Minimal cells are cultured using a method

Abstract

To provide a culture method capable of efficiently proliferating an extremely small number of cells.SOLUTION: A culture method of an extremely small number of cells includes the steps of: inputting a liquid culture medium LA of an amount at which a liquid level is positioned above openings 52H and a plurality of single cells C onto a culture plate 5 equipped with a plurality of micro grids 52 having the openings 52H on a top face to allow at least some micro grids 52 out of the plurality of micro grids 52 to hold the single cells C; removing the liquid culture medium LA in the culture plate 5 until the liquid level of the liquid culture medium LA approximately matches a height position of the openings 52H of the micro grids 52; and injecting sealing liquid 7 for preventing evaporation of the liquid culture medium LA onto the culture plate 5 to seal an area above the openings 52H.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This invention relates to a method for culturing extremely small numbers of cells, which involves seeding and culturing a very small number of cells relative to the culture area. [Background technology]

[0002] In some cases, it is necessary to culture cells in a minimal culture environment, where only a very small number of single cells or cells of a single origin are seeded and cultured in a culture area. In the development of antibody drugs that specifically bind to and remove foreign antigens such as virus-infected cells or cancer cells, culturing antibody-producing cells is essential. Culture of antibody-producing cells often takes place in a minimal culture environment. Various prior art methods exist for cell culture. For example, Patent Document 1 discloses a spheroid culture method using an alginate gel.

[0003] Screening is necessary to identify cells with high antibody production from among cultured cells. Patent Document 2 discloses a screening method in which cells cultured in a large number of wells are irradiated with light, and antibody production is evaluated based on the amount of fluorescence emitted from the wells. As the next step after screening, cells with high antibody production are cultured and proliferated to produce large amounts of antibodies. However, there are many cells that are difficult to proliferate even if they produce a lot of antibodies. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Special Publication No. 2021-511078 [Patent Document 2] Patent No. 6461580 [Overview of the project] [Problems that the invention aims to solve]

[0005] The objective of the present invention is to provide a culture method that enables the efficient proliferation of single cells, such as antibody-producing cells, which require culture in a minimal culture environment. [Means for solving the problem]

[0006] A method for culturing minute cells according to one aspect of the present invention includes the steps of: placing a culture medium in an amount such that the liquid level is above the openings and a plurality of single cells into a container having a plurality of containment compartments having openings on the top surface, thereby holding the single cells in at least some of the containment compartments; removing the culture medium from the container until the liquid level of the culture medium substantially coincides with the height of the openings of the containment compartments; and pouring a sealing solution for preventing evaporation of the culture medium into the container to seal the area above the openings.

[0007] According to this embodiment, after holding the cells in the containment section, the culture medium is removed and the opening is sealed with a sealing solution, thereby preventing evaporation of the culture medium inside the containment section and creating a closed culture environment for each containment section. It has been confirmed that culturing single cells in a small culture area promotes proliferation. Therefore, by culturing single cells as described above, it is possible to improve the efficiency of proliferation even in an extremely small culture environment.

[0008] In the culture method described above, it is desirable that the single cell is a single cell capable of producing antibodies.

[0009] In the culture method described above, the step of retaining the single cell may include the step of pouring a culture medium into the container in advance in an amount such that the liquid level is above the opening, and the step of dispensing the single cell, picked up from another container along with the culture medium using a tip, into the container.

[0010] According to this embodiment, single cells with high antibody-producing ability can be selected and picked from another container, and these can be held in the container's holding section for culture and proliferation. In other words, cell lines with superior antibody-producing ability can be cultured in an environment that is more conducive to proliferation.

[0011] In the above culture method, it is desirable that the sealing liquid has air permeability. In this case, it is more desirable that the sealing liquid is embryo culture oil.

[0012] According to this aspect, even if the opening of the accommodating portion is sealed with the sealing liquid, the medium in the accommodating portion can be communicated with the atmosphere, so that the culture environment in the accommodating portion can be maintained soundly.

[0013] In the above culture method, the accommodating portion has an opening area of 1.0×10 -3 ~1.0×10 -1 mm 2 , and a volume of 2.0×10 -5 ~1.0×10 -2 mm 3 and is preferably selected from the range of sizes.

[0014] According to this aspect, the culture region of a single cell can be sufficiently miniaturized, and a large number of single cells can be independently cultured on a limited plate area. Therefore, the efficiency of culturing and proliferating single cells can be improved.

[0015] In the above culture method, the container includes a large partition portion that divides a relatively large-sized region of the container, and a small partition portion that further subdivides the inside of the large partition portion, and it is desirable that the small partition portion is the accommodating portion.

[0016] According to this aspect, it becomes possible to utilize the cell type and culture solution by changing them in units of the large partition portion, and it becomes possible to diversify the culture.

Advantages of the Invention

[0017] According to the present invention, it is possible to provide a culture method capable of efficiently growing single cells that require culturing in an extremely small culture environment, such as antibody-producing cells.

Brief Description of the Drawings

[0018] [Figure 1]FIG. 1 is a diagram showing a process flow of a method for culturing high antibody-producing cells according to an embodiment of the present invention. [Figure 2] FIG. 2(A) is a plan view with an enlarged view showing the structure of a culture plate, and FIG. 2(B) is a cross-sectional view taken along line IIB-IIB of FIG. 2(A). [Figure 3] FIG. 3 is a schematic cross-sectional view showing a process of seeding a single cell from a suction pipette onto a culture plate and holding the single cell in a microgrid. [Figure 4] FIG. 4 is a schematic cross-sectional view showing a process of establishing a minimal culture environment by aspirating a culture medium. [Figure 5] FIG. 5 is a schematic cross-sectional view showing a situation where an opening of a microgrid is sealed with a sealing liquid. [Figure 6] FIG. 6 is an image showing the growth status of a single cell during a culture period in a culture plate. [Figure 7] FIG. 7 is an image showing the growth status of a single cell using a culture method of a comparative example. [Figure 8] FIG. 8 is an image showing the growth status of a single cell using a culture method of a comparative example.

Mode for Carrying Out the Invention

[0019] Hereinafter, embodiments of a method for culturing minimal cells according to the present invention will be described in detail based on the drawings. The culture method of the present invention is directed to culturing in a minimal culture environment in which a very small number of single cells are seeded with respect to the cell culture area. Note that the “single cell” as used in this specification includes single-origin cells in which a single cell has proliferated. Generally, single cells tend to be difficult to culture in a minimal culture environment, and the present invention enables efficient culturing of single cells in such an environment. Examples of single cells include single cells having the ability to produce recombinant proteins, such as CHO cells and B cells, which are expected to be used in the production of antibody pharmaceuticals and antibody production (Single Cell). Hereinafter, an example of culturing antibody-producing cells in a minimal culture environment will be shown.

[0020] [Overall Flow of Culture Process] First, the overall flow of the antibody-producing cell culture method according to this embodiment will be explained with reference to the process flow shown in Figure 1. The culture method of this embodiment includes steps S1 to S5 which are carried out sequentially. First, a large number of antibody-producing single cells are created using a predetermined method (step S1). Next, the created single cells are seeded together with liquid culture medium into a culture plate 5 (Figure 2) equipped with numerous cell containment compartments (step S2).

[0021] Next, liquid culture medium is aspirated from culture plate 5 to establish a microculture environment in which single cells are cultured in individual cell containments (step S3 / Figure 4). Furthermore, the top surface of culture plate 5 is sealed with a sealing solution to prevent evaporation of the culture medium (step S4 / Figure 5). After that, the single cells are cultured in the cell containments for a predetermined number of days (step S5 / Figure 6). Steps S1 to S5 described above will be explained in detail below.

[0022] [Step S1: Creation of antibody-producing single cells] In step S1, for example, antibody production ability is conferred to a single cell to be cultured by introducing a predetermined gene into the single cell. An example of a single cell is an immune cell, a B cell, and an example of an antibody produced is a monoclonal antibody produced by a single type of B cell. Gene introduction can be carried out by chemical methods using carrier molecules such as cationic lipids, physical methods such as electroporation, or biological methods such as viral vectors.

[0023] The gene-transformed single cells described above may be used directly as seeding targets in the next step S2, or cell lines with high antibody production capacity may be selected from the single cells and used as seeding targets. In this case, the single cells created in step S1 are cultured in a microplate or the like for a predetermined period to allow for antibody production. Then, a liquid medium containing a detection antibody that binds to the antibody produced by the single cells is added to the microplate to identify single cells with high antibody production capacity. The single cells identified as high antibody-producing cell lines are picked using a micropipette or the like to create a cell suspension, and seeding is performed in step S2.

[0024] [Step S2: Seeding Cells onto a Culture Plate] In step S2, the single cells created in step S1 are seeded onto a culture plate for culturing. FIG. 2(A) is a plan view with an enlarged view showing the structure of the culture plate 5, and FIG. 2(B) is a cross-sectional view taken along line IIB-IIB of FIG. 2(A). The culture plate 5 includes a grid 51 composed of recesses matrix-arranged on one side of a flat substrate, and a microgrid 52 composed of micro-sized recesses matrix-arranged within each grid 51.

[0025] The grid 51 is a large division part that divides a relatively large-sized area of the culture plate 5. In FIG. 2, a rectangular grid 51 in top view divided by vertical and horizontal grid plates is exemplified. Instead of this, a structure in which circular well-type grids 51 in top view are arranged in a honeycomb shape or a matrix shape may also be used. The microgrid 52 is a small division part that further subdivides the inside of each grid 51. The microgrid 52 is formed on the bottom plate of the grid 51 and is a rectangular recess in top view partitioned by side plates lower than the grid plates partitioning the grid 51. This microgrid 52 may also be in a circular well-type in top view.

[0026] The microgrid 52 has an opening on the top surface and serves as a housing part for holding single cells. Taking an example of the size of the microgrid 52, one side is 200 μm and the depth is 100 μm. The culture plate 5 is a plate provided with a large number of housing parts partitioned into such micro-sizes. The microgrid 52 is preferably set to a size that can form a micro-culture space. For example, the opening area is 4.0×10 -2 ~1.0×10 -1 mm 2 , and the volume is 4.0×10 -3 ~1.0×10 -2 mm 3 selected from the range, more preferably the opening area is 1.0×10 -3 ~1.0×10 -1 mm 2 and the volume is 2.0×10 -5 ~1.0×10 -2 3 ​It can be set to a size selected from a range.

[0027] Figure 3 is a schematic cross-sectional view showing the seeding of single cells C onto a culture plate 5. Figure 3 shows a cross-sectional view of one grid 51 of the culture plate 5 shown in Figure 2. Grid 51 has a grid bottom plate 511 that forms the bottom surface and grid side plates 512 that form the sides. Each microgrid 52 is partitioned by a common grid bottom plate 511 and side plates 521 that form the individual sides.

[0028] For seeding, a cell suspension of 2L is prepared, containing antibody-producing single cells C created in step S1 in liquid medium LA. The cell suspension of 2L is placed in a dispensing container 21 and injected into each grid 51 of the culture plate 5. This injection ensures that at least some of the numerous microgrids 52 within grid 51 hold liquid medium LA and one or more single cells C. The dilution of single cells C by the cell suspension of 2L can be set to such an extent that, for example, one single cell C is assigned to every 20 to 25 microgrids 52, given the extremely small culture environment.

[0029] Prior to seeding single cells C, a predetermined amount of liquid medium LA is added to the culture plate 5. As shown in Figure 3, the amount of liquid medium LA added is such that the liquid level reaches above the top 522 of the side plate 521 that partitions the microgrid 52. In other words, an amount of liquid medium LA such that the liquid level is above the top opening of the microgrid 52 is pre-filled into the grid 51 of the culture plate 5. Note that the top 522 of each microgrid 52 is at the same height. As the liquid medium LA, a normal culture medium containing inorganic salts, glucose, amino acids and other growth factors, and additives such as antibiotics and growth promoters may be used. For example, CH150 medium (product name of Z-MEP Corporation) can be suitably used as the liquid medium LA.

[0030] Subsequently, single cells C from the 2L cell suspension are seeded from the dispensing container 21 onto the culture plate 5. This seeding ensures that at least some of the microgrids 52 of the multiple microgrids 52 on a single grid 51 hold one or more single cells C.

[0031] [Step S3: Establishment of a micro-culture environment by aspiration of culture medium] Step S3 is a process in which the liquid culture medium LA in the culture plate 5 is aspirated to establish an independent single-cell culture environment for each microgrid unit of 52. The culture environment established here is a culture environment with an extremely small culture area. Figure 4 is a schematic cross-sectional view showing the operation of step S3. In Figure 4, the liquid culture medium LA in the grid 51 is shown being aspirated by the suction tip 24.

[0032] The suction by the suction tip 24 is performed from the state shown in Figure 3 until the liquid level of the liquid medium LA in grid 51 is such that the top 522 of the side plate 521 of microgrid 52 is exposed. That is, the liquid medium LA in grid 51 is removed until the liquid level of the liquid medium LA is approximately at the same height as the opening 52H of microgrid 52. This suction prevents the liquid medium LA in one microgrid 52 from mixing with the liquid medium LA in other microgrids 52. In other words, a minimal culture environment for a single cell C is formed, consisting of the liquid medium LA in each individual microgrid 52. The amount of liquid medium LA in one microgrid 52 is, for example, 4 nanoliters.

[0033] In a very small culture environment where only a few single cells C (1 to 10) are introduced into a large culture environment, the single cells C are difficult to proliferate. For example, even if liquid culture medium LA is poured into grid 51 (where the side plate 521 is removed to eliminate the microgrid compartments in Figure 4) and single cells C are introduced and given a predetermined culture period, the single cells C are difficult to proliferate. On the other hand, when 1 to 10 single cells C are introduced into a very small culture environment with about 4 nanoliters of liquid culture medium LA and cultured, the proliferation of the single cells C tends to be promoted, partly because the cells tend to grow adjacent to each other. In the cell seeding step S2, it is advantageous for the liquid level of the liquid culture medium LA to be above the opening 52H, as this allows the single cells C to be retained in the microgrid 52 with a single dispensing operation. By aspirating the culture medium in the subsequent step S3, a very small culture environment suitable for culturing and proliferating a small number of single cells C, isolated in microgrid units of 52, can be established.

[0034] [Process S4; Microgrid sealing] Step S4 is the step of pouring the sealing solution 7 into the culture plate 5 to seal the upper part of the opening 52H of the microgrid 52. Figure 5 is a schematic cross-sectional view showing the state in which the opening 52H of the microgrid 52 is sealed with the sealing solution 7. The lower surface of the sealing solution 7 is in contact with the top 522 of the side plate 521 of the microgrid 52, blocking the opening 52H. In other words, the liquid culture medium LA and single cells C are confined within a single microgrid 52 by the sealing solution 7. As the sealing solution 7, for example, embryo culture oil consisting of light liquid paraffin can be used.

[0035] The required function of the sealing solution 7 is to prevent the evaporation of the liquid culture medium LA within the microgrid 52. Since the liquid culture medium LA contains water, if the sealing solution 7 is not present, the water will evaporate. As a result, during the culture period in step S5, problems such as a decrease or depletion of the amount of liquid culture medium LA within the microgrid 52, and changes in the culture medium conditions such as osmotic pressure and pH may occur. By sealing the opening 52H with the sealing solution 7, which has an evaporation prevention function, the evaporation of the liquid culture medium LA during the culture period can be suppressed.

[0036] Another desirable function of the sealing solution 7 is permeability. If the sealing solution 7 is permeable, even if the opening 52H of the microgrid 52 is sealed, the liquid culture medium LA inside the microgrid 52 can be connected to the atmosphere. Therefore, the culture environment of single cells C inside the microgrid 52 can be maintained in a healthy state. The embryo culture oil mentioned above is suitable as the sealing solution 7 because it possesses both the function of preventing evaporation and the function of permeability. In addition to embryo culture oil, other liquids or semi-liquids (gels) that have at least the function of preventing evaporation may be used as the sealing solution 7. Needless to say, it is necessary that the specific gravity is lighter than that of the liquid culture medium LA.

[0037] The formation of the sealing solution layer 7 allows the microculture environment established in step S3 to be maintained throughout the culture period. In other words, not only is evaporation of the liquid culture medium LA within the microgrid 52 prevented, but foreign matter contained in the outside air, such as minute dust particles, mold spores, and bacteria, is also prevented from entering the microgrid 52. Furthermore, it has the advantage of preventing the diffusion of active substances emitted by single cells C cultured within the microgrid 52 and promoting the proliferation of single cells C.

[0038] [Step S5; Cell culture] Step S5 is a process in which single cells C are cultured for a predetermined culture period while the openings 52H of the microgrid 52 are sealed with sealing solution 7 as shown in Figure 5. In other words, this is a process in which antibodies are produced by culturing and growing the single cells C created in step S1 for a predetermined period. During this culture period, liquid culture medium containing growth factors is replenished in grid 51.

[0039] Figure 6 is an image showing the proliferation of single cells C during the culture period in culture plate 5. "Day 1" in the figure refers to the state on day 1 from the start of the second culture. Figure 6 shows images of a portion of the microgrid 52 of one grid 51 on days 1, 4, 5, 6, 8, 11, and 18 from the start of the culture. Looking at the mutation status of single cells C in the grid GA of interest among the multiple microgrids 52, it can be seen that they proliferate day by day. The rapid proliferation of single cells C around the grid GA of interest between days 11 and 18 is due not only to the longer culture period, but also to the fact that single cells C that proliferated from the grid GA of interest entered adjacent grids when the liquid culture medium was replenished.

[0040] Figures 7 and 8 show images of the proliferation of single cells using the culture method of the comparative example. Figure 7 is an image of Comparative Example 1, in which the culture medium aspiration in step S3 (Figure 4) was not performed, and the sealing with embryo culture oil in step S4 (Figure 5) was not performed, meaning that culture was performed immediately after cell seeding in step S2 (Figure 3). Figure 7 shows images from day 1, day 6, and day 11 from the start of culture. Looking at the proliferation of single cells C in grid GA1, one of the multiple microgrids 52, it can be seen that no significant proliferation occurred between day 1 and day 11.

[0041] Figure 8 shows images of Comparative Example 2, in which the culture medium aspiration in step S3 (Figure 4) was omitted, and the sealing with embryo culture oil in step S4 was performed. In other words, after cell seeding in step S2 (Figure 3), the sealing solution 7 was poured in when the liquid level of the liquid medium LA was higher than the top 522, and the culture was performed. Figure 8 also shows images from day 1, day 6, and day 11 of the culture. Looking at the proliferation status of single cells C in grid GA2, one of the multiple microgrids 52, it can be seen that no significant proliferation occurred between day 1 and day 11.

[0042] [Effects and Effects] The antibody-producing cell culture method according to the present invention, as described above, produces the following effects. Specifically, after holding single cells C in the microgrid 52 of the culture plate 5, the liquid medium LA is removed and the opening 52H is sealed with sealing solution 7. This prevents evaporation of the liquid medium LA in the microgrid 52 and allows the construction of a very small culture environment enclosed by the microgrid 52 units. By culturing single cells C in a narrow culture area, the proliferation of the single cells C is promoted. Therefore, by culturing single cells C with antibody-producing ability as described above, the proliferation efficiency of single cells C can be improved, and consequently, a large amount of antibodies can be produced. [Explanation of Symbols]

[0043] C single cell LA liquid culture medium (culture medium) 23 Suction Tips 5. Culture plate (container) 51 Grid (Large Section) 52 Microgrid (Housing Section / Small Compartment Section) 52H opening 7 Sealing liquid

Claims

1. A process of placing a culture medium in an amount such that the liquid level is above the opening and a single cell into a container having multiple storage compartments with openings on the top surface, thereby causing one or more of the single cells to be held in at least some of the storage compartments. The steps include removing the culture medium from the container until the liquid level of the culture medium is approximately equal to the height of the opening of the containment section, A method for culturing minute cells, comprising the step of pouring a sealing solution for preventing evaporation of the culture medium into the container and sealing the top of the opening, The aforementioned storage section has an opening area of ​​1.0 × 10⁻³ to 1.0 × 10⁻¹ mm² and a volume of 2.0 × 10⁻⁵ to 1.0 × 10⁻² mm³, A method for culturing extremely small cells, wherein the culture medium and the single cell are placed in the container as a cell suspension containing the single cell in the culture medium, and the degree of dilution of the single cell by the cell suspension is such that one single cell is allocated to 20 to 25 of the containers.

2. In the method for culturing minute cells according to claim 1, A method for culturing a very small number of cells, wherein the single cell is a single cell capable of producing antibodies.

3. In the method for culturing minute cells according to claim 1 or 2, The step of retaining the single cell is, The process involves first pouring a culture medium into the container in an amount such that the liquid level is above the opening, A method for culturing a very small number of cells, comprising the step of discharging the cell suspension into the container.

4. In the method for culturing minute cells according to claim 1 or 2, A method for culturing extremely small cells, wherein the sealing solution is permeable.

5. In the method for culturing minute cells according to claim 1 or 2, A method for culturing minute cells, wherein the sealing solution consists of embryo culture oil.

6. In the method for culturing minute cells according to Claim 1, The container includes a large compartment that divides a relatively large area of ​​the container, and a small compartment that further subdivides the inside of the large compartment. A method for culturing a very small number of cells, wherein the small compartment is the containment section.

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