Method for culturing cells, and method for producing cells

Abstract

This invention provides a cell culture method that enables appropriate adherent culture when switching between spheroid culture and adherent culture in a single culture vessel filled with culture medium. [Solution] In a method of culturing cells using a culture vessel filled with culture medium, the culture vessel has a cell-non-adhesion culture surface treated to prevent cell adhesion and a cell-adhesion culture surface treated to prevent cell adhesion. The adhesion factor necessary for adhering cells to the cell-adhesion culture surface is added at the latest while the cells are being cultured on the cell-non-adhesion culture surface. After culturing the cells on the cell-non-adhesion culture surface, the cells are moved to the cell-adhesion culture surface and cultured thereafter.

Description

Technical Field

[0001] The present invention relates to a method for culturing cells using a culture vessel and a method for producing cells.

Background Art

[0002] In recent years, in a culture technique for differentiating iPS cells into target cells, when differentiating into cells of the nervous system or the like, after performing spheroid culture of cells and then performing adherent culture, or after performing adherent culture of cells and then performing spheroid culture, it is known that the differentiation rate is improved and the yield is increased.

[0003] In order to switch between different culture methods such as spheroid culture and adherent culture, conventionally, it has been necessary to separately use dedicated culture vessels suitable for each culture method. For example, when performing spheroid culture, it is necessary to use a dedicated spheroid formation container, and when performing adherent culture, it is necessary to use a dish or bag for adherent culture.

[0004] However, when using a dedicated culture vessel for each different culture method, there is a problem that the man-hours for transferring the vessels increase. Such a process of transferring the vessels is more difficult and time-consuming as the culture scale is larger. In addition, there is also a problem that the risk of contamination by bacteria and foreign substances increases by including an operation of transferring the vessel during culture. Furthermore, there is also a problem that the yield decreases due to the recovery loss when transferring the vessel.

[0005] Therefore, in order to solve these problems, the present inventors developed a closed culture vessel (double-sided culture vessel) having both a culture surface for performing spheroid culture and a culture surface for performing adherent culture inside a single culture vessel, and made it possible to switch the culture method by changing the orientation of the culture vessel.

Prior Art Documents

[0006] [Patent Document 1] Patent No. 4543212 [Patent Document 2] Patent No. 7384018 [Overview of the project] [Problems that the invention aims to solve]

[0007] However, when spheroid culture of cells was performed using such a double-sided culture vessel, followed by adherent culture, a problem arose: the spheroids obtained from spheroid culture did not migrate to adherent culture. Specifically, after culturing iPS cells as spheroids in a double-sided culture vessel with the spheroid culture surface facing the bottom for two days, the vessel was then rotated so that the adhesion culture surface faced the bottom, and adhesion factors were added to help the spheroids adhere to the culture surface. After culturing for one day, it was found that the spheroids did not adhere to the culture surface, indicating that proper adhesion culture could not be performed.

[0008] Therefore, the inventors diligently researched how to solve these problems and succeeded in performing both spheroid culture and adherent culture using a double-sided culture vessel. Specifically, we discovered that by adding cell adhesion factors for adherent culture to a double-sided culture vessel at the spheroid culture stage, spheroid culture proceeds without inhibition, and spheroids adhere to the culture surface during adherent culture, allowing for proper adherent culture. This discovery led to the completion of the present invention.

[0009] Herein, Patent Document 1 discloses a cell culture method comprising the steps of: aggregating cells in each of the first culture sections of a polyhedral container, in which a plurality of recesses forming first culture sections are recessed on one surface of the inner wall of the polyhedral container, to form a cell aggregate of a predetermined size restricted by the size of the first culture section; transferring the cell aggregate to a second culture section which is larger in size than the first culture section and has the bottom surface of an inner wall other than the surface in which the first culture section is formed within the polyhedral container, or a recess formed in an inner wall other than the surface in which the first culture section is formed within the polyhedral container, and having an opening larger in size than the opening of the recess that is the first culture section; and culturing the cells in the second culture section.

[0010] Furthermore, Patent Document 2 discloses a cell culture vessel comprising an inner wall surface defining a closed space, and a first inlet and a first outlet communicating with the closed space, wherein the inner wall surface has a first surface on which wells and grooves connected to the wells are formed, and a second surface facing the first surface so as to be able to contact and separate from the first surface, and when the first surface and the second surface are in contact, the first inlet and the first outlet are connected to a channel defined by the grooves and the second surface.

[0011] However, these studies did not describe or suggest switching between different culture methods, such as spheroid culture and adherent culture, within a single culture vessel. Of course, the method of adding adhesion factors at the spheroid culture stage to enable proper adhesion culture was not disclosed.

[0012] The present invention has been made in view of the above circumstances, and aims to provide a method for culturing cells and a method for producing cells that enable appropriate adherent culture when switching between spheroid culture and adherent culture in a method of culturing cells using a single culture vessel filled with culture medium. [Means for solving the problem]

[0013] To achieve the above objective, the present invention provides a cell culture method in which cells are cultured using a culture vessel filled with culture medium, wherein the culture vessel has a cell non-adhesion culture surface that has been treated to prevent cell adhesion and a cell adhesion culture surface that has been treated to prevent cell adhesion, and the adhesion factor necessary for adhering the cells to the cell adhesion culture surface is added at the latest while the cells are being cultured on the cell non-adhesion culture surface, and after culturing the cells on the cell non-adhesion culture surface, the cells are moved to the cell adhesion culture surface and cultured by adhering the cells to the cell adhesion culture surface.

[0014] Furthermore, it is preferable that the cell culture method of the present invention involves adding the adhesion factor to the culture medium before filling the culture vessel with the culture medium, or adding it to the culture medium when filling the culture vessel with the culture medium. Furthermore, it is preferable that the cell culture method of the present invention is a method in which the cells cultured on the cell-non-adherent culture surface are pluripotent stem cells.

[0015] Furthermore, it is preferable that the cell culture method of the present invention is a method in which the cells cultured on the cell adhesion culture surface are ectodermal cells differentiated from the pluripotent stem cells. Furthermore, it is preferable that the cell culture method of the present invention is a method in which the culture vessel is filled with the culture medium and the culture medium is in contact with the non-adherent cell culture surface and the adherent cell culture surface.

[0016] Furthermore, it is preferable that the cell culture method of the present invention be a method in which the adhesion factor is a laminin 511E8 fragment. Furthermore, it is also preferable that the cell culture method of the present invention be a configuration method that combines various methods of cell culture as described above.

[0017] Furthermore, the present invention provides a method for producing cells by culturing the cells using the cell culture method described in any of the above-mentioned methods. [Effects of the Invention]

[0018] According to the present invention, there is provided a method for culturing cells using a single culture vessel filled with a medium, which enables appropriate adherent culture when switching between spheroid culture and adherent culture, and a method for producing cells.

Brief Description of Drawings

[0019] [Figure 1] It is a schematic diagram showing a culture vessel that can be used in the method for culturing cells according to an embodiment of the present invention. [Figure 2] It is a diagram showing a specific example of a culture vessel that can be used in the method for culturing cells according to an embodiment of the present invention. [Figure 3] It is an explanatory diagram of the usage method of a culture vessel that can be used in the method for culturing cells according to an embodiment of the present invention. [Figure 4] It is an explanatory diagram showing a hypothesis for the reason why spheroids did not adhere to the cell adhesion culture surface when adherent culture was performed after spheroid culture using a culture vessel that can be used in the method for culturing cells according to an embodiment of the present invention. [Figure 5] It is an explanatory diagram showing the steps of the method for culturing cells according to an embodiment of the present invention. [Figure 5] [Figure 6] It is a diagram showing the test results that spheroids did not adhere to the cell adhesion culture surface when using a culture vessel that can be used in the method for culturing cells according to an embodiment of the present invention. [Figure 7] It is a diagram showing the test results that spheroids adhered to the cell adhesion culture surface by the method for culturing cells according to an embodiment of the present invention. [Figure 6]

Embodiments for Carrying Out the Invention

[0020] Hereinafter, embodiments of the method for culturing cells and the method for producing cells of the present invention will be described in detail. However, the present invention is not limited to the specific contents of the following embodiments and the examples described later.

[0021] The cell culture method of this embodiment is a method of culturing cells using a culture vessel filled with culture medium, characterized in that the culture vessel has a cell-non-adhesion culture surface treated for cell non-adhesion and a cell-adhesion culture surface treated for cell adhesion, the adhesion factors necessary for adhering cells to the cell-adhesion culture surface are added at the latest while the cells are being cultured on the cell-non-adhesion culture surface, and after culturing the cells on the cell-non-adhesion culture surface, the cells are moved to the cell-adhesion culture surface and cultured by adhering the cells to the cell-adhesion culture surface.

[0022] As shown in Figure 1, the culture container 1 that can be used in the cell culture method of this embodiment can be configured as a closed-system culture bag formed by heat-sealing the periphery of a first film 10 and a second film 11, for example. The culture container 1 that can be used in the cell culture method of this embodiment is not limited to a culture bag, but may be a rigid container.

[0023] Furthermore, in this embodiment, the culture vessel 1 is equipped with a cell-non-adhesion culture surface 101 that has been treated to prevent cell adhesion and a cell-adhesion culture surface 111 that has been treated to prevent cell adhesion. This double-sided culture vessel allows cells to be cultured without adhering them to the culture surface using the cell-non-adhesion culture surface 101, and after that, cells can be further cultured by adhering them to the culture surface using the cell-adhesion culture surface 111.

[0024] The cell non-adherent culture surface 101 is provided on the first film 10 (cell non-adherent culture surface side film) side, and the cell adherent culture surface 111 is provided on the second film 11 (cell adherent culture surface side film) side.

[0025] The cell non-adhesion treatment can be a process of coating with a cell-low adhesion polymer, thereby forming a cell non-adhesion coating layer M on the first film 10, and the surface of the cell non-adhesion coating layer M is provided as a cell non-adhesion culture surface 101. Examples of low-adhesion cell polymers include phospholipid polymers, polyvinyl alcohol derivatives, phospholipid-polymer complexes, polyhydroxyethyl methacrylate, polyvinyl alcohol, agarose, chitosan, polyethylene glycol, and albumin. These may also be used in combination.

[0026] Furthermore, it is preferable that at least a portion of the cell-non-adherent culture surface 101 has a fine uneven pattern structure. The shape of the recesses in the fine uneven pattern structure is not particularly limited, but it is preferable that they be of a size suitable for culturing spheroids in the recesses.

[0027] The cell adhesion treatment can be, for example, excimer treatment, corona treatment, plasma treatment, etc., and the surface of the second film 11 treated in this way is provided as the cell adhesion culture surface 111.

[0028] The culture vessel 1 is equipped with a port 12, which allows for the injection and discharge of culture media, reagents, and other substances. Furthermore, the culture vessel 1 is filled with culture medium 2, and the culture medium 2 is in contact with the cell non-adherent culture surface 101 and the cell adherent culture surface 111. A specific example of culture vessel 1 that was actually manufactured is shown in Figure 2.

[0029] As shown in Figure 3, in the cell culture method of this embodiment, the culture vessel 1 can be used to culture cells 3 with the cell non-adherent culture surface 101 (spheroid-forming surface) facing the bottom. Furthermore, in the cell culture method of this embodiment, the culture vessel 1 is positioned with the cell adhesion culture surface 111 (adhesion culture surface) facing the bottom, and cells 3 are cultured by adhering them to the cell adhesion culture surface 111, thereby obtaining extended cells 3'.

[0030] The cells 3 cultured on the cell-non-adherent culture surface 101 can be, for example, pluripotent stem cells such as iPS cells. Furthermore, the cells 3' cultured on the cell adhesion culture surface 111 can be, for example, ectodermal cells differentiated from pluripotent stem cells such as iPS cells.

[0031] As detailed in Experiment 1 described later, when spheroid culture of cell 3 was performed in culture vessel 1 followed by adherent culture, the spheroids obtained from spheroid culture did not migrate to adherent culture. In other words, after culturing cell 3 on the non-adherent culture surface 101, an attempt was made to perform adherent culture on the adherent culture surface 111, but adherent culture could not be performed properly.

[0032] Specifically, after culturing iPS cell spheroids with the non-adherent culture surface 101 facing the bottom for two days, the cell-adherent culture surface 111 was placed facing the bottom, and adhesion factors were added to allow the spheroids to adhere to the culture surface. After culturing for one day, the spheroids did not adhere to the culture surface.

[0033] The inventors have formulated the hypothesis shown in Figure 4 regarding the reason for this. In other words, the culture vessel 1 is filled with culture medium 2, and the culture medium 2 is in contact with the cell adhesion culture surface 111 of the second film 11. While culture medium 2 contains culture medium-derived protein 4, it is possible that during the 2-day spheroid culture, the culture medium-derived protein 4 adsorbed to and denatured the cell adhesion culture surface 111, coating the cell adhesion culture surface 111. Therefore, although adhesion factors were added to adhere the spheroids to the culture surface, we hypothesized that the adsorption of adhesion factor 5 to the cell adhesion culture surface 111 was inhibited by denatured culture medium-derived protein 4'.

[0034] Based on this hypothesis, the inventors attempted a culture method in which adhesion factor 5 is added to the culture vessel 1 from the start of spheroid culture. In other words, adhesion factor 5, which is not normally necessary for spheroid formation, was added in advance for the subsequent adhesion culture. As a result, as shown in Experiment 2 described later, it was found that spheroid culture proceeded without inhibition, and that spheroids adhered to and spread on the culture surface during adherent culture, allowing for proper adherent culture.

[0035] In other words, as shown in Figure 5, according to the cell culture method of this embodiment, using a culture vessel 1 which is a double-sided culture vessel filled with culture medium 2, adhesion factors 5 necessary for adhering cells 3 to the cell adhesion culture surface 111 are added at the latest while cells 3 are being cultured on the non-cell adhesion culture surface 101, and after culturing cells 3 on the non-cell adhesion culture surface 101, cells 3 are moved to the cell adhesion culture surface 111, and cells 3 are adhered to the cell adhesion culture surface 111 to obtain extended cells 3'.

[0036] In other words, according to the cell culture method of this embodiment, adhesion factors 5 can be adsorbed onto the cell adhesion culture surface 111 in advance of adhesion culture. Therefore, it is possible to solve the problem of the present invention, in which adhesion culture could not be performed properly after culturing cells 3 on the non-cell adhesion culture surface 101 in the culture vessel 1 and then performing adhesion culture. Furthermore, according to this embodiment of cell culture method, by using a single culture vessel, it is possible to improve the differentiation rate through spheroid culture, which facilitates cell differentiation, and to increase the cell yield through adherent culture.

[0037] In the cell culture method of this embodiment, the adhesion factor 5 may be added to the culture medium 2 before filling the culture vessel 1 with the culture medium 2, or it may be added to the culture medium 2 when filling the culture vessel 1 with the culture medium 2. Furthermore, in the cell culture method of this embodiment, adhesion factor 5 may be added while the cells 3 are being cultured on the cell-non-adherent culture surface 101.

[0038] In the cell culture method of this embodiment, the adhesion factor 5 can be appropriately changed depending on the type of cells to be cultured, but for example, it can be a laminin 511E8 fragment. Furthermore, the cell production method of this embodiment is characterized by producing cells 3 by culturing cells using the cell culture method described above.

[0039] As described above, according to the cell culture method and cell manufacturing method of this embodiment, adhesion factors can be adsorbed onto the cell adhesion culture surface in advance prior to adhesion culture. Therefore, in a method of culturing cells using a single culture vessel filled with culture medium, it is possible to perform adhesion culture appropriately when switching between spheroid culture and adhesion culture. [Examples]

[0040] [Test 1] This section describes a test that demonstrated that adherent culture could not be properly performed when adherent culture was performed after spheroid culture of cells using a double-sided culture vessel. As a double-sided culture container, a closed-system culture bag was prepared by heat-sealing the perimeter of a first film 10 and a second film 11 made of polyethylene, as shown in Figure 3.

[0041] Specifically, the first film 10 was subjected to a cell-non-adhesion coating treatment using MPC coating. The second film 11 was subjected to a cell-adhesion treatment using excimer treatment, followed by an aging process in which it was stored at 50°C for more than 10 days. The excimer treatment was performed using a xenon excimer lamp (wavelength 172 nm) in a gas atmosphere where nitrogen gas and air were mixed in a 19:1 ratio. The distance between the excimer lamp and the film surface was 3-4 mm, and the film was transported at a speed of 2 mm / second.

[0042] Then, the processed sides of the first film 10 and the second film 11 were bonded together, the port 12 was sandwiched between these films, and the surrounding area was heat-sealed to form a culture bag. Furthermore, a fine uneven pattern structure (each recess: hemispherical shape with a diameter of 500 μm and a depth of 250 μm) was formed on the processed surface side of the first film 10 by vacuum pressure forming. The capacity of this culture bag was 20 mL.

[0043] Next, 15 mL of Stemfit AK02N (Ajinomoto Co., Inc.) and 15 μL of 10 mM Y-27632 (Fujifilm Wako Pure Chemical Corporation) were injected into the culture bag, and human pluripotent stem cells (human iPS cells: 1231A3 strain) were cultured in 1.8 × 10⁶ cells. 6 Cells were seeded in Cells (100 Cells / well), and spheroid culture was performed with the cell-non-adherent culture surface 101 facing the bottom.

[0044] After 2 days of incubation, 5 mL of Stemfit AK02N (Ajinomoto Co., Inc.) and 5 μL of 10 mM Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) were injected into the culture bag, and 0.5 μg / cm³ of laminin 511-E8 (Nippi Corporation) was added. 2 It was added. Then, the cell adhesion culture surface 111 was placed on the bottom side and adhesion culture was performed.

[0045] After culturing for another day, the culture medium in the culture bag was changed, and 20 mL of Stemfit AK02N (Ajinomoto Co., Inc.) was injected into the culture bag. Furthermore, microscopic observations were performed on the cell-non-adherent culture surface 101 on the bottom side on day 2, and on the cell-adherent culture surface 111 on days 2 and 3. The micrograph on day 3 was taken before the culture medium was changed. The same procedure was followed for the following tests. The results are shown in Figure 6.

[0046] On the second day, it can be seen that spheroid cells 3 (approximately 100 μm in size) have formed in each recess on the non-adherent cell culture surface 101. It can be seen that spheroid cells 3 have migrated to the cell adhesion culture surface 111 on day 2 after the culture bag was inverted.

[0047] On the other hand, on the third day, spheroid cells 3 were still present on the cell adhesion culture surface 111 in their original shape, indicating that they had not transitioned to adherent culture. Thus, it was found that even if adhesion factors necessary for cell adhesion to the cell adhesion culture surface are added at the start of adherent culture, adherent culture cannot be properly performed.

[0048] [Exam 2] This section describes a test demonstrating that, using the cell culture method of this embodiment, adherent culture could be properly performed when spheroid culture of cells was performed using a double-sided culture vessel followed by adherent culture. For the double-sided culture vessel, we prepared the same closed-system culture bag used in Experiment 1.

[0049] Next, 20 mL of Stemfit AK02N (Ajinomoto Co., Inc.) and 20 μL of 10 mM Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) were injected into the culture bag, and 0.5 μg / cm³ of laminin 511-E8 (Nippi Corporation) was added. 2 It was added. Furthermore, human pluripotent stem cells (human iPS cells: strain 1231A3) were divided into 1.8 × 10⁻¹⁴ units. 6 Cells were seeded in Cells (100 Cells / well), and spheroid culture was performed with the cell-non-adherent culture surface 101 facing the bottom.

[0050] After culturing for two days, the cells were cultured with the cell adhesion culture surface 111 facing downwards. After culturing for another day, the culture medium in the culture bag was changed, and 20 mL of Stemfit AK02N (Ajinomoto Co., Inc.) was injected into the culture bag. Furthermore, microscopic observations were performed on the cell-non-adherent culture surface 101 on the bottom side on day 2, and on the cell-adherent culture surface 111 on days 2 and 3. The results are shown in Figure 7.

[0051] On the second day, it can be seen that spheroid cells 3 (approximately 100 μm in size) have formed in each recess on the non-adherent cell culture surface 101. It can be seen that spheroid cells 3 have migrated to the cell adhesion culture surface 111 on day 2 after the culture bag was inverted.

[0052] Furthermore, it can be seen that on the cell adhesion culture surface 111 on the third day, spheroid cells 3' have been formed, which are spheroid cells that have adhered to and extended onto the cell adhesion culture surface 111. Thus, it has become clear that, according to the cell culture method of this embodiment, adhesive culture can be properly performed by adding adhesion factors necessary for adhering cells to the cell adhesion culture surface to the culture bag prior to adhesive culture.

[0053] The present invention is not limited to the embodiments and examples described above, and it goes without saying that various modifications can be made within the scope of the present invention. For example, the cell-non-adherent culture surface of the culture vessel does not need to be provided with a fine recessed pattern structure, as long as spheroid culture can be performed. Alternatively, spheroid culture may be performed first, followed by adherent culture, and then spheroid culture again. This method eliminates the need for separate culture vessels for spheroid culture and adherent culture, as the cultures can be performed in the same vessel, thus reducing the risk of contamination by bacteria and other microorganisms. [Industrial applicability]

[0054] The present invention can be suitably used when switching between spheroid culture and adherent culture using a single culture vessel. [Explanation of Symbols]

[0055] 1 Culture vessel 10 First Film 101 Cell non-adhesion culture surface 11 Second film 111 Cell adhesion culture surface 12 ports 2 Culture medium 3. Spheroid cells 3' Extended spheroid cells 4. Proteins derived from culture medium 4' Denatured culture medium-derived protein 5 Adhesion factor M cell non-adherent coating layer

Claims

1. In a method of culturing cells using a culture vessel filled with culture medium, The culture vessel has a cell-non-adhesion culture surface that has been treated to prevent cell adhesion and a cell-adhesion culture surface that has been treated to prevent cell adhesion inside, The adhesion factors necessary for adhering the cells to the cell adhesion culture surface are added at the latest while the cells are being cultured on the non-cell adhesion culture surface. After culturing the cells on the non-adherent culture surface, the cells are transferred to the cell-adherent culture surface, and then cultured with the cells adhered to the cell-adherent culture surface. A method for culturing cells characterized by the following features.

2. The cell culture method according to claim 1, characterized in that the adhesion factor is added to the culture medium before filling the culture vessel with the culture medium, or when filling the culture vessel with the culture medium.

3. The cell culture method according to claim 1 or 2, characterized in that the cells cultured on the cell-non-adherent culture surface are pluripotent stem cells.

4. The cell culture method according to claim 3, characterized in that the cells cultured on the cell adhesion culture surface are ectodermal cells differentiated from the pluripotent stem cells.

5. A method for culturing cells according to claim 1 or 2, characterized in that the culture vessel is filled with the culture medium, and the culture medium is in contact with the cell-non-adherent culture surface and the cell-adherent culture surface.

6. A method for culturing cells according to claim 1 or 2, characterized in that the adhesion factor is a laminin 511E8 fragment.

7. A method for producing cells, characterized by producing cells by culturing the cells using the cell culture method described in claim 1 or 2.

Images