JP5113812B2 - Colony picking device, colony picking method, and fusion cell clone preparation method - Google Patents

Description

  The present invention relates to a colony picking method, a colony picking apparatus, and a fusion cell clone preparation method for producing a monoclonal fusion cell that retains a desired differentiation trait.

Conventionally, monoclonal fusion cells that retain the desired differentiation traits such as antibody production and physiologically active substance production have been produced by the following production methods.
For example, a case of producing a clone of a hybridoma that produces a monoclonal antibody will be described.
(A) Cell fusion of a B cell contained in a spleen cell of an animal immunized by inoculating a target antigen with a tumor cell such as a myeloma cell line using a cell fusion inducer such as polyethylene glycol, Producing a plurality of hybridomas;
(B) Select a plurality of hybridomas prepared above in a selective medium such as a medium containing HAT (hypoxanthine, aminopreten and thymidine) placed in each well of a 96-well (well) plate (for example, 100 to 200 μL / well). Culturing and obtaining one or more colonies having a plurality of colonies derived from each hybridoma;
(C) A colony group having a hybridoma colony that produces an antibody that binds to the target antigen is selected from one or more colony groups obtained by a flow cytometry method such as FACS (fluorescence activated cell sorting), Screening by ELISA (enzyme-linked immunosorbent assay) or the like (generally referred to as first screening),
(D) a step of culturing a colony group of hybridomas contained in positive wells obtained in the primary screening in a 24-well plate (for example, 1 mL / well) and culturing;
(E) A step of screening a hybridoma colony group cultured in a 24-well plate by a flow cytometry method such as FACS method or ELISA method (generally called second screening). ,
(F) a step of collecting a hybridoma colony from a positive well obtained by secondary screening, and performing cloning by a limiting dilution method,
(G) a step of screening whether the cloned colony is derived from a single cell,
Are sequentially performed to produce a fusion cell such as a monoclonal hybridoma that produces a monoclonal antibody (see, for example, Non-Patent Document 1).

  However, in a conventional production method, for example, a positive well obtained by screening in the step (C) or the step (E) is, for example, a hybridoma (fused cell) that retains an undesired differentiation trait. ) And a number of unnecessary hybridomas such as hybridomas that do not retain the desired differentiation trait. In some cases, the hybridoma that retains the desired differentiation trait is lost. In addition, the removal of the chromosome sometimes caused the disappearance of the hybridoma retaining the desired differentiation trait. Furthermore, a series of operations for culturing, screening and cloning of fused cells, which require one set for 7 to 14 days, must be repeated several times before obtaining a single cell clone, which is labor intensive, time consuming and financially intensive. It was.

  In addition to these, in the conventional production methods, the number of specimens of hybridoma (fused cell) clones that retain the desired differentiation trait is extremely high in the screening of the steps (C) and (E). In many cases, it takes about half a year to obtain a single cell clone of a hybridoma that retains the desired differentiation trait. Thus, labor, time, and financial burden are large.

  The present invention has been made in view of the above-mentioned problems, and it is possible to obtain a fused cell that retains the desired differentiation trait more reliably than before, and labor and time for producing such a fused cell. It is another object of the present invention to provide a colony picking method, a colony picking apparatus, and a fused cell clone preparation method capable of reducing a financial burden.

The colony picking apparatus according to the present invention that has solved the above-described problems is a fusion cell from a colony of fused cells contained in a well that was positive in the result of primary screening performed after cell fusion and selective culture in a selective medium. A colony picking apparatus used for obtaining a clone, wherein one of the fused cell colonies contained in the well is selected, and the fused cell contained in the colony is determined as the size of the fused cell. A picking means for picking with a capillary having a tip having an inner diameter 1 to 40 times larger than the above, a means for injecting a fused cell picked with the capillary into an individual medium, and the well It includes a mounting table for fixing and placing the culture vessel, and a mounting table tilting mechanism that tilts the mounting table diagonally. .

The colony picking method according to the present invention is a method for obtaining a clone of a fused cell from a fused cell colony contained in a well in which a primary screening result performed after cell fusion and selective culture in a selective medium is positive. a colony picking method performed, the fused cells contained in the colonies by selecting one colony from the colonies of fused cells contained within the wells using colony picking device according to claim 1 It is seen containing a picking step of pins Kkingu, and seeded included step of pre Kipi Kkingu fusion cells Komu plated on the respective medium, a, in the picking process, picking the fusion cells to tilt the mounting table diagonally It is characterized by that.

The fusion cell clone production method according to the present invention produces a clone of a fusion cell from a colony of fusion cells contained in a well that was positive in the result of primary screening after cell fusion and selective culture in a selective medium. A method for preparing a fused cell clone for performing the selection, wherein the colony picking device according to claim 1 is used to select one colony from the fused cell colonies contained in the well and to be included in the colony. and picking process of picking a fusion cell, and Maki inclusive process Komu seeded fused cells were the picking in a separate medium, only including the culturing step, the culturing the crowded but fused cells were plated in a separate medium, in the picking process The above-mentioned fused cells are picked by tilting the mounting table obliquely .

  According to the colony picking method according to the present invention, a single colony is selected from colonies of fused cells contained in a well having a positive primary screening result, and the fused cells contained in the colony are picked. Since the picked fused cells are placed in a separate medium that can be individually cultured, the survival competition with the fused cells that retain the desired differentiation trait or the like during the passage is lost, or the chromosomes It is possible to prevent the fusion cells that retain the desired differentiation trait from being lost. Moreover, since it can prevent that the number of the specimens of the fusion cell clone which hold | maintains the target differentiation trait becomes very large, an effort, time, and a financial burden can be reduced.

  According to the colony picking apparatus according to the present invention, since the picking means and the swallowing means are provided, one colony is selected from the colonies of the fused cells contained in the well in which the primary screening result is positive. The selected fused cells contained in the colony are picked by a tubule having a tip having an inner diameter 1 to 40 times the size of the fused cell, and the fused cells picked by the tubule are selected. It can be infused into a separate medium prepared for individual culturing. Therefore, the fusion cells that retain the desired differentiation trait disappear due to the loss of competition for survival with fusion cells that retain the desired differentiation trait, etc., or the loss of chromosomes, while continuing the passage. Can be prevented. Moreover, since it can prevent that the number of the specimens of the fusion cell clone which hold | maintains the target differentiation trait becomes very large, an effort, time, and a financial burden can be reduced.

  According to the fusion cell clone preparation method of the present invention, a single colony is selected from the colonies of the fusion cells contained in the wells in which the primary screening result is positive, and the fusion cells contained in the colonies are selected. The picked fused cells are soaked in an individual medium prepared so that they can be individually cultured, and cultured. In other words, because the cells are individually cultured without scaling up the colonies of the fused cells in the positive wells, the fused cells retain the desired differentiation trait as the passage continues, for example due to the slow growth rate. It is possible to prevent the fusion cells that retain the desired differentiation trait from disappearing due to loss of the competition with the human body or the like, or the loss of chromosomes. Therefore, it is possible to reliably obtain a fused cell that retains the desired differentiation trait. Moreover, since it can prevent that the number of the specimens of the fusion cell clone which hold | maintains the target differentiation trait becomes very large, an effort, time, and a financial burden can be reduced.

(A) is a flowchart which shows the content of the process of the colony picking method which concerns on this invention, (b) is a flowchart which shows the internal use of the process of the fusion cell clone preparation method concerning this invention. (A) is a perspective view explaining the specific structural example of the colony picking apparatus based on this invention, (b) is the enlarged view to which the A part of (a) was expanded. (A) And (b) is explanatory drawing explaining the structural example of a container position exchange means. (A) And (b) is a conceptual diagram explaining a mode that the picked fused cell is moved to a separate culture medium.

Hereinafter, the colony picking method, the colony picking apparatus, and the fused cell clone preparation method according to the present invention will be described in detail with reference to the drawings as appropriate.
The present invention relates to a colony picking method, a colony picking apparatus, and a fusion cell clone preparation method for producing a monoclonal fusion cell that retains the desired differentiation trait. If there is no particular description of the best mode for carrying out the invention and examples of necessary operations, fusion cell handling methods, fusion cell cloning methods, etc., for example, Non-Patent Document 1 and commonly used standards A method described in a general protocol collection, or a method obtained by modifying or modifying them can be used. In addition, when using commercially available reagent kits and measuring devices, unless otherwise explained, protocols attached to them can be used. Further, those skilled in the art can easily reproduce the present invention from the description of the present specification and, for example, the descriptions of Non-Patent Document 1 and standard protocol collections.

First, the colony picking method according to the present invention will be described.
As shown in FIG. 1 (a), the colony picking method according to the present invention is a method in which fused cells contained in a well that has been positively subjected to a primary screening result after cell fusion and selective culture in a selective medium. A colony picking method for obtaining a fusion cell clone from a colony, which comprises a picking step S10 and a winding step S20.
In addition, each process of the immunization before performing picking process S10, cell fusion, the selective culture by a selective medium, and a primary screening can be performed by the method normally performed.

  Immunization can be performed, for example, by preparing a phage on which an antigen is displayed by the phage display method, isolating the displayed antigen by a conventional method, and immunizing an immunized animal such as a mouse. It can also be carried out by immunizing an immunized animal such as a mouse with an antigen purified by a conventional method. In immunization, complete Freund's adjuvant, incomplete Freund's adjuvant, or the like can be used.

  Cell fusion can be performed, for example, by cell fusion of a biological cell retaining a desired differentiation trait and a tumor cell. In addition, as long as the biological cell holding the target differentiation character is used, any cell can be used as long as it holds the target differentiation character. For example, when the target differentiation trait is antibody production that produces a monoclonal antibody that reacts with a specific antigen, the spleen cell (B cell) of the animal immunized by inoculating the animal with the target antigen Can be used. In addition, for example, when the target differentiation trait is the production of a physiologically active substance that produces a physiologically active substance such as lymphokine, animal thymus cells (T cells) can be used. The inoculation of the target antigen to the animal and the acquisition of spleen cells from the immunized animal can be performed by conventional methods.

  As animals for obtaining the above-mentioned cells, mice, rats, rabbits, goats, etc., and these knockout or / and transgenic animals prepared as necessary can be used. For example, when using a mouse, it is preferable to use a BALB / c strain mouse. This is because myeloma cell lines widely used as tumor cells for producing fusion cells originate from BALB / c strain mice. Therefore, the differentiation traits obtained from BALB / c strain mice Advantages of good compatibility with retained biological cells, and the resulting fused cells can be propagated in the peritoneal cavity of BALB / c mice. This is because there is a merit that it can be produced and that it is easy to obtain. From the same viewpoint, for example, when using a rat, it is preferable to use a low strain. Needless to say, the animals for obtaining the cells are not limited to those described above, and animals other than the animals described above and strains other than the strains described above can also be used.

  In addition, as a tumor cell used for producing a fusion cell, any tumor cell can be used as long as it grows in vivo. It is preferable to use tumor cells that cannot survive in a selective medium with or without a specific composition and have a low ability to produce immunoglobulins. As such tumor cells, for example, thymidine kinase (TK) or hypoxanthine guanine phosphoribosyltransferase (HGPRT) is deficient and nucleic acid cannot be biosynthesized by the salvage pathway out of two nucleic acid synthesis pathways. A mouse myeloma cell line NS-1 that can biosynthesize nucleic acids only by the novo pathway can be suitably used. Needless to say, the tumor cells that can be used in the present invention are not limited thereto. For example, rabbit myeloma cells can also be used when the desired differentiation trait is the production of a rabbit monoclonal antibody that reacts with a specific antigen.

  For example, fusion between a living cell and a tumor cell that retains a differentiation trait is performed by, for example, a polyethylene glycol method using polyethylene glycol having an average molecular weight of about 1000 to 6000 and a concentration of about 30 to 50%, a cell fusion method using Sendai virus, It can be performed by any cell fusion technique such as pulse electrofusion.

Here, the medium that can be used for preparing cell fusion varies depending on the fused cells to be prepared. For example, in the case of a fused cell prepared using a mouse, RPMI1640 medium containing HAT, HT (hypoposome). RPMI 1640 medium with xanthine and thymidine) RPMI 1640 medium with or without 10% FCS (fetal bovine serum) or 20% FBS (fetal bovine serum) can be used. Moreover, you may use what added the appropriate cell growth factor to these culture media. The fused cells are cultured, for example, under 5% CO ₂ concentration at 37 ° C. until the colonies in the wells can be distinguished (diameter of about 100 to 1400 μm). During this time, the culture medium may be allowed to stand or may be rocked by a rocking device or the like as necessary.

  The fused cells obtained by the above-described operation contain a considerable number of fused cells that do not retain the desired differentiation trait. Therefore, selective culture using a selective medium is performed in order to efficiently detect fused cells that retain the desired differentiation trait.

  The selective culture using the selective medium can be performed by using a selective medium having a specific composition. As the selective medium having a specific composition, a medium containing HAT and the like can be preferably used as in the case of producing fused cells. For example, when a myeloma cell line capable of biosynthesizing nucleic acids only by the de novo pathway described above is used as a tumor cell, it contains aminopreten, which is an inhibitor of nucleic acid biosynthesis by the de novo pathway. Nucleic acid biosynthesis cannot be performed by the pathway, and nucleic acid biosynthesis by the salvage pathway cannot be performed because TK or HGPRT is deficient as described above. Therefore, tumor cells that have not fused with living cells that retain differentiation traits cannot grow in a medium containing HAT. On the other hand, living cells that retain a differentiation trait that has not fused with tumor cells are normal cells and can grow in a medium containing HAT for a while, but cannot survive for a long time. It will be killed. As described above, by using a selective medium having a specific composition, it is possible to selectively grow (selective culture) a fused cell in which a living cell retaining a differentiation trait and a tumor cell are fused and obtain this appropriately and easily. Is possible. Note that the selective medium that can be used in the present invention is not limited to this, and any medium can be used as long as it allows selective culture of fused cells.

The selective culture using the selective medium may be performed using, for example, a 96-well plate to which an appropriate amount of the selective medium is added per well, but is not limited thereto. For example, the cells may be cultured in a culture vessel having 6, 12, 24, and 48 wells.
Selective culture by such selective medium, the fused cells produced during the same conditions, e.g., 5% CO ₂ concentration under can be done by culturing until about can be distinguished colonies in the wells at 37 ° C.. During the culturing period, half or all of the selective medium may be exchanged as necessary, and the well plate may be swung at an appropriate speed. In this way, a colony group having a plurality of colonies derived from the prepared fused cells is formed in each well of the 96-well plate.

In the primary screening, a colony group having a colony of a fusion cell that retains a desired differentiation trait is selected from a colony group derived from a fusion cell obtained by selective culture in a selective medium.
This primary screening corresponds to what is called primary screening in a conventional method for producing a hybridoma. For example, a flow cytometry method, which is a cell measurement method using a fluid, particularly a fluorescence method. The target fused cells stained with antibodies can be put in a liquid flow, passed through the focal point of the laser beam, the fluorescence emitted by each cell is measured, and only specific cells remain alive aseptically. It can be carried out by a possible FACS method (fluorescence activated cell sorting). In the case of the FACS method, it can be performed by a droplet charging method or a cell capture method. In addition, the primary screening is not limited to the above-described method, and can also be performed by, for example, ELISA (enzyme-linked immunosorbent assay).

  When such a primary screening is performed, for example, a well including a colony group having a colony of a fusion cell that retains a desired differentiation trait in a 96-well plate can be detected as a positive well.

  And picking process S10 in this invention is a process of selecting one colony from the colonies of the fused cell contained in the well in which the result of the primary screening was positive and picking the fused cell contained in the colony. It is.

  Here, a method for producing a clone of a fusion cell such as a general hybridoma will be described. All the fusion cells positive in the primary screening were cultured in a larger system such as a 24-well plate. Later, secondary screening and cloning are performed. The reason is that the prepared fused cell colonies are extremely small at about 140 μm in diameter at this stage, making it extremely difficult to handle, and the prepared fused cell clones may disappear. This is because it is desirable to increase the fusion cell clones produced to an amount that can be cryopreserved as soon as possible.

On the other hand, in the present invention, for example, immediately after performing selective culture in a 96-well plate, only the fused cells contained in the colonies of the fused cells obtained by performing selective culture are individually picked and picked. Only the fused cells are isolated from other fused cells and cultured, that is, transferred to an isolated medium prepared so that they can be individually cultured, that is, transferred to individual media. Therefore, in the present invention, since a cloning operation for obtaining a single cell clone is performed from this stage, a fusion cell clone that retains the target differentiation trait is not produced by the clone of the fusion cell that retains the desired differentiation trait. The possibility of disappearing due to the loss of survival competition can be eliminated. As a result, the number of positive wells that contain colonies of fused cells that retain the desired differentiation trait is increased in the screening process that can be performed later, compared to the conventional method. Since the cloning work for obtaining the above is performed, the burden of cloning that can be performed thereafter can be reduced. The screening step will be described in the fused cell clone preparation method described later.
As described above, since the size of the colonies formed in the wells of the 96-well plate immediately after the primary screening is very small, it is extremely difficult to individually pick colonies of the prepared fused cells. In this application, it can carry out easily by using the colony picking apparatus mentioned later. The colony picking apparatus will be described in detail later.

  In this picking step S10, if one colony can be selected from the colonies of the fused cells contained in the wells in which the primary screening result is positive, and the fused cells contained in the colonies can be picked. Well, it doesn't necessarily mean you have to get fused cells from one colony in a single picking operation. For example, since two colonies are very close to each other or overlap, there is a possibility that two or more colony fusion cells may be picked by a single picking operation. In such a case, the picked fused cells may be once dispersed in another medium before being dispersed in an individual medium, and then dispersed in an individual medium, and then infused into an individual medium.

The next soaking step S20 is a step of soaking the picked fused cells into individual media prepared so that they can be individually cultured. Such a medium is sufficient if the fused cells soaked can grow. Accordingly, the medium may be the same as or different from the medium used at the time of preparing the fused cells. For example, a medium containing HAT, a medium containing HT, or RPMI 1640 medium containing or not containing 10% FCS (fetal calf serum) can be used. Moreover, you may use what added the appropriate cell growth factor to these culture media. The fused cells soaked in the medium are cultured to the extent that the colonies in the wells can be distinguished at 37 ° C., for example, at a concentration of 5% CO ₂ as in the case of producing the fused cells. During this time, the culture medium may be allowed to stand or may be rocked by a rocking device or the like as necessary.

  The medium to which the colonies are picked and transferred in the entrapment step S20 may be a new medium added to each well of the 96-well plate, for example. As this new medium, a medium containing HAT can be used as described above. In addition, the culture medium added to each well of the culture container which has 6, 12, 24, 48 wells with larger capacity | capacitance, and the culture medium added to individual culture containers, such as a petri dish, may be sufficient.

Next, a method for producing a fused cell clone according to the present invention will be described.
The fused cell clone preparation method according to the present invention includes the above-described colony picking method according to the present invention as a part of the process, and the result of primary screening performed after cell fusion and selective culture in a selective medium is performed. This is a method for producing a clone of fused cells from a colony of fused cells contained in a well that was positive.
The fused cell clone production method according to the present invention includes a picking step S10, a winding step S20, and a culturing step S30. Note that the picking step S10 and the punching step S20 are exactly the same as the picking step S10 and the punching step S20 of the colony picking method, and thus detailed description thereof is omitted.

  The culturing step S30 is a step of culturing the fused cells soaked in an individual medium. As already explained, since an operation that also serves as a cloning operation is performed in the picking step S10, the fusion cell strain cultured in the culturing step S30 is very likely to be a single cell clone.

  Prior to the picking step S10, a pre-picking screening step S3 for detecting a fused cell colony showing a positive among the colonies of the fused cell contained in the positive well may be performed. The pre-picking screening step S3 corresponds to so-called secondary screening. In this way, since the colonies of the fused cells showing positive can be narrowed down, the number of times of picking in the picking step S10 can be reduced. The pre-picking screening step S3 can be performed by a flow cytometry method such as the FACS method, an ELISA method, or the like.

  Moreover, it is preferable to perform cloning process S25 which clones a fused cell after winding process S20. As described above, although the cloning operation is also performed in the picking step S10, there is no guarantee that the cell line of the fused cell obtained by culturing is a cell line consisting of a single cell clone. The cloning step S25 may be performed in order to obtain confirmation that a cell line composed of a single cell clone retaining a trait has been reliably established. Any method may be used for the cloning step S25 as long as a single cell clone can be obtained. For example, the cloning step S25 can be performed by a limiting dilution method.

  Further, after the culture step S30, a post-culture screening step S33 for confirming a positive reaction of the cultured fused cells is preferably performed. The post-culture screening step S33 corresponds to a so-called tertiary screening if the pre-picking screening step S3 is performed before this, and corresponds to a so-called secondary screening if the pre-picking screening step S3 is not performed. In this way, it is possible to confirm that the fused cells showing a positive result in the post-culture screening step S33 retain the target differentiation trait and to selectively obtain the fused cells. Therefore, the cloning step S35 described later. Thus, the fusion cell can be easily cloned. The post-culture screening step S33 can be performed by a flow cytometry method such as FACS method or an ELISA method.

  Further, it is more preferable that after the post-culture screening step S33, a cloning step S35 for cloning the fused cells that are positive as a result of the post-culture screening is performed. In this way, a colony (cell line) composed of a single cell clone of a fused cell retaining the target differentiation character can be obtained more reliably.

Next, the colony picking apparatus according to the present invention will be described.
The colony picking apparatus 1 according to the present invention is for obtaining a clone of a fused cell from a colony of fused cells contained in a well that is positive in the result of a primary screening performed after cell fusion and selective culture in a selective medium. A colony picking apparatus used for the above-mentioned, which is an apparatus for carrying out the picking step S10 and the entrapment step S20 of the above-described colony picking method according to the present invention, and has picking means and entrainment means. is doing.

The picking means selects one colony from the colonies of the fused cells contained in the well and picks the fused cells contained in the colonies, and has substantially the same action as the above-described picking step S10. Play.
In addition, the sowing means is soaked into an individual medium so that the picked fused cells can be individually cultured, and has substantially the same effect as the above-described soaking process S20.

  A specific example of the colony picking apparatus 1 according to the present invention is shown in FIG. As shown to (a) of FIG. 2, the colony picking apparatus 1 is mainly comprised including the microscope 2, the mounting base 3, the thin tube body 4, and the pressure control apparatus 5. As shown in FIG.

  The microscope 2 is for enlarging and observing a colony of fused cells. Further, by using the microscope 2, it is possible to reliably perform the operation using the thin tube body 4. Any microscope 2 can be used as long as it can observe colonies and fused cells in an enlarged manner. The magnification should just have at least about 400 times. As such a microscope 2, an optical microscope etc. can be used, for example.

  The mounting table 3 is a culture vessel containing a colony of fused cells provided under the microscope 2, a 96-well plate containing newly prepared individual media for individually culturing picked fused cells, etc. The culture container is fixed and placed. The means for fixing these culture vessels may be fixed as long as they are fixed, and any means may be used.

  As shown in FIGS. 2 (a) and 2 (b), the tubule body 4 has a tip 41 for picking fused cells. The distal end portion 41 is more preferable because the smaller the inner diameter of the distal end portion 41, the more the fused cells can be picked individually. The inner diameter of the tip 41 is about 1 to 40 times, more preferably about 1 to 20 times, and still more preferably about 1.5 to 15 times the size of the fused cell to be picked (about 10 to 18 μm in diameter). Even more preferably, it may be about 1.5 to 10 times. That is, the inner diameter of the tip portion 41 is preferably about 10 to 400 μm, for example. In addition, even if the inner diameter of the tip portion 41 is within this range, if it is formed as large as 400 μm, there is a possibility of picking a plurality of fused cells from within one colony. If it is picked from one colony, it is highly probable that it is a proliferating cell derived from the same fusion cell, and a single cell clone can be obtained by screening and cloning in a later step. It will not be.

If the inner diameter of the tip 41 is less than about 10 μm, the fused cells may be damaged when picking. On the other hand, when the inner diameter of the distal end portion 41 of the thin tubular body 4 exceeds about 400 μm, it becomes difficult to pick up the fused cells of a plurality of colonies when picking the fused cells.
Further, it is preferable that the thickness of the tip portion 41 is smaller because it is easier to pick fused cells from a colony formed in the vicinity of the wall of the well. Therefore, the thickness of the tip 41 is preferably about 50 to 400 μm, for example.
Such a thin tube 4 can be produced by heating and stretching a glass tube or a plastic tube.

In addition, since the capillary body 4 is provided with at least the tip 41 perpendicular to the mounting table 3, even if it is a small well such as a 96-well plate, as shown in FIG. It is possible to operate so as not to contact the wall or the like. Moreover, by doing in this way, even if it is a very small colony, a fused cell can be picked correctly.
Further, since the narrow tube body 4 is held by a holder 43 having a movable portion 42 that is movable at least in the vertical direction with respect to the mounting table 3, the movable portion 42 is moved manually or electrically to lower the narrow tube body 4. If it is moved in the direction, it becomes possible to accurately pick the fused cell colony, and if the moving part 42 is moved to move the tubule 4 upward, the picked fused cell is transferred to another medium. Will be able to.

The pressure control device 5 is connected to the proximal end portion 44 of the thin tube body 4 via the connection means 51 and controls the inside of the thin tube body 4 to a negative pressure and a positive pressure. The pressure may be controlled so long as one or a plurality of fused cells can be sucked up from the fused cell colony and discharged to another medium. Note that it is not preferable that the pressure control by the pressure control device 5 cannot be precisely performed, or that the pressure control speed is too rapid, because the possibility of picking fused cells of other unintended colonies increases.
As the pressure control device 5, any device can be used as long as it can control the pressure in the thin tube body 4 connected via the connection means 51. For example, a peristaltic pump can be suitably used. The connection means 51 may be a hollow tube made of silicon or the like and has a small inner diameter as much as possible so that the pressure in the connection means 51 can be precisely controlled.

  In the colony picking apparatus 1 according to the present invention, when the holding tool 43 has a movable portion 42a that can move the thin tube body 4 in the front-rear and left-right directions in a direction horizontal to the mounting table 3, It is more preferable because the range of motion is widened and picking work and swallowing work can be easily performed. Here, it is preferable that the holder 43 having the movable portion 42a is such that the distal end portion 41 of the thin tubular body 4 does not shake during the picking operation or the entrapment operation of the fused cells. As such a holder 43, for example, it can be mentioned that one manipulator manufactured by Narimo Scientific Instruments Laboratory Co., Ltd. is produced or used in combination as necessary.

  In addition, the mounting table 3 can move the mounting table 3 in the front / rear / left / right direction with respect to a specified position of the mounting table 3 or a container moving mechanism (not shown) that moves the culture vessel such as a 96 well plate in the front / rear / left / right direction A mounting table moving mechanism (not shown) or the like may be provided. Further, the mounting table 3 may be provided with a mounting table tilting mechanism (not shown) that tilts the mounting table 3 that is normally kept horizontal at a slight angle. When these mechanisms are provided, it is possible to facilitate the picking operation and the entrapment operation as in the case of expanding the movable range of the thin tube body 4. In particular, when the mounting table tilting mechanism is provided, the culture vessel can be tilted slightly obliquely. As a result, the fused cells can be picked from the colonies at the wall of the well. The container moving mechanism and the mounting table moving mechanism are interlocked with an appropriate means capable of moving the position of the culture vessel or the mounting table 3 in the front-rear and left-right directions, for example, an operation knob arranged at the user's hand. It can be realized by means such as manual or electric rack and pinion. The mounting table tilting mechanism can be realized by providing one or a plurality of electric, hydraulic or pneumatic actuators at arbitrary positions so that the mounting table 3 tilts obliquely.

In addition, the colony picking apparatus 1 according to the present invention, as shown in FIGS. 3 (a) and (b), in order to infuse the individually picked fused cells into individual media prepared so that they can be individually cultured, The container position exchanging means 6 for exchanging the position P ₁ of the culture container having the colony group used for culturing the fused cells and the position P ₂ of the culture container to which the individual medium has been added is described above. 3 may be provided. As such a container position exchanging means 6, for example, a rotating table 61 (see FIG. 3A) that rotates circularly in the in-plane direction of the mounting table 3 or an in-plane of the mounting table 3 is used. A horizontal moving plate 62 (see FIG. 3 (b)) that moves horizontally in the direction can be cited, and used for culturing fused cells at predetermined positions P ₁ and P ₂ on the rotating plate 61 or on the horizontal moving plate 62. The culture vessel having a colony group and the culture vessel to which the individual medium is added may be fixed.

As shown in FIG. 3A, the turntable 61 can be switched between the position P ₁ and the position P ₂ of the culture vessel by rotating in a circular direction in the in-plane direction of the mounting table 3. Further, as shown in FIG. 3B, the horizontal moving board 62 moves horizontally in the in-plane direction of the mounting table 3, so that the position P ₁ and the position P _{2 of the} culture vessel indicated by the solid line in the figure. Can be interchanged with each other at positions P ₁ and P ₂ indicated by broken lines in FIG.
If these container position exchanging means 6 are provided, the tubule can be obtained simply by exchanging the position P ₁ of the culture container having the colony group and the position P ₂ of the culture container to which the individual medium is added. The fused cells picked at 4 can be reliably and easily inoculated into individual media.

Next, the usage aspect of the colony picking apparatus 1 which concerns on this invention is demonstrated.
First, cells are fused by a conventional method, selectively cultured in a selective medium, subjected to primary screening, and a culture vessel such as a 96-well plate having wells with positive results is placed on the mounting table 3. And fix.

Then, while observing the colony group in the well that was positive with the microscope 2, as shown in FIG. 2 (b), carefully insert the tip 41 of the capillary 4 held by the holder 43 into the well. Insert the tip 41 into contact with one colony arbitrarily selected from the colony group. For example, as shown in FIGS. 4A and 4B, one colony of a fused cell is selected from the colony group numbered “B5”, “C2”, “C13”, etc., and the tip 41 is brought into contact therewith. Then, the inside of the tubule 4 connected to the connecting means 51 is made negative pressure by the pressure control device 5, and one or a plurality of fused cells are sucked up and picked from one selected colony. The movable unit 42 and, if necessary, the movable unit 42a, the above-described container movable mechanism, the above-described mounting table movable mechanism, the above-described mounting table tilting mechanism, the container position exchanging means 6, and the like, After the tip 41 is moved onto the medium in the well of the culture vessel to which the medium has been added, the tip 41 is carefully lowered downward to bring the tip 41 into contact with the individual medium. And the inside of the thin tube body 4 connected with the connection means 51 by the pressure control apparatus 5 is made into a positive pressure, and the fused cells can be infused into the medium by discharging the picked fused cells onto the medium.
Thereafter, the fused cells entrained in the medium are cultured under predetermined conditions, and secondary screening, cloning, and the like are performed by a conventional method, whereby a cell line composed of a single cell clone can be obtained (FIG. 1 (b)). reference).

  As described above, the colony picking device 1 according to the present invention is connected to the thin tube body 4 and the thin tube body 4 held by the holder 43 having the movable portion 42 movable at least in the vertical direction with respect to the mounting table 3. The pressure control device 5 is used to implement a picking means (picking step S10) for picking the fused cells, and each of the tubules 4 picked with the fused cells and the individual prepared in another culture vessel The movable part 42 to be moved on the culture medium, and the movable part 42a, the container movable mechanism, the mounting table moving mechanism, the mounting table tilting mechanism, the container position exchanging means 6 and the like, if necessary, are connected to the thin tube body 4. By having the pressure control device 5, the picking-up means (the pouring step S20) for picking up the fused cells that have been picked up onto the individual medium prepared in the other culture vessel described above is realized. To have. Needless to say, the means for embodying the picking means and the entraining means is not limited to this, and can be embodied by other configuration examples.

Next, an example that exhibits the effect of the present invention will be described in comparison with a comparative example that does not exhibit the effect of the present invention.
(Comparative example)
In the comparative example, cloning of a fusion cell (hybridoma) that produces a monoclonal antibody of protein α with a low expression level in vivo and a slow growth rate of the fusion cell was attempted according to the technique described in Non-Patent Document 2.

  That is, in this comparative example, a baculovirus (BV) in which the full-length DNA of the protein α was introduced into the knockout mouse (protein αKO mouse) of the protein α crossed with the gp64 transgenic mouse and the protein α was displayed on the surface. ) Was purified by a conventional method and immunized 3 times every 2 to 4 weeks. Immunization was carried out by intraperitoneal administration, and the dose was 0.1 mg in Bradford value per animal.

Anti-serum was FACS-evaluated for each immunization, cell fusion was performed by a conventional method using mice that differed in anti-serum evaluation, a fused cell (hybridoma) was obtained, and primary screening was performed by FACS. Approximately 500 positive wells could be obtained from one 96-well plate. As the medium in the 96-well plate, RPMI1640 medium supplemented with 20% FBS was used and cultured under conditions of 5% CO ₂ and 37 ° C.

  The medium in a positive well was transferred to a well of a 24-well plate, scaled up, and subjected to secondary screening by the FACS method. The Geo Mean was about 1/5 to 1/10 that of the primary screening. Declined. In other words, a single cell that produces an anti-protein α antibody because a fused cell that produces an anti-protein α antibody contained in a positive well has lost its survival competition with a fused cell that does not produce the anti-protein α antibody. A cell clone could not be obtained.

Example 1
In Example 1, the preparation of the fused cells and the operation for the primary screening were performed under the same conditions as in the comparative example. The medium in the 96-well plate was the same as that in the comparative example and cultured under the same conditions as in the comparative example. As a result of the primary screening, approximately the same number (480) of positive wells as in the comparative example could be obtained.

  In Example 1, the colony picking apparatus 1 shown in FIG. 2 is used to select one colony from the colonies of the fused cells contained in the wells that were positive in the primary screening, and the fused cells contained in the colonies. Were picked and placed in a separate medium (using a 96-well plate) prepared so that the picked fused cells could be cultured individually. This operation was performed for all colonies contained in positive wells that could be obtained by cell fusion. In addition, the same culture medium as the comparative example was used as an individual culture medium.

  Here, the colony picking apparatus 1 used in Example 1 is produced by combining a plurality of manipulators manufactured by Narimo Scientific Instrument Research Co., Ltd., as described in the section of the embodiment for carrying out the invention. It has a configuration. That is, the colony picking device 1 is mainly configured by including a microscope 2, a mounting table 3, a thin tube body 4, and a pressure control device 5 (see FIG. 2 and corresponding portions of the present specification). A detailed description of the configuration of the colony picking apparatus 1 is omitted here).

Then, the fused cells soaked in individual media were cultured at 37 ° C. under a 5% CO ₂ concentration until the colonies in the wells could be distinguished. The number of days of culture was about 3 to 5 days for the fast growing colony group, and about 7 to 10 days for the slow growing colony group. When the fused cells cultured in individual media were subjected to the secondary screening using the Cell ELISA method, 71 positive wells could be obtained. Further, 71 positive wells obtained were subjected to the third screening by the FACS method and cloned by the limiting dilution method. As a result, positive wells (positive clones) could be obtained for 5 wells.

In Example 1, it took about 2 weeks in total until the final 5 positive wells were obtained after producing the fused cells. This is approximately 2 / in comparison with the conventional method in which secondary screening and cloning are performed after the primary screening is performed and then scaled up in a 24-well plate (usually, it takes about 3 to 4 weeks). Only 3 to 1/2 days or less were required. Moreover, the only well plate used was a 96 well plate.
Therefore, according to Example 1, it is possible to obtain a fused cell that retains the desired differentiation trait more reliably than before, and the labor, time, and money for producing such a fused cell compared to the conventional method. It was possible to reduce the burden.

(Example 2)
In Example 2, experiments were performed under exactly the same conditions except that immunization was performed under conditions different from those in Example 1. In immunization in Example 1, full-length DNA of protein α was introduced as an antigen, BV on which protein α was presented on the surface was purified by a conventional method and administered intraperitoneally (dosage is the Bradford value per mouse) The purified protein α (dose is 0.005 mg as a Bradford value per mouse) was administered by subcutaneous administration in the vicinity of the tumor together with complete Freund's adjuvant.

  As a result of the primary screening, approximately the same number (480) of positive wells as in Example 1 could be obtained. One colony is selected from the colonies of the fused cells contained in the positive well, and the fused cells contained in the colony are picked with the colony picking apparatus 1 in the same manner as in Example 1 so that the picked fused cells can be cultured individually. Cultivated in a separate medium prepared in the above. As in Example 1, the number of culture days was about 3 to 5 days for the fast growing colony group, and about 7 to 10 days for the slow growing colony group. When the cultured fusion cells were subjected to a secondary screening using the Cell ELISA method, 53 positive wells could be obtained. Further, the 53 positive wells obtained were subjected to the third screening by the FACS method and cloned by the limiting dilution method. As a result, positive wells (positive clones) could be obtained for 5 wells.

  Also in Example 2, in the same manner as in Example 1, it took about 2 weeks in total until the final five positive wells were obtained after the fusion cells were prepared. That is, Example 2 can also obtain a fused cell that retains the desired differentiation trait more reliably than in the past, and labor, time, and money for producing such a fused cell as compared with the conventional method. It was possible to reduce the burden.

Kremer, L., Marquez, G., Methods Mol. Biol., “Generation of monoclonal antibodies against chemokine receptors”, 239, 2004, p.243-260. Ryoichi Saitoh et al., Journal of Immunological Methods, "Viral envelope protein gp64 transgenic mouse facilitates the generation of monoclonal antibodies against exogenous membrane proteins displayed on baculovirus", 322, 2007, p.104-117.

S10 Picking Step S20 Rolling Step S3 Pre-Picking Screening Step S25 Cloning Step S30 Culture Step S33 Post-Culturing Screening Step S35 Cloning Step 1 Colony Picking Device 2 Microscope 3 Mounting Table 4 Tubular Body 41 Tip 42, 42a Movable Part 43 Holder 44 Base part 5 Pressure control device 51 Connection means 6 Container position replacement means 61 Rotating board 62 Horizontal moving board

Claims (7)

A colony picking apparatus used for obtaining a fused cell clone from a fused cell colony contained in a well in which a primary screening result performed after cell fusion and selective culture in a selective medium is positive. ,
One colony is selected from the colonies of the fused cells contained in the well, and the fused cells contained in the colony are arranged with a tip having an inner diameter of 1 to 40 times the size of the fused cells. Picking means for picking with a provided thin tube,
Means for injecting the fused cells picked by the tubules into an individual medium;
A mounting table for fixing and mounting the culture vessel containing the well;
A mounting table tilting mechanism that tilts the mounting table diagonally;
A colony picking apparatus comprising: A colony picking method performed to obtain a fused cell clone from a fused cell colony contained in a well in which a primary screening result performed after cell fusion and selective culture in a selective medium is positive,
A picking step of selecting one colony from the colonies of the fused cells contained in the well using the colony picking apparatus according to claim 1, and picking the fused cells contained in the colonies;
A step of instilling the picked fused cells into a separate medium;
Including
In the picking step, the colony picking method is characterized by picking the fused cells by tilting the mounting table obliquely. A fusion cell clone production method for producing a clone of a fusion cell from a colony of fusion cells contained in a well that has been positive after a primary screening performed after cell fusion and selective culture in a selective medium. ,
A picking step of selecting one colony from the colonies of the fused cells contained in the well using the colony picking apparatus according to claim 1, and picking the fused cells contained in the colonies;
A step of instilling the picked fused cells into a separate medium;
A culture step of culturing the fused cells in individual media;
Only including,
In the picking step, the fused cell clone producing method, wherein the fused cell is picked by tilting the mounting table obliquely .   4. The pre-picking screening step of detecting a fusion cell colony showing a positive among the colonies of the fusion cell contained in the positive well before the picking step. Fusion cell clone production method.   The method for producing a fused cell clone according to claim 3 or 4, wherein a cloning step for cloning the fused cell is performed after the entrapment step.   The method for producing a fused cell clone according to claim 3 or 4, wherein a post-culture screening step for confirming a positive reaction of the cultured fused cells is performed after the culturing step.   The method for producing a fused cell clone according to claim 6, wherein a cloning step for cloning a fused cell having a positive post-cultivation screening result is performed after the post-culture screening step.

Images