorescence photograph showing the absence of cell nuclei within slices of axon bundles.

In this experiment, motor neurons derived from human iPS cells (409B2 cell line) were used. Motor neuron cell body spheroids were seeded and cultured in the first chamber 12a. Motor neuron and cell body spheroids were obtained in the same manner as described above. In addition, their culture was also performed by the same method as described above. As shown in FIG. 11( a ), after 10 days of culture, one of the motor neurons containing large growing and elongated axonal bundles was extracted from module 11 of culture plate 10 , i.e. removed. . Then, the axonal bundles were sliced to obtain thin slices of the axonal bundles suitable for observation of proteins existing inside. As shown in FIG. 11(d), it was confirmed that the protein was observed inside the axonal bundle. In addition, as shown in FIG. 11(e), it was confirmed that no cell nucleus was present inside the axonal bundle, and that the axonal bundle was a pure collection of axons.

As described above, according to the present embodiment, spheroids of cell bodies integrated with axon bundles can be taken out from each module 11 of the culture plate 10 . Therefore, by slicing the axonal bundle and observing the proteins inside it, it is possible to analyze and identify proteins present in the axonal bundle, and to screen and identify neurological diseases. Furthermore, screening for drugs effective against neurological diseases can be performed. On the other hand, in the conventional method of culturing motor neurons, it is not possible to obtain a cell tissue in which cell bodies and axonal bundles are integrated, and cell bodies and axonal bundles can be obtained outside the conventional culture apparatus. It was difficult to observe the inside of each.

Next, the results of an experiment conducted by the inventor using the culture plate 10 according to the present embodiment to extend grown axons will be described.

FIG. 12 is a photograph of an axon bundle used for extension in this embodiment. In the figure, (a) is a photograph of spheroids with axonal bundles before stretching outside the culture plate, and (b) is a photograph of spheroids with axonal bundles after stretching outside the culture plate.

In this experiment, motor neurons derived from human iPS cells (409B2 cell line) were used. Motor neuron cell body spheroids were seeded and cultured in the first chamber 12a. Motor neuron and cell body spheroids were obtained in the same manner as described above. In addition, their culture was also performed by the same method as described above. As shown in FIG. 12( a ), after culturing, one of the motor neurons containing cell body spheroids and large growing and elongated axonal bundles was taken out of the module 11 of the culture plate 10 . Its total length was 3.1 [mm]. When the proximal end and the distal end of the axon bundle are pinched with forceps and pulled, the axon bundle extends to a length of 6.4 [mm] as shown in FIG. 12(b). rice field. Axonal bundles were confirmed to have twice the extensibility.

As described above, according to the present embodiment, the axonal bundles integrated with the spheroids of the cell body can be extracted, ie taken out, from each module 11 of the culture plate 10 . The axonal bundle can then be mechanically stretched, as confirmed by the experiments described above. Therefore, axonal bundles of motor nerve cells can be transplanted by previously culturing desired types of motor nerve cells that are integrated with the axonal bundles. It has been difficult to test and evaluate the physical properties of axonal bundles in the body. Various experiments can be performed.

Next, the results of a second experiment conducted by the inventor using the culture plate 10 according to the present embodiment to extend grown axons will be described.

FIG. 12A is a diagram for explaining the results of a second experiment of extending axons according to this embodiment. In the figure, (a) is a schematic diagram explaining a method of stretching axon bundles, (b) is a photograph showing changes in extension of axon bundles, and (c) is the degree of extension of axon bundles and axons. FIG. 10 shows the relationship between the tension applied to the bundle, (d) is a TEM image of the cross section of the axon bundle before stretching, and (e) is the TEM image of the cross section of the stretched axon bundle.

In this experiment, motor neurons derived from human iPS cells (409B2 cell line) were used. Motor neuron cell body spheroids were seeded and cultured in the first chamber 12a. Motor neuron and cell body spheroids were obtained in the same manner as described above. In addition, their culture was also performed by the same method as described above. After culturing, one of the motor neurons, which grew large and contained an elongated axonal bundle, was extracted, ie removed, from module 11 of culture plate 10 . Then, as shown in FIG. 12A (a), the distal end of the axon bundle is wrapped around the tip (free end) of a glass capillary (thin glass tube) to which the proximal end is fixed to fix the axon bundle. The proximal end (spheroid side end) was pinched and pulled with forceps to apply tension to the axonal bundle. Then, the tensioned glass capillary bends elastically, and the tip of the capillary is displaced according to the magnitude of the tension. Therefore, the magnitude of the tension can be calculated from the amount of displacement of the tip. In FIG. 12A(a), L is the length of the axonal bundle before tension is applied, F is the tension applied to the axonal bundle, and ΔL is the amount of change in the length of the axonal bundle due to the application of tension (extension length), and Δx is the amount of displacement of the tip of the glass capillary due to the application of tension. In FIG. 12A(b), an asterisk (*) indicates the tip of the glass capillary, and a triangle (▴) indicates a point obtained by dividing the axon bundle into four parts in the longitudinal direction. The three photographs in FIG. 12A(b) are arranged so that the degree of extension of the axons progresses toward the bottom. Furthermore, FIG. 12A (c) is a graph showing the measurement results of the relationship between the degree of axonal extension and the magnitude of the applied tension, in which the horizontal axis is the degree of axonal extension (%) and the vertical axis is the tension. represents the magnitude (μN) of . Furthermore, FIGS. 12A (d) and (e) show transmission electron micrograph images, i.e., TEM images, of cross-sections of axonal bundles before and after stretching. It was confirmed that the cross section of the axon contained therein was deformed.

As described above, according to the present embodiment, the axonal bundles integrated with the spheroids of the cell body can be extracted, ie taken out, from each module 11 of the culture plate 10 . Then, by applying tension to the axon bundle while the distal end of the axon bundle is wrapped around the tip of the glass capillary and fixed, the relationship between the extension degree of the axon bundle and the magnitude of the applied tension is measured. can be used to measure the elastic properties (elastic modulus) of axonal bundles.

Next, the results of calcium imaging experiments on grown axons performed by the inventor using the culture plate 10 according to the present embodiment will be described.

FIG. 13 is a photograph of motor neurons used for calcium imaging in this embodiment. In the figure, (a) is a photograph of spheroids with axonal bundles outside the culture plate before calcium application, and (b) is a photograph of spheroids with axonal bundles after calcium application outside the culture plate. .

In this experiment, motor neurons derived from human iPS cells (409B2 cell line) were used. Motor neuron cell body spheroids were seeded and cultured in the first chamber 12a. Motor neuron and cell body spheroids were obtained in the same manner as described above. In addition, their culture was also performed by the same method as described above. After culturing, one of the motor neurons, which grew large and contained an elongated axonal bundle, was extracted, ie removed, from module 11 of culture plate 10 . Then, as shown in FIG. 13(a), the motor neurons including the cell body spheroids and axonal bundles were subjected to fluorescence treatment. Subsequently, when 1 mol of KCl was applied to the cell body spheroids, the fluorescence became stronger as a result of the electrophysiological activity induced by KCl stimulation, as shown in FIG. 13(b). It was confirmed that the application of KCl electrophysiologically activates motor neurons, including cell body spheroids and axonal bundles.

As described above, according to the present embodiment, motor neurons including cell body spheroids and axonal bundles can be taken out from each module 11 of the culture plate 10 . And, as confirmed by the previous experiments, motor neurons can be used for calcium imaging. Therefore, where in vitro evaluation of electrophysiological activity on axonal bundles has been difficult, by pre-culturing the desired type of motor neuron integrated with axonal bundles, the It is possible to observe how stimuli are transmitted through axons, which can be applied to the development of neural prosthesis technology.

Next, the results of an experiment conducted by the inventor to measure action potential changes in grown axons using the culture plate 10 according to the present embodiment will be described.

FIG. 13A is a diagram explaining the results of an experiment for measuring action potential changes in this embodiment. In the figure, (a-1) is a schematic diagram showing a method of applying electrical stimulation to spheroids with axonal bundles, and (a-2) is calcium when electrical stimulation is applied to spheroids with axonal bundles. Imaging photographs, (a-3) photographs showing changes in fluorescence intensity when electrical stimulation is applied to spheroids with axonal bundles, each of (b-1) to (b-3) shows cut axons FIG. 10 is a diagram and a photograph respectively corresponding to (a-1) to (a-3) after the above.

In this experiment, motor neurons derived from human iPS cells (409B2 cell line) were used. Motor neuron cell body spheroids were seeded and cultured in the first chamber 12a. Motor neuron and cell body spheroids were obtained in the same manner as described above. In addition, their culture was also performed by the same method as described above. After culturing, one of the motor neurons, which grew large and contained an elongated axonal bundle, was extracted, ie removed, from module 11 of culture plate 10 . Then, as shown in FIG. 13A(a-1), electrodes provided on both sides of the spheroid of the cell body of the motor neuron applied electrical stimulation to the cell body side to measure the action potential on the axon side. Thereafter, as shown in FIG. 13A (b-1), after cutting the axonal bundle, electrical stimulation was similarly applied to the cell body side, and action potentials on the axonal side were measured. Figures 13A (a-2) and 13A (b-2) are photographs of calcium imaging using Fluo-4AM as a calcium measurement reagent when electrical stimulation is applied before and after axonal bundle cutting. are shown, respectively. In addition, the results of measuring the temporal change in fluorescence intensity of Fluo-4AM when electrical stimulation was applied before and after cutting the axonal bundle are shown in FIGS. 13A (a-3) and 13A (b-3). , respectively, are shown.

Thus, it was confirmed that the electrical stimulation applied to the cell body was transmitted to the axonal bundle. In addition, the electrophysiological conduction velocity from the cell body to the axon terminal can be measured based on the temporal change in fluorescence intensity that changes in response to electrical stimulation.

Next, various examples of arrangement of the first and second chambers 12a, 12b and the channel 13 of the module 11 in this embodiment will be described.

FIG. 14 is a schematic diagram showing various examples of the culture module in this embodiment. In the figure, (a) to (g) respectively show different types of arrangement of chambers and channels.

In this embodiment, the arrangement of first and second chambers 12a, 12b and channel 13 in module 11 is necessarily a pair of first and second chambers connected by channel 13, as shown in FIGS. It is not limited to including chambers 12a, 12b. According to the example shown in FIG. 14( a ), the module 11 includes two first chambers 12 a each seeded with cell body spheroids, one second chamber 12 b and one chamber 12 a each. It contains two channels 13 connecting one with the second chamber 12b. According to the example shown in FIG. 14(b), the module 11 comprises one first chamber 12a in which the spheroids of the cell bodies are seeded, two second chambers 12b, the first chamber 12a and the second chamber 12b, respectively. and two channels 13 connecting one of the chambers 12b. According to the example shown in FIG. 14(c), the module 11 comprises two first chambers 12a each seeded with cell body spheroids, one second chamber 12b and two first chambers 12a. It includes one channel 13 branched into two on the way so as to connect with the second chamber 12b. According to the example shown in FIG. 14(d), the module 11 includes one first chamber 12a in which cell body spheroids are seeded, two second chambers 12b, the first chamber 12a and two second chambers 12b. It includes one channel 13 branched into two on the way so as to connect with the chamber 12b. According to the example shown in FIG. 14(e), the module 11 includes one first chamber 12a in which spheroids are seeded with cell bodies, four second chambers 12b, each of the first chamber 12a and the second chamber 12b. and four channels 13 connecting with one of the chambers 12b.

As described above, according to this embodiment, when nerve cells are seeded and cultured in the first chamber 12a, axons grow in the channel 13 and extend toward the second chamber 12b. When skeletal muscle is seeded in the second chamber 12b, the distal end of the axon and the skeletal muscle join in the second chamber 12b. Furthermore, as shown in FIG. 14(f), it can be applied to a central nervous system model by seeding cell body spheroids inside both the first chamber 12a and the second chamber 12b. Further, as shown in FIG. 14(g), spheroids of cell bodies of lower motor neurons were seeded in the first chamber 12a, and spheroids of cell bodies of upper motor neurons were seeded in the second chamber 12b. , by seeding skeletal muscle in the other second chamber 12b, it can be applied to a system including lower motor nerves, upper motor nerves and skeletal muscle. Therefore, by preparing various culture modules 11, it is possible to obtain various forms of junctional relationships between nerve cells, axons, and skeletal muscles in vitro, thereby enabling various experiments and research on drugs effective against neurological diseases. can be used for

Next, a method for separating the axon from the cell body in the module 11 of the culture plate 10 according to the present embodiment and extracting the axon and the cell body separately will be described.

FIG. 15 is a diagram showing a step of separating an axon from a cell body in this embodiment. In the figure, each of (a-1) to (a-5) is a photograph showing the upper side of the module, each of (b-1) to (b-5) is (a-1) to (a-5) ) are schematic cross-sectional views of modules respectively corresponding to FIG.

According to this embodiment, as shown in Figs. They can be extracted separately by separating from First, as shown in FIGS. 15(a-1) and (b-1), cell bodies of motor neurons are seeded and cultured in the first chamber 12a of the module 11, thereby forming axonal bundles. It grows and extends through the channel 13 from the first chamber 12a to the second chamber 12b. Subsequently, as shown in FIGS. 15(a-2) and (b-2), the cutter 21 separates the axon bundle from the cell body. In this case, since at least the top plate 16 is preferably made of a soft material such as PDMS, the cutter 21 can smoothly cut the axon bundles together with the top plate 16 . Cell bodies are then removed, ie picked up, from the first chamber 12a by aspiration using a pipette 22, as shown in FIGS. 15(a-3) and (b-3). Subsequently, as shown in FIGS. 15(a-4) and (b-4), the remaining axonal bundles flow into the channel 13 along with the flow of the culture solution 18 generated by spraying using the pipette 22. from the second chamber 12b. Finally, the axon bundle is removed, ie picked up, from the second chamber 12b by aspiration using a pipette 22, as shown in FIGS. 15(a-5) and (b-5).

As described above, according to the present embodiment, the axonal bundle can be separated from the cell body within the module 11 and only the axonal bundle can be extracted. This makes it possible to analyze proteins and RNA that exist only within axonal bundles. Such analysis is an important process for analyzing neurological diseases.

Next, the usefulness of the device according to the present embodiment for developing drugs effective against amyotrophic lateral sclerosis (ALS) will be described.

FIG. 16 is a schematic diagram showing the cause of ALS, FIG. 17 is a photograph showing the results of the stress test in this embodiment, and FIG. 18 is a photograph showing the results of co-culturing with glial cells in this embodiment, and FIG. 19 is a photograph of axon bundles extending from cell body spheroids with and without glial cells in this embodiment. . In FIG. 17, (a) is a photograph of axon bundles without stress, (b) is a photograph of axon bundles with ER stress, and (a) is a photograph of glial cells in FIG. (b) is a photograph of cell body spheroids in the absence of glial cells, and (a) is a photograph of axon bundle spheroids in the presence of glial cells in FIG. , (b) is a photograph of axonal bundle spheroids in the absence of glial cells.

As shown in FIG. 16, ALS disease includes toxicity of glutamate, hyperexcitability, glial toxicity, mitochondrial dysfunction, disruption of axonal transport. ), physical damage, oxidative stress, endoplasmic reticulum (ER) stress, formation of inclusion bodies, and the like. If even one of these causes can be artificially reproduced, it will lead to the discovery of an effective drug for ALS. In order to reproduce these causes, the inventor conducted several experiments using the culture plate 10 according to this embodiment.

A first experiment was performed to confirm the effect of ER stress on motor neurons using thapsigargin. In the first experiment, motor neurons derived from human iPS cells (409B2 cell line) were used. Motor neuron cell body spheroids were seeded and cultured in the first chamber 12a. Motor neuron and cell body spheroids were obtained in the same manner as described above. Cultivation was also performed in the same manner as described above. After culturing, motor neurons with well-grown and elongated axonal bundles were treated with a culture medium containing 1.5 [mol] of thapsigargin for 5 hours. Then, after 6 days, evaluation of the axonal bundle morphology as shown in FIG. compared to went. Axon orientation was also evaluated as shown in FIG. 17A. It can be said that the axons become less oriented when stressed, that is, the axons are degenerated. It was confirmed that motor neurons subjected to ER stress undergo morphological changes.

To confirm that motor neurons can be co-cultured with glial cells using the culture plate 10 according to the present embodiment, since the toxicity of glial cells to ALS due to mutation of a specific gene has been discussed. A second experiment was carried out. In a second experiment, motor neurons derived from human iPS cells (409B2 cell line) were used. Motor neuron cell body spheroids obtained by culturing in the first chamber 12a were mixed with glial cells and then cultured. Subsequently, motor neurons were stained with markers Hoechst, Tuj 1 and GFAP (Glia Fibrillary Acidic Protein). FIG. 18(a) shows motor neurons mixed with glial cells, and FIG. 18(b) shows motor neurons without glial cells. Since GFAP is a marker for identifying glial cells, it was confirmed that the glial cells were uniformly distributed within the spheroids of the cell bodies. Furthermore, after culturing for 10 days, even when glial cells were mixed with motor neurons as shown in FIG. As in the unmixed case, axonal bundles grew and extended from the soma spheroids into channels 13 . Thus, since it was confirmed that a co-culture system with glial cells can be obtained, the co-culture system with glial cells can be used for reproduction of neurodegeneration due to glial toxicity and drug screening for the development of drugs effective against ALS. Application can be expected.

Next, the results of observing the axon bundle grown using the culture plate 10 according to the present embodiment in comparison with the cell body will be described.

FIG. 20 is a diagram showing the results of observation of axon bundles grown in the channels of the culture plate in this embodiment, and FIG. 20A shows nerve cells containing axon bundles grown in the channels of the culture plate in this embodiment. FIG. 20B is another photograph showing a cross section of nerve cells including axonal bundles grown in the channels of the culture plate in this embodiment. In FIG. 20, (a-1) to (a-3) are photographs showing cross sections of cell bodies stained with various markers, and (b-1) to (b-3) are each of (a-1 ) to (a-3) are photographs showing cross sections of axonal bundles, (c) is a transmission electron micrograph of the cross section of axonal bundles, and (d) is protein analysis of cell bodies and axonal bundles. FIG. 20A shows the results, in which (a-1) and (a-2) are photographs showing cross sections of cell bodies stained with various markers, (b-1) and (b-2), respectively. (a-1) and (a-2) are photographs showing cross-sections of axon bundles corresponding to each, and in FIG. 20B, (a-1) to (a-3) are each stained with various markers. Each of (b-1) to (b-3) is a photograph showing a cross section of the axonal bundle corresponding to (a-1) to (a-3).

Here, motor neurons derived from human iPS cells (409B2 cell line) were used. Motor neuron cell body spheroids were seeded and cultured in the first chamber 12a. Motor neuron and cell body spheroids were obtained in the same manner as described above. In addition, their culture was also performed by the same method as described above. After culturing, one of the motor neurons, which grew large and contained an elongated axonal bundle, was extracted, ie removed, from module 11 of culture plate 10 . Cell body spheroids and axonal bundles were then stained with markers Hoechst, Tau1, Synapsin1 and Map2. Fig. 20 (a-1) to (a-3) show, respectively, Hoechst and Tau 1-stained cell body cross-sections, Hoechst, Tau 1 and Synapsin 1-stained cell body cross-sections, Hoechst, Map 2 and Synapsin 20(b-1)-(b-3) are cross-sections of cell bodies stained by 1, and axons stained corresponding to FIGS. 20(a-1)-(a-3), respectively. It is a cross section of the bundle. 20A (a-1) and (a-2) are respectively a cross section of the cell body stained with Tau 1 and a cross section of the cell body stained with Hoechst and Tau 1, and FIG. 20A (b-1). and (b-2) are cross-sections of axonal bundles stained to correspond to FIGS. 20A (a-1) and (a-2), respectively. Furthermore, FIG. 20B (a-1) to (a-3) are respectively a section of the cell body stained with Hoechst, a section of the cell body stained with Map 2, and a section of the cell body stained with Synapsin 1, FIGS. 20B(b-1) to (b-3) are cross sections of the axonal bundles stained corresponding to FIGS. 20B(a-1) to (a-3), respectively. Since the axons are bundled, cross-sections of the axons can be easily observed by immunostaining.

Also, FIG. 20(c) is a TEM image of a cross section of an axonal bundle, that is, a photograph taken by a transmission electron microscope. Since the axons are bundled, cross-sections of multiple axons can be easily observed with a transmission electron microscope. As a result, the state of mitochondria, synapses, and microtubules in axons can be observed, and can be used to evaluate the effects of drugs.

Furthermore, FIG. 20(d) shows cell body spheroids stained with markers Map2, Nucleoporin, Tau1, Synapsin1 and Synaptophysin by a known Western blotting method (Western blot analysis). and the results of protein analysis of axonal bundles. [kDa] indicates the unit kilodalton. Since the axons are bundled, it is possible to efficiently collect a sample of only the axons, and analyze specific proteins of the axons. In FIG. 20(d), a comparison of the cell body spheroid sample and the axon bundle sample reveals that the cell body markers Map 2 and Nucleoporin are negative in the axon bundle sample. Therefore, it can be seen that the axonal bundle sample does not contain cell bodies, ie, the axonal bundle sample has high axonal purity. This is shown in FIGS. 20(b-1) to (b-3), FIGS. 20A(b-1) and (b-2), and FIGS. 20B(b-1) to (b-3). In the immunostaining, the axonal bundle samples were found to be negative for Map 2, a cell body marker, and Hoechst, a nuclear stain. Since Synapsin 1 exists throughout the nerve, it is observed both in axonal bundles and cell bodies.

Next, the results of observing the axon bundle grown using the culture plate 10 according to the present embodiment with a scanning electron microscope will be described.

FIG. 21 is a photograph showing the result of observation with a scanning electron microscope of the surface of the axon bundle grown in the channels of the culture plate in this embodiment. In the figure, (a-1) and (a-2) are low-magnification and high-magnification photographs when no stress is applied, (b-1) and (b-2) are each of (a-1 ) and (a-2), respectively.

Axon bundles are similar to those used in the observations shown in FIG. Since the axons are bundled, the surface of the axonal bundles can be easily observed with a scanning electron microscope. In addition, since the axons are bundled and extend in the same direction, for example, when stress (the examples shown in FIGS. 21(b-1) and (b-2) are oxidative stress), The change is clear.

Next, morphological changes when stress is applied to axon bundles grown using the culture plate 10 according to the present embodiment will be described.

FIG. 22 is a photograph showing axon bundles when stress is applied in the channels of the culture plate in this embodiment, and FIG. 23 is a photograph showing axon bundles when stress is applied in the channels of the culture plate in this embodiment. is a graph showing the experimental results of the morphological change of. In FIG. 22, (a) is a photograph when no stress is applied, (b) is a photograph when oxidative stress is applied, and (c) is a photograph when oxidative stress and an antioxidant are applied.

Here, human iPS cell (409B2 cell line)-derived motor neurons were seeded on a non-adhesive culture plate 10 to prepare cell body spheroids. Subsequently, after culturing the cell body spheroids for 10 days, they were seeded in the first chamber 12 a of the module 11 of the culture plate 10 . Then, on the 30th day of culture, the following treatments (1) to (3) were performed.
(1) control PBS (Phosphate Buffered Saline) washing >>> culture medium
( ₂ ) H2O2 _3hour treatment >>>PBS washing>>>culture medium
(3) H ₂ O ₂ 3 hour treatment >>> PBS washing >>> culture medium containing Edaravone
Two days after the treatment, the mice were immunostained with the marker Tau 1 and evaluated. The evaluation item is Directionality: axon directionality. Specifically, the directionality of the object in FIG. 22 was measured. In this case, 0 degrees is the flow path (channel 13) direction. Since the axons are bundled and extend in the same direction, for example, when oxidative stress is applied within the channel 13 as shown in FIG. As described above, morphological changes when oxidative stress and an antioxidant (Edaravone), which is used as a drug for neurological diseases, are applied simultaneously, can be compared. The results of the evaluation experiment shown in FIG. 23 show that when oxidative stress is applied, axons that extend in the direction of the axons are degenerated, so that the directionality in the direction of the flow path (0 degrees) is reduced, resulting in a complicated structure. It shows that the shape changes. As an evaluation of morphological changes, when examining the directionality of the objects shown in FIG. Next, the difference in orientation could be seen in the order of when only oxidative stress was applied. From this, it can be seen that the stress received by nerve cells can be evaluated based on image processing as morphological changes in axons.

Moreover, from the orientation evaluation results shown in FIG. 23 and FIG. A change or degeneration in the shape of a corn or axonal bundle can be quantified and quantitatively evaluated by using an index of orientation. In other words, it is possible to quantify the morphological degeneration of axons or axonal bundles by orientation analysis, and thereby to quantitatively evaluate the morphological degeneration of axons or axonal bundles.

Next, myelination of axon bundles grown using the culture plate 10 according to the present embodiment will be described.

FIG. 24 is a diagram showing myelination of axons in the module of the culture plate in this embodiment. In the figure, (a) is a schematic diagram explaining the myelination of neurons in the module, (b) is a photograph of an axon without Schwann cells, and (c) is an axon with Schwann cells. is a photograph of

When recreating myelin (myelin sheath) in the body in vitro, evaluation was complicated by conventional methods because the soma of neurons and the soma of Schwann cells and oligoderonocytes are mixed (each It was difficult to discern the cell bodies of the cells.). On the other hand, when the culture plate 10 of the present embodiment is used, as shown in FIG. 24(a), cell bodies of nerve cells are placed in the channel 13 of the module 11 and in the second chamber 12b on the opposite side. In the absence of , it is easy to observe how axons and myelinating cells behave. Specifically, the IMS32 cell line was seeded and cultured in the first chamber 12a, and Schwann cells, which are cells that myelinate after the axons were bundled, were seeded. Myelination can be readily observed in the example shown in FIG. 24(c) where Schwann cells are present compared to the example shown in FIG. 24(b) where Schwann cells are not present.

Next, the adaptability of the device according to this embodiment will be described.

FIG. 25 is a schematic diagram showing applicable fields of the device according to the present embodiment, and FIG. 26 is a schematic diagram showing other applicable fields of the device according to the present embodiment. In FIG. 25, (a) is a diagram showing application to drug screening, and (b) is a diagram showing application to a phenotype device.

By using the culture plate 10 according to the present embodiment, as shown in FIG. 25(a), screening for a co-culture system of neural tissue having thick axonal bundles, that is, nerve cells and other tissues It can be performed. In addition, as shown in FIG. 25(b), phenotypes can be examined comprehensively by using patient-derived tissues. The culture plate 10 can be applied to development of diagnostic agents by phenotype analysis. As shown in FIG. 26, neural tissue with thick axonal bundles cultured using the culture plate 10 according to this embodiment can be used for the development and testing of medical devices such as pacing systems. . In addition, it can be expected to be used for transplantation.

In each experiment in the present embodiment, only the case of using human iPS cell-derived motor neurons was described. can also be used.

It should be noted that this disclosure describes features of preferred exemplary embodiments. Various other embodiments, modifications and variations within the scope and spirit of the claims appended hereto will naturally occur to those skilled in the art upon reviewing the disclosure herein. be.

The present disclosure can be applied to devices and methods suitable for culturing neurons.

10 culture plate 11 module 12a first chamber 12b second chamber 13 channel 17 sealing member 18 culture solution

Claims (2)

A method of culturing neurons having axons, comprising:
culture medium (18) in one first chamber (12a), one second chamber (12b) and one channel (13) connecting said first chamber (12a) and second chamber (12b) wherein said first chamber (12a), second chamber (12b) and channel (13) are contained in a module (11) arranged in a culture plate (10);
seeding neuronal spheroids inside both the first chamber (12a) and the second chamber (12b), respectively;
culturing the nerve cells such that an axonal bundle grows and extends within each channel (13) and the spheroids are connected to each other by the axonal bundle;
How to prepare. applying a stress or drug to the axonal bundle;
measuring the orientation of the stressed or drug-applied axonal bundle;
A method for quantifying morphological degeneration of axonal bundles by orientation analysis,
Said axonal bundle is a channel (13) connecting one first chamber (12a) capable of receiving neuronal cell bodies and one second chamber (12b) capable of receiving neuronal cell bodies. an axonal bundle received in and extending into said channel (13) and connecting together spheroids seeded respectively within both said first chamber (12a) and said second chamber (12b),
The orientation is the direction in which the channel (13) extends,
A method for quantifying morphological degeneration of axonal bundles by orientation analysis.

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