Method for culturing cells for producing recombinant proteins

Abstract

A method for culturing cells capable of producing recombinant proteins, comprising: a step for injecting a cell suspension, which contains a plurality of single cells capable of producing recombinant proteins in a culture medium, into a plate provided with a plurality of housing parts divided into small sizes, and holding the culture medium and one single cell in each of at least some of the plurality of housing parts; a step for gelling the culture medium in the accommodating part; a step for imparting a recombinant protein production period to the single cells in the accommodation unit; and a step for adding, to the plate, a liquid containing a detection protein capable of binding to a recombinant protein produced by the single cell in the gelled culture medium.

Description

Technical Field

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

[0002] When developing antibody drugs that have the effect of specifically binding to antigens of foreign substances such as virus-infected cells and cancer cells and removing the above foreign substances, the culture of cells that can produce antibodies is indispensable. As a method for culturing cells, there are various existing technologies. For example, in Patent Document 1, although it is a culture technique for spheroids, a culture 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. In Patent Document 2, a screening method is disclosed 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. However, in the method of inoculating cells in a general well, culturing them, and obtaining the fluorescence amount, it is sometimes difficult to perform screening efficiently.

[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 method for culturing cells that produce recombinant proteins, which can efficiently identify cell lines that produce a large amount of recombinant proteins.

[0009] A method for culturing cells that produce recombinant proteins according to one aspect of the present invention includes: injecting a cell suspension in which a plurality of single cells capable of producing recombinant proteins are contained in a medium into a plate having a plurality of accommodation parts divided into minute sizes, and causing at least some of the plurality of accommodation parts to each hold a medium and a single cell; a step of gelifying the medium in the accommodation part; a step of giving a recombinant protein production period to the single cell in the accommodation part; and a step of adding a liquid containing a detection protein capable of binding to the recombinant protein produced by the single cell in the gelified medium to the plate. Brief Description of the Drawings

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

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

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

[0013] Figure 3 is a schematic cross-sectional view showing the state of inoculating cells onto the first plate.

[0014] Figure 4 is a schematic cross-sectional view showing the step of gelifying the culture medium within the microgrid.

[0015] Figure 5 is a schematic cross-sectional view showing the step of subjecting a single cell to the first culture during antibody production.

[0016] Figure 6A is a schematic cross-sectional view showing the state of adding a liquid culture medium containing an antibody for detection to the first plate.

[0017] Figure 6B is a schematic cross-sectional view showing the state of washing.

[0018] Figure 7A is a top view showing the step of determining the microgrid that holds a single cell with a high antibody production amount.

[0019] Figure 7B is a top view showing the step of determining the microgrid that holds a single cell with a high antibody production amount.

[0020] Figure 8A is a schematic cross-sectional view showing the state of picking up a single cell using a suction tip.

[0021] Figure 8B is a schematic view showing the step of transferring the picked-up single cell to the second plate.

[0022] Figure 9 is a schematic cross-sectional view showing the step of ejecting a single cell from the suction tip onto the second plate and holding the single cell in the microgrid.

[0023] Figure 10 is a schematic cross-sectional view showing the step of establishing a minimal culture environment by attracting the culture medium.

[0024] Figure 11 is a schematic cross-sectional view showing the state where the opening of the microgrid is sealed with a sealing liquid.

[0025] Figure 12 is an image showing the proliferation state of a single cell during the second culture in the second plate.

[0026] Figure 13 is an image showing the proliferation state of a single cell using the culture method of the comparative example.

[0027] Figure 14 These are images of the proliferation status of single cells using the culture method of the comparative example. Detailed implementation mode

[0028] Hereinafter, based on the accompanying drawings, the implementation modes of the method for culturing cells that produce recombinant proteins of the present invention will be described in detail. In the present invention, the object to be cultured is a single cell that produces recombinant proteins. In the implementation modes shown below, as an example of the cells that produce recombinant proteins, single cells (Single Cell) such as CHO cells and B cells that can be expected to be used in the manufacture of antibody drugs and antibody production are exemplified. The culture method shown in this implementation mode is generally as follows: after culturing the prepared single cells for a certain period, single cells with excellent antibody production ability are screened, and the single cells are further cultured to proliferate, thereby producing a large amount of antibodies.

[0029] [Overall process of the culture step]

[0030] First, with reference to Figure 1 The overall process of the culture method of the antibody-producing cells in this implementation mode is described according to the shown step flow. The culture method of this implementation mode includes steps S1 to S9 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 onto a first plate 1 having a plurality of cell accommodation parts together with a culture medium ( Figure 2A 、 Figure 2B )(step S2). Then, after gelling the culture medium ( Figure 4 ), the first culture of culturing the single cells in the above cell accommodation part for a specified number of days is carried out (step S3 / Figure 5 ).

[0031] After the first culture, a detection antibody that binds to the antibody produced by the above single cells is added ( Figure 6A ), and screening for determining high antibody-producing cell lines is carried out ( Figure 7A 、 Figure 7B )(step S4). The determined high antibody-producing cell lines are picked up using a pipette tip 23 and transferred to a second plate 5 having a plurality of cell accommodation parts (step S5 / Figure 8A 、 Figure 8B ). Then, the picked single cells as high antibody-producing cell lines are ejected from the pipette tip 23 into the second plate 5 filled with a liquid culture medium (step S6 / Figure 9 ).

[0032] Next, the liquid culture medium is aspirated from the second plate 5 to establish a minimal culture environment for culturing single cells in each cell accommodation part, that is, a culture environment with a minimal culture area (step S7 / Figure 10)。Furthermore, the top surface of the second plate 5 is sealed with a sealing liquid for preventing evaporation of the culture medium (step S8 / Figure 11 )。Then, a second culture is performed in the above cell accommodation part for a specified number of days for culturing single cells (step S9 / Figure 12 )。Hereinafter, steps S1 to S9 described above will be described in detail respectively.

[0033] [Step S1: Production of antibody-producing single cells]

[0034] In step S1, for example, by introducing a specified gene into a single cell to be cultured, the single cell is given the ability to produce antibodies. As the single cell, a B cell of an immune cell can be exemplified, and as the produced antibody, a monoclonal antibody produced by a single type of B cell can be exemplified. The introduction of the gene can be carried out, for example, by chemical methods such as transfection, physical methods such as electroporation, or biological methods such as viral vectors. For the single cell into which the gene has been introduced, a method of culturing in a normal culture medium for 2 days and then replacing the above culture medium with a selection medium suitable for the single cell to be cultured is preferably adopted. Of course, the single cell can also be cultured with a selection medium immediately after gene introduction.

[0035] [Step S2: Seeding cells onto the first plate]

[0036] In step S2, in order to perform the first culture, the single cells produced in step S1 are seeded onto a culture plate. Figure 2A is a top view with an enlarged view showing the structure of the first plate 1 (plate) as an example of the above culture plate, Figure 2B is Figure 2A a sectional view taken along line IIB-IIB. The first plate 1 includes a grid 11 composed of recesses arranged in a matrix on a single surface of a flat substrate and a micro-grid 12 (accommodation part) composed of micro-sized recesses arranged in a matrix within each grid 11.

[0037] The grid 11 is a large partition part that divides a relatively large area of the first plate 1. In FIG. 2, a grid 11 that is rectangular in a top view divided by horizontal and vertical grid plates is exemplified. Instead, a structure in which circular hole-shaped grids 11 are arranged in a honeycomb shape or a matrix shape can also be adopted. The micro-grid 12 is a small partition part that further subdivides the inside of each grid 11. The micro-grid 12 is a recess formed on the bottom plate of the grid 11, divided by side plates lower than the grid plates that divide the grid 11, and rectangular in a top view. The micro-grid 12 can also be a circular hole type in a top view.

[0038] The microgrid 12 serves as a housing section for holding single cells. 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 having a plurality of housing sections divided into such minute sizes. The microgrid 12 is preferably set to a size capable of forming a minute culture space. For example, it can be set to a size within the range where the opening area is selected from 4.0×10 -2 ~1.0×10 -1 mm 2 and the volume is selected from 4.0×10 -3 ~1.0×10 -2 mm 3 More preferably, it can be set to a size within the range where the opening area is selected from 1.0×10 -3 ~1.0×10 -1 mm 2 and the volume is selected from 2.0×10 -5 ~1.0×10 -2 mm 3 .

[0039] Figure 3 is a schematic cross-sectional view showing the state of inoculating single cell C into the first plate 1. At the time of inoculation, a cell suspension 2L prepared in step S1 and containing a plurality of single cells C capable of producing antibodies in the liquid medium LA is prepared. The cell suspension 2L is housed in a dispensing container 21, and the liquid is injected into each grid 11 of the first plate 1. By this injection, the liquid medium LA and one single cell C are held in at least a part of the plurality of microgrids 12 within the grid 11. Of course, there are also microgrids 12 into which a plurality of single cells C enter or in which single cells C are not held. For example, when there are 475 microgrids 12 in one grid 11 and 400 single cells are inoculated in this grid 11, in terms of probability theory, about 1 / 3 of the microgrids 12 become grids holding one single cell C.

[0040] In the cell suspension 2L, in addition to the liquid medium LA, a gel material for immobilizing the liquid medium LA is also incorporated. As the liquid medium LA, a usual culture solution containing inorganic salts, glucose, amino acids and other growth factors, antibiotics, growth promoting factors and other additive components in an aqueous medium can be used. For example, the CH150 medium (trade name of Gimep Co., Ltd.) can be preferably used as the liquid medium LA. As the gel material, there is no particular limitation as long as it can immobilize the liquid medium LA, and alginate is preferably used. The liquid medium LA containing alginate has the property of gelling rapidly by adding a gelling agent containing, for example, calcium ions and magnesium ions, and thus has the advantage of being able to rapidly complete the subsequent gelling step.

[0041] [Step S3: First culture]

[0042] Step S3 is a step of culturing a single cell C in the microgrid 12 for a certain period after gelifying the liquid medium LA in the microgrid 12 and endowing the single cell with an antibody production period. Figure 4 It is an enlarged cross-sectional view of the microgrid 12 and is a schematic diagram showing the step of gelifying the liquid medium LA in the microgrid 12. Each microgrid 12 is demarcated by a bottom plate 121 and a side plate 122.

[0043] After injecting the liquid medium LA containing alginate into the first plate 1, a gelling agent is added. As the gelling agent, for example, CaCl2 can be used. By adding the gelling agent, the liquid medium LA in the microgrid 12 becomes a gel medium LB. The single cell C in the microgrid 12 is immobilized by the gel medium LB. The height of the gel medium LB is preferably adjusted to be the same as the top 123 of the side plate 122 or a slightly lower height. Thereby, a state in which a single cell C is encapsulated in the gel medium LB of one microgrid 12 can be formed. It should be noted that the height of the gel medium LB can be located at a position slightly higher than the top 123, and the gel medium LBs of adjacent microgrids 12 can be connected to each other.

[0044] Figure 5 It is a schematic cross-sectional view showing the step of the first culture for endowing the single cell with an antibody production period. After adding the gelling agent and allowing it to stand for a certain time (for example, 30 minutes) to convert the liquid medium LA into the gel medium LB, the liquid medium LA is injected into the first plate 1. That is, the upper part of the microgrid 12 having the gel medium LB is covered with the liquid medium LA. In this state, the single cell C is cultured for the first time with a specified antibody production period, whereby cell proliferation and antibody production of the single cell C can be carried out in each microgrid 12. A camera 13 is arranged on the top surface of the first plate 1. During the first culture period, the microgrids 12 of the first plate 1 are photographed by the camera 13 for status monitoring. The first culture period is, for example, about 4 to 8 days.

[0045] Figure 6A It is a diagram schematically showing the state several days after the first culture. Here, a state in which the single cell C in the microgrid 12 has produced antibody 3 is shown. During the first culture period, a replacement operation of the liquid medium LA is carried out. The replacement liquid medium LA contains a detection antibody 4 that can bind to the antibody 3 produced by the single cell C in the gel medium LB. Figure 6A It shows the state in which the liquid medium LA containing the detection antibody 4 is added to the first plate 1 and a part of the detection antibody 4 binds to the antibody 3.

[0046] The replacement of the liquid medium LA is carried out in the following washing manner: The liquid medium LA injected into the first plate 1 is aspirated, and a liquid medium LA added with the detection antibody 4 is newly injected. Figure 6B It is a schematic cross-sectional view showing the above washing condition. First, Figure 5 The initial liquid medium LA shown is aspirated by a suction head (not shown). Then, a replacement liquid medium LA added with the detection antibody 4 is prepared. The replacement liquid medium LA is held by the medium supply pipette tip 22 and, as Figure 6B shown, is injected onto the gel medium LB of the microgrid 12. Such washing is carried out about 1 to 3 times during the first culture.

[0047] The dilution rate of the detection antibody 4 in the replacement liquid medium LA is preferably selected within a range where an excessive amount that cannot bind to the antibody 3 is not excessively generated. If the detection antibody 4 is contained in excess, a large amount of the detection antibody 4 floats in the liquid medium LA on the upper layer of the gel medium LB, and sometimes it is impossible to accurately evaluate the production amount of the antibody 3 of each single cell C.

[0048] [Step S4: Screening of high antibody-producing cell lines]

[0049] Step S4 is a step of applying a trigger operation for reacting the detection antibody 4 added in Step S3 and determining the microgrid 12 that holds the single cell C with a large antibody production amount. In the present embodiment, an example is shown in which light is irradiated on the first plate 1 as the above trigger operation to cause the detection antibody 4 to fluoresce. In the microgrid 12 that holds the single cell C with a large production amount of the antibody 3, more of the detection antibody 4 that binds to these antibodies 3 also exists, and the fluorescence intensity becomes larger. Therefore, the cell line with a large antibody production amount can be determined only by evaluating the fluorescence intensity of the detection antibody 4 in units of the microgrid 12.

[0050] Figure 7A and Figure 7B is a top view showing the step of determining the microgrid 12 that holds the single cell C with a large antibody production amount. Figure 7A It shows the holding state of the single cell C in the microgrid 12 at the initial stage of the first culture as the antibody production period. Addresses of n rows and m columns are assigned to the microgrids 12 arranged in a matrix. A single cell C is held in each of the grids G1 at n1m4, G2 at n2m1, G3 at n2m3, G4 at n3m3, and G5 at n5m4 among the n-row and m-column microgrids 12. In the grid G6 at n4m1, multiple (two) single cells C have been held since the beginning of the first culture.

[0051] Figure 7B It shows the state after the end of the first culture. Except for the grid G4, the single cell C has proliferated. In addition,Figure 7B It shows a state in which light of a specified wavelength is irradiated onto the first plate 1 from a light source (not shown in the figure), and fluorescence FL is generated from the detection antibody 4. In grids G3 and G5, fluorescence FL with a luminescence area exceeding the opening area of the micro-grid 12 is generated, and it can be seen that single cells C with a large antibody production amount are cultured. On the other hand, regarding grid G1, although the single cell C proliferates into 4 cells, only fluorescence FL with a luminescence area smaller than the opening area of the micro-grid 12 is generated. The fluorescence FL of grids G2 and G4 is also small.

[0052] The amount of antibody production is evaluated based on the degree of generation of fluorescence FL in each micro-grid 12. As evaluation indexes, for example, (1) the generation range of fluorescence FL, (2) the average brightness of fluorescence FL, and (3) the maximum brightness of fluorescence FL can be exemplified. The generation range of fluorescence FL is the ratio of the luminescence area of the fluorescence FL generated from the micro-grid 12 to the opening area of the micro-grid 12. In Figure 7B the example of, the fluorescence FL of grids G3 and G5 has a luminescence area exceeding the opening area of each micro-grid 12, and is evaluated as a high antibody production amount. On the other hand, the luminescence areas of the fluorescence FL of grids G1, G2, and G4 are narrow, and their antibody production amounts are evaluated as low. Based on such a generation range of fluorescence FL, the average brightness and the maximum brightness are further considered. The antibody production amount of grids with higher average brightness and maximum brightness is highly evaluated. Here, grids G3 and G5 can be determined as "micro-grids 12 holding high antibody-producing cell lines".

[0053] It should be noted that grid G6, which has held multiple single cells C from the beginning of the antibody production period, is excluded from the above-mentioned determined objects. In an overholding grid in which multiple single cells C are held in one micro-grid 12, there may be a mixed presence of high antibody-producing cell lines and non-producing or low antibody-producing cell lines. In this case, it may occur that the latter is picked in the subsequent step S5. Therefore, from the beginning of the first culture, the first plate 1 is monitored by the camera 13 ( Figure 5 ), and even if fluorescence FL with a high generation degree is generated, overholding grids such as grid G6 are excluded from the evaluation objects of the antibody production amount. Thereby, it is possible to accurately determine the micro-grid 12 holding single cells C with a large antibody production amount.

[0054] [Step S5: Picking of high antibody-producing cell lines]

[0055] In step S5, the single cell C accommodated in the micro-grid 12 of the grid determined to have a high antibody production amount in the previous step S4 is picked by the suction head 23 and moved to the second plate 5. That is, the picking is performed in units of micro-grids 12. Figure 8A is a schematic cross-sectional view showing the state of picking the single cell C by the suction head 23,Figure 8B It is a schematic diagram showing the step of transferring the picked single cell C to the second plate 5.

[0056] The suction head 23 has a front end opening 23T at its lower end for sucking and discharging the single cell C. As Figure 8A shown, the front end opening 23T of the suction head 23 is aligned with the single cell C as the suction target in the XY direction, and the suction head 23 descends so that the front end opening 23T enters the microgrid 12. Then, by generating a negative pressure at the front end opening 23T, the single cell C as the suction target and the gel culture medium LB are sucked into the suction head 23. The XY coordinates of the single cell C can be obtained from the captured image of the first plate 1 using the camera 13 ( Figure 5 ). In the suction using this suction head 23, all the single cells C existing in one microgrid 12 determined to have a high antibody production amount can be sucked by a single suction operation, or a part of them can be sucked.

[0057] As Figure 8B shown, the suction head 23 is mounted on the head 61 of the head unit 6. The head unit 6 is a unit capable of moving in the horizontal direction (XY direction) along a guide rail (not shown). The head 61 is vertically movably mounted on the main body of the head unit 6 and has a lower end portion for mounting the suction head 23. Inside the main body of the head unit 6, a mechanism for generating negative pressure and positive pressure is built in at the above-mentioned lower end portion of the head 61.

[0058] By the XY movement of the head unit 6, the front end opening 23T of the suction head 23 is aligned with the single cell C as the suction target. The advance and retreat of the front end opening 23T relative to the microgrid 12 holding the single cell C as the suction target are achieved by the lifting of the head 61. The suction or discharge of the single cell C from the front end opening 23T is achieved by generating negative pressure or positive pressure at the above-mentioned lower end portion of the head 61. In the first plate 1, if the single cell C as the suction target is sucked by the suction heads 23 mounted on the respective heads 61, the head unit 6 moves above the second plate 5 (other container) for the second culture. As a device for automatically performing cell picking and cell movement as described above, for example, CELL HANDLER (trade name of Yamaha Motor Co., Ltd.) can be preferably used. Instead of such an automated device, it can also be set as follows: an operator uses a cell suction / discharge instrument such as a micropipette and performs cell picking and cell movement by manual operation.

[0059] [Step S6: Discharge of cells to the second plate]

[0060] Step S6 is a step of holding the single cell C picked by the suction head 23 in a predetermined housing portion of the second plate 5. Figure 9It is a schematic cross-sectional view showing the step of retaining the single cell C on the second plate 5 by the above-described ejection in step S6. Step S6 includes: a step of previously injecting a prescribed amount of liquid culture medium into the second plate 5; and a step of ejecting the single cell C picked up in the first plate 1 onto the second plate 5.

[0061] The second plate 5 is a container having a plurality of accommodating portions with openings on the top surface. As the second plate 5, a plate having the same structure as the first plate 1 described above with reference to FIG. 2 can be used. In Figure 9 FIG., a second plate 5 is illustrated which has a grid 51 that divides a relatively large-sized region of the second plate 5 and a micro-grid 52 that further divides the inside of the grid 51. Each micro-grid 52 is the above-described accommodating portion for retaining the single cell C. Figure 9 Shown is a cross-sectional view of one grid 51. 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 the shared grid bottom plate 511 and side plates 521 forming the respective side surfaces.

[0062] The micro-grid 52 that becomes the accommodating portion for retaining the single cell C preferably has the same size as the micro-grid 12 of the first plate 1. That is, the micro-grid 52 is preferably set to a size capable of forming a minute culture space. For example, it can be set to a size in which 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 in which 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

[0063] Before ejecting the single cell C, a prescribed amount of liquid culture medium LA is put into the second plate 5. As Figure 9 shown, the amount of the liquid culture medium LA put in is an amount such that the liquid level reaches a position above the top 522 of the side plate 521 that divides the micro-grid 52. In other words, a prescribed amount of the liquid culture medium LA in which the liquid level is located above the top surface opening of the micro-grid 52 is previously injected into the grid 51 of the second plate 5. It should be noted that the top 522 of each micro-grid 52 is located at the same height position.

[0064] Then, a single cell C in an amount of one micro-grid 12 selected as a high antibody-producing cell line is ejected from the suction head 23 onto the second plate 5. As​Figure 9 As shown, this ejection is achieved by opposing the front-end opening 23T of the suction head 23 to the opening of the mesh 51 and generating a positive ejection pressure at the front-end opening 23T. Of course, the front-end opening 23T may be projected into the upper layer of the liquid medium LA in the mesh 51 to perform the above-mentioned ejection, or the front-end opening 23T may be inserted into the interior of the micro-mesh 52 to perform the above-mentioned ejection. Through this ejection, one or more single cells C are retained in at least a part of the plurality of micro-meshes 52 provided in one mesh 51.

[0065] [Step S7: Establishment of an extremely small culture environment by medium suction]

[0066] Step S7 is a step of sucking the liquid medium LA of the second plate 5 and establishing a culture environment for the single cells C independent in units of the micro-meshes 52. 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 it shows the case where the liquid medium LA in the mesh 51 is sucked by the liquid suction head 24.

[0067] The above-mentioned suction by the liquid suction head 24 is performed from Figure 9 the state to the state where the top 522 of the side plate 521 of the micro-mesh 52 is exposed at the liquid level of the liquid medium LA in the mesh 51. That is, the liquid medium LA in the mesh 51 is removed until the liquid level of the liquid medium LA is substantially the same as the height position of the opening 52H of the micro-mesh 52. Through such suction, the liquid medium LA in one micro-mesh 52 does not mix with the liquid medium LA in other micro-meshes 52. That is, a culture environment with an extremely small culture area for the single cells C, which is composed of the liquid medium LA in each micro-mesh 52, is formed. The amount of the liquid medium LA in one micro-mesh 52 is, for example, 4 nanoliters.

[0068] In an extremely small culture environment in which 1 to about 10 or so few single cells C are introduced into a vast culture environment, it is difficult for the single cells C to proliferate. For example, in Figure 10In the case where liquid culture medium LA is injected into grid 51 of the partition from which side plate 521 has been removed and which has no micro-grid 52, and single cells C are introduced and given a prescribed culture period, it is difficult for these single cells C to proliferate. On the other hand, if 1 to about 10 single cells C are introduced and cultured in a culture environment where the liquid culture 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 cells C to be promoted. In the cell ejection in step S6, when the liquid level of the liquid culture medium LA is positioned above the opening 52H, the single cells C can be held in the micro-grid 52 by a single ejection operation, which is thus appropriate. By performing medium suction in the subsequent step S7, a culture environment suitable for the culture / proliferation of a small number of single cells C isolated in units of micro-grid 52 can be established.

[0069] [Step S8: Sealing of the micro-grid]

[0070] Step S8 is a step of injecting a sealing liquid 7 into the second plate 5 to seal above the opening 52H of the micro-grid 52. Figure 11 It 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, blocking the opening 52H. That is, the liquid culture medium LA and the single cells C are sealed within 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.

[0071] A function required of the sealing liquid 7 is to prevent the evaporation of the liquid culture medium LA within the micro-grid 52. Since the liquid culture medium LA contains moisture, in the absence of the sealing liquid 7, the above-mentioned moisture evaporates. Therefore, during the second culture in step S9, adverse conditions such as a reduction or exhaustion of the amount of the liquid culture medium LA within the micro-grid 52 and a change in the medium state such as osmotic pressure or pH occur. By sealing the opening 52H with the sealing liquid 7 having an evaporation prevention function, the evaporation of the liquid culture medium LA during the second culture can be suppressed.

[0072] 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 micro-grid 52 is sealed, the liquid culture medium LA within the micro-grid 52 can be communicated with the atmosphere. Therefore, the culture environment of the single cells C within the micro-grid 52 can be maintained in good condition. The above-mentioned embryo culture oil has both the above-mentioned evaporation prevention function and the above-mentioned air permeability, and is thus suitable as the sealing liquid 7. In addition to embryo culture oil, other liquids or semi-liquids (gels) having at least an evaporation prevention function can also be used as the sealing liquid 7. Of course, the specific gravity needs to be lighter than that of the liquid culture medium LA.

[0073] By forming a layer of the sealing liquid 7, it is possible to maintain the extremely small culture environment established in step S7 during the second culture. That is, it is possible not only to prevent the evaporation of the liquid culture medium LA in the microgrid 52, but also to 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 the active substances produced by the single cell C cultured in the microgrid 52 and promoting the proliferation of the single cell C.

[0074] [Step S9: Second culture]

[0075] Step S9 is a step of culturing the single cell C only for a specified culture period in a state where the opening 52H of the microgrid 52 is sealed with the sealing liquid 7 as Figure 11 described. That is, it is a step of further culturing the single cell C determined as a high antibody-producing cell line in step S4 only for a specified period in the second culture to proliferate it so as to produce a large amount of the antibody 3. During the second culture period, the liquid culture medium containing growth factors is filled into the grid 51.

[0076] Figure 12 is an image showing the proliferation state of the single cell C in the second culture in the second plate 5. "Day 1" in the figure refers to the state on the first day from the start of the second culture. In Figure 12 images of a part of the microgrid 52 included in one grid 51 on the 1st, 4th, 5th, 6th, 8th, 11th, and 18th days from the start of the second culture are shown. By observing the variation state of the single cell C in the target grid GA among a plurality of microgrids 52, it can be seen that it proliferates every day. It should be noted that the main reason for the rapid proliferation of the single cell C around the target grid GA between the 11th day and the 18th day is that in addition to the simple long culture days, the single cell C proliferated from the target grid GA enters the adjacent grid when the above liquid culture medium is filled.

[0077] Figure 13 and Figure 14 are images of the proliferation state of single cells using the culture method of the comparative example. Figure 13 is an image of Comparative Example 1 in a state where the medium suction in step S7 ( Figure 10 ) is not performed and the sealing with embryo culture oil in step S8 ( Figure 11 ) is not performed, that is, immediately after the cell ejection in step S6 ( Figure 9 ) a second culture is carried out. In Figure 13 images of the 1st, 6th, and 11th days from the start of the second culture are shown. By observing the proliferation state of the single cell C in the target grid GA1 among a plurality of microgrids 52, it can be seen that no significant proliferation occurred between the 1st day and the 11th day.

[0078] Figure 14 is a state in which, without performing the medium suction of step S7 ( Figure 10 ), the sealing using embryo culture oil of step S8 is performed, that is, a state in which the liquid level of the liquid medium LA is located at a position higher than the top 522 after the cell discharge ( Figure 9 ) in step S6, and the sealing liquid 7 is injected and the second culture is performed. Images of Comparative Example 2 are shown. In Figure 14 , images on the 1st day, 6th day, and 11th day from the start of the second culture are also shown. By observing the proliferation status of the single cell C in the attention grid GA2 among the plurality of microgrids 52, it can be seen that no significant proliferation occurs between the 1st day and the 11th day.

[0079] [Function and Effect]

[0080] According to the method for culturing antibody-producing cells of the present embodiment described above, the following function and effect are exerted. First, in the first culture before picking in step S5, after holding a single cell C in one microgrid 12 of the first plate 1, the liquid medium LA in the microgrid 12 is gelled. By the gelation of the liquid medium LA, a state in which one single cell C is enclosed in one microgrid 12 can be formed. In this state, by culturing the single cell C only for a specified antibody production period in the first culture, cell proliferation and the production of antibody 3 can be performed in each microgrid 12. Then, by adding the detection antibody 4 to the first plate 1, the antibody production amount can be evaluated in units of microgrids 12. Since the floating of the single cell C is suppressed by the gelation of the medium, a high antibody-producing cell line is retained in the microgrid 12 in which a large amount of the detection antibody 4 is detected. Thus, the culture of the single cell C, the production of antibody 3, and the detection of the antibody production amount can be performed in units of microgrids 12, so that a cell line with a high antibody production amount can be efficiently determined.

[0081] Next, in the second culture after picking in step S5, after holding the single cell C in the microgrid 52 of the second 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 very small culture environment enclosed in units of microgrids 52 can be constructed. By culturing the single cell C in a narrow culture area, the proliferation of the single cell C can be promoted. Therefore, by picking a single cell C having excellent antibody-producing ability and performing the second culture as described above, the efficiency of proliferation of the single cell C can be achieved, and thus a large amount of antibody 3 can be produced.

[0082] [Invention Included in the Above Embodiment]

[0083] The above-described embodiment includes an invention having the following configuration.

[0084] A method for culturing cells that produce recombinant proteins according to one aspect of the present invention includes: injecting a cell suspension containing a plurality of single cells capable of producing recombinant proteins in a medium into a plate having a plurality of accommodation parts divided into minute sizes, and causing at least a part of the plurality of accommodation parts to each hold a medium and a single cell; a step of gelling the medium in the accommodation part; a step of giving a period for producing recombinant proteins to the single cell in the accommodation part; and a step of adding a liquid containing a detection protein capable of binding to the recombinant protein produced by the single cell in the gelled medium to the plate.

[0085] According to this mode, after a single cell is held in one accommodation part, the medium in the accommodation part is gelled. By gelation, a state in which one single cell is enclosed in one accommodation part can be formed. In this state, the single cell is cultured only for a specified period for producing recombinant proteins, whereby cell proliferation and production of recombinant proteins can be carried out in each accommodation part. Then, by adding a detection protein to the plate, the amount of recombinant protein produced can be evaluated in units of accommodation parts. Since the floating of single cells is suppressed by the gelation of the medium, a cell line with a high ability to produce recombinant proteins is held in the accommodation part where a large amount of detection protein is detected. Thus, according to the above mode, culturing of single cells, production of recombinant proteins, and detection of the amount of recombinant protein produced can be carried out in units of accommodation parts, and thus a cell line with a large amount of recombinant protein produced can be efficiently determined.

[0086] In the above culturing method, it is preferable that the single cell capable of producing the recombinant protein is a single cell capable of producing an antibody, and the detection protein is a detection antibody.

[0087] According to this mode, culturing of single cells, antibody production, and detection of the amount of antibody produced can be carried out in units of accommodation parts, and thus a cell line with a large amount of antibody produced can be efficiently determined.

[0088] In the above culturing method, it is preferable that it further includes a step of applying a trigger operation for reacting the detection antibody and determining the accommodation part holding the single cell with a large amount of antibody production.

[0089] According to this mode, for example, by applying a trigger operation such as irradiating the plate with light to cause the detection antibody to fluoresce, a cell line with a large amount of antibody production can be determined only by detecting the amount of the detection antibody in units of accommodation parts based on the fluorescence intensity or the like.

[0090] In the above culturing method, it may further include a step of picking up the single cell held in the determined accommodation part and transferring it to another container.

[0091] According to this method, by picking up high antibody-producing cell lines and transferring them to other containers, for example, the cell lines can be further cultured to produce a large amount of antibodies.

[0092] In the above-mentioned culture method, it is preferable to monitor the above-mentioned plate during the production period of the above-mentioned antibody, and exclude the above-mentioned accommodation part that has held multiple single cells from the beginning of the production period from the above-mentioned determined objects.

[0093] When injecting the cell suspension into the plate, accommodation parts that hold only one single cell are found in multiple accommodation parts at a probabilistically roughly constant ratio. The remaining accommodation parts become non-holding accommodation parts that do not hold single cells or over-holding accommodation parts that hold multiple single cells. In the above-mentioned over-holding accommodation parts, high antibody-producing cell lines and non-producing or low antibody-producing cell lines may be mixed. According to the above-mentioned method, the above-mentioned over-holding accommodation parts are excluded from the determined objects, so that it is possible to accurately determine the accommodation parts that hold single cells with a large amount of antibody production.

[0094] In the above-mentioned culture method, it is preferable that the above-mentioned accommodation 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 of dimensions.

[0095] According to this method, it is possible to sufficiently miniaturize the culture area of single cells, and a large number of single cells can be independently cultured with a limited plate area. Therefore, it is possible to achieve the efficiency of culture and the efficiency of antibody production evaluation.

[0096] In the above-mentioned culture method, it is preferable that the above-mentioned plate includes a large partition part that divides a larger-sized area of the plate and a small partition part that further subdivides the inside of the large partition part, and the above-mentioned small partition part is the above-mentioned accommodation part.

[0097] According to this method, it is possible to change cell types, culture media, etc. in units of large partition parts, and it is possible to achieve diversification of culture.

[0098] In the above-mentioned culture method, it is preferable to inject a culture medium containing alginate into the above-mentioned plate.

[0099] The culture medium containing alginate, for example, has the property of gelling rapidly by adding calcium ions and magnesium ions. Therefore, according to the above method, the gelation step can be completed rapidly.

[0100] In the above-described culturing method, it preferably includes a step of introducing a gene into the above single cell to endow it with antibody-producing ability. After culturing the single cell into which the above gene has been introduced in a conventional medium for a predetermined period, the above conventional medium is replaced with a selection medium suitable for culturing the above single cell.

[0101] According to this method, although the reason has not been identified, it is possible to proliferate the single cells having antibody-producing ability well.

[0102] According to the present invention described above, it is possible to provide a culturing method capable of efficiently determining a cell line with a high production amount of a recombinant protein.

Claims

1. A method for culturing cells that produce recombinant proteins, comprising: injecting a cell suspension in which a culture medium contains a plurality of single cells capable of producing recombinant proteins into a plate having a plurality of accommodation parts divided into minute sizes, and causing at least a part of the plurality of accommodation parts to each hold the culture medium and a single cell; gelatinizing the culture medium in the accommodation part; assigning a period for producing recombinant proteins to the single cell in the accommodation part; and adding a liquid containing a detection protein capable of binding to the recombinant protein produced by the single cell in the gelatinized culture medium to the plate.

2. The culturing method of the cells for producing recombinant protein according to claim 1, wherein, The single cell capable of producing recombinant proteins is a single cell capable of producing an antibody, and the detection protein is a detection antibody.

3. The method for culturing cells producing recombinant proteins according to claim 2, wherein, It further includes a step of applying a trigger operation for reacting the detection antibody and determining the accommodation part holding the single cell with a large amount of antibody production.

4. The method for culturing cells for producing recombinant proteins according to claim 3, wherein, It further includes a step of picking up the single cell held in the determined accommodation part and transferring it to another container.

5. The method for culturing cells for producing recombinant proteins according to claim 3, wherein, During the production period of the antibody, the plate is monitored, and the accommodation part that has held a plurality of single cells from the beginning of the production period is excluded from the determined objects.

6. The method for culturing cells for producing recombinant proteins according to any one of claims 1 to 5, wherein, The receiving portion 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 terms of size.

7. The method for culturing cells for producing recombinant proteins according to claim 6, wherein, The plate includes a large partition part that divides a relatively large area of the plate and a small partition part that further subdivides the inside of the large partition part, and the small partition part is the accommodation part.

8. The method for culturing cells for producing recombinant proteins according to any one of claims 2 to 5, wherein, A culture medium containing alginate is injected into the plate.

9. The method for culturing cells that produce recombinant proteins according to any one of claims 2 to 5, wherein it includes a step of introducing a gene into the single cell to endow it with the ability to produce an antibody, after culturing the single cell into which the gene has been introduced in a conventional culture medium for a predetermined period, the conventional culture medium is replaced with a selection medium suitable for culturing the single cell.

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