Method for culturing recombinant protein-producing cells

Abstract

To provide a culture method that can efficiently identify cell lines with high recombinant protein production.SOLUTION: The method for culturing recombinant protein-producing cells includes the steps of: injecting 2 L of cell suspension, for example, containing a large number of single cells C capable of producing an antibody 3 in a liquid medium LA, into a first plate 1 equipped with a large number of microgrids 12 partitioned into minute sizes, and allowing at least some of the microgrids 12 among the large number of microgrids 12 to retain the liquid medium LA and one single cell C; converting the liquid medium LA in the microgrid 12 into a gel medium LB; providing an antibody production period to the single cells C in the microgrid 12; and adding a liquid containing a detection antibody 4 capable of binding to the antibody 3 produced by the single cells C in the gel medium LB to the first plate 1.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for culturing cells that produce recombinant proteins. [Background technology]

[0002] In developing antibody drugs that have the efficacy of specifically binding to antigens of foreign substances such as virus-infected cells and cancer cells and removing said foreign substances, the cultivation of antibody-producing cells is essential. Various prior art methods exist for culturing cells. For example, Patent Document 1 discloses a culture method using alginate gel, although it is a spheroid culture technique.

[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. However, in methods where cells are seeded and cultured in general-purpose wells and the amount of fluorescence is determined, it was sometimes difficult to perform screening efficiently. [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 object of the present invention is to provide a method for culturing recombinant protein-producing cells that can efficiently identify cell lines with high recombinant protein production. [Means for solving the problem]

[0006] A method for culturing recombinant protein-producing cells according to one aspect of the present invention includes the steps of: pouring a cell suspension containing a large number of single cells capable of producing recombinant proteins into a culture medium into a plate having a large number of compartments divided into micro-sized sections, and holding the culture medium and a single cell in each of at least some of the compartments; gelling the culture medium in the compartments; giving the single cell in the compartment a period for recombinant protein production; and adding a liquid to the plate containing a detection protein capable of binding to the recombinant protein produced by the single cell in the gelled culture medium.

[0007] According to this embodiment, after holding a single cell in one containment unit, the culture medium within the containment unit is gelled. Gelation creates a state in which one single cell is confined within each containment unit. In this state, by culturing the single cell for a predetermined recombinant protein production period, cell proliferation and recombinant protein production can be performed in each containment unit. Then, by adding a detection protein to the plate, the amount of recombinant protein produced can be evaluated on a unit-by-unit basis. Since the suspension of the single cell is suppressed by the gelling of the culture medium, a containment unit in which a large amount of detection protein is detected will contain a cell line with high recombinant protein production capacity. Thus, according to the above embodiment, single cell culture, recombinant protein production, and detection of recombinant protein production are possible on a unit-by-unit basis, making it possible to efficiently identify cell lines with high recombinant protein production.

[0008] In the culture method described above, it is desirable that the single cell capable of producing the recombinant protein is a single cell capable of producing an antibody, and that the detection protein is a detection antibody.

[0009] According to this embodiment, single-cell culture, antibody production, and detection of antibody production volume are possible within each containment unit, thus enabling efficient identification of cell lines with high antibody production.

[0010] In the above-described culturing method, it is desirable to further include a step of providing a trigger for reacting the detection antibody and identifying a storage section in which a single cell producing a large amount of antibody is retained.

[0011] According to this aspect, for example, by providing a trigger such as irradiating a plate with light to fluoresce the detection antibody and detecting the amount of the detection antibody per storage section based on the fluorescence intensity or the like, a cell line producing a large amount of antibody can be identified.

[0012] In the above-described culturing method, it may further include a step of picking the single cell retained in the identified storage section and transferring it to another container.

[0013] According to this aspect, by picking a high antibody-producing cell line and transferring it to another container, for example, it becomes possible to further culture the cell line and produce a large amount of antibody.

[0014] In the above-described culturing method, it is desirable to monitor the plate during the antibody production period, and exclude the storage sections in which a plurality of single cells have been retained from the beginning of the production period from the specific target.

[0015] When a cell suspension is poured into a plate, among a large number of storage sections, a storage section that holds only one single cell appears at a generally constant ratio probabilistically. The remaining storage sections become non-retaining storage sections that do not hold a single cell or over-retaining storage sections that hold a plurality of single cells. In the over-retaining storage sections, there may be a mixture of high antibody-producing cell lines and non-producing or low antibody-producing cell lines. According to the above aspect, since the over-retaining storage sections are excluded from the specific target, it is possible to accurately identify the storage sections in which single cells producing a large amount of antibody are retained.

[0016] In the above-described culturing method, the storage section 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 mm3 It is desirable to have a size selected from the range of.

[0017] According to this aspect, the culture area 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 the efficiency of evaluating the amount of antibody production can be improved.

[0018] In the above culture method, it is desirable that the plate includes a large partition portion that divides a relatively large-sized area of the plate, and a small partition portion that further subdivides the inside of the large partition portion, and the small partition portion is the accommodating portion.

[0019] According to this aspect, it becomes possible to utilize, such as changing cell types and culture solutions in units of large partition portions, and it becomes possible to diversify the culture.

[0020] In the above culture method, it is desirable that a medium containing alginate is injected into the plate.

[0021] A medium containing alginate has the property of rapidly gelling, for example, by adding calcium ions or magnesium ions. Therefore, according to the above aspect, the gelling process can be rapidly completed.

[0022] In the above culture method, it includes a step of introducing a gene into the single cell to impart antibody-producing ability, and after culturing the single cell introduced with the gene in a normal medium for a predetermined period, it is desirable to replace the normal medium with a selection medium suitable for culturing the single cell.

[0023] According to this aspect, although the cause has not been identified, single cells having antibody-producing ability can be well proliferated.

Effect of the Invention

[0024] According to the present invention, it is possible to provide a culture method capable of efficiently identifying a cell line that produces a large amount of recombinant protein. [Brief explanation of the drawing]

[0025] [Figure 1] Figure 1 is a diagram showing the process flow of a method for culturing antibody-producing cells according to an embodiment of the present invention. [Figure 2] Figure 2(A) is a plan view with an enlarged view showing the structure of the first plate, and Figure 2(B) is a cross-sectional view taken along the line IIB-IIB in Figure 2(A). [Figure 3] Figure 3 is a schematic cross-sectional view showing the process of seeding cells onto the first plate. [Figure 4] Figure 4 is a schematic cross-sectional view showing the process of gelling the culture medium within the microgrid. [Figure 5] Figure 5 is a schematic cross-sectional view showing the process of primary culture, which gives a single cell an antibody production period. [Figure 6] Figure 6(A) is a schematic cross-sectional view showing the state after adding the liquid culture medium containing the detection antibody to the first plate, and Figure 6(B) is a schematic cross-sectional view showing the state after washing. [Figure 7] Figures 7(A) and (B) are plan views illustrating the process of identifying microgrids containing single cells with high antibody production. [Figure 8] Figure 8(A) is a schematic cross-sectional view showing the process of picking single cells using a suction tip, and Figure 8(B) is a schematic diagram showing the process of transferring the picked single cells to a second plate. [Figure 9] Figure 9 is a schematic cross-sectional view showing the process of extruding a single cell from the aspiration tip to the second plate and retaining the single cell in a microgrid. [Figure 10] Figure 10 is a schematic cross-sectional view showing the process of establishing a microculture environment by aspiration of culture medium. [Figure 11] Figure 11 is a schematic cross-sectional view showing the microgrid openings sealed with a sealing solution. [Figure 12] Figure 12 is an image showing the proliferation status of single cells in secondary culture on the second plate. [Figure 13]Figure 13 shows an image of the proliferation status of single cells using the culture method of the comparative example. [Figure 14] Figure 14 shows an image of the proliferation status of single cells using the culture method of the comparative example. [Modes for carrying out the invention]

[0026] The embodiments of the culture method for recombinant protein-producing cells according to the present invention will be described in detail below with reference to the drawings. The target of culture in the present invention is a single cell that produces recombinant protein. In the embodiments shown below, as an example of recombinant protein-producing cells, single cells such as CHO cells and B cells, which are expected to be used in the manufacture of antibody drugs or to produce antibodies, are given as examples. The culture method shown in this embodiment is, broadly speaking, a method for producing a large amount of antibodies by culturing prepared single cells for a certain period of time, selecting single cells with excellent antibody production ability, and further culturing and proliferating those single cells.

[0027] [Overall flow of the 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 S9 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 the culture medium onto a first plate 1 (Figure 2) equipped with numerous cell containment compartments (step S2). Then, the culture medium is gelled (Figure 4), and a primary culture is performed in which the single cells are cultured in the cell containment compartments for a predetermined number of days (step S3 / Figure 5).

[0028] After the primary culture, a detection antibody that binds to the antibody produced by the single cell is added (Figure 6) to perform a screening to identify high antibody-producing cell lines (Figure 7) (Step S4). The identified high antibody-producing cell lines are picked with a suction tip 23 and transferred to a second plate 5 equipped with multiple cell containment compartments (Step S5 / Figure 8). Then, the single cells of the high antibody-producing cell lines that were picked are dispensed from the suction tip 23 into the second plate 5, which is filled with liquid culture medium (Step S6 / Figure 9).

[0029] Next, liquid culture medium is aspirated from the second plate 5 to establish a microculture environment in which single cells are cultured in individual cell containments, i.e., a culture environment with an extremely small culture area (Step S7 / Figure 10). Furthermore, the top surface of the second plate 5 is sealed with a sealing solution to prevent evaporation of the culture medium (Step S8 / Figure 11). After that, a secondary culture is performed in which single cells are cultured in the cell containments for a predetermined number of days (Step S9 / Figure 12). Steps S1 to S9 described above will be explained in detail below.

[0030] [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 such as transfection, physical methods such as electroporation, or biological methods such as viral vectors. It is desirable to culture the single cell into which the gene has been introduced in a standard culture medium for two days, and then replace the culture medium with a selective medium suitable for the single cell to be cultured. Of course, the single cell may be cultured in the selective medium immediately after gene introduction.

[0031] [Step S2; Cell seeding onto the first plate] In step S2, the single cells prepared in step S1 are seeded onto a culture plate for primary culture. Figure 2(A) is a plan view with an enlarged view showing the structure of a first plate 1 (plate) as an example of the culture plate, and Figure 2(B) is a cross-sectional view taken along line IIB-IIB in Figure 2(A). The first plate 1 includes a grid 11 consisting of matrix-arranged recesses on one side of a flat substrate, and microgrids 12 (container sections) consisting of matrix-arranged minute-sized recesses within each grid 11.

[0032] Grid 11 is a large compartment that divides a relatively large-sized area of the first plate 1. In FIG. 2, a rectangular grid 11 in top view, partitioned by vertical and horizontal grid plates, is illustrated. Instead of this, it may also be structured such that circular well-type grids 11 in top view are arranged in a honeycomb or matrix pattern. The microgrid 12 is a small compartment that further subdivides the inside of each grid 11. The microgrid 12 is a rectangular recess in top view, formed on the bottom plate of the grid 11 and partitioned by side plates that are lower than the grid plates partitioning the grid 11. This microgrid 12 may also be of a circular well type in top view.

[0033] The microgrid 12 serves as a housing for holding a single cell. As an example of the size of the microgrid 12, one side is 200 μm and the depth is 100 μm. The first plate 1 is a plate provided with a large number of housings partitioned into such minute sizes. It is desirable to set the microgrid 12 to a size capable of forming a minute 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, and 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 mm 3 and can be set to a size selected from the range.

[0034] Figure 3 is a schematic cross-sectional view showing the seeding of single cell C onto the first plate 1. For seeding, a cell suspension 2L is prepared, containing a large number of antibody-producing single cell C cells in liquid medium LA, as prepared in step S1. The cell suspension 2L is placed in a dispensing container 21 and injected into each grid 11 of the first plate 1. This injection ensures that at least some of the numerous microgrids 12 within grid 11 retain the liquid medium LA and a single single cell C. Of course, some microgrids 12 may contain multiple single cell C cells, or they may not retain any single cell C cells. For example, if there are 475 microgrids 12 in one grid 11, and 400 single cells are seeded into that grid 11, probabilistically, about 1 / 3 of the microgrids 12 will retain a single single cell C.

[0035] The 2L cell suspension contains liquid culture medium LA and a gel material to immobilize the liquid culture medium LA. As the liquid culture medium LA, a standard culture medium containing inorganic salts, glucose, amino acids, and other growth factors, as well as additives such as antibiotics and growth promoters, may be used. For example, CH150 medium (a product name of G-MEP Corporation) can be suitably used as the liquid culture medium LA. While there are no particular restrictions on the gel material as long as it can immobilize the liquid culture medium LA, it is preferable to use alginate. Liquid culture medium LA containing alginate has the advantage of rapidly gelling when a gelling agent containing, for example, calcium or magnesium ions is added, thus allowing for the rapid completion of the subsequent gelling process.

[0036] [Step S3; Primary culture] Step S3 is a step in which the liquid culture medium LA in the microgrid 12 is gelled, and then a single cell C is cultured in the microgrid 12 for a certain period of time to give the single cell an antibody production period. Figure 4 is an enlarged cross-sectional view of the microgrid 12, and is a schematic diagram showing the step of gelling the liquid culture medium LA in the microgrid 12. Each microgrid 12 is partitioned by a bottom plate 121 and a side plate 122.

[0037] After pouring the liquid medium LA containing alginate into the first plate 1, a gelling agent is added. For example, CaCl2 can be used as the gelling agent. Upon addition of the gelling agent, the liquid medium LA in the microgrid 12 becomes gel medium LB. The single cell C in the microgrid 12 is immobilized by the gel medium LB. It is desirable that the height of the gel medium LB be adjusted to the same height as the top 123 of the side plate 122, or slightly lower. This creates a state in which one single cell C is trapped within the gel medium LB of one microgrid 12. The height of the gel medium LB may be slightly higher than the top 123, or the gel medium LBs of adjacent microgrids 12 may be connected to each other.

[0038] Figure 5 is a schematic cross-sectional view showing the process of primary culture to give single cells an antibody production period. After adding a gelling agent and letting it stand for a certain period of time (e.g., 30 minutes) to convert the liquid medium LA to gel medium LB, liquid medium LA is poured into the first plate 1. In other words, the top of the microgrid 12 equipped with gel medium LB is covered with liquid medium LA. In this state, by primary culturing single cells C for a predetermined antibody production period, cell proliferation and antibody production can be performed on each microgrid 12. A camera 13 is positioned on the top surface of the first plate 1. During the primary culture period, the microgrid 12 on the first plate 1 is imaged by the camera 13 and its condition is monitored. The primary culture period is, for example, about 4 to 8 days.

[0039] Figure 6(A) schematically shows the state several days after the initial culture. Here, it shows the state in which a single cell C in the microgrid 12 has produced antibody 3. During the initial culture period, the liquid medium LA is replaced. The replacement liquid medium LA contains detection antibody 4 that can bind to antibody 3 produced by single cell C in gel medium LB. Figure 6(A) shows the state in which liquid medium LA containing detection antibody 4 has been added to the first plate 1, and some of the detection antibody 4 has bound to antibody 3.

[0040] The liquid culture medium LA is replaced by a washing method in which the liquid culture medium LA already injected into the first plate 1 is aspirated, and a new liquid culture medium LA containing the detection antibody 4 is injected. Figure 6(B) is a schematic cross-sectional view showing the washing process. First, the initial liquid culture medium LA shown in Figure 5 is aspirated using an aspiration tip (not shown). Then, the replacement liquid culture medium LA containing the detection antibody 4 is prepared. The replacement liquid culture medium LA is held in the culture medium supply tip 22 and injected onto the gel culture medium LB on the microgrid 12 as shown in Figure 6(B). This type of washing is performed approximately 1 to 3 times during the primary culture period.

[0041] The dilution ratio of detection antibody 4 in the replacement liquid medium LA should be selected to avoid excessive amounts of antibody 3 that cannot bind to it. If detection antibody 4 is present in excess, a large amount of detection antibody 4 will float in the liquid medium LA on top of the gel medium LB, making it difficult to accurately evaluate the amount of antibody 3 produced by individual single cells C.

[0042] [Step S4; Screening of highly antibody-producing cell lines] Step S4 is a step in which a trigger is provided to cause a reaction of the detection antibody 4 added in step S3, and a microgrid 12 containing single cells C that produce a large amount of antibody is identified. In this embodiment, the first plate 1 is irradiated with light as the trigger, and an example is shown in which the detection antibody 4 is made to fluoresce. In a microgrid 12 containing single cells C that produce a large amount of antibody 3, there will be a large amount of detection antibody 4 that binds to those antibodies 3, and the degree of fluorescence will increase. Therefore, by simply evaluating the degree of fluorescence of the detection antibody 4 in units of microgrid 12, cell lines that produce a large amount of antibody can be identified.

[0043] Figures 7(A) and (B) are plan views illustrating the process of identifying microgrids 12 that retain single cells C with high antibody production. Figure 7(A) shows the retention status of single cells C in microgrids 12 during the initial stage of primary culture, which is the antibody production period. The matrix-arranged microgrids 12 are assigned n x m column addresses. Of the n x m microgrids 12, grid G1 (n1m4), grid G2 (n2m1), grid G3 (n2m3), grid G4 (n3m3), and grid G5 (n5m4) each retain one single cell C. Grid G6 (n4m1) retains multiple (2) single cells C from the beginning of primary culture.

[0044] Figure 7(B) shows the state after the completion of the first culture. Except for grid G4, single cell C has proliferated. Figure 7(B) also shows the state when light of a predetermined wavelength is irradiated onto the first plate 1 from a light source (not shown) and fluorescence FL is generated from the detection antibody 4. Grids G3 and G5 generate fluorescence FL with an emission area exceeding the aperture area of ​​the microgrid 12, indicating that single cell C with high antibody production is being cultured. On the other hand, for grid G1, although single cell C has proliferated to 4 cells, only fluorescence FL with an emission area small compared to the aperture area of ​​the microgrid 12 is generated. Fluorescence FL in grids G2 and G4 is also small.

[0045] The amount of antibody production is evaluated by the degree of fluorescence (FL) generation in each microgrid 12. Examples of evaluation indicators include (1) the fluorescence FL generation range, (2) the average fluorescence FL brightness, and (3) the maximum fluorescence FL brightness. The fluorescence FL generation range is the ratio of the emission area of ​​the fluorescence FL generated from a microgrid 12 to the aperture area of ​​that microgrid 12. In the example in Figure 7(B), the fluorescence FLs in grids G3 and G5 have emission areas exceeding the aperture area of ​​their respective microgrids 12, and are evaluated as having high antibody production. On the other hand, the fluorescence FL emission areas of grids G1, G2, and G4 are narrow, and their antibody production is evaluated as low. Based on this fluorescence FL generation range, the average brightness and maximum brightness are further considered. Grids with higher average brightness and maximum brightness are evaluated as having high antibody production. Here, grids G3 and G5 can be identified as "microgrids 12 that retain high antibody-producing cell lines."

[0046] Furthermore, grid G6, which retained multiple single cells C from the beginning of the antibody production period, is excluded from the aforementioned specific targets. In over-retention grids where multiple single cells C are retained in a single microgrid 12, there is a possibility that highly antibody-producing cell lines and non-producing or low-antibody-producing cell lines are mixed together. In this case, the latter may be picked in the later step S5. For this reason, the first plate 1 is monitored with camera 13 (Figure 5) from the beginning of the primary culture, and over-retention grids such as grid G6 are excluded from the evaluation of antibody production, even if they produce a high degree of fluorescence FL. This makes it possible to accurately identify microgrids 12 that retain single cells C with high antibody production.

[0047] [Process S5; Picking of highly antibody-producing cell lines] In step S5, single cells C contained in the microgrid 12, which was identified in the previous step S4 as a grid with high antibody production, are picked using the aspiration tip 23 and transferred to the second plate 5. In other words, picking is performed in units of 12 microgrids. Figure 8(A) is a schematic cross-sectional view showing the situation of picking single cells C with the aspiration tip 23, and Figure 8(B) is a schematic diagram showing the process of transferring the picked single cells C to the second plate 5.

[0048] The suction tip 23 is equipped with a tip opening 23T at its lower end for aspirating and discharging single cells C. As shown in Figure 8(A), the tip opening 23T of the suction tip 23 is aligned in the XY direction with respect to the single cell C, which is the aspiration target, and the suction tip 23 is lowered so that the tip opening 23T enters the microgrid 12. Subsequently, by generating negative pressure at the tip opening 23T, the single cell C, which is the aspiration target, is aspirated into the suction tip 23 along with the gel medium LB. The XY coordinates of the single cell C can be determined based on the image of the first plate 1 captured by the camera 13 (Figure 5). With this suction using the suction tip 23, all single cells C present in one microgrid 12 identified as having a high antibody production rate may be aspirated in one turn, or only a portion of them may be aspirated.

[0049] As shown in Figure 8(B), the suction tip 23 is attached to the head 61 of the head unit 6. The head unit 6 is a unit that can move horizontally (XY direction) along a guide rail (not shown). The head 61 is mounted on the body of the head unit 6 so as to be able to move up and down, and has a lower end to which the suction tip 23 is attached. Inside the body of the head unit 6, a mechanism for generating negative and positive pressure is built into the lower end of the head 61.

[0050] The XY movement of the head unit 6 aligns the tip opening 23T of the suction tip 23 with the single cell C that is the suction target. The advancement and retraction of the tip opening 23T relative to the microgrid 12 holding the single cell C is achieved by raising and lowering the head 61. The suction or discharge of the single cell C from the tip opening 23T is achieved by generating negative or positive pressure at the lower end of the head 61. Once the single cell C of the suction target has been aspirated in the first plate 1 using the suction tip 23 attached to each head 61, the head unit 6 is moved above the second plate 5 (another container) where the secondary culture is performed. As a device that automatically performs cell picking and cell transfer as described above, for example, a CELL HANDLER (product name of Yamaha Motor Co., Ltd.) can be suitably used. Alternatively, the cell picking and cell transfer may be performed manually by an operator using a cell aspiration / discharge tool such as a micropipette.

[0051] [Step S6; Cell dispensing to the second plate] Step S6 is a step of holding the single cells C picked with the suction tip 23 in step S5 into a predetermined containment section of the second plate 5. Figure 9 is a schematic cross-sectional view showing the step of holding the single cells C into the second plate 5 by the dispensing in step S6. Step S6 includes the steps of pouring a predetermined amount of liquid culture medium into the second plate 5 in advance and dispensing the single cells C picked in the first plate 1 into the second plate 5.

[0052] The second plate 5 is a container having multiple containment compartments with openings on its upper surface. As the second plate 5, a plate having the same structure as the first plate 1 described earlier with reference to Figure 2 can be used. Figure 9 illustrates the second plate 5, which has a grid 51 that divides a relatively large area of ​​the second plate 5, and microgrids 52 that further subdivide the inside of the grid 51. Each microgrid 52 is the containment compartment that holds a single cell C. Figure 9 shows a cross-sectional view of one grid 51. The 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 individual sides.

[0053] The microgrid 52, which serves as the containment area for holding single cells C, should preferably have a similar size to the microgrid 12 of the first plate 1. That is, the microgrid 52 should preferably be sized to form a small culture space, for example, with an opening area of ​​4.0 × 10⁻⁶. -2 1.0 × 10 -1 mm 2 , volume is 4.0 × 10 -3 ~1.0×10 -2 mm 3 A size selected from the range, more preferably with an opening area of ​​1.0 × 10 -3 ~1.0×10 -1 mm 2 , volume is 2.0 × 10 -5 ~1.0×10 -2 mm 3 It can be set to a size selected from a range.

[0054] Prior to the dispensing of single cells C, a predetermined amount of liquid medium LA is added to the second plate 5. As shown in Figure 9, 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 second plate 5. Note that the top 522 of each microgrid 52 is at the same height.

[0055] Subsequently, a single cell C from one microgrid 12, selected as a high antibody-producing cell line, is dispensed from the aspiration tip 23 onto the second plate 5. This dispensing is achieved by facing the tip opening 23T of the aspiration tip 23 against the opening of the grid 51, as shown in Figure 9, and generating positive dispensing pressure at the tip opening 23T. Of course, the dispensing may also be performed by inserting the tip opening 23T into the upper layer of the liquid culture medium LA within the grid 51, or by inserting the tip opening 23T into the interior of the microgrid 52. As a result of this dispensing, one or more single cells C are held in at least some of the microgrids 52 of the multiple microgrids 52 provided on one grid 51.

[0056] [Step S7: Establishment of a micro-culture environment by aspiration of culture medium] Step S7 is a process in which the liquid culture medium LA from the second plate 5 is aspirated to establish an independent single-cell culture environment for 52 microgrid units. The culture environment established here is a culture environment with an extremely small culture area. Figure 10 is a schematic cross-sectional view showing the operation of step S7. In Figure 10, the liquid culture medium LA in the grid 51 is shown being aspirated by the aspirator tip 24.

[0057] The suction by the suction tip 24 is performed from the state shown in Figure 9 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 culture environment is formed in which the culture area for a single cell C is extremely small, consisting of the liquid medium LA in each microgrid 52. The amount of liquid medium LA in one microgrid 52 is, for example, 4 nanoliters.

[0058] 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 10) 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 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 dispensing step S6, it is advantageous if the liquid level of the liquid culture medium LA is above the opening 52H, as this allows the single cells C to be retained in the microgrid 52 with a single dispensing operation. By performing culture medium aspiration in the subsequent step S7, a culture environment suitable for culturing and proliferating a small number of single cells C, isolated in microgrid 52 units, can be established.

[0059] [Process S8; Microgrid sealing] Step S8 is the step of pouring the sealing solution 7 into the second plate 5 to seal the upper part of the opening 52H of the microgrid 52. Figure 11 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.

[0060] 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 second culture period in step S9, 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 second culture period can be suppressed.

[0061] 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.

[0062] The formation of the sealing solution layer 7 allows the microculture environment established in step S7 to be maintained during the second 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.

[0063] [Step S9; Secondary culture] Step S9 is a process in which single cell C is cultured for a predetermined culture period while the opening 52H of the microgrid 52 is sealed with sealing solution 7 as shown in Figure 11. In other words, this is a process in which single cell C, which was identified as a high antibody-producing cell line in step S4, is further cultured and grown for a predetermined period to produce a large amount of antibody 3. During the secondary culture period, liquid culture medium containing growth factors is replenished in grid 51.

[0064] Figure 12 is an image showing the proliferation status of single cells C in the second culture on plate 5. "Day 1" in the figure refers to the state on day 1 from the start of the second culture. Figure 12 shows images of a portion of the microgrid 52 on one grid 51 on days 1, 4, 5, 6, 8, 11, and 18 from the start of the second 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 proliferation increases 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 medium was replenished.

[0065] Figures 13 and 14 show images of the proliferation of single cells using the culture method of the comparative example. Figure 13 is an image of Comparative Example 1, in which the culture medium aspiration in step S7 (Figure 10) was not performed, and the sealing with embryo culture oil in step S8 (Figure 11) was not performed, meaning that secondary culture was performed immediately after cell dispensing in step S6 (Figure 9). Figure 13 shows images from day 1, day 6, and day 11 from the start of secondary 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.

[0066] Figure 14 shows images of Comparative Example 2, in which the culture medium aspiration in step S7 (Figure 10) was omitted, and the embryo culture oil sealing in step S8 was performed, meaning that after cell dispensing in step S6 (Figure 9), the sealing solution 7 was poured in when the liquid level of the liquid medium LA was higher than the top 522, and secondary culture was performed. In Figure 14, images from day 1, day 6, and day 11 from the start of secondary culture are also shown. 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.

[0067] [Effects and Effects] The antibody-producing cell culture method according to this embodiment, as described above, produces the following effects. First, in the primary culture before picking in step S5, a single cell C is held in one microgrid 12 of the first plate 1, and then the liquid culture medium LA in the microgrid 12 gels. The gelling of the liquid culture medium LA creates a state in which one single cell C is confined in one microgrid 12. In this state, by primary culturing the single cell C for a predetermined antibody production period, cell proliferation and antibody 3 production can be carried out in each microgrid 12. Then, by adding the detection antibody 4 to the first plate 1, the amount of antibody produced can be evaluated in units of microgrids 12. Since the suspension of single cell C is suppressed by the gelling of the culture medium, the microgrid 12 in which a large amount of detection antibody 4 is detected will contain a cell line with high antibody production. In this way, since it is possible to culture single cell C, produce antibody 3, and detect the amount of antibody produced in units of microgrids 12, cell lines with high antibody production can be efficiently identified.

[0068] Next, in the secondary culture after picking in step S5, single cells C are held in the microgrid 52 of the second plate 5, and then the liquid medium LA is removed and the opening 52H is sealed with sealing solution 7. This prevents evaporation of the liquid medium LA within the microgrid 52 and creates an extremely 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 picking single cells C with excellent antibody production ability and performing secondary culture as described above, the proliferation efficiency of single cells C can be improved, and consequently, a large amount of antibody 3 can be produced. [Explanation of Symbols]

[0069] C single cell LA liquid culture medium LB gel medium 1. First plate (plate) 11 Grid (Large Section) 12. Microgrid (Housing section / Small compartment section) 2L cell suspension 23 Suction Tips 3 Antibodies 4. Antibodies for detection 5. Second plate (other container)

Claims

1. A plate having numerous compartments divided into minute sections is infused with a cell suspension containing numerous antibody-producing single cells in a culture medium, thereby holding the culture medium and a single cell in each of at least some of the compartments. A step of gelling the culture medium in the aforementioned containment section, A step of giving the single cell in the containment section an antibody production period, The process involves adding a liquid containing a detection antibody capable of binding to the antibody produced by the single cell in the gelled culture medium to the plate, A step of providing a trigger to cause the aforementioned detection antibody to react, and identifying a containment section that holds a single cell with a high antibody production rate, The process includes picking a single cell held in the identified containment unit and transferring it to another container, The plate is monitored during the antibody production period, and any containment section in which multiple single cells were held from the beginning of the production period is excluded from the specific target. A method for culturing recombinant protein-producing cells.

2. In the method for culturing recombinant protein-producing cells according to Claim 1, The aforementioned storage section has an opening area of ​​1.0 × 10 －３ ~1.0 x 10 －１ mm ２ , volume is 2.0 × 10 －５ ~1.0 x 10 －２ mm ３ A method for culturing recombinant protein-producing cells having a size selected from a range.

3. In the method for culturing recombinant protein-producing cells according to Claim 2, The plate includes a large compartment that divides a relatively large area of ​​the plate, and a small compartment that further subdivides the inside of the large compartment. A method for culturing recombinant protein-producing cells, wherein the small compartment is the containment section.

4. In the method for culturing recombinant protein-producing cells according to Claim 1, A method for culturing recombinant protein-producing cells, wherein a culture medium containing alginate is injected into the plate.

5. In the method for culturing recombinant protein-producing cells according to Claim 1, The process includes introducing a gene into the aforementioned single cell to confer antibody production ability, A method for culturing recombinant protein-producing cells, comprising culturing a single cell into which the aforementioned gene has been introduced in a conventional medium for a predetermined period of time, and then replacing the conventional medium with a selective medium suitable for culturing the single cell.

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