JP6978045B2 - A system for culturing undifferentiated stem cells to induce differentiation of cardiomyocytes and a method for producing cardiomyocytes - Google Patents

Description

The present invention relates to a cell culture apparatus and its use. Specifically, the present invention relates to a cell culture apparatus, a cell culture system including the cell culture apparatus, and a method for producing myocardial cells using the cell culture apparatus or the cell culture system.

Cardiomyocytes in adults have lost proliferative activity. Therefore, in the case of severe myocardial infarction, cardiomyopathy, and other diseases, there has been a continuous situation in which heart transplantation has to be relied on. However, in recent years, research on pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) has progressed. From this, it is becoming possible to induce differentiation of these pluripotent stem cells to produce cardiomyocytes, and to use such induced cardiomyocytes in transplantation medicine.

So far, suspension culture systems (SCSs) have been used for the maintenance and proliferation of human iPS cells. With SCSs, the culture volume can be easily scaled up and the medium exchange is easy. Further, in SCSs, human iPS cells spontaneously aggregate cells and form an embryoid body (EB), and are therefore suitable for the generation of differentiated cells (see, for example, Non-Patent Document 1).

Hemmi N et al., “A Massive Suspension Culture System With Metabolic Purification for Human Pluripotent Stem Cell-Derived Cardiomyocytes.”, Stem Cells Translational Medicine, Vol. 3, No. 12, p1473-1483, 2014.

However, in a culture of undifferentiated stem cells using SCSs or a differentiation-inducing system such as the method described in Non-Patent Document 1, it is difficult to obtain a homogeneous EB because the cell mass is difficult to grow and the differentiation efficiency is unstable. be. In addition, since the optimum conditions differ for each cell line, it is necessary to examine the conditions for the rotation speed of the feathers. In addition, it is difficult to completely remove undifferentiated stem cells remaining in the system. Furthermore, when transplanting cells, it is common to change them into various microstructures such as spheroids, sheets, and fibers, and in that case, it is necessary to isolate the cells once using an enzyme or the like. There is. However, in the culture of undifferentiated stem cells by SCSs or the differentiation induction system, the adhesion between cells is strong by long-term culture, and isolation becomes extremely difficult.

The present invention has been made in view of the above circumstances, and provides a cell culture apparatus and a cell culture system in which undifferentiated stem cells are proliferated with high efficiency and the efficiency of inducing differentiation into cardiomyocytes is stable. Further, the present invention provides a method for producing cardiomyocytes using the cell culture apparatus or the cell culture system.

That is, the present invention includes the following aspects.
The system according to the first aspect of the present invention is a system for culturing undifferentiated stem cells and inducing differentiation of myocardial cells, in which the rate of the cell culture device, the gas supply device, and the gas supplied to the cell culture device is high. Each layer is provided with a control unit that controls the gas supply device so that it is constant at 20 mL / min or more and 60 mL / min or less in all layers, and the cell culture device has a liquid level of 2 mm or more and 10 mm or less. A culture solution storage unit capable of storing the culture solution set to be, a gas supply flow path through which the supplied gas flows, and the gas supply flow path are provided and stored in the culture solution storage unit. A gas discharge port for discharging the gas to the culture solution so as to be constant in all layers at the speed, a gas discharge port for discharging the gas discharged to the culture solution, and the above. comprising a gas discharge channel for the gas through the gas discharge port is flows, a culture vessel equipped with a four or more layers, and is formed by laminating the culture vessel 4 or more layers.

The culture vessel having four or more layers includes the gas supply channel of one culture vessel, the gas supply channel of another culture vessel, the gas discharge channel of one culture vessel, and the like. The gas discharge flow path of the culture container may be laminated so as to be communicatively connected to each other.

The culture solution storage section may be formed in a rectangular shape, and the gas supply flow path and the gas discharge flow path may be provided at both corners of one side of the culture solution storage section.

The method of manufacturing cardiomyocytes according to the second aspect of the present invention, using the system according to the first aspect, culturing undifferentiated stem cells, a method comprising a differentiation induction step of inducing differentiation into cardiomyocytes.

According to the cell culture apparatus and cell culture system of the above-described embodiment, undifferentiated stem cells can be proliferated with high efficiency and cardiomyocytes can be obtained with stable differentiation induction efficiency. According to the method for producing cardiomyocytes according to the above embodiment, a large amount of cardiomyocytes can be obtained from undifferentiated stem cells with stable differentiation-inducing efficiency.

It is a schematic block diagram (perspective view) which shows an example of the cell culture apparatus of this embodiment. It is a schematic block diagram (front view) which shows an example of the cell culture apparatus of this embodiment. It is a schematic block diagram which shows an example of the cell culture system of this embodiment. It is a schematic block diagram which shows the flow of the culture test of the human iPS cell in the reference example 1. FIG. 6 is an image showing the result of alkaline phosphatase staining of human iPS cells in Reference Example 1. 6 is an image showing the result of immunofluorescent staining of human iPS cells in Reference Example 1. The upper part is an image in a bright field, and the lower part is an image in which fluorescence is detected. The scale bar is 100 μm. It is a schematic block diagram which shows the flow of the culture test of the human iPS cell in Example 1. FIG. 3 is a graph showing the proliferation efficiency of human iPS cells in Reference Example 1 and Example 1. 6 is an image showing the result of immunofluorescent staining of human iPS cells in Example 1. The left is an image in the bright field, and the right is an image in which fluorescence is detected. The scale bar is 100 μm. _{3 is a graph showing changes over time in CO 2} partial pressure, O ₂ partial pressure, pH, glucose concentration and glutamine concentration in a culture medium in which human iPS cells were cultured under AGV and NGV conditions in Example 1. It is an image which shows the time-dependent change of the color of phenol red in PBS in Example 2. FIG. It is a graph which shows the gas replacement rate under the AGV condition in Example 2. FIG. It is a graph which shows the gas replacement rate under the NGV condition in Example 2. FIG. It is a schematic block diagram which shows the flow of the culture test of the human iPS cell in Example 2. FIG. It is a graph which shows the time-dependent change of the cell number of the human iPS cells under the AGV condition and the NGV condition in Example 2. FIG. 3 is a graph showing the proliferation efficiency of human iPS cells under AGV and NGV conditions in Example 2. 6 is an image showing the result of immunofluorescent staining of human iPS cells in Example 2. The upper part is an image in a bright field, and the lower part is an image in which fluorescence is detected. The scale bar is 100 μm. 3 is a graph showing changes over time _{in CO 2} partial pressure, O ₂ partial pressure, and pH in a culture medium in which human iPS cells were cultured under AGV and NGV conditions in Example 2. It is a schematic block diagram which shows the flow of the differentiation induction test from a human iPS cell to a cardiomyocyte in Example 3. FIG. 3 is a graph showing the cell viability of differentiated cells derived from human iPS cells cultured under each condition in Example 3. 3 is a graph showing the number of differentiated cells derived from human iPS cells cultured under each condition in Example 3. In the graph, "non-CMs" indicates the number of cells that are not cardiomyocytes, and "CMs" indicates the number of cells differentiated into cardiomyocytes. It is a graph which shows the ratio of the troponin T positive cell in the differentiated cell derived from the human iPS cell cultured under each condition in Example 3 (left), and the graph which shows the cell number of the human iPS cell-derived cardiomyocyte (right). 3 is a graph showing the results of FACS analysis using differentiated cells derived from human iPS cells cultured under each condition in Example 3. Above is a graph showing the results of FACS analysis using human iPS cells. The middle is a graph showing the results of FACS analysis using cells before metabolism selection. Below is a graph showing the results of FACS analysis using cells after metabolism selection. 6 is an image showing the results of immunofluorescent staining of differentiated cells derived from human iPS cells in Example 3. The left is a fluorescence detection image of cells before metabolism selection, and the right is a fluorescence detection image of cells after metabolism selection. The scale bar is 500 μm.

≪Cell culture device≫
1A (perspective view) and 1B (front view) are schematic configuration views showing an example of the cell culture apparatus of the present embodiment. Each configuration of the cell culture apparatus of this embodiment will be described in detail below with reference to FIGS. 1A and 1B.

The cell culture apparatus 100 shown in FIGS. 1A and 1B includes a plurality of culture containers 10. The culture container 10 includes a culture solution storage unit 1, a gas supply flow path 2, a gas discharge port 3, a gas discharge port 4, and a gas discharge flow path 5. Further, the cell culture device 100 is formed by stacking a plurality of culture containers 10. In FIGS. 1A and 1B, 10 layers of the culture vessel 10 are laminated, but the number of layers may be two or more, and the number of layers is not limited to this.

In the culture vessel 10, the culture solution storage unit 1 is for storing the culture solution 7 so as to have a predetermined liquid level.

In the culture solution storage unit 1, the liquid level height of the culture solution is preferably set to be 2 mm or more and 10 mm or less, more preferably 2 mm or more and 6 mm or less, and more preferably 2 mm or more. It is more preferable that it is set to be 4 mm or less.
When the liquid level height of the culture solution is at least the above lower limit value, the cells can be grown with higher efficiency in the culture solution while providing sufficient nutrition without drying the cells. On the other hand, when the value is not more than the above upper limit, the gas supplied to the culture solution can be dissolved with higher efficiency.

The shape of the culture solution storage portion 1 is not particularly limited, but it is preferable that the shape when viewed from the top surface is rectangular.

Further, it is preferable that the bottom surface of the culture solution storage portion 1 is coated with a known matrix capable of culturing undifferentiated stem cells. Known matrices include, for example, Matrigel (manufactured by Thermo Fisher Scientific), Laminin 511E8 (manufactured by Nippi), Laminin 511/521 (manufactured by Biolamina), Vitronectin (manufactured by Thermo Fisher) with reduced growth factors. Examples thereof include, but are not limited to, E-cadherin (manufactured by R & D) and Synthemax (manufactured by Corning).

The culture medium used in the cell culture apparatus of the present embodiment may contain components effective for cell growth, and examples thereof include various components such as amino acids, vitamins, inorganic salts, saccharides, and growth factors. .. In addition, it may contain components such as an antibiotic, a buffer solution, a chelating agent, and a phenol red indicator.

The culture medium may have, for example, the same composition as a general cell culture medium used as a conventional medium. Specific examples of the general cell culture medium used as the conventional medium include Dulvecco-modified Eagle's culture medium (DMEM) and MEM culture medium (eg, α-MEM, MEM [Hank's BSS]). Examples thereof include RPMI culture medium (for example, RPMI 1640 etc.), F12 culture medium, StemPro34, mTeSR1 and the like.

Further, the culture medium may have, for example, the same composition as a general cell culture medium used as an undifferentiated stem cell maintenance medium. Specific examples of the undifferentiated stem cell maintenance medium include StemFit medium, mTeSR® Essential 8® medium, StemSure® medium and the like.

In addition, in this specification, "undifferentiated stem cell" is used as a concept including pluripotent stem cell having pluripotent differentiation. Examples of undifferentiated stem cells include embryonic stem cells (ES cells), artificial pluripotent stem cells (iPS cells), and cells having differentiation pluripotency derived from these stem cells. It also includes various somatic stem cells. The "undifferentiated stem cell" is not particularly limited as long as it is a cell having pluripotency, and includes an unknown cell having the same properties as the ES cells and iPS cells exemplified above.

The fact that the cell is an undifferentiated stem cell can be determined from the presence or absence of properties specific to the undifferentiated stem cell, the expression of various markers specific to the undifferentiated stem cell, and the like. For example, as a property specific to undifferentiated stem cells, there is a property of having self-renewal ability and being able to differentiate into another type of cell having properties different from those of undifferentiated stem cells. In addition, teratoma-forming ability and chimeric mouse-forming ability are also mentioned as properties specific to undifferentiated stem cells.

Various markers specific to undifferentiated stem cells (hereinafter, may be referred to as “undifferentiated stem cell markers”) are factors specifically expressed in undifferentiated stem cells. Specific examples of the undifferentiated stem cell marker include Oct3 / 4, Nanog, Sox2, SSEA-1, SSEA-3, SSEA-4, TRA1-60, TRA1-81, Lin28, Fbx15 and the like. When the expression of at least one of these undifferentiated stem cell markers is observed, the cell can be determined to be undifferentiated stem cell. The undifferentiated stem cell marker may be used alone or in combination of two or more. In one embodiment, cells expressing Oct3 / 4 may be determined to be undifferentiated stem cells. The expression of the undifferentiated stem cell marker in the cells can be confirmed by using a known method such as RT-PCR or microarray.

The undifferentiated stem cell may be a mammalian undifferentiated stem cell, a rodent undifferentiated stem cell, a primate undifferentiated stem cell, or a human undifferentiated stem cell. good. An example of undifferentiated stem cells is undifferentiated stem cells derived from humans. More specific examples of undifferentiated stem cells include human iPS cells and human ES cells.

As used herein, the term "differentiated cell" refers to a cell that does not have the above-mentioned "undifferentiated stem cell" property. Differentiated cells can be cells derived and differentiated from undifferentiated stem cells, but do not have pluripotency. The differentiated cell may be, for example, a cell differentiated from an ES cell or a cell differentiated from an iPS cell. Examples of differentiated cells include myocardial cells, muscle cells, fibroblasts, nerve cells, immune cells such as lymphocytes, vascular cells, eye cells such as retinal pigment epithelial cells, blood cells such as giant nuclei and red blood cells, and others. Examples thereof include tissue cells and their precursor cells. Among them, cardiomyocytes are preferable as the cells induced and differentiated from undifferentiated stem cells using the cell culture apparatus of the present embodiment.

When cardiomyocytes are produced from undifferentiated stem cells, it is considered that as the differentiation into cardiomyocytes progresses, they differentiate into cardiomyocytes via undifferentiated mesoderm and cardiac mesoderm (or planned cardiomyocytes). Here, the undifferentiated mesoderm refers to a stage in which expression of a Brachyury protein specific to the undifferentiated mesoderm is recognized. On the other hand, in the heart germ layer (or planned myocardial cell), expression of a protein specific to an undifferentiated mesodermal such as Brachyury is observed, and in the same cell, a myocardial cell-specific protein such as Nkx2.5 or actinine is used. It means in cells that do not show expression. In addition, cardiac mesoderm (or planned cardiomyocytes) means cells that do not require the addition of new material to the culture medium and have the ability to differentiate exclusively into cardiomyocytes. Cardiomyocytes mean cells that are performing autonomous beatings. Cardiomyocytes express markers such as Nkx2.5, GATA4, actinin and the like.

When producing myocardial cells from undifferentiated stem cells, for example, differentiation and induction into myocardial cells may be performed by adding a substance that induces differentiation into myocardial cells to a culture medium for culturing undifferentiated stem cells. .. Examples of the substance that induces differentiation into cardiomyocytes include cytokines, adhesion molecules, vitamins, transcription factors, extracellular matrix derived from cardiomyocytes, BMP antagonists and the like. As shown in Examples described later, a substance that activates the Wnt signal and the BMP signal (for example, CHIR99021) is added in the early stage of differentiation, and a substance that suppresses the Wnt signal in the late stage of differentiation (for example, IWR1, Dkk, etc.). IWP2 etc.) is preferably added.

Examples of the cytokine include chromosomal DNA demethylating agents such as demethylase, 5-azacitidine, and DMSO; PDGF, fibroblast growth factor 8 (FGF-8), endoserin 1 (ET1), and midkine bone morphogenetic factor. 4 (BMP-4), G-CSF, Active A and the like can be mentioned.

Examples of the adhesion molecule include gelatin, laminin, collagen, fibronectin and the like.

Examples of the vitamin include retinoic acid and the like.

Examples of the transcription factor include Nkx2.5 / Csx, GATA4, MEF-2A, MEF-2B, MEF-2C, MEF-2D, dHAND, eHAND, TEF-1, TEF-3, TEF-5, Mesp1 and the like. Can be mentioned.

Examples of the BMP antagonist include noggin, chordin, fetuin, follistatin, sclerostin, dan, cerberus, gremlin, dante and the like.

The gas supply flow path 2 is for passing the supplied gas.

The gas supplied in the cell culture apparatus of the present embodiment may be a gas having a composition used for cell culture. Among them, as the gas to be supplied, the concentration of carbon dioxide in the gas is preferably 3% or more and 10% or less, more preferably 4% or more and 8% or less, and 5% or more and 6% or less. Is even more preferable. When the concentration of carbon dioxide in the gas is in the above range, cells can be proliferated with higher efficiency.

The composition of the supplied gas also includes oxygen (concentration in gas: for example, about 10% or more and 30% or less) and nitrogen (concentration in gas: for example, about 70% or more and 90% or less). Can be done.

The gas discharge port 3 is provided in the gas supply flow path 2 and is for discharging gas to the culture liquid 7 stored in the culture liquid storage unit 1. As a result, the gas is dissolved in the culture solution 7, and the cells can be proliferated with high efficiency.

The gas discharge port 4 is for discharging the gas discharged to the culture solution 7. As a result, the air in the culture vessel 10 can be replaced with high efficiency, and the composition of the air in the culture vessel 10 can be kept constant.

The gas discharge flow path 5 is for allowing gas to flow through the gas discharge port 4. As a result, the discharged gas is discharged to the outside of the culture system.

Further, in a plurality of culture containers, the gas supply flow path of one culture container and the gas supply flow path of another culture container may be independent of each other or may be connected to each other so as to be able to communicate with each other. Above all, it is preferable that the gas supply flow path of one culture container and the gas supply flow path of another culture container are connected to each other so as to communicate with each other. As a result, the composition of air between the culture vessels can be kept constant.

Further, in a plurality of culture containers, the gas discharge flow path of one culture container and the gas discharge flow path of another culture container may be independent of each other or may be connected to each other so as to be able to communicate with each other. Above all, it is preferable that the gas discharge flow path of one culture container and the gas discharge flow path of the other culture container are connected to each other so as to communicate with each other. As a result, the composition of air between the culture vessels can be kept constant.

Further, the positions of the gas supply channel 2 and the gas discharge channel 5 in the culture vessel 10 are not particularly limited, and when the shape of the culture solution storage section 1 as viewed from the top surface is rectangular, the culture solution storage section 1 has a rectangular shape. A gas supply flow path 2 and a gas discharge flow path 5 may be provided at the corners at both ends of one side. Alternatively, the gas supply flow path 2 and the gas discharge flow path 5 may be provided at the diagonal corners of the culture solution storage unit 1. Above all, when the shape of the culture solution storage unit 1 when viewed from the top surface is rectangular, the gas supply flow path 2 and the gas discharge flow path 5 are provided at the corners of both ends of one side of the culture solution storage unit 1. Is preferable. As a result, the air in the culture vessel 10 is appropriately replaced without causing the liquid level to undulate in the culture vessel 10 due to the flow of air, and the cells can be proliferated with higher efficiency.

≪Cell culture system≫
FIG. 2 is a schematic configuration diagram showing an example of the cell culture system of the present embodiment. Each configuration of the cell culture system of the present embodiment will be described in detail with reference to FIG. 2.

The cell culture system A shown in FIG. 2 includes a cell culture device 100 and a gas supply device 200 according to the above embodiment.

The gas supply device 200 is connected to the gas supply flow path 2 so as to communicate with each other. The gas supply device 200 includes a carbon dioxide cylinder 11a, an oxygen cylinder 11b and a nitrogen cylinder 11c, a valve 12a, a valve 12b and a valve 12c, and a pump 13. By controlling the valves 12a, 12b and 12c in the gas supply device 200, the concentrations of carbon dioxide, oxygen and nitrogen in the gas supplied to the cell culture device 100 are within the range shown in the cell culture device. As described above, carbon dioxide, oxygen and nitrogen can be supplied from the carbon dioxide cylinder 11a, the oxygen cylinder 11b and the nitrogen cylinder 11c, respectively.

Further, as shown in FIG. 2, the valves 12a, 12b and 12c may be electrically connected to the computer as the control unit 300 and may operate based on a command from the computer. .. The control unit 300 controls the valves 12a, 12b and 12c so that the concentrations of carbon dioxide, oxygen and nitrogen in the gas supplied to the cell culture apparatus 100 are within the ranges shown in the cell culture apparatus. , Carbon dioxide cylinder 11a, oxygen cylinder 11b and nitrogen cylinder 11c can supply carbon dioxide, oxygen and nitrogen, respectively.

Further, although not shown in FIG. 2, at least one of the culture vessels 10 may be provided with a dissolved gas concentration detector (not shown). Further, the dissolved gas concentration detector may be electrically connected to a computer as a control unit 300. As a result, the dissolved gas concentration in the culture solution detected by the dissolved gas concentration detector can be transmitted to the control unit 300. For example, when the concentration of the dissolved gas in the culture solution is out of the range shown in the above cell culture apparatus, the carbon dioxide cylinder is controlled by controlling the valves 12a, 12b and 12c so as to be within the range. Carbon dioxide, oxygen and nitrogen can be supplied from 11a, the oxygen cylinder 11b and the nitrogen cylinder 11c, respectively, to control the concentration of the dissolved gas in the culture broth.

Further, although not shown in FIG. 2, the pump 13 may be electrically connected to a computer as a control unit 300 and may operate based on a command from the computer. As a result, the control unit 300 can control the pump 13 to control the speed of the supplied gas.

The rate of the gas supplied is preferably 5 mL / min or more and 100 mL / min or less, more preferably 10 mL / min or more and 80 mL / min or less, and 20 mL / min or more and 60 mL / min or less. It is more preferable to have. When the gas velocity is in the above range, the air in the culture vessel 10 can be replaced with higher efficiency.

Further, although not shown in FIG. 2, the gas discharge flow path may be provided with a pump for discharging gas. As a result, the gas in the culture apparatus 100 can be discharged to the outside of the culture system more efficiently. Further, the pump for discharging this gas may also be electrically connected to the computer as the control unit 300 and may be operated based on a command from the computer.

≪Manufacturing method of cardiomyocytes≫
In the method for producing myocardial cells according to an embodiment of the present invention, undifferentiated stem cells are cultured using the cell culture apparatus according to the above embodiment or the cell culture system according to the above embodiment to induce differentiation into myocardial cells. It is a method including a differentiation induction step.

According to the method for producing cardiomyocytes of the present embodiment, a large amount of cardiomyocytes can be obtained from undifferentiated stem cells with stable differentiation-inducing efficiency. The method for producing cardiomyocytes according to this embodiment will be described in detail below with reference to FIGS. 1A, 1B and 2.

[Differentiation induction step]
First, the undifferentiated stem cells 8 are seeded in the culture vessel 10. The number of cells to be seeded is not particularly limited, and may be, for example, 1.0 × 10 ³ cells / cm ² or more and 5.0 × 10 ³ cells / cm ² or less. Moreover, as the undifferentiated stem cell, the same thing as those exemplified in the said cell culture apparatus can be mentioned.

Then, the culture is started using the culture solution 7 containing the substance that induces the differentiation into cardiomyocytes. Examples of the substance and culture medium that induce differentiation into cardiomyocytes include those similar to those exemplified in the above cell culture apparatus. The culture medium can be changed every 1 day or more and 3 days or less.

In the differentiation induction step, it is preferable to positively supply gas to the cell culture apparatus 100. Examples of the gas to be supplied include the same as those exemplified in the above cell culture apparatus. Above all, the concentration of carbon dioxide in the supplied gas is preferably 3% or more and 10% or less, more preferably 4% or more and 8% or less, and further preferably 5% or more and 6% or less. .. When the concentration of carbon dioxide in the gas is in the above range, cells can be proliferated with higher efficiency.

The rate of the supplied gas is preferably 5 mL / min or more and 100 mL / min or less, more preferably 10 mL / min or more and 80 mL / min or less, and 20 mL / min or more and 60 mL / min or less. The following is more preferable. When the gas velocity is in the above range, the air in the culture vessel 10 can be replaced with higher efficiency.

The culture temperature can be, for example, 30 ° C. or higher and 40 ° C. or lower. The culture time is preferably 7 days or more and 14 days or less. When the culture time is within the above range, the induction of differentiation from undifferentiated stem cells to cardiomyocytes can be completed by culturing.

When the cell culture system A shown in FIG. 2 is used, the composition and speed of the supplied gas can be automatically controlled by a computer as the control unit 300.

The method for producing cardiomyocytes of the present embodiment may include other steps in addition to the differentiation induction step. Examples of other steps include a step of purifying differentiated cells, a step of recovering differentiated cells, and the like. When these steps are added, these steps are performed after the above-mentioned differentiation induction step.

Examples of the cardiomyocyte purification step and recovery step include International Publication No. 2006/022377 (Reference 1), International Publication No. 2007/088744 (Reference 2), and International Publication No. 2016/010165 (Reference 3). The method described in 1 may be applied to the purification step. Further, as the recovery step, a centrifugation method or the like may be applied.

In the cardiomyocytes obtained by the production method of the present embodiment, for example, the differentiated cell ratio represented by differentiated cells / (undifferentiated stem cells + differentiated cells) × 100 may be 50% or more, and may be 70% or more. It may be 80% or more, 90% or more, and 95% or more. In addition, dead cells are not included in the above number of cells.

Furthermore, in another embodiment, the present invention provides cardiomyocytes produced by the method for producing cardiomyocytes including the above-mentioned differentiation induction step.

Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to the following Examples.

[Reference Example 1] Culturing of human iPS cells using a cell culture device consisting of one layer 1. Preparation of human iPS cells Human iPS cell lineage (253G4) was obtained from the Kyoto University iPSC Research and Application Center.

2. 2. Culture of human iPS cells Next, 1 × 10 ⁶ human iPS cells (culture area: 632 cm ² , a plate coated with a growth factor-reducing matrix gel) (manufactured by Thermo Fisher Scientific) were seeded. (See FIG. 3A). As the culture broth, 120 mL of modified Stem Fit medium (manufactured by Ajinomoto Co., Inc.) was used and replaced every other day.

The composition of the modified Stem Fit medium is 7 kinds of growth factors including 21 kinds of amino acids, 10 kinds of vitamins, 5 kinds of trace minerals and basic fibroblast growth factor (bFGF). The 21 kinds of amino acids include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, and L-glutamic acid. With L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine. be. The 10 vitamins are L-ascorbic acid, cobalamin, biotin, folic acid, I-inositol, niacinamide, calcium D-pentitate, pyridoxine, riboflavin and thioamine hydrochloride. The five trace minerals are copper (II) sulfate, iron (II) sulfate, iron (II) nitrate, zinc sulfate and sodium selenite. The modified Stem Fit medium contains 8 times the amount of most of the components for large-scale culture as compared with the normal Stem Fit medium.

In addition, the cells were cultured in a normal gas incubator for 7 days. The cultured cells were then collected and resuspended in modified Stem Fit medium (Ajinomoto) (containing 10 μM Y27632 (Wako Pure Chemical Industries, Ltd.)) on a plate coated with a growth factor-reduced matrigel. did. Then, the number of cells was counted using a Vi-Cell cell counter (manufactured by Beckman Coulter). Cell numbers after 7 days is 7.9 × 10 ⁸ cells (3.0 × 10 ⁵ cells / cm ^2), proliferation efficiency of the cells was 187 times. The proliferation efficiency of human iPS cells is the ratio of the total number of cells in the cell culture device after 7 days to the number of seeded cells in the cell culture device.

3. 3. Confirmation of traits of human iPS cells (1) Alkaline phosphatase (ALP) staining Human iPS cells cultured for 7 days in "1." were stained using a Leukocyte Alkaline Phosphatase kit (manufactured by Sigma). The results are shown in FIG. 3B.

From FIG. 3B, all colonies were ALP-positive, and the pluripotency state was maintained. Also, as shown in FIG. 3B, human iPS cells were uniformly cultured throughout the plate.

(2) Detection of undifferentiated stem cell markers Human iPS cells cultured for 7 days in "1." were fixed with 4% paraformaldehyde for 20 minutes. The cells were then infiltrated with 0.1% Triton X-100 (manufactured by Sigma) at room temperature for 5 minutes. Then, as the primary antibody, the cells were incubated overnight at 4 ° C. using the following a) to c).
a) 1/200 dilution of mouse anti-TRA1-60 (Millipore) b) 1/200 dilution of mouse anti-TRA1-81 (Millipore) c) 1/200 of mouse anti-SSEA4 (Millipore) Diluted product

The cells were then washed 3 times with PBS containing n0.1% Tween 20. Then, as a secondary antibody, Alexa Fluor 488/594 anti-mouse IgG was used and incubated at room temperature for 1 hour.

Then, nuclear staining was performed with Hoechst 33342 (manufactured by Thermo Fisher Scientific). Stained cells were detected using a fluorescence microscope (Axio Obsave, manufactured by Carl Zeiss). The results are shown in FIG. 3C. The upper part is an image in a bright field, and the lower part is a fluorescence detection image. In FIG. 3C, the scale bar is 100 μm both above and below.

From FIG. 3C, strong expression of undifferentiated stem cell markers was observed in all colonies.

From the above, it was strongly suggested that a 2D culture system using a cell culture device consisting of one layer is suitable for maintaining pluripotency in human iPS cells.

[Example 1] Culturing of human iPS cells using a cell culture device consisting of four layers 1. Preparation of human iPS cells A human iPS cell line (253G4) was prepared in the same manner as in "1." of Reference Example 1.

2. 2. Human iPS cell culture then human iPS cells (253G4) 1 × 10 ⁶ cells / 1 layer (a total of 4 × 10 ⁶ cells) of 4-layer plates (culture area: 632cm ² × 4 layers = 2528cm ^2, reduced growth factor The seeds were sown on a plate coated with Matrigel (manufactured by Thermo Fisher Scientific) (see FIG. 4A). As the culture broth, a modified Stem Fit medium (manufactured by Ajinomoto Co., Inc.) 500 mL / 4 layer was used and replaced every other day. In addition, a multi-layer gas incubator (MG-70M, manufactured by TAITEC) (hereinafter, may be referred to as "AGV") is used _{to actively aerate 5% CO 2-} containing gas at a rate of 50 mL / min per layer. The cells were cultured at 37 ° C. for 7 days. In addition, as a control, cells were cultured at 37 ° C. for 7 days using a normal gas incubator (hereinafter, may be referred to as “NGV”). The cultured cells were then collected and resuspended in modified Stem Fit medium (Ajinomoto) (containing 10 μM Y27632 (Wako Pure Chemical Industries, Ltd.)) on a plate coated with a growth factor-reduced matrigel. did. Then, the number of cells was counted using a Vi-Cell cell counter (manufactured by Beckman Coulter).

As a result, the proliferation efficiency of human iPS cells using the 4-layer cell culture device was 179 times, which was almost the same as that when the 1-layer cell culture device was used (187 times) (Fig.). See 4B).

3. 3. Confirmation of human iPS cell traits (1) Detection of undifferentiated stem cell markers Human iPS cells cultured for 7 days in "1." were fixed with 4% paraformaldehyde for 20 minutes. The cells were then infiltrated with 0.1% Triton X-100 (manufactured by Sigma) at room temperature for 5 minutes. Then, as the primary antibody, the cells were incubated overnight at 4 ° C. using the following a) to d).
a) 1/200 dilution of rabbit anti-NANOG (ReproCELL) b) 1/200 dilution of mouse anti-OCT4 (Santa Cruz Biotechnology) c) 1/200 of mouse anti-TRA1-60 (Millipore) Diluted d) 1/200 diluted product of mouse anti-SSEA4 (manufactured by Millipore)

The cells were then washed 3 times with PBS containing 0.1% Tween 20. Then, as a secondary antibody, Alexa Fluor 488/594 anti-mouse IgG or Alexa Fluor 488/594 anti-rabbit IgG was used and incubated at room temperature for 1 hour.

Then, nuclear staining was performed with Hoechst 33342 (manufactured by Thermo Fisher Scientific). Stained cells were detected using a fluorescence microscope (Axio Obsave, manufactured by Carl Zeiss). The results are shown in FIG. 4C. The image on the left is a bright field image, and the image on the right is a fluorescence detection image. In FIG. 4C, the scale bar is 100 μm on both the left and right sides.

From FIG. 4C, strong expression of undifferentiated stem cell markers was observed in all colonies.

4. Bio profile analysis AGV or CO ₂ partial pressure of the culture solution in 3, 5 and 7 days after the start of culture in the NGV conditions in the culture medium _{(pCO 2), O} 2 partial pressure _(pO 2), pH, glucose concentration And glutamine concentration was measured using BioProfile 400 Analyzer (manufactured by Nova Biomedical). The results are shown in FIG. 4D.

From FIG. 4D, the _{partial pressure of CO 2} in the culture medium was stable under AGV conditions at any of the time points 3, 5 and 7 days after the start of the culture. On the other hand, under NGV conditions, the _{partial pressure of CO 2} in the culture medium increased significantly over time.

Further, in any conditions, pH of the culture decreases, O ₂ partial pressure in the culture medium did not change significantly. In addition, under either condition, the glucose concentration and the glutamine concentration in the medium decreased significantly with the passage of time.

[Example 2] Culturing of human iPS cells using a cell culture device consisting of 10 layers 1. Preparation of human iPS cells A human iPS cell line (253G4) was prepared in the same manner as in "1." of Reference Example 1.

2. 2. Confirmation of CO ₂ permeability in each layer Fill a cell culture device consisting of 10 layers with 2 L of PBS (containing 20 mg of phenol red), and use a multi-layer gas incubator (MG-70M, manufactured by TAITEC) at 50 mL / layer. _{A 5% CO 2-} containing gas was actively aerated into the cell culture apparatus at 37 ° C. for 1 hour at a rate of 1 minute. In addition, photographs were taken every 10 minutes from the start. The results are shown in FIG.

From FIG. 5, under AGV conditions, the red color changed to yellow 1 hour after the phenol red in PBS. From this, it was confirmed that _{CO 2} was sufficiently supplied to the culture broth.

3. 3. The confirmation AGV conditions of gas exchange rate, multi-layer gas incubator for cell culture apparatus comprising a 10-layer (MG-70M, manufactured by TAITEC Co., Ltd.) using a positively _{N 2} gas in the 50 mL / min per layer rate Ventilated. In addition, under NGV conditions, a cell culture device consisting of 10 layers was allowed to stand under a _{hypoxic state (1% O 2).} A sensor chip (SP-PSt3-YAU-D5, manufactured by PreSens) and an optical fiber (POF-1MSA, manufactured by PreSens) are placed on the fifth layer of a cell culture apparatus consisting of 10 layers under AGV and NGV conditions. did. The oxygen concentration in the layer was measured using Fibox 3 (manufactured by PreSens). The results are shown in FIGS. 6A (AGV conditions) and 6B (NGV conditions).

From FIG. 6A, the gas exchange was completed in 4 hours under AGV conditions. On the other hand, from FIG. 6B, under NGV conditions, the gas exchange could not be completed within 24 hours.

4. Human iPS cell culture plates are then of human iPS cells (253G4) 1 × 10 ⁶ cells / 1 Layers (total 1 × 10 ⁷ cells) 10 layers (culture area: 632cm ² × 10 layers = 6320cm ^2, reduced growth factor The seeds were sown on a plate coated with Matrigel (manufactured by Thermo Fisher Scientific) (see FIG. 7A). As the culture broth, 1200 mL / 10 layers of modified Stem Fit medium (manufactured by Ajinomoto Co., Inc.) were used and replaced every other day. In addition, a multi-layer gas incubator (MG-70M, manufactured by TAITEC) (hereinafter, may be referred to as "AGV") is used _{to actively aerate 5% CO 2-} containing gas at a rate of 50 mL / min per layer. The cells were cultured at 37 ° C. for 7 days. In addition, as a control, cells were cultured at 37 ° C. for 28 days using a normal gas incubator (hereinafter, may be referred to as “NGV”). Human iPS cells were washed with D-PBS every week (7 days) and subcultured using Actase (registered trademark) (manufactured by Thermo Fisher Scientific) as a cell dispersant.

In addition, cells were collected 7, 14, 21 and 28 days after the start of culture, and modified Stem Fit medium (manufactured by Ajinomoto Co., Inc.) (10 μM Y27632 (manufactured by Wako Pure Chemical Industries, Ltd.) on a plate coated with a growth factor-reduced matrigel. ) Containing) cells were resuspended. Then, the number of cells was counted using a Vi-Cell cell counter (manufactured by Beckman Coulter). FIG. 7B shows the time course of the cell number of human iPS cells using a cell culture device consisting of 10 layers under AGV and NGV conditions. In addition, the proliferation efficiency of human iPS cells 7 days after the start of culture under AGV and NGV conditions is shown in FIG. 7C.

From FIG. 7B, the proliferation of human iPS cells was more stable under AGV conditions than under NGV conditions.

From Figure 7C, the AGV conditions, cell number of human iPS cells after 7 days from the initiation of the culture is 1.7 × 10 ⁹ cells were often cell number approximately 44% than the NGV conditions.

5. Confirmation of traits of human iPS cells (1) Detection of undifferentiated stem cell markers For human iPS cells cultured under AGV conditions using the same method as in (1) of "3." of Example 1, undifferentiated stem cell markers. (NANOG, OCT4, TRA1-60 and SSEA4) were detected. The results are shown in FIG. 7D. In FIG. 7D, the scale bar is 100 μm both above and below.

From FIG. 7D, strong expression of undifferentiated stem cell markers was observed in all colonies.

6. In the same manner as in "4." bio profile analysis Example 1 in the culture medium, CO ₂ partial pressure of the culture solution in 3, 5 and 7 days after the start of culture in AGV conditions and NGV conditions (PCO ₂ ), O ₂ partial pressure (pO ₂ ) and pH were measured. The results are shown in FIG. 7E.

From FIG. 7E, the pH of the culture broth decreased under any of the conditions as in Example 1. On the other hand, the O ₂ partial pressure was significantly reduced under NGV conditions, unlike Example 1.

Further, _{it was confirmed that the partial pressure of CO 2} was stable under AGV conditions, as in the case of culturing using a normal 10 cm dish. On the other hand, under the NGV condition, the CO _{2 partial pressure was significantly lower than the CO 2} partial pressure under the AGV condition 3 days after the start of the culture, and increased significantly with the passage of time.

From the above, it was clarified that in the culture of human iPS cells using a cell culture device consisting of 10 layers, human iPS cells can be efficiently cultured under AGV conditions.

[Example 3] Production of cardiomyocytes derived from human iPS cells using a cell culture device consisting of one layer, four layers or ten layers. Preparation of human iPS cells A human iPS cell line (253G4) was prepared in the same manner as in "1." of Reference Example 1. Human iPS cell lines (201B7 and Ff14) were also obtained from the Kyoto University iPSC Research and Application Center. Human iPS cell lines (253G4, 201B7 and Ff14) were subjected to 1-layer or 10-layer plates (plates coated with growth factor-reducing matrigel) under NGV conditions 4 days before the start of differentiation induction (manufactured by Thermo Fisher Scientific). ), And under AGV conditions, seeded on a 4-layer or 10-layer plate (a plate coated with a growth factor-reducing matrix gel) (manufactured by Thermo Fisher Scientific), and 90% of the cells were concentrated on the day when differentiation induction was started. Was pre-cultured to reach (see FIG. 8A).

2. 2. Induction of differentiation of human iPS cells into cardiomyocytes Next, B27 (insulin-free (-)) (Thermo Fisher Scientific), 6 μM CHIR99021 (Wako Pure Chemical Industries, Ltd.) and 1 ng / mL bone morphogenetic protein 4 The culture solution was exchanged with RPMI (manufactured by Wako Pure Chemical Industries, Ltd.) containing (manufactured by R & D Systems) and cultured for 1 day. Then, from the 1st day to the 2nd day from the start of differentiation induction, the culture solution was exchanged with RPMI containing B27 (insulin-free (−)) and cultured. Then, from the 3rd day to the 5th day from the start of differentiation induction, the culture solution was exchanged with RPMI containing B27 (insulin-free (−)) and 5 μM IWR1 (manufactured by Sigma-Aldrich) and cultured. Then, on the 6th day from the start of differentiation induction, the culture solution was exchanged with RPMI containing B27 (insulin-free (−)) and cultured. Then, on the 7th day from the start of differentiation induction, the culture solution was exchanged with MEMα containing 5% fetal bovine serum (FBS, manufactured by Hyclone) to maintain the culture. Then, on the 10th day from the start of differentiation induction, the number of dissociated single cells and the cell viability were evaluated by trypan blue staining using ViCell (manufactured by Beckman Coulter). The results are shown in FIG. 8B (cell viability) and FIG. 8C (cell number). In FIG. 8C, "non-CMs" indicates the number of cells undifferentiated into cardiomyocytes, and "CMs" indicates the number of cells differentiated into cardiomyocytes.

From FIG. 8B, the cell viability was 95% or more under all conditions.

From FIG. 8C, when a cell culture device having one layer (NGV condition), four layers (AGV condition) or ten layers (AGV condition) was used, the total number of cells after induction of differentiation from human iPS cells was calculated. They were 1.5 × 10 ⁶ pieces, 6.7 × 10 ⁸ pieces and 1.5 × 10 ⁹ pieces, respectively.

3. 3. Purification of human iPS cell-derived cardiomyocytes by metabolic selection 12 to 14 days after the start of differentiation induction, B27 (insulin-free (-)) -containing RPMI was removed from human iPS cell-derived differentiated cells, and together with D-PBS. Insulin was incubated for 3 minutes. The cells were then dissociated using 0.25% trypsin-EDTA (manufactured by Nacalai Tesque). Then, the dissociated cells were collected and suspended in MEMα containing 5% FBS, and a type 1 collagen-coated 15 cm dish (manufactured by IWAKI), a fibronectin-coated 15 cm dish (manufactured by Sigma), or a layer coated with fibronectin. The cells were seeded in a cell culture device (manufactured by Thermo Fisher Scientific). Then, using MEMα containing 5% FBS, the cells were cultured for 1 to 2 days. MEMα containing 5% FBS was then removed and incubated with D-PBS for 3 minutes. Then, the culture solution was replaced with glucose-free and glutamine-free DMEM (manufactured by Ajinomoto Co., Inc.) (containing 4 mM L-lactic acid and 0.1% BSA (manufactured by Thermo Fisher Scientific)), and the cells were cultured for 3 to 6 days. The culture medium was replaced every 2 to 3 days to eliminate dead cells. Human iPS cell-derived cardiomyocytes purified by this metabolic selection were cryopreserved and used for immunofluorescent staining and FACS analysis.

4. Evaluation of Differentiation Efficiency Cells were collected 10 days after the start of differentiation induction and analyzed using immunofluorescent staining and flow cytometry. Specifically, it is as shown below.

(1) Differentiated cells derived from human iPS cells 10 days after the start of differentiation induction recovered by immunofluorescent staining "2." and derived from human iPS cells purified by metabolic selection obtained in "3." Myocardial cells were each fixed with 4% paraformaldehyde for 20 minutes. The cells were then infiltrated with 0.1% Triton X-100 (manufactured by Sigma) at room temperature for 5 minutes. It was then incubated overnight at 4 ° C. with a 1/500 dilution of mouse anti-α-actinin (manufactured by Sigma) as the primary antibody.

The cells were then washed 3 times with PBS containing 0.1% Tween 20. Then, as a secondary antibody, Alexa Fluor 488/594 anti-mouse IgG was used and incubated at room temperature for 1 hour.

Then, nuclear staining was performed with Hoechst 33342 (manufactured by Thermo Fisher Scientific). Stained cells were detected using a fluorescence microscope (Axio Obsave, manufactured by Carl Zeiss). The results are shown in FIG. 8F. The left is a fluorescence detection image of cells before metabolism selection, and the right is a fluorescence detection image of cells after metabolism selection. In FIG. 8F, the scale bar is 500 μm on both the left and right sides.

From FIG. 8F, fluorescence of α-actin, which is a myocardial marker, was confirmed in cells before and after metabolism selection. In particular, fluorescence of α-actin was remarkably confirmed in the cells after selection of metabolism.

(2) FACS analysis Differentiated cells derived from human iPS cells 10 days after the start of differentiation induction recovered in "2." and human iPS cell-derived myocardium purified by metabolic selection obtained in "3." Each cell was fixed with 4% paraformaldehyde for 20 minutes. The cells were then infiltrated with 0.1% Triton X-100 (manufactured by Sigma) at room temperature for 5 minutes. Then, as a primary antibody, a 1/200 diluted solution of mouse anti-myocardial troponin T (manufactured by Thermo Fisher Scientific) was used and incubated overnight at 4 ° C.

The cells were then washed with PBS containing 0.1% Tween 20. Then, as a secondary antibody, Alexa488 donkey anti-mouse IgG (manufactured by Thermo Fisher Scientific) was used, and the mixture was incubated at room temperature for 2 hours.

The cells were then analyzed using FACS (Gallios, manufactured by Beckman Coulter). The results are shown in FIG. 8E. Above is a graph showing the results of FACS analysis using human iPS cells. The middle is a graph showing the results of FACS analysis using cells before metabolism selection. Below is a graph showing the results of FACS analysis using cells after metabolism selection.

Further, FIG. 8D is calculated from the result of the FACS analysis of FIG. 8E. The left is a graph showing the ratio of troponin T-positive cells in the differentiated cells derived from human iPS cells, and the right is a graph showing the number of cardiomyocytes derived from human iPS cells.

From FIG. 8D, there was no significant difference in the efficiency of cardiomyocyte differentiation between AGV and NGV conditions. On the other hand, the number of cardiomyocytes was higher under AGV conditions than under NGV conditions. It was speculated that this is because the cell proliferation efficiency in the lower layers of 6 or less of the 10 layers is low under the NGV condition.

From FIG. 8E, among the cells before metabolism selection, the ratio of human iPS cell-derived cardiomyocytes (troponin T-positive cells) was about 80%. On the other hand, in the cells after selection of metabolism, the ratio of human iPS cell-derived cardiomyocytes (troponin T-positive cells) was 97% or more, and it was confirmed that troponin T was expressed in almost all cells.

From the above, it was clarified that in the culture of human iPS cells using a cell culture device consisting of 10 layers, differentiation can be efficiently induced from human iPS cells to cardiomyocytes under AGV conditions. Furthermore, it was revealed that a large amount of cardiomyocytes can be obtained.

According to the cell culture apparatus and cell culture system of the present embodiment, undifferentiated stem cells can be proliferated with high efficiency and cardiomyocytes can be obtained with stable differentiation induction efficiency. According to the method for producing cardiomyocytes of the present embodiment, a large amount of cardiomyocytes can be obtained from undifferentiated stem cells with stable differentiation-inducing efficiency.

1 ... Culture solution storage, 2 ... Gas supply flow path, 3 ... Gas discharge port, 4 ... Gas discharge port, 5 ... Gas discharge flow path, 7 ... Culture solution, 8 ... Undifferentiated stem cells, 10 ... Culture container, 11a ... Carbon dioxide cylinder, 11b ... Oxygen cylinder, 11c ... Nitrogen cylinder, 12a, 12b, 12c ... Valve, 13 ... Pump, 100 ... Cell culture device, 200 ... Gas supply device, 300 ... Control unit (computer), A ... Cell Culture system.

Claims (4)

Cell culture device and
Gas supply device and
A control unit that controls the gas supply device so that the rate of gas supplied to the cell culture device is constant at 20 mL / min or more and 60 mL / min or less per layer in all layers.
Equipped with
The cell culture device is
A culture solution storage unit capable of storing a culture solution set so that the liquid level height is 2 mm or more and 10 mm or less, and a culture solution storage unit.
The gas supply flow path through which the supplied gas flows, and
A gas discharge port provided in the gas supply flow path and discharging the gas to the culture liquid stored in the culture liquid storage unit at the speed so as to be constant in all layers.
A gas discharge port for discharging the gas discharged to the culture solution, and
A gas discharge flow path through which the gas flows through the gas discharge port, and
With 4 or more layers of culture containers equipped with
The culture is one formed by laminating four layers or more containers, a system for inducing differentiation of cardiomyocytes cultured undifferentiated stem cells. The culture vessel having four or more layers includes the gas supply channel of one culture vessel, the gas supply channel of another culture vessel, the gas discharge channel of one culture vessel, and the like. The system according to claim 1, wherein the gas discharge flow path of the culture container is laminated so as to be communicatively connected to each other. The culture solution storage portion is formed in a rectangular shape and has a rectangular shape.
The system according to claim 1 or 2, wherein the gas supply flow path and the gas discharge flow path are provided at the corners of both ends of one side of the culture solution storage unit, respectively. A method for producing cardiomyocytes , comprising a differentiation-inducing step of culturing undifferentiated stem cells and inducing differentiation into cardiomyocytes using the system according to any one of claims 1 to 3.

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