JPWO2011052281A1 - Neural spheroid network construction method - Google Patents

Abstract

A method for constructing a neural spheroid network in which two or more spheroids containing nerve cells are connected by neurites extended from each spheroid, the method comprising culturing two or more spheroids arranged in close proximity.

Description

  The present invention relates to a method for constructing a network of nerve cells using cultured nerve cells.

  In recent years, research on cell patterning has been actively conducted on cells derived from various tissues. For example, a method of micropatterning cells on the surface of a biochip has been proposed (Surface Science, 25, pp.290-295, 2004). ). Attempts have been made to construct neural networks by patterning cells one by one using neurons (The Institute of Electrical Engineers of Japan C, 127, pp.1575-1580, 2007). However, neuronal cells have low cell viability and low circuit formation efficiency, so they are limited to two-dimensional patterning using a small number of neurons (J. Neurosci., Methods, 117, pp. 123 -131, 2002; J. Neurosci. Methods, 160, pp. 317-326, 2007), further improvement is necessary in order to form a practical neural network by applying cell patterning technology. As an example of such a method, a method for constructing a hippocampal neural network (Sensors and Actuators B: Chemical, 99, 156-162, 2004) that is gradually complicated on an on-chip multielectrode array or a neuron on a bead. A method of forming a three-dimensional neural circuit including a process of attaching and arranging cells (Nat. Methods, 5, pp.735-740, 2008) has been reported.

  On the other hand, spheroids are agglomerates of cells and have a three-dimensional structure that is closer to living tissue than normal cultured cells that spread in a planar shape, and thus are attracting attention in fields such as regenerative medicine. A variety of reports have been made on the spheroid culture system that creates a three-dimensional aggregated state of a large number of cells as an excellent in vitro cell culture system from the viewpoint of maintaining cell polarity and cell-cell interactions. . For example, spheroids of cells such as pancreatic cells, osteoblasts and hepatocytes have been reported (for example, Pancreas, 25, pp. 71-77, 2002; Biochem. Biophys. Res. Comm., 322, pp. 684- 692, 2004; Biochem. Biophys. Res. Comm., 161, pp. 385-391, 1989), all of which have been reported to exhibit biological responses different from those of monolayer culture systems. Also reported are methods for preparing spheroids of uniform size from pancreatic cells (Proc. Of MEMS, pp.423-426, 2009) and methods for creating larger tissues by appropriately arranging spheroids by patterning. (Biomaterials, 30, pp.2164-2174, 2009).

  Regarding neuronal spheroids, for example, it has been reported that neural stem cells prepared from embryonic day 14 mouse cerebral cortex tissue form spheroids on honeycomb films with a pore size (3 μm) smaller than the size of neural stem cells. On the honeycomb film having a pore diameter of 5 μm or more, neural stem cells differentiate into nerve cells, the number of protrusions and protrusion branches coming out of the nerve cells are controlled by the pore diameter of the honeycomb film, and particularly when the pore diameter is 10 μm, It has also been reported that it stretches along the trunk of the chemical (Chemical Industry, 57, pp.27-35, 2006). However, conventionally, no method has been reported for constructing a neural network using neuronal spheroids.

IEEJ Transaction C, 127, pp.1575-1580, 2007 J. Neurosci., Methods, 117, pp.123-131, 2002 J. Neurosci. Methods, 160, pp.317-326, 2007 Sensors and Actuators B: Chemical, 99, 156-162, 2004 Nat. Methods, 5, pp.735-740, 2008 Pancreas, 25, pp.71-77, 2002 Biochem. Biophys. Res. Comm., 322, pp. 684-692, 2004 Biochem. Biophys. Res. Comm., 161, pp.385-391, 1989 Proc. Of MEMS, pp.423-426, 2009 Biomaterials, 30, pp.2164-2174, 2009

An object of the present invention is to provide a method for constructing a network of nerve cells using cultured nerve cells.
More specifically, it is an object of the present invention to provide a method for easily constructing a huge neural network using neuronal spheroids.

  As a result of diligent research to solve the above-mentioned problems, the present inventors have cultured a plurality of spheroids including nerve cells in proximity or in contact with each other, so that axons are extended from the nerve cells contained in each spheroid. And found that a neural network is efficiently constructed between spheroids. In addition, by culturing the above spheroids in each well of a microchamber having a large number of wells arranged two-dimensionally, a neural network is constructed between the spheroids, and a culture containing a huge patterned neural spheroid network is obtained. It has been found that it can be easily prepared, and that the above-mentioned neural spheroid network can be engrafted in the brain and exert its neural function by transferring and culturing this culture on the brain surface. The present invention has been completed based on the above findings.

  That is, according to the present invention, there is provided a method for constructing a neural spheroid network in which two or more spheroids containing nerve cells are connected by neurites extended from each spheroid, and the two or more spheroids arranged in close proximity are cultured. There is provided a method comprising the steps of:

  According to a preferred embodiment of the above method, the above method wherein the spheroid contains glial cells; the above method wherein the spheroid contains neural stem cells; and one spheroid is cultured in each well using a chamber having a plurality of wells The above method; the above method wherein the ratio of well diameter, depth, and distance between adjacent wells is in the range of about 1: 1: 1 to 1: 1: 3; the well diameter is 50 to 300 μm The above method; the above method using a polydimethylsiloxane chamber as the chamber is provided.

  Another aspect of the present invention provides a neural spheroid network in which two or more spheroids containing nerve cells are connected by neurites extended from each spheroid. As a preferred embodiment of the present invention, there is provided the above network in which spheroids contain glial cells; and the above network in which spheroids contain neural stem cells. Furthermore, network complexes comprising two or more neural spheroid networks are also provided by the present invention.

  In addition, a culture containing the neural spheroid network, preferably a culture containing the neural spheroid network in a chamber, more preferably a culture containing the neural spheroid network in a polydimethylsiloxane chamber is provided by the present invention. .

  From still another aspect, according to the present invention, there is provided a method for transplanting the above-described neural spheroid network into a tissue, wherein the neural spheroid network is a tissue of an animal including a human, preferably a nervous tissue, more preferably a central nervous system tissue. Particularly preferably, there is provided a method comprising the step of applying to cranial nerve tissue. As a preferred embodiment of the above method, the nerve spheroid network formed in the chamber is attached to animal tissue, preferably nerve tissue, more preferably central nervous system tissue, particularly preferably cranial nerve tissue, and then the chamber is peeled off. There is provided a method comprising the steps of:

  By the method of the present invention, a neural network can be easily constructed using spheroids containing nerve cells. For example, a huge neural spheroid network can be constructed by culturing spheroids containing nerve cells using a chamber having a large number of wells. Moreover, the nerve spheroid network obtained in this way can be transcribed to, for example, cranial nerve tissue and the like, and this technique can be used as, for example, nerve regeneration therapy.

It is the figure which showed typically the process of forming a spheroid in a well using the chamber which has a some well. It is a phase contrast photograph of a spheroid (derived from cerebral cortex cells, culture day 1) formed in a well using a chamber having a plurality of wells. Three types of chambers with a large number of wells in a grid pattern (well diameter: 50/100 / 150μm, well depth: 50/100 / 150μm, distance between wells: 100/200 / 300μm) using polydimethylsiloxane Schematic diagram of manufacturing method ((a) to (e)), and electron microscope of the resulting SU-8 mold (bottom right diagram of (c-2)) and PDMS chamber (right diagram of (e-2)) It is a photograph. It is the figure which showed the preferable aspect of the method of this invention typically. It is the figure which showed typically the method to construct | assemble the nerve spheroid network complex which combined 2 or more types of nerve spheroid networks. It is the figure which showed typically the method of transcribe | transferring a nerve spheroid network to a cranial nerve tissue. In this method, the nerve spheroid network formed in the PDMS chamber can be affixed to the cerebrum, cerebellum, olfactory bulb, etc. together with the chamber, and then the PDMS chamber can be peeled to transplant the nerve spheroid network to the brain tissue surface. It is the photograph which collect | recovered the nerve spheroid (2nd culture | cultivation day) formed in the PDMS chamber from the chamber, and observed under the phase contrast microscope. The numbers in the figure indicate the diameter of the well, and the non-coat dish indicates the state of culture on an uncoated culture dish. It is the figure which showed distribution of the magnitude | size of the spheroid formed in the PDMS chamber. The vertical axis represents the number of spheroids, and the horizontal axis represents the area of the spheroids measured by taking a phase contrast micrograph of the spheroids taken out from the wells of the PDMS chamber. The results of cell staining of spheroids formed in wells having a diameter of 100 μm are shown. (a) Green color indicates live cells, red color indicates dead cells, (b) Upper image is an immunocytochemical staining image of the marker protein MAP2 (green; MAP2), and lower image is MAP2 (green) and glial cells Shows immunocytochemical staining images of marker proteins GFAP (red) and Hoechst (nuclear staining; blue). It is the figure which showed that the connection of the neurite was formed between the spheroids on the 14th day, and the nerve spheroid network was constructed | assembled by culture | cultivating the adjacent spheroid. (b) MAP2 immunocytochemical staining image (green: MAP2, blue: nuclear staining with Hoechst), (c) phase contrast microscopic image of neural spheroid network taken out from PDMS chamber on day 14 of culture, (d) PDMS chamber Phase-contrast micrograph after culturing the more detached neural spheroid network for 24 hours (upper figure), Live / Dead assay fluorescence acquisition image (lower figure, green: live cell, red: dead cell), (e) From PDMS chamber The extracted nerve spheroid network is folded and immunostained images are shown (green; MAP2, red; GFAP, blue; nuclear staining). It is the figure which showed the result of having transferred the nerve spheroid network constructed | assembled by culturing in a PDMS chamber for 14 days to the glass substrate. It is the figure which showed the immuno-staining result of the nerve spheroid network transcribe | transferred on the glass substrate. (b) According to the method shown in FIG. 5, two types of neural spheroid networks are labeled with cell tracker green (1st) and cell tracker orange (2nd), respectively, and transferred onto a glass substrate (primary neural spheroid network (1st ): Green, secondary neural spheroid network (2nd): Red), (c) Immunocytochemical staining image of the transcribed neural spheroid network. In the figure, Hoechst indicates nuclear staining (blue), MAP2 indicates an immunocytochemical staining image of the neuronal marker protein MAP2 (green), and Neurofilament indicates a neuronal axon marker (red). It is the figure which showed the result of having imaged Ca ^<2+ > the nerve spheroid network transcribe | transferred to the glass substrate. It is the figure which showed the result of having formed the spheroid from a rat cerebral cortex cell, and having transcribe | transferred the neural spheroid network obtained after 14-day culture | cultivation to the right brain surface of a rat fetus. It is the figure which showed the result of having transcribe | transferring the nerve spheroid network to the rat cerebral cortex tissue surface, and analyzing the function of the transcribed network. (A) and (B) show the state of transcription (transplant), and (C) and (D) show the state of the transcribed neural spheroid network ((D) is an enlarged photograph of (C)). (E) shows Ca ²⁺ imaging of neural spheroid network transcribed on rat cerebral cortical tissue surface, (F) is a graph showing Ca ²⁺ changes in numbered regions in neural spheroid network (E) . (G) is a schematic diagram showing the process by which neurites develop from neural spheroid networks transcribed in front of cerebral cortical tissues and form synapses with neurons in cerebral tissues, and (H) is the surface of cerebral tissues. The transcribed neural spheroid network (PHA-L labeled neurons) is shown. The arrow indicates the transcribed neural spheroid network. (I) shows a state in which PHA-L is transferred from a neuron of a neurospheroid network to a neuron in a cerebral tissue via a synapse (labeled with PHA-L in the cerebral tissue). Neuron that has been treated). It is the figure which showed preparation of the multilayer nerve spheroid network, and transcription | transfer to the surface of a cerebral cortex tissue. In the figure, A is a schematic diagram of multilayer neural spheroid network creation, B is a photograph showing the state of the multilayer neural spheroid network created on the PDMS chamber, and C is a peeled multilayer neural spheroid network from the PDMS chamber. D shows a multilayer neural spheroid network transcribed on the surface of rat cerebral cortex.

  The method of the present invention is a method for constructing a neural spheroid network in which two or more spheroids including nerve cells are connected by neurites extended from each spheroid, and the two or more spheroids arranged in close proximity are connected to each other. It includes a step of culturing. According to a preferred embodiment of the present invention, the spheroids contain glial cells and / or the spheroids contain neural stem cells.

  As used herein, “spheroid” means a cell mass formed by aggregation of cells. The shape of the spheroid is not particularly limited, but is generally preferably a spherical shape, a rugby ball shape, an oval shape, or the like. However, the spheroid may be a cube, a rectangular parallelepiped, a cylinder, a conical triangular pyramid, or the like, and an appropriate spheroid shape can be selected in accordance with the purpose of the neural spheroid network construction.

  The size of the spheroid is not particularly limited. For example, when a substantially spherical spheroid is used, a spheroid having a diameter of 30 to 500 μm, preferably 50 to 400 μm, and more preferably about 50 to 200 μm can be used. The size of the spheroid can be estimated from the area of the spheroid by, for example, taking a phase contrast micrograph of the spheroid.

  Spheroids include neurons, but in addition to glial cells and neural stem cells, spheroids may contain embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) that have pluripotency. Good. Spheroids containing neurons can also be prepared by inducing differentiation using spheroids of ES cells or iPS cells. The number of nerve cells contained in the spheroid is appropriately determined according to the size of the spheroid. For example, in the case of a substantially spherical spheroid having a diameter of about 50 μm, the number is 10 or less.

  Spheroids can include glial cells. The number of glial cells is not particularly limited, but in the case of a substantially spherical spheroid having a diameter of about 50 μm, it is about 2 to 50 per spheroid. For example, for spheroids containing nerve cells and glial cells, the ratio of nerve cells to glial cells is not particularly limited, but the number of glial cells is about 2 to 100 times, preferably about 1 to 50 times that of nerve cells. Preferably, it contains one or more neurons and glial cells. More preferably, it contains about 10 to several hundred neurons and about 10 to 1,000 glial cells per spheroid having a diameter of about 100 to 150 μm.

  For the formation of spheroids, for example, if cells are collected from any region of fetal brain tissue of fetal age 14 to 20 days, such as cerebrum, hippocampus, cerebellum, olfactory bulb, etc., and cultured under conditions suitable for spheroid formation Good. As neurons for forming spheroids, primary cultured neurons are preferably used. Cells collected from the cerebral cortex include excitatory or inhibitory neurons as neurons, either or both of which can be used. In addition, the cells collected from the cerebral cortex may contain glial cells and undifferentiated neural stem cells. Cells from the cerebral cortex can generally be collected from, for example, a fetal brain of about 14-20 days of gestation, but the origin of the cells used for spheroid formation is not limited to this particular embodiment. . Sensory cells (such as olfactory nerve cells and vomeronasal nerve cells) and other tissue cells (pancreatic cells, liver cells, fibroblasts, vascular endothelial cells, Myofibroblasts, cardiomyocytes, etc.), and these cells may be combined with nerve cells to form spheroids.

  Neuronal spheroids are sometimes called neurospheres when they contain undifferentiated neural stem cells. In general, a neurosphere means a cell mass that contains cells that have been differentiated from embryonic brains of about 12-14 days of gestation and mainly contains undifferentiated neural stem cells. Needless to say, it is included in the spheroids of the present specification. Spheroids can also be formed using cells other than cerebral cortical cells. For example, spheroids may be formed using cells derived from the cerebellar cortex.

  According to the method of the present invention, by culturing two or more spheroids arranged close to each other, a neurite extends from each spheroid and a spheroid network in which each spheroid is connected by a neurite can be constructed. it can. In this specification, the term “proximity” used for the arrangement of spheroids includes not only a case where spheroids are arranged at an appropriate distance but also a case where two or more spheroids are in contact. The term “neurite” as used herein includes dendrites and axons.

  In arranging two or more spheroids close to each other, the size and shape of the spheroids may be different from each other, but it is preferable to arrange spheroids having the same shape and a uniform size. From this point of view, it is preferable to form one spheroid in each well using a chamber having a plurality of wells of the same size, but it is possible to put two or more spheroids in each well. In this case, the two or more spheroids may be of the same type or may be two or more different types of spheroids. For example, two or more spheroids derived from different brain tissues can be placed in one well, for example, spheroids derived from the cerebrum and spheroids derived from the hippocampus, or derived from cells collected from spheroids derived from nerve cells and other tissues Spheroids can also be placed in one well.

Methods for forming spheroids in wells are disclosed, for example, in MEMS, pp. 423-426, 2009, the entire disclosure of which is incorporated herein by reference.
Hereinafter, although the method of this invention is demonstrated concretely about said preferable aspect, the method of this invention is not limited to the method of using said chamber.

  The size of the well in the chamber is not particularly limited. For example, the diameter of the well is about 50 to 400 μm, preferably about 100 to 150 μm, and it is preferable that the well has a depth substantially the same as the diameter of the well. The arrangement pattern of the plurality of wells is not particularly limited, but the distance between adjacent wells is preferably about 1OO to 600 μm. In a particularly preferred embodiment, the well diameter is about 100 to 150 μm, the well depth is substantially the same as the well diameter, and the distance between adjacent wells is about twice the well diameter.

  For example, the ratio of the well diameter, the well depth, and the distance between adjacent wells is preferably in the range of about 1: 1: 1 to 1: 1: 3, preferably about 1: 1: 2. Is particularly preferred. When the well diameter and depth are approximately the same, a substantially spherical spheroid is efficiently formed in the well. By setting the distance between adjacent wells to be about twice the well diameter, one spheroid is transformed into another spheroid. Elongation of the neurite into the is performed efficiently. If the diameter of the well is larger than the depth, the spheroid may rise from the well and the spheroid may not be formed efficiently. In addition, if the diameter of the well is smaller than the depth, the spheroid may sink to the bottom of the well and the neural network may not be formed efficiently. When the distance between adjacent wells is about the same as the well diameter, neurites formed between the spheroids may rise, and when the distance between adjacent wells exceeds 3 times the well diameter, neurites are connected The efficiency of formation may be reduced.

  A process of forming spheroids in a well using a chamber having a plurality of wells is schematically shown in FIG. As one embodiment of the present invention, when cortical cells containing neurons and glial cells are collected and seeded in a chamber, the cells settle in the well (step 1)), and the contacting cells adhere to each other in the well. First (step 2)), cells that have not settled in the well after 20 to 60 minutes are washed away by means such as medium exchange, and when the culture is continued for 24 hours, spheroids are formed in the well (step 3)). A phase contrast photograph of the spheroid (cultured day 1) obtained from cerebral cortical cells in this way is shown in FIG. It can be seen that the cells adhere to form spheroids. Cerebral cortical cells can be cultured according to the methods described in, for example, Journal of Pharmacy (Folia Pharmacol. Jpn.), 124, pp. 11-17, 2004 and Developmental Brain Res., 152, pp. 99-108, 2004. Although it can be performed, the culture medium, temperature conditions, and the like may be appropriately changed as necessary.

  The chamber used in the method of the present invention is not particularly limited, and a chamber in which a plurality of wells are arranged in an arbitrary pattern can be used. Spheroid formation efficiency, efficiency when exfoliating the obtained neural spheroid network, etc. From this point of view, it is preferable to use a chamber made of polydimethylsiloxane, for example. The well arrangement pattern is not particularly limited, and is typically a regular pattern such as a grid pattern, a regular pattern such as a regular triangle or regular hexagon aggregate pattern, or an arbitrary pattern such as an irregular pattern. Can be arranged. Also, wells of different sizes can be arranged.

  As an example, many wells in a grid pattern with polydimethylsiloxane (each well has the same size and shape: well diameter: 50/100/150 μm, well depth: 50/100/150 μm, distance between wells: 100 FIG. 3 shows a schematic diagram of a method for producing three types of chambers having / 200/300 μm). Spin coat SU-8 100 (Microchem) on a silicon wafer (step (a)), place a mask and irradiate with ultraviolet rays (step (b)), develop and wash the SU-8 pattern (pattern ( (SU-8 mold) is formed (step (c)). Thereafter, polydimethylsiloxane (PDMS) is poured and solidified to recover the PDMS chamber from the SU-8 mold (step (e)). The photo of (c-2) is a SU-8 mold (well diameter: 100 μm, well depth: 100 μm, distance between wells: 200 μm), (e-2) shows the resulting PDMS chamber (well (Aperture: 100 μm).

  In carrying out the method of the present invention, it is generally preferable to form spheroids derived from the same type of cerebral cortical cells in each well, but if necessary, different neurons in one or more wells Spheroids containing can also be formed. For example, spheroids derived from cerebral cortical cells and spheroids derived from cerebellar cortical cells may be formed in different wells. Alternatively, cerebral cortex cell-derived spheroids can be formed in one or a plurality of wells, and ES or iPS cell spheroids can be formed in the other wells.

  By continuing to culture by forming one spheroid in each well of the chamber, a neurite extends from each spheroid, and a spheroid network in which spheroids are connected by neurites is formed. In general, culture may be performed for about 1 to 2 weeks. Between each spheroid, about 10 or more, preferably about 20 or more neurites are connected. Thus, since it has connection by many neurites, the nerve spheroid network obtained by the method of this invention has the characteristic that it is hard to raise | generate a network damage by physical irritation | stimulation, a load, etc. The obtained neural spheroid network can be easily peeled off from the chamber by using the tip of the chip or a spatula, and the neural spheroid network thus obtained can be folded as necessary. An example according to a preferred embodiment of the method of the present invention is shown schematically in FIG. In this manner, a three-dimensional neural spheroid network can be constructed by the method of the present invention.

  A network combining two or more types of neural spheroid networks can also be constructed by the above method. A schematic diagram is shown in FIG. A PDMS chamber in which a neural spheroid network (primary neural spheroid network) was formed by spheroid culture was adhered to a glass substrate coated with polyethyleneimine (step (1)), and cultured at 37 ° C. for 24 hours (step ( 2)), the nerve spheroid network is peeled from the PDMS chamber and the nerve spheroid network transferred to the glass substrate is prepared (step (3)), and another nerve spheroid network (secondary nerve spheroid network) is formed. Adhering the PDMS chamber so as to overlap the primary nerve spheroid network transferred on the glass substrate (step (4)), and peeling the PDMS chamber on the secondary nerve spheroid network side (step (5)), 2 Network complexes can be constructed that contain different types of neural spheroid networks. In addition, as shown schematically in FIG. 16A, it is possible to produce a multilayer network complex by combining two neural spheroid networks vertically. It will be easily understood that this technique can be used to construct a complex of three or more types of neural networks, and that this method is useful as a method for constructing a three-dimensional neural spheroid network.

  The neural spheroid network obtained by the above method is not only a spheroid physically connected by a neurite, but also a neural network in which each spheroid is functionally linked, and a neuron contained in one spheroid is another It is physiologically synchronized with the neurons contained in the spheroids. The state of physiological synchronization between spheroids can be confirmed by, for example, dynamically imaging calcium ion uptake, and one example is shown in Example 3 of the example.

  By attaching this neural spheroid network to, for example, tissue of mammals including humans, the neural spheroid network can be transplanted into the tissue. The tissue to be transferred is not particularly limited, and examples thereof include nerve tissue and muscle tissue. As the nerve tissue, for example, it can be transferred to a central nervous system tissue, preferably a cranial nerve tissue. This method may be performed by peeling the nerve spheroid network from the chamber with tweezers or the like and attaching it to the target tissue surface, but preferably the nerve spheroid network formed in the chamber is attached to the target tissue surface together with the chamber, Thereafter, the chamber is peeled off and a method of transplanting the nerve spheroid network onto the target tissue surface can be employed. A schematic diagram of the method is shown in FIG.

  By transplanting the neural spheroid network obtained by the method of the present invention, it becomes possible to restore the neural circuit in brain tissue damaged by cerebral infarction, cerebral hemorrhage, cerebral contusion, etc., for example. In addition, for example, in neurodegenerative diseases such as Alzheimer's disease and neurological diseases such as Parkinson's disease, a neural spheroid network obtained by the method of the present invention is transplanted to complement a neural circuit whose function is reduced or stopped. It becomes possible to treat these diseases. Furthermore, by transplanting the neural spheroid network obtained by the method of the present invention, a neural circuit that is genetically deficient in a disease that is congenitally deficient in a neural circuit, for example, a disease such as a congenital color vision abnormality It is also possible to construct a neural circuit that complements. These methods have been patterned on conventionally proposed neural activation methods, such as the injection of neural cells, glial cells, and neural stem cells (Lancet, 373, pp.2055-2066, 2009) and temperature sensitive surfaces. It can be used instead of, or together with, a method of transplanting cell sheets (Advanced Materials, 21, pp.1-6, 2009).

EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, the scope of the present invention is not limited to the following Example.
Example 1
According to the method shown in Fig. 3, wells of the same size in a grid pattern with polydimethylsiloxane (well diameter: 50/100 / 150μm, well depth: 50/100 / 150μm, distance between wells: 100/200 / 300μm) Three types of chambers were manufactured. Using this chamber, cerebral cortical cells were seeded according to the method shown in FIG. 1, and after 20 to 60 minutes, the medium was changed to wash out cells outside the well, and then the culture was continued for 24 hours to form spheroids. The culture conditions were as described in JP Pharmacol. Jpn., 124, pp. 11-17, 2004, and a medium with 10% FBS and B27 (GIBCO) added to DMEM / F12 medium was used. Incubation was performed at 37 ° C. under 5% CO ₂ , and the medium was changed every 2-3 days.

  FIG. 7 shows a phase contrast micrograph of the spheroids (cultured day 2) formed in the PDMS chamber. The numbers in the figure indicate the diameter of the well, and the non-coat dish indicates the state of culture on an uncoated culture dish. In the uncoated culture dish, cells could not adhere to each other and a non-uniform cell mass was formed, but spherical spheroids were formed in the wells of the chamber. FIG. 8 shows the size distribution of spheroids formed in the PDMS chamber. The vertical axis represents the number of spheroids, and the horizontal axis represents the area of the spheroids measured by taking a phase contrast micrograph of the spheroids taken out from the wells of the PDMS chamber. FIG. 9 shows the results of cell staining (live / dead assay) of spheroids formed in wells having a diameter of 100 μm. Green indicates live cells and red indicates dead cells. In the figure, the upper figure of (b) shows the immunocytochemical staining image (green; MAP2) of the neuronal marker protein MAP2 (microtubule-associated protein 2), and the lower figure shows the neuronal marker protein MAP2 (green) and glia. An immunocytochemical staining image with cell marker proteins GFAP (glial marker protein) (red) and Hoechst (nuclear staining; blue) is shown.

  Around 5 days in culture, neurites (axial splits, dendrites, etc.) were elongated from the spheroids, and on 14 days, a neural spheroid network was formed in which neurites extended from adjacent spheroids were connected (FIG. 10). On the fifth day of culture, the cells coming out of the well were mainly glial cells, and most of the nerve cells were not out of the well.

Example 2
The neural spheroid network constructed by performing culture in the PDMS chamber for 14 days according to the method of Example 1 can be transferred to the glass substrate by attaching the neural spheroid network to the glass substrate and incubating for 24 hours, and then peeling the PDMS chamber. (Fig. 11). The neural spheroid network transferred to the glass substrate did not peel off even when medium was changed or pipetting was performed. When immunostaining of the neuronal spheroid network transferred to the glass substrate was performed, the shape of each spheroid was clearly identified, and it was confirmed that the transferred spheroid secured its position in the PDMS chamber (Fig. 12 (a)).

  According to the method shown in FIG. 5, two types of nerve spheroid networks were transferred to a glass substrate, and fluorescent photographs of the two types of transferred nerve spheroid networks were taken (FIG. 12 (b)). The primary nerve spheroid network (1st, green) and the secondary nerve spheroid network (2nd, red) were labeled with the cell tracker. As a result, it was confirmed that two types of nerve spheroid network complexes were formed on the glass substrate.

Example 3
One type of neural spheroid network was transferred to a glass substrate by the method of Example 2, and Ca ²⁺ imaging was performed. Spontaneous Ca ²⁺ vibration of each spheroid (spontaneous Ca ²⁺ oscillations) are synchronized (13), a neurological function as a whole nerve spheroids network formed is connected to functional by spheroids It became clear that Furthermore, in a large neural spheroid network, there was a time difference in spontaneous Ca ²⁺ oscillations between spheroids that were far apart. This means that as the distance increases, synchronization is delayed due to the transmission speed between networks.

Example 4
According to the method of Example 1, spheroids were formed from rat cerebral cortical cells using a PDMS chamber (well diameter: 100 μm, well depth: 100 μm, distance between wells: 200 μm), and a neural spheroid network was obtained after 14 days of culture. . In accordance with step 3 (transfer on the rat brain) in the conceptual diagram shown in FIG. 6, the PDMS chamber was pasted so that the neural spheroid network was in direct contact with and in close contact with the surface of the rat cerebrum (19 days old). The culture was continued with the medium added, and the PDMS chamber was peeled off after 24 hours. When the transcribed neural spheroid network was labeled with Cell tracker red and a fluorescent photograph was taken, the connection by spheroids and neurites (dendrites and axons) was not deformed or destroyed even after 24 hours. It was confirmed that the nerve spheroid network was engrafted on the surface of the cerebrum in the same shape (FIG. 14, 24 hours after transcription and enlarged view). 48 hours after the transcription of the neural spheroid network, glial cells migrate from the transcribed neural spheroid network to the cerebral tissue, and the dendrites and axons of the neural cells inside the neural spheroid also extend toward the rat fetal brain. Was confirmed (FIG. 14, 48 hours after transcription).

Example 5
The neurospheroid network was transcribed on the surface of rat cerebral cortex tissue by the method of Example 4, and the function of the transcribed network was analyzed. Figures 15 (A) and (B) show the state of transcription (transplant), (C) and (D) show the state of the transcribed neural spheroid network ((D) is an enlarged photograph of (C). is there). Ca ²⁺ imaging (pseudo-color image) of neural spheroid network transcribed on rat cerebral cortex tissue surface (after culturing for 8 days after transcription) is shown in (E) and numbered in neural spheroid network (E) The graph of Ca ²⁺ change in the region is shown in (F). Even on the 8th day after transcription, spontaneous intracellular Ca ²⁺ changes in the neurons were observed, indicating that neuronal activity continued.

  Fig. 15 (G) is a schematic diagram showing the process by which neurites develop from neural spheroid networks transcribed on the surface of cerebral cortical tissues and form synapses with neurons in cerebral tissues, (H) is cerebral tissue The neural spheroid network transcribed on the surface (the neuron labeled with the antegrade transporter PHA-L) is shown. A neural spheroid network with arrows transcribed. Since the antegrade transporter PHA-L is transferred from the neurons in the neural spheroid network to the neurons in the cerebral tissue via the synapse, the nerve cells are labeled, and the inside of the nerve spheroid network transcribed on the surface of the cerebral tissue These neurons were found to be synapse with neurons in cerebral tissue (Figure (I), arrows indicate neurons labeled with PHA-L in cerebral tissue).

Example 6
According to the method of Example 1, spheroids were formed from rat cerebral cortical cells using a PDMS chamber (well diameter: 100 μm, well depth: 100 μm, distance between wells: 200 μm), cultured for 14 days, and neural spheroid network (1st NSN ) This neural spheroid network (1st NSN) was brought into close contact with another neural spheroid network (2nd NSN) obtained in the same manner (FIG. 16A (i)), and the culture was continued for 24 hours. After that, the PDMS chamber of the 1st NSN was removed (Fig. The network was transferred to the surface of cerebral cortex tissue. In FIG. 16, (C) shows a state in which the multi-layer nerve spheroid network is peeled off from the PDMS chamber, and (D) shows the multi-layer nerve spheroid network transferred to the surface of rat cerebral cortex tissue.

Claims (8)

A method for constructing a neural spheroid network in which two or more spheroids containing nerve cells are connected by neurites extended from each spheroid, the method comprising culturing two or more spheroids arranged in close proximity. 2. The method according to claim 1, wherein the spheroid comprises glial cells and / or neural stem cells. 3. The method of claim 2, wherein the ratio of well diameter, depth, and distance between adjacent wells ranges from about 1: 1: 1 to 1: 1: 3. The method according to claim 2 or 3, wherein the well has a diameter of 50 to 400 µm. The method according to any one of claims 2 to 4, wherein a chamber made of polydimethylsiloxane is used as the chamber. A neural spheroid network in which two or more spheroids containing nerve cells are connected by neurites extending from each spheroid. The neural spheroid network according to claim 6, which is a network complex including two or more types of neural spheroid networks. The nerve spheroid network according to claim 6 or 7 for use in nerve transplantation.