阅读说明：本技术 细胞容纳容器及其生产方法 (Cell container and method for producing the same ) 是由 仲山智明 荒谷知行 腰冢慎之介 宫冈敦史 盐野入桃子 矢本梨惠 北泽智文 于 2021-03-12 设计创作，主要内容包括：本发明的名称为细胞容纳容器及其生产方法。提供了包括神经细胞和培养基的细胞容纳容器,其中神经细胞粘着于细胞容纳容器的培养表面,神经细胞与培养表面之间的粘着面积为0.5mm～(2)以上/80,000个神经细胞,并且培养基中葡萄糖的浓度为1g/L以上。(The invention relates to a cell container and a method for producing the same. There is provided a cell-containing vessel comprising a neural cell and a culture medium, wherein the neural cell adheres to a culture surface of the cell-containing vessel, and an adhesion area between the neural cell and the culture surface is 0.5mm 2 The number of nerve cells is 80,000 or more, and the concentration of glucose in the medium is 1g/L or more.)

1. A cell containment vessel comprising:

a neural cell; and

a culture medium is used for culturing the bacteria,

wherein the neural cells adhere to the culture surface of the cell containment vessel,

the adhesion area between the nerve cells and the culture surface is 0.5mm²Above 80,000 nerve cells, and

the concentration of glucose in the culture medium is more than 1 g/L.

2. The cell containment vessel of claim 1, wherein an electrode array is placed on the culture surface.

3. The cell containment vessel of claim 1 or 2, wherein the neural cells are derived from stem cells.

4. The cell containment vessel of claim 3, wherein the stem cells are human cells.

5. A method for producing a cell containment vessel, the method comprising:

incubating a container comprising neural cells and a culture medium under culture conditions while replacing the culture medium at a predetermined time,

wherein the concentration of glucose in the medium is maintained above 1g/L for a predetermined period of time.

6. The production method according to claim 5, wherein the incubation is performed for 30 days or more.

7. The production method according to claim 5 or 6,

wherein the neural cells adhere to the culture surface of the cell-containing vessel, and

the adhesion area between the nerve cells and the culture surface was 3mm²Above 80,000 nerve cells.

8. The production method according to claim 7, wherein an electrode array is placed on the culture surface.

9. The production method according to any one of claims 5 to 8, wherein the neural cells are derived from stem cells.

10. The production method according to claim 9, wherein the stem cell is a human cell.

Technical Field

The present invention relates to a cell-containing container and a method for producing the same. Priority is claimed based on Japanese patent application No. 2020-061251, filed on 30/3/2020, and Japanese patent application No. 2021-010564, filed on 26/1/2021, the contents of which are incorporated herein by reference.

Description of the Related Art

The action potential of a nerve cell can be used to assess efficacy or toxicity relative to the nerve cell. As one of methods for detecting and evaluating action potential of nerve cells, an evaluation method using a Micro Electrode Array (MEA) is known. The MEA is an array of tiny electrodes placed on a substrate where cells are cultured, and the electrical activity of the cells can be detected.

However, it is known that when nerve cells derived from human iPS cells are cultured on MEA and action potentials are detected, it is necessary to culture the nerve cells at a higher density and for a longer period of time than in the case of using nerve cells derived from animals other than humans.

Background

Disclosure of Invention

The inventors have found that when iPS-derived neural cells are cultured at high density for a long time, the cells may aggregate and be separated from the culture surface of the culture vessel.

For example, patent document 1 (japanese unexamined patent application, first publication No. 2018-. The cell culture apparatus includes a culture tank (culture tank) for culturing living cells, an online monitoring apparatus, a cell state determination apparatus having a sterile sampling apparatus, an analyzer, a data collection apparatus, a data analyzer and a cluster analysis function apparatus (cluster analysis function), and an operation control compensator (operation control compensator) having a reference function of a cell reaction model for each cluster, and the like. However, patent document 1 does not describe the aggregation of nerve cells in the case of culturing nerve cells at a high density for a long time, nor does it describe specific parameters necessary to inhibit such aggregation of nerve cells.

An object of the present invention is to provide a technique for inhibiting nerve cell aggregation.

The cell-containing container according to the present invention comprises: a neural cell; and a culture medium, wherein the nerve cells are adhered to the culture surface of the cell-holding container, and the adhesion area between the nerve cells and the culture surface is 0.949 to 28.2mm²80,000 nerve cells, and the concentration of glucose in the culture medium is 1g/L or more.

The method for producing a cell-containing vessel according to the present invention comprises the steps of: incubating a container comprising neural cells and a culture medium under culture conditions while replacing the culture medium at a predetermined time, wherein the concentration of glucose in the culture medium is maintained above 1g/L for a predetermined period of time.

According to the present invention, a technique for inhibiting nerve cell aggregation can be provided.

Drawings

Fig. 1 is a graph showing the measurement results of the change with time of the glucose concentration in the neural cell culture medium in experimental example 1.

Fig. 2A to 2C show representative micrographs obtained by imaging the neural cells from the 53 th day (DIV53) of the seeded neural cells in experimental example 2.

Fig. 3A shows a representative micrograph of the neural cells evaluated as aggregation level 1 in experimental example 3. Fig. 3B shows a representative micrograph of the neural cells evaluated as aggregation level 2 in experimental example 3. Fig. 3C shows a representative micrograph of the neural cells evaluated as aggregation level 3 in experimental example 3. Fig. 3D shows a representative micrograph of the neural cells evaluated as aggregation level 4 in experimental example 3.

Fig. 4 is a graph showing the change in the aggregation level of nerve cells with time in experimental example 4.

Fig. 5 is a graph showing the results obtained by examining the relationship between the concentration of glucose in the medium and the aggregation level of neural cells in experimental example 5.

Detailed Description

[ cell Container ]

The present invention provides a cell-containing container according to one embodiment, comprising: spirit of the inventionPassing through cells; and a culture medium, wherein the nerve cells are adhered to the culture surface of the cell-holding container, and the adhesion area between the nerve cells and the culture surface is 0.5mm²The number of nerve cells is 80,000 or more, and the concentration of glucose in the medium is 1g/L or more.

As described in the examples below, the inventors have found that there is a relationship between the accumulation of neural cells and the concentration of glucose in the culture medium. In addition, it was clarified that the glucose concentration in the medium may be less than 1g/L in the case of culturing the neural cells according to the conventional protocol. Furthermore, it was found that the aggregation of nerve cells can be suppressed by maintaining the glucose concentration in the medium at 1g/L or more.

When the aggregation of nerve cells is inhibited, the action potential of nerve cells can be satisfactorily detected and evaluated using MEA or the like. Moreover, when the aggregation of nerve cells is inhibited, the state of nerve cells can be satisfactorily evaluated by other analytical means (e.g., immunostaining).

The cell container according to the present embodiment is a culture of neural cells in a culture container. The culture vessel may be a vessel generally used for cell culture, and a culture dish (dish) and a well plate are illustrative examples thereof. The diameter of the culture dish, the number of holes in the well plate, and the like may be appropriately selected depending on the application.

In the cell housing container of the present embodiment, the electrode array may be placed on the culture surface of the container. That is, the cell container of the present embodiment may be an MEA plate. The number of electrodes in the MEA or the like may be appropriately selected depending on the application.

The organic material and the inorganic material described below are illustrative examples of the material of the culture surface of the culture vessel. One of these may be used alone, and two or more thereof may be used in combination.

The organic material is not particularly limited and may be appropriately selected according to the purpose. Polyethylene terephthalate (PET), Polystyrene (PS), Polycarbonate (PC), triacetyl cellulose (TAC), Polyimide (PI), ninon (Ny), Low Density Polyethylene (LDPE), Medium Density Polyethylene (MDPE), vinyl chloride, vinylidene chloride, polyphenylene sulfide, polyethersulfone, polyethylene naphthalate, polypropylene, acrylic materials (such as urethane acrylate), cellulose, silicone-based materials, such as Polydimethylsiloxane (PDMS), polyvinyl alcohol (PVA), metal alginates, such as calcium alginate, polyacrylamide, methyl cellulose, and gel-like materials (such as agarose) are illustrative examples.

The inorganic material is not particularly limited and may be appropriately selected according to the purpose, and glass and ceramic are illustrative examples thereof.

The culture surface of the culture vessel may be coated with a coating agent. Coating agents generally used for cell culture may be suitably used, and collagen, Matrigel (registered trademark, Corning), geltrex (thermo Fisher scientific), PLO (Sigma-Aldrich), PDLO (Sigma-Aldrich), fibronectin, fibrinogen, gelatin, Polyethyleneimine (PEI), laminin, and the like are illustrative examples thereof.

The nerve cells contained in the cell-containing container of the present embodiment may be cells collected from a living body or cells that have been established and cultured. In addition, from the viewpoint of easily obtaining a desired cell population containing a large number of nerve cells, cells can be differentiated from stem cells. That is, the neural cells may be derived from stem cells.

Embryonic stem cells (ES cells), induced pluripotent stem cells, mesenchymal stem cells, umbilical cord blood-derived stem cells, neural stem cells, and the like are illustrative examples of the stem cells. Nuclear-transferred embryonic stem cells (ntES cells), induced pluripotent stem cells (iPS cells), and the like are illustrative examples of the induced pluripotent stem cells. Bone marrow mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, and the like are illustrative examples of mesenchymal stem cells. Among them, the stem cell is preferably an iPS cell.

iPS cells can be derived from healthy humans or patients with various neurological diseases. In addition, cells that have undergone various gene edits can be used. For example, cells that have been engineered by gene editing to have genes that are causative or risk factors of various nervous system diseases can be used.

In the case where iPS cells are derived from patients with various neurological diseases, iPS cells can be used to construct disease models of the nervous system. The neurological diseases are not particularly limited, and neurodegenerative diseases, autism, epilepsy, Attention Deficit Hyperactivity Disorder (ADHD), schizophrenia, bipolar disorder, and the like are illustrative examples thereof. Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, etc. are illustrative examples of neurodegenerative diseases.

The animal species from which the neural cells are derived is not particularly limited, and humans, monkeys, dogs, cows, horses, sheep, pigs, rabbits, mice, rats, guinea pigs, and hamsters are illustrative examples thereof. Among them, human is preferable.

Also, one kind of nerve cell may be used alone, or a mixture of two or more kinds of nerve cells may be used. Nerve cells can be roughly classified into, for example, peripheral nerves and central nerves. Sensory nerve cells, motor nerve cells, and autonomic nerve cells are illustrative examples of peripheral nerves. Intervening nerve cells and projection neurons are illustrative examples of central nerves. Cortical neurons, hippocampal neurons, amygdala neurons, etc., are illustrative examples of projection neurons. In addition, central nerve cells can be roughly classified into excitatory neurons and inhibitory neurons. Glutamate-operated neurons mainly responsible for excitatory transmission in the central nervous system, GABA (gamma-aminobutyric acid) -operated neurons mainly responsible for inhibitory transmission, and the like are illustrative examples thereof.

Cholinergic neurons, dopaminergic neurons, noradrenergic neurons, 5-hydroxytryptotaminergic neurons, histaminergic neurons, and the like are illustrative examples of other neurons that release neuromodulators.

The cell container according to the present embodiment can contain astrocytes, microglia, and the like together with nerve cells. The adhesion area between the nerve cells and the culture surface of the culture vessel was 0.5mm²Above, preferably 0.949mm²Above, more preferably 3mm²Above, still more preferably 3.14mm²Above 80,000 nerve cells. Furthermore, the culture table of the nerve cells and the culture vesselThe upper limit of the adhesion area between the faces is preferably about 28.2mm²。

In addition, it is preferable that the nerve cell is mature according to the purpose. For example, it is preferable that one of the marker genes of tubulin β 3, MAP2, NeuN, 160kDa neurofilament, 200kDa neurofilament, NSE, PSD93 and PSD95 is positively expressed.

As the medium contained in the cell-containing vessel of the present embodiment, a medium suitable for the cells to be used can be appropriately selected and used as long as the concentration of glucose in the medium is 1g/L or more.

The medium in which the essential components are added to the basal medium is a specific illustrative example of the medium. DME Medium (Dulbecco's Modified Eagle's Medium, DMEM), Ham F12 Medium (Ham nutrient mixture F12), D-MEM/F12 Medium, McCoy's 5A Medium, Eagle's MDM Medium (Eagle's essential Medium, EMEM), alpha MEM Medium (alpha Modified Eagle's essential Medium, alpha MEM), MEM Medium (essential Medium), RPMI1640(Roswell Park mental Institute-1640) Medium, Iscove Modified Dulbecco's Medium (IMDM), MCDB131 Medium, William Medium E, IPL41 Medium, Fischer Medium, M199 Medium, high performance Medium 199, Sterno 34 (produced by Thermo Scientific), X-Chem 10 (produced by Chemb), X-Chem 15 (produced by Chem), VImS 15 (produced by StemSem), StemSem technology VO 3000, StemS technology, StemS 3000, StemS technology, StemS essential Medium, and StemS, Stemline II (manufactured by Sigma-Aldrich), QBSF-60 (manufactured by Quality Biological), StemPro hESC SFM (manufactured by Thermo Fisher Scientific), Essential8 (registered trademark) medium (manufactured by Thermo Fisher Scientific), mTeSR1 or mTeSR2 medium (manufactured by Stem Cell Technologies), Rero FF or Rero FF2 (manufactured by Reprocell), PSGro hESC/iPSC medium (manufactured by System Biosciences), NutriStem (registered trademark) medium (manufactured by Biological Industries), CSTI-7 medium (manufactured by Science Laboratory), MesenPRO RS medium (manufactured by Thermo Scientific), MF-mesenchymal (registered trademark) Stem Cell growth medium (manufactured by thermal Laboratory), Thermsf-Pro medium (manufactured by Toxico Biological), example II, Fisher Co 900. One of these may be used alone, or two or more thereof may be used in combination.

In addition, examples of additives to be added to the basal medium include those generally used for culturing nerve cells. Component N (Elixargen Scientific), component G2 (Elixargen Scientific), N2 supplement (Thermo Fisher Scientific), iCell neuron supplement B (CDI), iCell nervous system supplement, B-27plus (Thermo Fisher Scientific), and the like are illustrative examples thereof.

[ method for producing cell-containing vessel ]

The present invention provides a method for producing a cell containment vessel according to one embodiment, the method comprising the steps of: incubating a container comprising neural cells and a culture medium under culture conditions while replacing the culture medium at a predetermined time, wherein the concentration of glucose in the culture medium is maintained above 1g/L for a predetermined period of time.

The cell container described above can be produced by the production method of the present embodiment. In addition, as will be described below in examples, a cell container that inhibits the aggregation of nerve cells can be produced by the production method of the present embodiment.

In the production method of the present embodiment, the vessel (culture vessel), the neural cells, and the culture medium are the same as described above. Specifically, for example, the electrode array may be placed on the culture surface of the cell-containing vessel. In addition, the neural cells may be derived from stem cells. Further, the stem cell may be a human cell.

Changing 10% to 100% by volume of the total amount of the medium 1 to 10 times per week is an illustrative example of changing the medium at a predetermined time. The time for medium change can be, for example, 1 to 8 times per week, 1 to 5 times per week, or 1 to 3 times per week. Preferably, the time period from a medium change to the next medium change is substantially constant.

Further, the amount of the medium to be replaced in one medium replacement may be 10% to 80% by volume of the total amount of the medium, 10% to 50% by volume of the total amount of the medium, and 10% to 30% by volume of the total amount of the medium.

In addition, the culture conditions may be those commonly used for culturing nerve cells, 37 ℃ and 5% CO₂Are illustrative examples thereof. In addition, the oxygen concentration may be set to 0% to 1%.

The incubation period may be appropriately set according to the purpose. For example, in the case where an action potential is detected in a nerve cell that has been induced to differentiate from a human iPS cell, the incubation period may be, for example, 30 days or more, for example, 40 days or more, for example, 50 days or more, for example, 60 days or more, and, for example, 70 days or more from the time point at which the nerve cell dissociated into a single cell is seeded in the culture vessel.

In the production method of the present embodiment, the concentration of glucose in the medium is maintained at 1g/L or more for a predetermined period of time. Here, the predetermined period of time may be the entire period during which the neural cells are cultured under the culture conditions, or may be, for example, at least 20 days, for example, 22 days, for example, 24 days from the start of the culture. In addition, the concentration of glucose in the medium is preferably maintained at 0.2g/L or more for at least 30 days from the start of culture, for example, 35 days from the start of culture, and for example, 40 days from the start of culture. Here, the start of culture refers to a point of time at which neural cells dissociated into single cells are seeded in a culture vessel.

In the production method of the present embodiment, the nerve cells are adhered to the culture surface of the cell-accommodating container, and the adhesion area between the nerve cells and the culture surface is preferably 0.5mm²Above, preferably 0.949mm²Above, more preferably 3mm²Above, still more preferably 3.14mm²Above 80,000 nerve cells. Furthermore, the upper limit of the adhesion area between the nerve cells and the culture surface of the culture vessel is preferably about 28.2mm²。

[ examples ]

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[ Experimental example 1]

(examination of the change in glucose concentration in the culture Medium with time)

Culturing human nerve cells. In addition, in the process, the change in the glucose concentration in the medium with time was measured. Cell culture medium was changed 3 times per week. In addition, the amount of medium to be replaced was changed for comparison.

As the human neural cell, a gabaergic neuron (elixigen Scientific) derived from human iPSC was used.

As the cell culture vessel, an MEA plate (model "M768-tMEA-48W", axis Biosystems) having a micro-electrode array (MEA) was used. The culture surface of the MEA plate was coated with Polyethyleneimine (PEI) and laminin (Thermo Fisher Scientific). The volume of each well of the culture vessel was 300. mu.L.

From the day of seeding with nerve cells (day 0 in vitro, hereinafter sometimes referred to as "DIV 0") to day 5 (hereinafter sometimes referred to as "DIV 5", and the same applies hereinafter), Quick-neuron (TM) GABA-sustainable Medium (Quick-neuron (TM) GABAergic Maintenance Medium) (product name) (Elixargen Scientific) was used as the Medium. The glucose concentration in the medium was 5 g/L. In DIV6 and beyond, "Complete Brainpys Medium (product name) (CDI) was used as the Medium. The glucose concentration in the medium was 0.5 g/L.

The medium was changed as follows. First, 250. mu.L, 150. mu.L or 50. mu.L of the medium was aspirated from the culture vessel at a time. Subsequently, a new medium having the same volume as the suction medium is added. The medium was changed 3 times per week. Aspirated media was used to measure glucose concentration. FLEX2(Nova Biomedical co., Ltd.) which is a media composition analyzer was used to measure glucose concentration.

FIG. 1 is a graph showing the measurement results of the change in glucose concentration in a medium with time. The horizontal axis shows the number of days of culture from the inoculation of the neural cells, and the vertical axis shows the glucose concentration (g/L) in the medium.

As a result, it was confirmed that the glucose concentration in the medium was maintained at the highest level every time 50. mu.L of the medium was changed (3 times per week). The glucose concentration in the medium at day 22 after seeding of the neural cells was about 1.4g/L at each replacement of 50. mu.L of the medium. In addition, the concentration of glucose in the medium was about 0.2g/L at day 40 after the inoculation of the neural cells.

On the other hand, in the case of replacing 150. mu.L of the medium each time (3 times per week), the glucose concentration in the medium on day 22 after the inoculation of the neural cells was about 0.4 g/L. In addition, the concentration of glucose in the medium was about 0g/L at day 40 after the inoculation of the neural cells.

In addition, each time 250 u L medium (3 times per week) change condition, after inoculation of neural cells on the 22 th day in the culture medium glucose concentration is about 0.3 g/L. In addition, the concentration of glucose in the medium was about 0g/L at day 40 after the inoculation of the neural cells.

[ Experimental example 2]

(examination of the level of aggregation of nerve cells)

Each of the neural cells cultured in experimental example 1 was observed with a microscope and the aggregation level thereof was evaluated. The evaluation criteria for aggregation level are as follows. As the level of aggregation increases, the adhesion area between the nerve cells and the culture surface becomes smaller, and the nerve cells tend to detach from the culture surface.

< evaluation criteria for aggregation level >)

1: the adhesion area between the nerve cells and the culture surface was 3.14mm²Above and 28.2mm²80,000 nerve cells.

2: the adhesion area between the nerve cells and the culture surface was 0.949mm²Above and 3.14mm²80,000 nerve cells.

3: the adhesion area between the nerve cells and the culture surface was 0.196mm²Above and 0.949mm²80,000 nerve cells.

4: the adhesion area between the nerve cells and the culture surface was 0mm²Above and 0.196mm²80,000 nerve cells.

Fig. 2A to 2C show representative micrographs of each neural cell at day 53 (DIV53) from the seeding of the neural cells. Figure 2A shows a micrograph of nerve cells obtained by replacing 250 μ Ι _ of medium each time (3 times per week). Figure 2B shows a micrograph of nerve cells obtained by replacing 150 μ Ι _ of medium each time (3 times per week). Figure 2C shows a micrograph of nerve cells obtained by replacing 50 μ Ι _ of medium each time (3 times per week).

As a result, the level of aggregation of the neural cells of DIV53 obtained by replacing 250 μ L of the medium each time (3 times per week) was 4. In addition, the level of aggregation of nerve cells of DIV53 obtained by replacing 150. mu.L of the medium each time (3 times per week) was 3. In addition, the level of aggregation of nerve cells of DIV53 obtained by changing 50. mu.L of the medium each time (3 times per week) was 1.

From the results, it was seen that neural cells cultured under the condition of keeping the glucose concentration in the medium high tend to have a low aggregation level. More specifically, it was confirmed that at day 22 after seeding of the neural cells, the neural cells having a glucose concentration of 1g/L or more in the medium tended to have a low aggregation level. Furthermore, it was confirmed that at day 40 after seeding of the neural cells, the neural cells having a glucose concentration of 0.2g/L or more in the medium tended to have a low aggregation level.

[ Experimental example 3]

(examination of the level of aggregation of nerve cells and action potential)

In the same manner as in experimental example 1, the nerve cells cultured for 42 days from the seeding of the nerve cells were observed with a microscope, and the aggregation level thereof was evaluated. The evaluation criteria for aggregation level were the same as in experimental example 2. In addition, action potential of each nerve cell was measured and evaluated using a microelectrode array. The evaluation criterion of the action potential is as follows, and the detectability of the action potential that can be detected is determined.

< evaluation criteria for action potential >)

A: the action potential can be detected well.

B: an action potential can be detected.

C: the action potential cannot be detected.

Figure 3A shows a representative micrograph of neural cells assessed as aggregation level 1. Figure 3B shows a representative micrograph of neural cells assessed as aggregation level 2. Figure 3C shows a representative micrograph of neural cells assessed as aggregation level 3. Figure 3D shows a representative micrograph of neural cells assessed as aggregation level 4. In addition, table 1 below shows the evaluation results of aggregation level and action potential.

[ Table 1]

Level of aggregation	1	2	3	4
					Determining detectability of action potentials	A	B	C	C

As a result, it was clarified that action potential could be detected well when using the neural cell evaluated as aggregation level 1.

[ Experimental example 4]

(examination of the Change in the level of aggregation of nerve cells with time)

Changes in the aggregation level of each neural cell cultured in the same manner as in experimental example 1 were observed with time. The evaluation criteria for aggregation level were the same as in experimental example 2.

FIG. 4 is a graph showing the change over time of the aggregation level of nerve cells obtained by replacing 250. mu.L, 150. mu.L or 50. mu.L of the medium each time (3 times per week). As a result, it was confirmed that the neural cells obtained by replacing 50 μ L of the medium each time (3 times per week) maintained the aggregation level 1 even on day 56 after the inoculation of the neural cells.

On the other hand, it was recognized that after the 38 th day after seeding of the neural cells, the aggregation level of the neural cells obtained by replacing 150. mu.L of the medium every time (3 times per week) was increased. In addition, it was recognized that after 31 days after seeding of the neural cells, the aggregation level of the neural cells obtained by replacing 250. mu.L of the medium each time (3 times per week) was increased.

The results show that the lower the glucose concentration in the medium, the higher the aggregation level of the neural cells tends to be. In the related art, culture conditions of neural cells obtained by replacing 150 μ L of the medium every time (3 times per week) are generally employed. On the other hand, it was clarified that in the case of the culture conditions of neural cells obtained by replacing 50. mu.L of the medium every time (3 times per week), the aggregation level of neural cells can be kept at a low level.

[ Experimental example 5]

(examination of the relationship between the glucose concentration in the culture Medium and the aggregation level of neural cells)

In the same manner as in experimental example 1, the neural cells were cultured by changing the amount of the replaced medium. In addition, the relationship between the glucose concentration of the nerve cells in the medium on day 22 from the inoculation of the nerve cells (DIV22) and the aggregation levels of the nerve cells on day 31 from the inoculation of the nerve cells (DIV31), day 38 (DIV38), day 45 (DIV45) and day 56 (DIV56) was examined.

Fig. 5 is a diagram showing the examination result. In fig. 5, the horizontal axis shows the glucose concentration (g/L) of the neural cells in the medium at day DIV22, and the vertical axis shows the aggregation level of the neural cells evaluated by the same evaluation criteria as experimental example 2.

As a result, it was clarified that the aggregation level of the nerve cells tended to be kept low even at day 31 (DIV31), day 38 (DIV38), day 45 (DIV45) and day 56 (DIV56) when the glucose concentration of the nerve cells in the medium was 1g/L or more at day 22 from the inoculation of the nerve cells (DIV 22).

On the other hand, it was confirmed that when the glucose concentration of the neural cells in the medium was less than 1g/L on day 22 from the inoculation of the neural cells (DIV22), the aggregation level of the neural cells tended to increase on days 31 (DIV31), 38 (DIV38), 45 (DIV45) and 56 (DIV 56).

The present invention includes the following aspects.

[1] A cell containment vessel comprising:

a neural cell; and

a culture medium is used for culturing the bacteria,

wherein the nerve cells adhere to the culture surface of the cell containment vessel,

the adhesion area between the nerve cells and the culture surface was 0.5mm²Above 80,000 nerve cells, and

the concentration of glucose in the medium is 1g/L or more.

[2] The cell-containing vessel according to [1], wherein the electrode array is placed on a culture surface.

[3] The cell-containing container according to [1] or [2], wherein the neural cell is derived from a stem cell.

[4] The cell holding container according to [3], wherein the stem cell is a human cell.

[5] A method for producing a cell containment vessel, the method comprising:

incubating a container comprising neural cells and a culture medium under culture conditions while replacing the culture medium at a predetermined time,

wherein the concentration of glucose in the medium is maintained above 1g/L for a predetermined period of time.

[6] The production method according to [5], wherein the cultivation is carried out for 30 days or more.

[7] The production method according to [5] or [6], wherein the nerve cells are adhered to the culture surface of the cell-holding vessel, and

the adhesion area between the nerve cells and the culture surface was 3mm²Above 80,000 nerve cells.

[8] The production method according to [7], wherein the electrode array is placed on a culture surface.

[9] The production method according to any one of [5] to [8], wherein the neural cell is derived from a stem cell.

[10] The production method according to [9], wherein the stem cell is a human cell.

While preferred embodiments of the present invention have been described and illustrated above, it should be understood that these are examples of the present invention and should not be considered as limiting. Additions, omissions, substitutions, and other modifications can be made without departing from the scope of the present invention. Accordingly, the invention is not to be seen as limited by the foregoing description, but is only limited by the scope of the appended claims.