Method for culturing very few cells

Abstract

A method for culturing very few cells, comprising: a step in which a culture medium and a plurality of single cells are introduced into a container provided with a plurality of accommodation parts having an opening in the top surface thereof in an amount such that the liquid level is positioned above the opening, and the single cells are held in at least some of the plurality of accommodation parts; a step of removing the culture medium in the container until the liquid level of the culture medium substantially coincides with the height position of the opening part of the accommodating part; and a step in which a sealing liquid for preventing evaporation of the culture medium is injected into the container, and the upper part of the opening is sealed.

Description

Technical Field

[0001] The present invention relates to a method for culturing a very small number of cells in which a very small number of cells are inoculated relative to the culture area and cultured. Background Art

[0002] There are times when it is necessary to culture in a very small culture environment in which a very small number of single cells or single-origin cells are inoculated and cultured relative to the culture area, which is the area for culturing cells. When developing an antibody drug having the effect of specifically binding to an antigen of a foreign substance such as a virus-infected cell or a cancer cell and removing the foreign substance, culturing of cells capable of producing an antibody is indispensable. Culturing of antibody-producing cells is mostly culturing in a very small culture environment. As a method for culturing cells, there are various existing technologies. For example, in Patent Document 1, although it is a culturing technique for spheroids, a culturing method using an alginate gel is disclosed.

[0003] It is necessary to screen for cells with a high antibody production amount from the cultured cells. Patent Document 2 discloses a screening method in which cells cultured in a plurality of wells are irradiated with light, and the antibody production amount is evaluated based on the amount of fluorescence generated from the wells. As the next step of the screening, an operation of culturing cells with a high antibody production amount to proliferate them and produce a large amount of antibodies is performed. However, there are many cells that are difficult to proliferate even if the antibody production amount is large.

[0004] Prior Art Documents

[0005] Patent Documents

[0006] Patent Document 1: Japanese Patent Application Laid-Open No. 2021-511078

[0007] Patent Document 2: Japanese Patent No. 6461580 Summary of the Invention

[0008] An object of the present invention is to provide a culturing method capable of efficiently proliferating single cells that require culturing in a very small culture environment, such as antibody-producing cells.

[0009] A method for culturing a very small number of cells according to one aspect of the present invention includes: a step of holding at least a part of the plurality of accommodating portions with the single cells by introducing a medium in an amount such that the liquid level is located above the opening portion into a container having a plurality of accommodating portions with an opening portion on the top surface and a plurality of single cells; a step of removing the medium in the container until the liquid level of the medium is substantially the same as the height position of the opening portion of the accommodating portion; and a step of injecting a sealing liquid for preventing evaporation of the medium into the container to seal the upper portion of the opening portion. Brief Description of the Drawings

[0010] Figure 1 It is a diagram showing the process flow of the method for culturing high antibody-producing cells according to an embodiment of the present invention.

[0011] Figure 2A It is a top view with an enlarged view showing the structure of the culture plate.

[0012] Figure 2B It is Figure 2A A sectional view taken along the line IIB-IIB.

[0013] Figure 3 It is a schematic sectional view showing the steps of inoculating a single cell from a suction head into the culture plate and having the microgrid hold the single cell.

[0014] Figure 4 It is a schematic sectional view showing the steps of establishing a minimal culture environment by medium suction.

[0015] Figure 5 It is a schematic sectional view showing the state of sealing the opening of the microgrid with a sealing liquid.

[0016] Figure 6 It is an image showing the proliferation state of a single cell during culture in the culture plate.

[0017] Figure 7 It is an image of the proliferation state of a single cell using the culture method of the comparative example.

[0018] Figure 8 It is an image of the proliferation state of a single cell using the culture method of the comparative example. Detailed Embodiments

[0019] Hereinafter, embodiments of the method for culturing a minimal number of cells of 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 an extremely small number of single cells are inoculated with respect to the cell culture area. It should be noted that the "single cell" as referred to in this specification also includes cells formed by the proliferation of a single cell, that is, clonal cells. Generally, single cells tend to be difficult to culture in a minimal culture environment, and the present invention can efficiently culture single cells in such an environment. As single cells, examples 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 drugs and produce antibodies (Single Cell). Hereinafter, an example of culturing antibody-producing cells in a minimal culture environment will be shown.

[0020] [Overall Process of the Culture Step]

[0021] First, refer to Figure 1The following step process illustrates the overall process of the method for culturing antibody-producing cells of the present embodiment. The culturing method of the present embodiment includes steps S1 to S5 implemented in sequence. First, a large number of single cells capable of producing antibodies are produced using a specified method (step S1). Next, the produced single cells are inoculated together with a liquid medium into a culture plate 5 having a plurality of cell accommodation portions ( Figure 2A , Figure 2B )(step S2).

[0022] Next, the liquid medium is aspirated from the culture plate 5 to establish a minimal culture environment for culturing single cells in each cell accommodation portion (step S3 / Figure 4 ). Furthermore, the top surface of the culture plate 5 is sealed with a sealing liquid for preventing evaporation of the medium (step S4 / Figure 5 ). Then, the single cells are cultured in the above-mentioned cell accommodation portions for a specified number of days (step S5 / Figure 6 ). Hereinafter, steps S1 to S5 described above will be detailed respectively.

[0023] [Step S1: Production of antibody-producing single cells]

[0024] In step S1, for example, by introducing a specified gene into the single cells to be cultured, the ability to produce antibodies is imparted to the single cells. As the single cells, B cells of immune cells can be exemplified, and as the produced antibodies, monoclonal antibodies produced by a single type of B cell can be exemplified. The introduction of the gene can be performed, for example, by a chemical method using a carrier molecule such as cationic lipid, a physical method such as electroporation, or a biological method such as a viral vector.

[0025] The single cells into which the gene has been introduced as described above can be directly used as the inoculation target in the next step S2, but cells with high antibody-producing ability can also be selected from the above single cells as the inoculation target. In this case, the single cells produced in step S1 are cultured in a microplate or the like for a specified period to undergo the antibody production period. Then, a liquid medium containing a detection antibody that binds to the antibody produced by the above single cells is added to the above microplate to identify single cells with high antibody-producing ability. The single cells determined to be high antibody-producing cell lines are picked up using a micropipette or the like to prepare a cell suspension, and inoculation in step S2 is performed.

[0026] [Step S2: Inoculation of cells into the culture plate]

[0027] In step S2, for culturing, the single cells produced in step S1 are inoculated into the culture plate. Figure 2A is a top view with an enlarged view showing the structure of the culture plate 5, Figure 2B is Figure 2AIIB-IIB line cross-sectional view. The culture plate 5 includes a grid 51 composed of recesses arranged in a matrix on a single surface of a flat substrate, and a micro-grid 52 composed of micro-sized recesses arranged in a matrix within each grid 51.

[0028] The grid 51 is a large partition part that divides the culture plate 5 into larger-sized regions. In Figure 2A it, a grid 51 that is rectangular in plan view and divided by horizontal and vertical grid plates is illustrated. Instead, it may also be a structure in which grid 51 with a circular hole shape in plan view is arranged in a honeycomb or matrix shape. The micro-grid 52 is a small partition part that further subdivides the inside of each grid 51. The micro-grid 52 is a recess that is formed on the bottom plate of the grid 51, divided by side plates that are lower than the grid plates dividing the grid 51, and is rectangular in plan view. The micro-grid 52 may also have a circular hole shape in plan view.

[0029] The micro-grid 52 has an opening at the top surface and serves as a housing part for holding single cells. If an example of the size of the micro-grid 52 is given, one side is 200 μm and the depth is 100 μm. The culture plate 5 is a plate having a plurality of housing parts divided into such micro-sized sizes. The micro-grid 52 is preferably set to a size capable of forming a micro culture space. For example, it can be set to a size where the opening area is selected from the range of 4.0×10 -2 ~1.0×10 -1 mm 2 and the volume is selected from the range of 4.0×10 -3 ~1.0×10 -2 mm 3 and more preferably set to a size where the opening area is selected from the range of 1.0×10 -3 ~1.0×10 -1 mm 2 and the volume is selected from the range of 2.0×10 -5 ~1.0×10 -2 mm 3 range.

[0030] Figure 3 is a schematic cross-sectional view showing the state of inoculating single cells C into the culture plate 5. In Figure 3 it, a cross-sectional view of one grid 51 of the culture plate 5 shown in Figure 2B is shown. The grid 51 has a grid bottom plate 511 forming the bottom surface and grid side plates 512 forming the side surfaces. Each micro-grid 52 is divided by a common grid bottom plate 511 and side plates 521 forming each side surface.

[0031] At the time of inoculation, 2 L of a cell suspension in which the liquid medium LA contains single cells C capable of producing antibodies, prepared in step S1, is prepared. The 2 L of cell suspension is accommodated in a dispensing container 21, and the grids 51 of the culture plate 5 are filled with the liquid. By this filling, the liquid medium LA and one or more single cells C are retained in at least a part of the plurality of microgrids 52 within the grid 51. Since the dilution degree of the single cells C by the 2 L of cell suspension is an extremely small culture environment, for example, it can be set to the extent that one single cell C is allocated to 20 to 25 microgrids 52.

[0032] Before inoculating the single cells C, a predetermined amount of the liquid medium LA is introduced into the culture plate 5. As Figure 3 shown, the amount of the liquid medium LA introduced is such that the liquid level reaches a position above the top 522 of the side plate 521 that divides the microgrid 52. In other words, the liquid medium LA in an amount such that the liquid level is located above the top opening of the microgrid 52 is pre-injected into the grid 51 of the culture plate 5. It should be noted that the top 522 of each microgrid 52 is located at the same height position. As the liquid medium LA, a normal culture solution containing inorganic salts, glucose, amino acids and other growth factors, and additives such as antibiotics and growth promoting factors in an aqueous medium can be used. For example, it is preferable to use the CH150 medium (trade name of Gimep Co., Ltd.) as the liquid medium LA.

[0033] Then, the single cells C in the 2 L of cell suspension are inoculated from the dispensing container 21 into the culture plate 5. By this inoculation, one or more single cells C are retained in at least a part of the plurality of microgrids 52 included in one grid 51.

[0034] [Step S3: Establishment of an extremely small culture environment by medium suction]

[0035] Step S3 is a step of sucking the liquid medium LA of the culture plate 5 and establishing a culture environment for single cells C independent in units of microgrids 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 it, the situation where the liquid medium LA within the grid 51 is sucked by the pipette tip 24 is shown.

[0036] By the above suction using the pipette tip 24, from Figure 3The state is advanced until the liquid surface of the liquid medium LA in the grid 51 exposes the top 522 of the side plate 521 of the micro-grid 52. That is, the liquid medium LA in the grid 51 is removed until the liquid surface of the liquid medium LA is approximately at the same height position as the opening 52H of the micro-grid 52. By such suction, the liquid medium LA in one micro-grid 52 does not mix with the liquid medium LA in other micro-grids 52. That is, a minimal culture environment for the single cell C composed of the liquid medium LA in each micro-grid 52 is formed. The amount of the liquid medium LA in one micro-grid 52 is, for example, 4 nanoliters.

[0037] In a very small culture environment where 1 to about 10 single cells C are introduced into a vast culture environment, it is difficult for the single cell C to proliferate. For example, in Figure 4 , even if the liquid medium LA is injected into the grid 51 of the partition without the micro-grid 52 by removing the side plate 521, the single cell C is introduced and allowed to undergo a prescribed culture period, it is difficult for the single cell C to proliferate. On the other hand, if 1 to about 10 single cells C are introduced and cultured in a very small culture environment where the liquid medium LA is about 4 nanoliters, the cells are likely to be adjacent to each other, and there is a tendency for the proliferation of the single cell C to be promoted. In the cell seeding in step S2, when the liquid surface of the liquid medium LA is located above the opening 52H, the single cell C can be held in the micro-grid 52 by a single ejection operation, so it is appropriate. By performing medium suction in the subsequent step S3, a minimal culture environment suitable for the culture / proliferation of a small number of single cells C isolated in units of micro-grids 52 can be established.

[0038] [Step S4: Sealing of the micro-grid]

[0039] Step S4 is a step of injecting the sealing liquid 7 into the culture plate 5 to seal the upper part of the opening 52H of the micro-grid 52. Figure 5 is a schematic cross-sectional view showing the state where the opening 52H of the micro-grid 52 is sealed by the sealing liquid 7. The lower surface of the sealing liquid 7 contacts the top 522 of the side plate 521 of the micro-grid 52 to block the opening 52H. That is, it becomes a state where the liquid medium LA and the single cell C are enclosed in one micro-grid 52 by the sealing liquid 7. As the sealing liquid 7, for example, embryo culture oil composed of light liquid paraffin or the like can be used.

[0040] The function required of the sealing liquid 7 is the function of preventing the evaporation of the liquid culture medium LA in the microgrid 52. Since the liquid culture medium LA contains moisture, in the absence of the sealing liquid 7, the above moisture will evaporate. Therefore, during the culture period in step S5, adverse conditions such as a decrease or exhaustion in the amount of the liquid culture medium LA in the microgrid 52, and changes in the state of the culture medium such as osmotic pressure or pH occur. By sealing the opening 52H with the sealing liquid 7 having the evaporation prevention function, the evaporation of the liquid culture medium LA during the culture period can be suppressed.

[0041] Another desired function of the sealing liquid 7 is air permeability. If the sealing liquid 7 has air permeability, even if the opening 52H of the microgrid 52 is sealed, the liquid culture medium LA in the microgrid 52 can be communicated with the atmosphere. Therefore, the culture environment of the single cell C in the microgrid 52 can be maintained in good condition. The above-mentioned embryo culture oil has both the above evaporation prevention function and the above air permeability, so it is suitable as the sealing liquid 7. In addition to the embryo culture oil, other liquids or semi-liquids (gels) having at least the evaporation prevention function can also be used as the sealing liquid 7. Of course, the specific gravity needs to be lighter than the liquid culture medium LA.

[0042] By forming a layer of the sealing liquid 7, the extremely small culture environment established in step S3 can be maintained during the culture period. That is, it can not only prevent the evaporation of the liquid culture medium LA in the microgrid 52, but also inhibit the intrusion of foreign substances contained in the external gas, such as minute dust, mold spores, bacteria, etc. into the microgrid 52. In addition, it also has the advantages of being able to prevent the diffusion of active substances produced by the single cell C cultured in the microgrid 52 and promoting the proliferation of the single cell C.

[0043] [Step S5: Cell culture]

[0044] Step S5 is a step of culturing the single cell C for only a specified culture period in a state where the opening 52H of the microgrid 52 is sealed with the sealing liquid 7 as Figure 5 described. That is, it is a step of proliferating the single cell C produced in step S1 by culturing it for only a specified period to produce an antibody. During this culture period, a liquid culture medium containing growth factors is filled into the grid 51.

[0045] Figure 6 is an image showing the proliferation status of the single cell C during the culture period in the culture plate 5. "Day 1" in the figure refers to the state on the first day starting from the second culture. In Figure 6Images of a part of the micro-grid 52 included in the grid 51 are shown for the 1st, 4th, 5th, 6th, 8th, 11th, and 18th days from the start of cultivation. By observing the variation status of the single cell C in the grid of interest GA among the multiple micro-grids 52, it can be seen that the cells proliferate every day. It should be noted that between the 11th day and the 18th day, the main reason for the rapid proliferation of the single cells C around the grid of interest GA is not only the long cultivation days, but also that the single cells C proliferated from the grid of interest GA enter the adjacent grids when filling the above liquid medium.

[0046] Figure 7 and Figure 8 are images of the proliferation status of single cells using the cultivation method of the comparative example. Figure 7 is an image of Comparative Example 1 in a state where the medium aspiration in step S3 ( Figure 4 ) is not performed and the sealing with embryo culture oil in step S4 ( Figure 5 ) is not performed, that is, immediately after the cell seeding in step S2 ( Figure 3 ). In Figure 7 , images of the 1st, 6th, and 11th days from the start of cultivation are shown. By observing the proliferation status of the single cell C in the grid of interest GA1 among the multiple micro-grids 52, it can be seen that no significant proliferation occurred between the 1st day and the 11th day.

[0047] Figure 8 is an image of Comparative Example 2 in a state where the medium aspiration in step S3 ( Figure 4 ) is not performed and the sealing with embryo culture oil in step S4 is performed, that is, after the cell seeding in step S2 ( Figure 3 ), and the culture is carried out in a state where the liquid level of the liquid medium LA is higher than the top 522. In Figure 8 , images of the 1st, 6th, and 11th days from the start of cultivation are also shown. By observing the proliferation status of the single cell C in the grid of interest GA2 among the multiple micro-grids 52, it can be seen that no significant proliferation occurred between the 1st day and the 11th day.

[0048] [Function and effect]

[0049] The method for culturing antibody-producing cells of the present invention described above has the following effects. That is, after maintaining the single cell C in the microgrid 52 of the culture plate 5, the liquid medium LA is removed and the opening 52H is sealed with the sealing liquid 7. Thereby, evaporation of the liquid medium LA in the microgrid 52 can be prevented, and a microculture environment enclosed in units of the microgrid 52 can be constructed. By culturing the single cell C in a narrow culture area, proliferation of the single cell C can be promoted. Therefore, by culturing the single cell C having the ability to produce antibodies as described above, the efficiency of proliferation of the single cell C can be achieved, and antibodies can be produced in large quantities.

[0050] [The invention included in the above-described embodiment]

[0051] The embodiment described above includes an invention having the following configuration.

[0052] A method for culturing a very small number of cells according to one aspect of the present invention includes: a step of maintaining the single cells in at least a part of the plurality of accommodating portions by introducing a medium and a plurality of single cells in an amount such that the liquid level is located above the opening in a container having a plurality of accommodating portions with openings on the top surface; a step of removing the medium in the container until the liquid level of the medium is substantially the same as the height position of the opening of the accommodating portion; and a step of injecting a sealing liquid for preventing evaporation of the medium into the container and sealing the upper part of the opening.

[0053] According to this method, after the cells are maintained in the accommodating portion, the medium is removed and the opening is sealed with the sealing liquid, whereby evaporation of the medium in the accommodating portion can be prevented, and a culture environment enclosed in units of the accommodating portion can be constructed. It has been confirmed that proliferation can be promoted by culturing single cells in a narrow culture area. Therefore, by culturing single cells as described above, even in a very small culture environment, the efficiency of proliferation can be achieved.

[0054] In the above-described culturing method, a preferred mode is that the single cell is a single cell capable of producing an antibody.

[0055] In the above-described culturing method, the step of maintaining the single cells may include: a step of previously injecting a medium in an amount such that the liquid level is located above the opening into the container; and a step of discharging the single cells picked up together with the medium from another container using a pipette tip into the container.

[0056] According to this method, single cells having high antibody-producing ability can be screened and picked up in another container and maintained in the accommodating portion of the container for culture and proliferation. That is, cell lines having excellent antibody-producing ability can be cultured in an environment where proliferation is easier.

[0057] In the above-described culture method, it is preferable that the sealing liquid has air permeability. In this case, it is more preferable that the sealing liquid is embryo culture oil.

[0058] According to this method, even if the opening of the housing part is sealed with the sealing liquid, the culture medium in the housing part can be communicated with the atmosphere, so that the culture environment in the housing part can be maintained healthy.

[0059] In the above-described culture method, it is preferable that the housing part has an opening area selected from the range of 1.0×10 -3 ~1.0×10 - 1 mm 2 and a volume selected from the range of 2.0×10 -5 ~1.0×10 -2 mm 3 in size.

[0060] According to this method, the culture area of single cells can be sufficiently miniaturized, and a large number of single cells can be independently cultured with a limited plate area. Therefore, the efficiency of culturing / proliferating single cells can be achieved.

[0061] In the above-described culture method, it is preferable that the container includes a large partition part that divides a large-sized area of the container and a small partition part that further subdivides the inside of the large partition part, and the small partition part is the housing part.

[0062] According to this method, it is possible to change cell types, culture media, etc. in units of large partition parts and achieve diversification of culture.

[0063] According to the present invention described above, a culture method can be provided that can efficiently proliferate single cells that require culturing in a minimal culture environment, such as antibody-producing cells.

Claims

1. A method for culturing a very small number of cells, comprising: a step of introducing a medium in an amount such that the liquid level is above the opening portion and a plurality of single cells into a container having a plurality of accommodating portions with opening portions on the top surface, so that at least a part of the plurality of accommodating portions hold the single cells; a step of removing the medium in the container until the liquid level of the medium is substantially flush with the height position of the opening portion of the accommodating portion; and a step of injecting a sealing liquid for preventing evaporation of the medium into the container to seal the upper portion of the opening portion.

2. The method for culturing a very small number of cells according to claim 1, wherein, The single cell is a single cell capable of producing an antibody.

3. The method for culturing extremely few cells according to claim 1 or 2, wherein The step of holding the single cell includes: a step of previously injecting a medium in an amount such that the liquid level is above the opening portion into the container; and a step of discharging the single cell picked up together with the medium from another container using a pipette tip into the container.

4. The method for culturing extremely few cells according to claim 1 or 2, wherein, The sealing liquid has air permeability.

5. The method for culturing a very small number of cells according to claim 1 or 2, wherein, The sealing liquid contains embryo culture oil.

6. The method for culturing extremely few cells according to claim 1, wherein, The receiving portion has a size with an opening area selected from the range of 1.0×10 -3 to 1.0×10 -1 mm 2 and a volume selected from the range of 2.0×10 -5 to 1.0×10 -2 mm 3 .

7. The method for culturing extremely few cells according to claim 6, wherein, The container includes a large partitioning portion that divides a relatively large area of the container and a small partitioning portion that further subdivides the inside of the large partitioning portion, and the small partitioning portion is the accommodating portion.

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