JP6762088B2 - Cell separation device and cell separation method - Google Patents

Description

The present invention relates to a cell separation device and a cell separation method for selectively separating and collecting antibody-producing cells from a plurality of candidate cells.

In recent years, there have been an increasing number of cases in which useful substances such as pharmaceuticals and physiologically active substances are produced by biochemical methods using cultured cells. Useful substances and predetermined genes involved in their production are introduced into various host cells such as animal cells, microbial cells, and plant cells, and the obtained recombinant cells are cultured on a large scale to make the useful substances efficient. Production is being carried out.

Conventionally, recombinant cells have been subjected to biological methods using viruses and phages, chemical methods typified by lipofection methods, polymer vector methods, etc., electroporation methods, heat shock methods, particle gun methods, etc. It is produced by various methods such as physical methods represented by. The mainstream methods are the lipofection method and electroporation method for animal cells, the electroporation method and heat shock method for bacterial cells, and the particle gun method, electroporation method, and transduction method using Agrobacterium for plant cells. ..

In such gene transfer methods, the copy number of the gene introduced into the host cell often varies. Therefore, it is not uncommon for the genetically modified cells produced to have individual differences in the ability to produce useful substances. Therefore, in general, screening is performed to select cells having a high ability to produce useful substances from a plurality of candidate cells subjected to gene transfer.

Conventionally, in selecting a predetermined cell from a candidate cell group, a technique utilizing the action of a temperature-responsive polymer has been studied. For example, Patent Document 1 describes a plurality of cell adhesion regions in which a temperature-responsive polymer whose cell adhesion state and cell non-adhesion state change in response to temperature is fixed on the surface, and a plurality of cells. Using a cell array sorter equipped with a heat generating element provided corresponding to each of the adhesion regions, the process of adhering cells to cell adhesion spots and culturing cells with the heat generating element heated, and the characteristics of the cells A cell sorting method including a step of evaluating and identifying cells to be collected and a step of stopping the heat generation of the heat generating element corresponding to the cell adhesion spot and collecting the cells is disclosed (claim 18 and the like). reference).

Japanese Unexamined Patent Publication No. 2011-101626

By using a temperature-responsive polymer as in the technique described in Patent Document 1, it is possible to freely control the adhesion and release of cells. At this time, since a reagent such as trypsin is not required to exfoliate the cells adhering to the incubator, it is possible to prevent the candidate cells from being damaged by the reagent or the like. That is, if a temperature-responsive polymer is used, there is a possibility that a predetermined cell can be easily and safely selected from the candidate cell group. However, when selecting cells with high production ability of useful substances from the gene-introduced candidate cell group, the isolation operation of separating a single cell from the candidate cell group and the production ability of useful substances are evaluated. It is also necessary to carry out a culture operation in which candidate cells are proliferated to the extent possible, and a measurement operation in which the number of cells, the amount of production of useful substances, etc. are measured.

The number of candidate cells that are subjected to a gene transfer operation and subsequently recovered is usually in the majority. Therefore, when comprehensive screening is required, there arises a problem that labor required for each operation such as cell isolation and the amount of culture medium used for cell culture become enormous. In addition, each operation must be performed aseptically, and each time a cell is isolated, it takes a long time to culture. As a result, this type of screening is inefficient in terms of time and cost.

In addition, when measuring the production amount of useful substances, when sampling some cells, the equivalence between the cells whose production ability is evaluated by sampling and the cells actually used for the production of useful substances is guaranteed. There is a need to. Therefore, it is often necessary to perform an additional culturing operation for culturing a plurality of lines or an operation for introducing a marker gene in advance. These additional operations also contribute to reducing the efficiency of screening.

In addition, in recent years, among useful substances, the demand for manufacturing antibody drugs has been increasing. Floating cells such as CHO (Chinese Hamster Ovary) cells have also come to be used for the production of antibodies such as chimeric antibodies, humanized antibodies, and human antibodies. Therefore, unlike the technique described in Patent Document 1, a screening means corresponding to the selective separation and recovery of suspended cells is also required.

Therefore, an object of the present invention is to provide a cell separation device and a cell separation method capable of selecting cells having a high antibody-producing ability from a candidate cell group with less damage by a simple step.

In order to solve the above problems, the cell separator according to the present invention is a cell separator that selectively separates and recovers antibody-producing cells from a plurality of candidate cells, and an incubator having a plurality of wells can be installed. A seeding means for seeding candidate cells into a culture solution held in each of the plurality of wells, a temperature control means for controlling the temperature of the culture solution, and holding in the plurality of wells. A means for adding a reagent for adding an antimembrane protein antibody to each of the culture broths and a means for collecting the culture broth held in each of the plurality of wells are provided, and the wells of the incubator have. On the inner wall surface, a protein binding site composed of a molecule capable of binding to an antibody and a three-dimensional structure reversibly changed by a temperature change across the phase transition temperature, changing the binding affinity between the protein binding site and the antibody. A temperature-responsive site composed of a molecular chain is bound, the seeding means seeds candidate cells expressing a membrane protein in each of the plurality of wells, and the reagent addition means is the seeded candidate. An anti-membrane protein antibody capable of binding to the membrane protein is added to the culture solution in which the cells are cultured, and the temperature control means sets the liquid temperature of the culture solution held in each of the plurality of wells as the candidate. The culture temperature of the cells is changed to a temperature exceeding the phase transition temperature of the temperature-responsive site, and the recovery means collects the culture solution held in each of the plurality of wells and seeds the candidate cells. Among them, antibody-producing cells that are not bound to the protein-binding site and are suspended in the culture solution are selectively selected, leaving non-antibody-producing cells bound to the protein-binding site via the antimembrane protein antibody. It is characterized in that it is separated and collected.

The cell separation method according to the present invention is a cell separation method that selectively separates and recovers antibody-producing cells from a group of candidate cells, and has a protein binding site composed of a molecule capable of binding to an antibody and a phase transition. A plurality of wells in which a temperature-responsive site composed of a molecular chain whose three-dimensional structure is reversibly changed by a temperature change across temperatures and changes the binding affinity between the protein binding site and the antibody is bound to the inner wall surface. Candidate cells expressing the membrane protein are seeded in each, the seeded candidate cells are cultured, and an antimembrane protein antibody capable of binding to the membrane protein is added to the culture solution held in each of the plurality of wells. Upon addition, the temperature of the culture solution held in each of the plurality of wells is changed from the culture temperature of the candidate cells to a temperature exceeding the phase transition temperature of the temperature-responsive site so that the candidate cells can be added. The expressed membrane protein and the antimembrane protein antibody are bound, and the protein binding site and the antimembrane protein antibody, and the protein binding site and the antibody produced by the candidate cell are bound, respectively. , The culture solution held in each of the plurality of wells is collected, and among the seeded candidate cells, antibody-producing cells that are bound to the protein binding site via the antimembrane protein antibody are left. Therefore, the antibody-producing cells that are not bound to the protein binding site and are suspended in the culture solution are selectively separated and recovered.

According to the present invention, it is possible to provide a cell separation device and a cell separation method capable of selecting cells having a high antibody-producing ability from a candidate cell group with less damage by a simple step.

It is a block diagram which shows the schematic structure of the cell separation apparatus which concerns on one Embodiment of this invention. It is a figure which shows an example of the shape of an incubator. It is a figure which shows schematic by enlarging the inner wall surface of the well which an incubator has. It is a figure which shows the principle of the cell separation method which concerns on one Embodiment of this invention. It is a figure which shows the principle of the cell separation method which concerns on one Embodiment of this invention.

The cell separation device and the cell separation method according to the embodiment of the present invention will be described below. The same reference numerals are given to common configurations in the following figures, and duplicate description will be omitted.

FIG. 1 is a block diagram showing a schematic configuration of a cell separation device according to an embodiment of the present invention.
As shown in FIG. 1, the cell separation device 100 according to the present embodiment includes an operation room (operation unit) 101, a heat block (temperature control means) 102 for an incubator, a pipette device (sewing means) 103, and suction. It includes a nozzle (recovery means) 104, a culture solution recovery tank 105, a supply system (reagent addition means) 106, a culture chamber 107, a gas exchange device 108, and a temperature control device 109.

The cell separation device 100 can selectively separate and collect cells having the ability to produce an antibody (antibody-producing cells) from a plurality of candidate cells (candidate cell groups) whose antibody-producing ability is unknown. It is a device. Specifically, as the candidate cell, a cell population or the like that has undergone a gene transfer operation for introducing an antibody gene or the like is provided.

The gene transfer operation is an operation for introducing a desired gene into a target host cell, and is generally performed by targeting a large number of cells. The ability to produce antibodies due to the number of copies of the introduced gene, the way the gene is integrated, the gene mutation, etc., among the large number of cells recovered after the gene transfer operation such as antibody genes There is a possibility that cells with high antibody, cells with low ability to produce antibody, cells lacking ability to produce antibody (antibody non-producing cells), etc. are mixed.

The cell separator 100 selects a large number of such cells collected after the gene transfer operation, and produces cells having the ability to produce an antibody based on the antibody-producing ability of each cell and an antibody. Select cells with high ability. The cell separation device 100 is particularly suitable for primary screening of a relatively large number of candidate cells collected immediately after the step of gene transfer operation.

The operation room 101 of the cell separation device 100 is provided so that an incubator P having a plurality of wells can be installed. The candidate cell group to be selected is cultured in each of the plurality of wells, and undergoes various operations in the operation room 101 to be selected based on the individual antibody-producing ability. Here, the incubator P and the principle of the cell separation method using the incubator P will be described.

FIG. 2 is a diagram showing an example of the shape of the incubator. FIG. 2A is a perspective view showing an example of the shape of the incubator, and FIG. 2B is a view showing a well included in the incubator.
As shown in FIG. 2A, the incubator P is in the form of a so-called well plate. The incubator P has a plurality of wells 10 on the upper surface of the flat plate-shaped substrate 9. The wells 10 are provided with the same shape and the same dimensions as each other, and are arranged in a regular arrangement on the upper surface of the substrate 9.

The incubator P can be installed in the operation chamber 101 of the cell separation device 100, and has a size suitable for the heat block 102 provided in the operation chamber 101. As an example, the incubator P having the form shown in FIG. 2A is provided with a length (L) of 130 mm, a width (W) of 85 mm, and a height (H) of 10.5 mm. The number of wells 10 formed on the upper surface of the substrate 9 is 96 holes of 8 × 12. The upper surface of the substrate 9 can be covered with a lid (not shown) that prevents contamination inside the well 10.

The well 10 is recessed as a cylindrical small hole on the upper surface of the substrate 9. The well 10 of the form shown in FIG. 2B is provided with an inner diameter (φ) of 3 mm and a depth (D) of 9.3 mm as an example. The bottom wall 120a of each well 10 is parallel to the upper and lower surfaces of the substrate 9. The inner wall (1) of each well 10 of the incubator P, that is, the bottom wall 120a and the side wall 120b, is surface-modified to enable selective separation and recovery of antibody-producing cells. That is, the bottom wall 120a and the side wall 120b are surface-modified molecular supports (1) described later (see FIG. 3).

FIG. 3 is an enlarged view schematically showing the inner wall surface of the well of the incubator. FIG. 3 (a) is a diagram showing an inner wall surface in a state in which surface-modified molecules are compatible with the aqueous phase, and FIG. 3 (b) is a diagram showing an inner wall surface in a state in which surface-modified molecules are phase-separated. It is a figure which shows.
As shown in FIGS. 3A and 3B, the inner wall (support) 1 of each well 10 of the incubator P has a protein binding site 2, a temperature-responsive site 3, and a hydrophilic site 4. Are combined.

The protein binding site 2, the temperature responsive site 3 and the hydrophilic site 4 are usually immobilized only on the bottom wall 120a (see FIG. 2) of each well 10, but may be immobilized only on the side wall 120b. However, it may be fixed to both the bottom wall 120a and the side wall 120b.

The protein binding site 2 is composed of a molecule having a binding ability to an antibody. Specifically, the binding ability of the protein binding site 2 is specific to an antibody and difficult to bind to a substance other than an antibody. As will be described later, the protein binding site 2 specifically binds to an antibody produced by antibody-producing cells, an anti-membrane protein antibody added to a culture solution, and the like. Then, it exhibits an action of capturing non-antibody-producing cells bound to the anti-membrane protein antibody so as to be attached to the inner wall (1) of the well 10 from the floating state in the aqueous phase.

The temperature-responsive site 3 is composed of a molecular chain whose three-dimensional structure is reversibly changed by a temperature change across the phase transition temperature. Specifically, the temperature-responsive site 3 has a highly hydrophilic and soluble state that is compatible with the aqueous phase and an aggregated state that is highly hydrophobic and phase-separated from the aqueous phase as the temperature changes over the phase transition temperature. A phase transition can be reversibly made between and.

The changes in hydrophilicity and hydrophobicity of the temperature-responsive portion 3 may be such that the measured values are displaced to the extent that the three-dimensional structure is reversibly changed. Specifically, as the measured values for determining hydrophilicity and hydrophobicity, solubility in water, acid dissociation constant, distribution ratio between aqueous phase and organic phase, contact angle and the like can be used. The distribution ratio is generally expressed by dividing the concentration of a substance dissolved in the octanol phase by the concentration of the substance dissolved in the aqueous phase in a two-phase system of an octanol phase and an aqueous phase.

The temperature-responsive portion 3 has a predetermined stable three-dimensional structure as shown in FIG. 3A when it is compatible with the aqueous phase. At this time, the main chain of the temperature-responsive site 3 is oriented from the inner wall (1) of the well 10 toward the aqueous phase side, and the protein binding site 2 is exposed to the aqueous phase side and has a binding affinity for a ligand, that is, an antibody. Becomes high. On the other hand, in the phase-separated state, as shown in FIG. 3 (b), it aggregates on the inner wall (1) side of the well 10. At this time, the protein binding site 2 causes steric hindrance and has a low binding affinity for the antibody. That is, the temperature-responsive site 3 mutually changes the binding affinity between the protein binding site 2 and the antibody due to the change in the three-dimensional structure accompanying the phase transition.

Generally, as a temperature-responsive polymer, a polymer showing a lower critical solution temperature (LCST) and a polymer showing an upper critical solution temperature (UCST) are known. .. A polymer showing the lower limit critical solution temperature shows hydrophilicity and dissolves in the aqueous phase in the low temperature range lower than the lower limit critical solution temperature, and shows hydrophobicity and insolubilizes in the high temperature range higher than the lower limit critical solution temperature for phase separation. Has the property of On the other hand, the polymer showing the upper limit critical solution temperature shows hydrophilicity and dissolves in the aqueous phase in the high temperature range higher than the upper limit critical solution temperature, and shows hydrophobicity and insolubilizes in the low temperature range lower than the upper limit critical solution temperature. Has the property of phase separation. As the temperature-responsive portion 3, any of the polymer showing the lower limit critical solution temperature and the polymer showing the upper limit critical solution temperature can be applied.

The hydrophilic site 4 is composed of a molecule or a molecular chain having a hydrophilic group. The hydrophilic site 4 is bound to the temperature responsive site 3 as a side chain in FIG. When the temperature-responsive site 3 is in a state of being compatible with the aqueous phase (see FIG. 3A), the hydrophilic site 4 is oriented so as to surround the main chain of the temperature-responsive site 3. That is, by forming an appropriate hydration state, the three-dimensional structure of the temperature-responsive site 3 is stabilized, and the binding affinity of the protein binding site 2 for the antibody is improved. On the other hand, when the temperature-responsive portion 3 is in a phase-separated state (see FIG. 3 (b)), it is distributed in the surroundings so as to cover the temperature-responsive portion 3 in the aggregated state. Therefore, it exhibits an action of preventing the antibody or the like from non-specifically adsorbing to the temperature-responsive site 3 in the aggregated state through hydrophobic interaction.

In the operation room 101 of the cell separation device 100 shown in FIG. 1, a plurality of wells 10 in which such a protein binding site 2, a temperature-responsive site 3, and a hydrophilic site 4 are immobilized on an inner wall surface are used. Then, the antibody-producing cells are selected from the candidate cell group. The cell separation method carried out in the cell separation apparatus 100 specifically selects antibody-producing cells through a cell seeding step, a cell culture step, an antibody addition step, a temperature stimulation step, and a culture solution recovery step. It is to be separated and collected.

4 and 5 are diagrams showing the principle of the cell separation method according to the embodiment of the present invention. 4 (a) and 4 (b) are states in which candidate cells are seeded in wells, FIGS. 4 (c) and 4 (d) are states in which seeded candidate cells are cultured, and FIGS. 5 (a) and 5 (b) are shown. , A state in which the temperature-responsive site is phase-transferred and an anti-membrane protein antibody is added, and FIGS. 5 (c) and 5 (d) are diagrams showing a state in which the anti-membrane protein antibody, the protein binding site, and the like are bound to each other. ..

Note that FIGS. 4 (a) and 4 (c) and 5 (a) and 5 (c) show the process in a certain well 10 in chronological order, and FIGS. 4 (b) and 4 (d) show the process. , And FIGS. 5 (b) and 5 (d) show the process in the well 10 different from that in chronological order. 4 (a), 4 (c), 5 (a), 5 (c) show well 10, where non-antibody-producing cells are present, FIG. 4 (b), FIG. 4 (d), FIG. 5 (b) and FIG. 5 (d) correspond to wells 10 in which antibody-producing cells are present, respectively. The temperature-responsive site 3 is a lower limit critical solution that separates from the aqueous phase in a culture temperature range such as around 37 ° C and is compatible with the aqueous phase in a low temperature range lower than the culture temperature of about 20 ° C to 30 ° C. The molecular chain indicating the temperature is illustrated.

In the cell seeding step, as shown in FIGS. 4A and 4B, candidate cells C expressing the membrane protein P10 are seeded in each of the plurality of wells 10. Each well 10 has a protein binding site 2 composed of a molecule capable of binding to an antibody, and the three-dimensional structure is reversibly changed by a temperature change across the phase transition temperature, and the binding affinity between the protein binding site 2 and the antibody is changed. The temperature-responsive site 3 made of a molecular chain that can be allowed to be formed is previously immobilized on the inner wall surface.

In the cell seeding step, in detail, candidate cells C expressing the membrane protein P10 and a predetermined culture medium are added to each well 10. For example, cells are collected after a gene transfer operation for introducing an antibody gene or the like, and a predetermined amount of a cell suspension of a large number of collected candidate cells C is taken and injected into each well 10. In addition, a predetermined amount of culture solution is injected into each well 10. At this time, the liquid temperature in the well 10 is preferably set to a temperature at which the temperature-responsive portion 3 is in a phase-separated state, that is, a high temperature range higher than the lower limit critical solution temperature.

As the candidate cell C, for example, a cell subjected to a gene transfer operation to produce a monoclonal antibody or the like exhibiting a desired specificity, and a floating cell exemplified as a CHO cell, a HEK293 cell, a HeLa cell or the like is seeded. .. The number of cells seeded per single well is preferably a single cell in principle from the viewpoint of accurately evaluating the antibody-producing ability. That is, individual candidate cells C are seeded in a large number of wells 10 according to the number of cells to be selected.

The membrane protein P10 expressed by the candidate cell C is a protein having an extracellular domain capable of an antigen-antibody reaction with a corresponding anti-membrane protein antibody having a good binding affinity, a glycoprotein, or the like. There are no restrictions on the functions. It may be a membrane protein naturally expressed on the cell surface of candidate cell C at the time of separation and recovery, or it may be a membrane protein artificially expressed by gene modification. Further, the membrane protein may be derived from an appropriate species such as human and mouse. Specific examples of the membrane protein include MHC class I, MHC class II, CD29, CD40, CD44, CD90, CD105, CD166 and the like.

As the culture medium, an appropriate growth medium can be used depending on the cell type of the candidate cell C. However, from the viewpoint of accurately evaluating the antibody-producing ability, it is preferable that the culture medium does not contain the antibody in advance, and it is preferable to use a synthetic medium containing no antibody, a serum-free medium, or the like. For example, when the candidate cell C is a CHO cell, an antibody-free CD-CHO medium or the like is preferably used.

Subsequently, in the cell culture step, as shown in FIGS. 4 (c) and 4 (d), candidate cells C seeded in each of the plurality of wells 10 are cultured. When the seeded candidate cell C does not have the ability to produce an antibody, the well 10 remains in a state where the antibody is not present in the culture medium, as shown in FIG. 4 (c). On the other hand, when the seeded candidate cell C has the ability to produce an antibody, the well 10 shows an antibody showing a desired specificity in the culture medium, as shown in FIG. 4 (d). Ab is produced.

In the cell culture step, in detail, the candidate cell C is cultured in a culture temperature range that does not exceed the phase transition temperature of the temperature-responsive site 3, for example, around 37 ° C. Further, by supplying a gas containing carbon dioxide, the incubator P is maintained in a carbon dioxide gas atmosphere having a predetermined concentration of about 5%. Further, the incubator P may be shaken, or the humidity may be adjusted to an atmosphere of a predetermined humidity. The culturing time can be set to an appropriate time as long as the seeded candidate cells C proliferate to an extent in which the antibody-producing ability can be evaluated. For example, it is preferably 3 days or more and 10 days or less, and more preferably 3 days or more and 5 days or less in that the antibody is sufficiently produced and the proportion of living cells is kept high.

Subsequently, in the antibody addition step, as shown in FIGS. 5 (a) and 5 (b), the membrane protein P10 in which the candidate cell C is expressed in the culture medium held in each of the plurality of wells 10 is specific. Anti-membrane protein antibody A1 capable of binding to is added. When the seeded candidate cell C does not have the ability to produce an antibody, the well is in a state in which only the anti-membrane protein antibody A1 is present as an antibody, as shown in FIG. 5 (a). On the other hand, when the seeded candidate cell C has the ability to produce an antibody, the well is added with the antibody Ab having the desired specificity, as shown in FIG. 5 (b). It becomes a state in which the anti-membrane protein antibody A1 is mixed. That is, the binding of the antibody to the protein binding site 2 competes between the produced antibody Ab and the added anti-membrane protein antibody A1.

In the antibody addition step, in detail, a predetermined amount of the anti-membrane protein antibody A1 is added to each well 10 so as to be equivalent. If the amount of the anti-membrane protein antibody A1 added is insufficient, sufficient competition with the produced antibody Ab cannot occur. On the other hand, if the amount added is excessive, non-specific adsorption may occur or specific interaction may be inhibited. Therefore, the specific addition amount for each well 10 should be an appropriate amount in consideration of the number of proliferated candidate cells C, the expression level of the membrane protein P10, the binding amount of the protein binding site 2, and the like. Is preferable.

The anti-membrane protein antibody A1 specifically binds to the membrane protein P10 expressed by the candidate cell C by an antigen-antibody reaction, while reacting to the inner wall (1) of the well 10 and the temperature-responsive site 3 and the like. Is preferably an antibody that is difficult to bind. Specifically, the anti-membrane protein antibody A1 may be F (ab) ₂ , F (ab') _2, or the like, but from the viewpoint of ensuring the binding property to each of the membrane protein P10 and the protein binding site 2. From the above, an immunoglobulin having a Fab region and an Fc region is preferable. As the anti-membrane protein antibody A1, a plurality of types corresponding to each of the plurality of types of membrane proteins P10 expressed by the candidate cell C may be added, but it is preferable to add one type of monoclonal antibody.

In the temperature stimulation step, as shown in FIGS. 5A and 5B, the temperature of the culture solution held in each of the plurality of wells 10 is adjusted from the culture temperature of the candidate cell C seeded and proliferated. The temperature-responsive site 3 undergoes a phase transition by changing the temperature to a temperature that exceeds the phase transition temperature of the responsive site 3. That is, when the temperature-responsive site 3 shows the lower limit critical solution temperature, the culture solution ranges from a culture temperature range such as around 37 ° C to a low temperature range exceeding the phase transition temperature of the temperature-responsive site 3, for example, around 20 ° C. Reduce the liquid temperature of. Then, the temperature-responsive site 3 is changed to a state of being compatible with the aqueous phase, and the protein binding site 2 is made capable of binding to the antibody.

After going through the antibody addition step and the temperature stimulation step, as shown in FIGS. 5C and 5D, the membrane protein P10 expressing the candidate cell C and the added anti-membrane protein antibody A1 are bound to each other. be able to. In addition, along with such binding, the protein binding site 2 immobilized on the well 10 and the added anti-membrane protein antibody A1, and the protein binding site 2 immobilized on the well 10 and the candidate cell C are produced. Each of these antibodies can be bound to the antibody Ab.

Then, when the candidate cell C does not have the ability to produce an antibody, only the anti-membrane protein antibody A1 is specific to the protein binding site 2 in the well 10 as shown in FIG. 5 (c). It becomes a state of being combined with. That is, the non-antibody-producing cells bind to the protein binding site 2 immobilized on the inner wall (1) of the well 10 via the anti-membrane protein antibody A1 bound to the membrane protein P10, and from the suspended state in the aqueous phase. It is captured by the state of adhesion to the inner wall (1).

On the other hand, when the candidate cell C has the ability to produce an antibody, the well 10 is produced by the candidate cell C with respect to the protein binding site 2 as shown in FIG. 5 (d). The antibody Ab and the added anti-membrane protein antibody A1 are in a state of competing and binding. That is, although the anti-membrane protein antibody A1 capable of binding to the membrane protein P10 expressed by the antibody-producing cell can specifically bind to some protein binding sites 2, many protein binding sites 2 The antibody Ab, which is produced by antibody-producing cells and has the property of not binding to the membrane protein P10, specifically binds to the protein. As a result, the antibody-producing cells can bind to the protein binding site 2 immobilized on the inner wall (1) of the well 10 even though the anti-membrane protein antibody A1 is bound to the membrane protein P10. Instead, it remains suspended in the culture solution.

The antibody addition step and the temperature stimulation step can be performed regardless of whether the step is prior or subsequent. Further, the antibody addition step and the temperature stimulation step may be performed substantially at the same time. However, from the viewpoint of precisely competing for antibody binding to the protein binding site 2, it is preferable to perform the temperature stimulation step after performing the antibody addition step. The binding reaction time of the anti-membrane protein antibody A1 and the protein binding site 2 is preferably about 15 minutes or more and 5 hours or less, and preferably about 1 hour or more and about 3 hours or less. Since the anti-membrane protein antibody A1 added in an appropriate amount has a specific binding ability to the membrane protein P10, it binds to most of the candidate cells C present in each well 10 after a predetermined reaction time. ..

Subsequently, in the culture solution recovery step, the culture solution held in each of the plurality of wells 10 is collected and bound to the protein binding site 2 of the seeded candidate cells C via the antimembrane protein antibody A1. Selectively select antibody-producing cells (see FIG. 5 (d)) that are not bound to the protein binding site 2 and are suspended in the culture medium, leaving the non-antibody-producing cells (see FIG. 5 (c)). Separate and collect. For example, when the culture solution of each well 10 is sucked with a weak force, only the antibody-producing cells remaining in the floating state in the culture solution are collected together with the culture solution, whereas the inner wall (1) of the well 10 is recovered. Non-antibody-producing cells that are in an attached state are eliminated without being recovered.

In the culture broth recovery step, if the culture broth recovered from one of the plurality of wells 10 contains a large number of cells, the cells existing in the well 10 are the protein binding sites. It means that the antibody-producing cells were suspended without being captured by 2. In the cell separation device 100 shown in FIG. 1, based on the selection principle using such an incubator P, the antibody-producing cells are semi-automatically separated and recovered from a plurality of candidate cells.

The operation room 101 included in the cell separation device 100 is a sterile room in which the incubator P can be carried in and out. The inside of the operation chamber 101 is aseptically supplied and exhausted through a HEPA filter or the like. The operation room 101 is provided with a heat block (temperature controlling means) 102 for an incubator, a pipette device (sowing means) 103, and a suction nozzle (collecting means) 104. The operation of the heat block 102, the pipette device 103, the suction nozzle 104, and the like is automatically controlled by the controller. Further, the operation room 101 is provided with a germicidal lamp, a steam sterilizer, and the like capable of sterilizing the room for each operation.

The heat block 102 makes each well 10 of the incubator P heatable and coolable by a Peltier element. The heat block 102 is provided with a guide according to the shape and size of the incubator P, and the incubator P is detachably attached to the heat block 102 along the guide. The heat block 102 can regulate the liquid temperature of the well 10 from at least the culture temperature range of the candidate cells to a temperature exceeding the phase transition temperature of the temperature-responsive site 3. Further, the heat block 102 can be provided so as to be swingable, and the attached incubator P is shake-cultured as necessary.

The pipette device 103 has a mobile nozzle to which a disposable tip can be attached, and is provided so that a fixed amount of liquid can be automatically sucked and discharged. The pipette device 103 is additionally provided with a tip rack. A chip having a capacity of about several hundred μL prepared in the chip rack is mounted by being press-fitted into the nozzle. Then, the pipette device 103 to which the tip is mounted moves in the operation chamber 101 to inject a predetermined reagent into each well 10 of the incubator P, and the used tip is replaced every operation.

The suction nozzle 104 is movably provided with respect to the wells 10 of the incubator P so that the liquid held in each well 10 can be automatically sucked. The suction nozzle 104 includes, for example, multiple nozzles in which a plurality of nozzles are linearly arranged in parallel corresponding to the row or column of the well 10 included in the incubator P. Each nozzle is individually communicated with the culture solution recovery tank 105 via a tube or the like, and the culture solution or the like sucked through the nozzle is individually collected in the culture solution recovery tank 105.

The supply system 106 can aseptically supply reagents such as cell suspension 111, medium reagent 112, anti-membrane protein antibody reagent 113, and pH adjustment reagent 114, which have been carried in and kept cold, to the operation room 101. .. The reagents supplied to the operation chamber 101 are automatically opened and sucked into the pipette device 103. Then, after the solution is injected into the well 10 of the incubator P, it is closed and re-stored. Alternatively, each reagent is aseptically sent to the pipette device 103 provided in the operation chamber 101 by a perista pump, a membrane pump, or the like. There may be a heat retaining mechanism in the process of sending the reagents supplied to the operation room 101 to the well 10 of the incubator P.

The culture chamber 107 is a sterile chamber in which the incubator P can be carried in and out of the operation chamber 101. A door that can be opened and closed is provided between the culture chamber 107 and the operation chamber 101, and operations, cultures, etc. in each chamber are performed in a state of being isolated from each other. In the culture chamber 107, a gas exchange device 108 for exchanging atmospheric gas for carbon dioxide gas or the like, a temperature control device 109, a humidifying dish, a temperature sensor, a humidity sensor, an incubator rack on which the incubator P is placed, and the like are installed. .. The culture chamber 107 is provided so that the temperature and humidity can be controlled, and the atmosphere is adjusted to a carbon dioxide gas atmosphere having a predetermined concentration, and the incubator P carried in is cultured in a predetermined atmosphere. ..

The cell separation device 100 includes a control unit (not shown). The control unit controls a display unit that displays the operation status, the state of the culture chamber 107, etc., an input unit consisting of a keyboard, a mouse, etc. for inputting instructions by the operator, and automatic operations related to separation and collection and culture. It is configured to include a CPU, ROM, RAM, memory, etc. for storing and executing programs and the like. When the operator supports the start of separation and recovery, the control unit sets the heat block 102, the pipette device 103, the suction nozzle 104, the gas exchange device 108, the temperature control device 109, and the incubator P according to the program stored in the ROM. Controls the robot arm, etc. that transports the

In the separation and recovery of antibody-producing cells in the cell separation device 100, first, the incubator P that has been sterilized in advance is attached to the heat block 102 provided in the operation room 101. Then, the pipette device 103 injects a predetermined amount of the medium reagent 112 supplied from the supply system 106 into each well 10 of the incubator P under the control of the control unit. Further, the pipette device 103 sucks a predetermined amount of the cell suspension 111 in which the candidate cell group is suspended, and seeds the candidate cell C in each of the plurality of wells 10 of the incubator P. The number of candidate cells C seeded per well 10 is adjusted by, for example, the suction amount and the discharge amount of the cell suspension 111.

Next, the incubator P is covered with a lid by a robot arm or the like and transported to the culture chamber 107. The atmosphere of the culture chamber 107 is adjusted to a culture temperature range such as around 37 ° C. and a predetermined gas atmosphere according to the control by the control unit. Then, the candidate cells C seeded in each well 10 are cultured in a predetermined culture temperature range and a gas atmosphere until a predetermined number of cells is reached. After that, the incubator P is transported to the operation chamber 101 again and reattached to the heat block 102 maintained in a temperature range not exceeding the phase transition temperature of the temperature responsive site 3.

Next, the pipette device 103 adds the anti-membrane protein antibody reagent 113 supplied from the supply system 106 to the culture medium in which the candidate cells are cultured under the control of the control unit. The anti-membrane protein antibody reagent 113 is a solution of a phosphate buffer solution or the like in which an anti-membrane protein antibody A1 capable of binding to a membrane protein expressed by a candidate cell is dissolved together with a salt such as NaCl or an antiseptic. ..

Next, the heat block 102 changes the temperature of the culture solution held in each of the plurality of wells 10 of the incubator P from the culture temperature of the candidate cells to the phase transition of the temperature-responsive site 3 under the control of the control unit. Change to a temperature that exceeds the temperature. Then, the temperature of the culture solution is maintained at a temperature at which the temperature-responsive site 3 is phase-separated, and the antibody-producing cells (see FIG. 5 (c)) are subjected to the protein binding site 2 via the anti-membrane protein antibody A1. To combine with. That is, the non-antibody-producing cells are captured in a state of being attached to the inner wall (1) of the well 10. At this time, the pipette device 103 may add the pH adjusting reagent 114 supplied from the supply system 106 to the culture solution and operate it so as to promote the phase transition by pH stimulation, if necessary.

Next, the suction nozzle 104 collects the culture solution held in each of the plurality of wells 10 under the control of the control unit, and among the seeded candidate cells, the anti-membrane protein antibody A1 is applied to the protein binding site 2. The antibody-producing cells that are not bound to the protein binding site 2 and are suspended in the culture solution are selectively separated and recovered, leaving the antibody-producing cells that are bound via the cells. The culture broth held in each well 10 is sucked in the same amount by each nozzle of the suction nozzle 104 at the same time, and is collected in the culture broth recovery tank 105 for each well through a tube or the like.

The number of cells in the collected culture medium is measured by microscopic observation, turbidity measurement, and the like. Of the collected culture medium, antibody-producing cells are contained in the culture medium in which a certain number or more of floating cells are present. When the separation / recovery operation is completed, the control unit displays the measurement result or the like on the display unit and ends the control program.

The incubator P mounted on the cell separation device 100 can be in an appropriate form as long as the principle of the above cell separation method is not impaired.

The incubator P is provided in a rectangular flat plate shape in FIG. However, the shape of the incubator P can be provided in other shapes such as a disk shape and an elliptical plate shape. Further, the incubator P can be provided with appropriate dimensions such as, for example, a thickness of 50 cm square or more and 200 cm square or less and 5 mm or more and 20 mm or less.

The incubator P can be formed of, for example, an organic material such as resin, an inorganic material such as glass, or an appropriate material such as a metal material. In the incubator P, the substrate 9 and the wells 10 may be formed by a combination of these various materials, but usually, the incubator P is integrally molded in a state in which a plurality of wells 10 are recessed. Resin materials and glass materials are preferable from the viewpoint of moldability of the material and modification of other molecules by covalent bonds. Therefore, the incubator P, that is, the well 10, may be formed of at least one of the resin material and the glass material. The incubator P may be either light-shielding or translucent (transparent), or may have spectroscopic transparency with respect to ultraviolet rays and the like.

Examples of the glass material applied to the incubator P include borosilicate glass and soda lime glass. Examples of the resin material applied to the incubator P include polystyrene, polycarbonate, polymethylmethacrylate, polyethylene terephthalate, polybutylene terephthalate, polyethylene, polypropylene, ethylene-propylene copolymer, polymethylpentene, and polytetrafluoroethylene. Examples thereof include polyacrylonitrile, acrylonitrile-butadiene-styrene copolymer, agarose, vinyl chloride, polyarylate, polysulfone, polyether sulfone, polyether ether ketone, polyetherimide and the like.

In FIG. 2, the wells 10 of the incubator P are recessed in a cylindrical shape, and the bottom wall 120a of each well 10 is parallel to the upper surface and the lower surface of the base body 9. However, the shape of the well 10 may be another shape such as a rectangular parallelepiped shape, a cube shape, or an elliptical pillar shape. Further, the bottom wall 120a of the well 10 may be inclined in one direction, or may be inclined in multiple directions such as a V shape. Further, the bottom wall 120a of the well 10 may have a hemispherical or U-shaped curvature so that the bottom wall 120a and the side wall 120b are connected by a curved surface. Further, it may be flat or porous, or a pillar-shaped or hemispherical structure may be arranged. The well 10 can be provided with appropriate dimensions such as an inner diameter of 2 mm or more and 10 mm or less and a depth of 4 mm or more and 14 mm or less.

The number of wells 10 included in the incubator P is 96 holes of 8 × 12 in FIG. However, for example, the number may be an appropriate number such as 48 holes such as 6 × 8, 112 holes such as 8 × 14, and 384 holes such as 16 × 24. In the preferred form of cell isolation method, a single candidate cell is seeded in a single well and the selected antibody-producing cells are harvested in units of 10 wells. Therefore, exhaustive screening is achieved by the total number of wells 10 rather than the number of wells 10 per incubator P. Therefore, the plurality of wells 10 may be covered by a plurality of incubators P.

The inner wall surface of the well 10 of the incubator P is preferably hydrophilized by surface treatment. This is to prevent the antibody produced by the cell and the anti-membrane protein antibody from being non-specifically adsorbed on the inner wall surface. The surface treatment may be a blocking agent such as bovine serum albumin (BSA), a physical treatment such as ultraviolet treatment or plasma treatment, or a coating such as vapor deposition. It may be a thing. Moreover, these processes may be combined.

As the protein binding site 2 immobilized on the well 10, a molecule having a specific binding ability to the constant region of immunoglobulin is preferable. As long as the protein binding site binds to the constant region of immunoglobulin, the entire antibody can be captured by the protein binding site 2 regardless of the specific cognitive ability of the antibody. Therefore, the anti-membrane protein antibody that binds to the membrane protein expressed by the candidate cell via the variable region can also be captured by the protein binding site 2. As a result, the protein binding site 2 makes it possible to reliably capture non-antibody-producing cells with high binding affinity, and the accuracy of selective separation and recovery of antibody-producing cells is enhanced.

Examples of the molecule constituting the protein binding site 2 include protein A, protein G, Fc receptor, anti-antibody (anti-IgG antibody) and the like, peptide fragments containing these binding domains, variants thereof, and binding thereof. Examples thereof include mimetics that mimic the ability and have the ability to bind to antibodies. The mimetic having the binding ability to the antibody may be either a peptide mimetic or a non-peptide mimetic.

Specific examples of the peptide fragment containing a binding domain include a fragment containing a dipeptide of Protein A derived from Staphylococcus aureus, Phe132-Tyr133. Specific examples of peptide mimetics include TG-19318, TG-19320 and the like. Specific examples of non-peptide mimetics include 22/8, TPN-BM, A2P, B14 and the like. As the molecule constituting the protein binding site 2, protein A, domain A of protein A, domain B, domain C, domain D, domain E or a variant thereof, or a mimetic capable of binding to an antibody is particularly preferable. ..

The protein binding site 2 is bound to the end of the main chain of the temperature responsive site 3 in FIGS. 3, 4 and 5. When the temperature-responsive site 3 is in a state of being compatible with the aqueous phase by binding to the end of the main chain of the temperature-responsive site 3 (see FIG. 3A), it is produced or added to the aqueous phase. The interaction between the antibody to be produced and the protein binding site 2 is likely to occur. On the other hand, when the temperature-responsive site 3 is in a phase-separated state (see FIG. 3 (b)), a large steric disorder occurs in the protein binding site 2 as compared with the state in which it is compatible with the aqueous phase. Since it becomes easy, the antibody in the aqueous phase is prevented from being trapped in the protein binding site 2. However, the protein binding site 2 may be directly bound to the inner wall surface of the well 10 or may be bound as a side chain of the temperature responsive site 3. Further, it may be bonded to a plurality of these bonding positions.

The binding between the protein binding site 2 and the inner wall surface of the well 10 or another molecule may be either covalent or non-covalent. For example, amide bond, ester bond, thioester bond, imine bond, siloxane bond, maleimide group bond, Diels-Alder reaction bond, click reaction bond, gold and thiol group bond and the like can be mentioned. Further, the temperature-responsive site 3 may be directly bound to the inner wall surface of the well 10 or another molecule, or may be indirectly bound via a linker. For example, various silane coupling agents, a bond between biotin and avidin, a bond between biotin and streptavidin, and the like can be used.

A preferred form of the temperature-responsive site 3 is a form having a molecular chain indicating the lower limit critical solution temperature. The temperature-responsive portion 3 showing the lower limit critical solution temperature preferably has a phase transition temperature in the range of 0 ° C. or higher and 40 ° C. or lower, more preferably 10 ° C. or higher and 40 ° C. or lower. It is more preferably in the range of 30 ° C. or lower. With a molecular chain exhibiting such a lower limit critical solution temperature, it is possible to change the protein binding site 2 to a state in which it can bind to an antibody by changing the temperature to a low temperature range where thermal denaturation or the like is unlikely to occur. Prior to that change, the protein binding site 2 can be placed in a higher temperature region. That is, after culturing the candidate cells under a relatively high culture temperature, there is an advantage that the protein binding site 2 can be continuously bound to the antibody in the same well with less heat denaturation.

As shown in FIG. 3A, the temperature-responsive site 3 immobilized on the well 10 preferably has a main chain having a spiral three-dimensional structure in a state of being compatible with the aqueous phase. In general, there are known examples in which the three-dimensional structure of a polymer is spirally stabilized by an intramolecular interaction such as a hydrogen bond. Such intramolecular interactions are relatively easily cleaved by an increase in temperature, and the three-dimensional structure tends to collapse easily. Therefore, the molecular chain having a spiral three-dimensional structure is advantageous in that the binding ability of the protein binding site 2 can be reliably changed with a temperature change exceeding the lower limit critical solution temperature.

As the temperature-responsive site 3, for example, polyamino acid, polypropylene glycol, polyethylene glycol, poly-N-isopropylacrylamide, poly-N, N-diethylacrylamide, polyvinylcaprolactam, polyvinylmethyl ether, polymethacrylic acid ester and the like are the main chains. Can be. These polymers may be a polymer composed of one kind of monomer, or may be a copolymer in which a plurality of types of monomers constituting each of these molecular chains are combined. Further, the copolymer may be any of a random type, a block type and a graft type.

The temperature-responsive site 3 preferably has a main chain of polyamino acids. The polyamino acid may be a polymer composed of one kind of amino acid or a copolymer composed of a plurality of types of amino acids. Further, it may be a polymer containing only one of the D-form and the L-form, or may be a hybrid thereof.

Examples of the amino acid constituting the temperature response site 3 include lysine, glutamic acid, aspartic acid, glutamine, asparagine, cysteine, alanine, leucine, methionine and the like. These amino acids generally easily form α-helix, and in particular, a polymer consisting of only one of the D-form and the L-form of these amino acids has a spiral three-dimensional structure in the main chain of the temperature response site 3. Is suitable for. Specific examples of the temperature response site 3 composed of amino acids include ε-polylysine valeric acid amide, ε-polylysine butyric acid amide, N-hydroxypentyl ε-polylysine, N-hydroxybutyl ε-polylysine and the like.

The temperature-responsive site 3 preferably has a number average molecular weight of 5 kDa or more, more preferably 10 kDa or more, and even more preferably 50 kDa or more. When the number average molecular weight of the temperature-responsive site 3 is in such a range, it is advantageous in that the reaction of binding the temperature-responsive site 3 to the substrate 9 of the incubator P can be easily performed.

The temperature responsive site 3 is attached to the bottom wall 120a of the well 10 in FIG. By binding to the bottom wall 120a of the well 10, when the temperature-responsive site 3 is in a state of being compatible with the aqueous phase (see FIG. 3A), the orientation of the main chain of the temperature-responsive site 3 is aligned. It is easy to stabilize. Therefore, the change in the binding ability of the protein binding site 2 due to the change in the three-dimensional structure is clarified between the state of being compatible with the aqueous phase and the state of being phase-separated (see FIG. 3 (b)), and the antibody-producing cells The accuracy of selective separation and recovery of is being improved. However, the temperature responsive site 3 may be bound to the protein binding site 2 in a state of being directly bound to the bottom wall 120a of the well 10. Further, it may be bonded to both of these bonding positions.

The binding between the temperature-responsive site 3 and the inner wall surface of the well 10 or other molecules can be either covalent or non-covalent, as in the binding between the protein binding site 2 and the inner wall surface of the well 10 or other molecules. Good. Further, the temperature-responsive site 3 may be directly bound to the inner wall surface of the well 10 or another molecule, or may be indirectly bound via a linker. For example, various silane coupling agents, a bond between biotin and avidin, a bond between biotin and streptavidin, and the like can be used.

As the molecule or molecular chain constituting the hydrophilic site 4, for example, an appropriate molecule or molecular chain having a hydrophilic group such as a hydroxy group, a carboxyl group, an amino group, a sulfonic acid group or a phosphoric acid group can be used. Specific examples of the hydrophilic site 4 include a polycondensation polymer obtained by polycondensing a polyol such as ethylene glycol and propylene glycol, a phospholipid, polyphosphoric acid, and the like. Among these, polyethylene glycol mainly composed of ethylene glycol is particularly preferable in that the hydrophilic site 4 can be easily bonded to the temperature responsive site 3 and the like.

The hydrophilic site 4 preferably has an average molecular weight of 100 or more and 1000 or less, more preferably 200 or more and 800 or less, and further preferably 400 or more and 600 or less. Further, in the hydrophilic part 4, the molar ratio of the temperature-responsive part 3 to the monomer is preferably 5% or more and 50% or less, more preferably 5% or more and 30% or less, and 8% or more. It is more preferably 15% or less. If such a hydrophilic site 4 is provided at a temperature-responsive site 3 or the like having polylysine as the main chain, a phase transition can occur near room temperature.

The hydrophilic site 4 is bound to the temperature responsive site 3 as a side chain in FIGS. 3, 4 and 5. However, the hydrophilic site 4 may be directly attached to the inner wall surface of the well 10. Further, it may be bonded to a plurality of these bonding positions.

In FIGS. 3, 4, and 5, the protein binding site 2, the temperature-responsive site 3, and the hydrophilic site 4 are immobilized on the inner wall surface of the well 10 included in the incubator P. However, the form may not include the hydrophilic part 4, or may have a hydrophobic part instead of a part or all of the hydrophilic part 4. Further, together with these molecules or molecular chains, it may have a site exhibiting other functions such as a pH response site.

The pH response site is composed of molecules and atomic groups whose properties change reversibly with a change in pH. By changing the local charge and ionization state of the pH response site by changing the pH of the culture solution, the hydrophilicity and hydrophobicity of the polymer can be significantly changed. Specifically, for example, by changing the hydration state by protonation of a carboxyl group or the like, deprotonation of an amino group or the like, the water solubility of the polymer can be changed, or the lower limit critical solution temperature or the upper limit critical solution can be changed. It is possible to raise or lower the temperature. That is, the binding affinity between the protein binding site 2 and the antibody can be rapidly changed by additionally providing a pH-responsive site.

Examples of the pH-responsive site include an amino group, an imino group, a phosphazene group, a guanidino group, a pyrrolyl group, a pyrazinyl group, an imidazolyl group, a pyridinyl group, a pyridadinyl group, a pyrimidinyl group, a triazinyl group, an indolyl group, an isoindryl group and a carbazolyl group. , Azacarbazolyl group and the like, and molecules having these substituents can be mentioned. Further, examples thereof include a carboxyl group, a phenol group, a dihydroxyboryl group, a sulfonic acid group, a phosphoric acid group, a phosphonic acid group, a phosphinic acid group and the like, and molecules having these substituents.

Hydrophobic sites are composed of molecules and molecular chains having hydrophobic groups. The hydrophobic site enhances the cohesiveness of the temperature-responsive site 3 and energetically stabilizes the temperature-responsive site 3 in the aggregated state. Therefore, it is possible to facilitate the phase transition of the temperature-responsive portion 3 from the non-aggregated state to the aggregated state by changing the temperature.

Examples of the hydrophobic site include methyl group, ethyl group, n-propyl group, iso-propyl group, n-butyl group, sec-butyl group, iso-butyl group, tert-butyl group, n-pentyl group and n. -Alkyl groups such as hexyl groups and molecules having these substituents can be mentioned.

The incubator P may be aseptically carried into the operation room 101 of the cell separation device 100 at the time of separation and recovery from outside the device and attached, or may be provided in the cell separation device 100 in advance. For example, a plurality of incubators P that have been sterilized in advance may be stored in a storage chamber adjacent to the operation chamber 101, and may be sequentially attached to the heat block 102 by a robot arm or the like at the time of separation and recovery. ..

According to the above cell separation method and the cell separation device 100 using the principle, the recovered antibody-producing cells are subjected to a culture operation, a temperature stimulus to the temperature-responsive site 3, and the like after the initial gene transfer operation. Since it has only been subjected to genetic modification, it is recovered in the state of live cells with little damage without undergoing any genetic modification such as introduction of a marker gene. In addition, it is not necessary to perform aseptic sampling of candidate cells, and a large number of candidate cells can be comprehensively screened with a small amount of culture medium. Therefore, it is time-efficient and cost-effective. Therefore, according to the cell separation device 100 and the cell separation method described above, it is possible to select cells having a high antibody-producing ability from the candidate cell group with less damage by a simple step. Since the selected cells are in a viable state and have little damage, they can be directly used for secondary screening for more precise selection, antibody production, and the like.

Further, according to the above cell separation device 100 and cell separation method, if various conditions such as the binding amount of the protein binding site and the addition amount of the anti-membrane protein antibody are unified for each of the plurality of wells, the cells can be contained in the culture solution. By comparing the ratio of the number of cells existing in the floating state among a large number of wells, it is possible to determine not only the presence or absence of antibody-producing ability but also the superiority or inferiority of antibody-producing ability. For example, when the proportion of cells remaining attached to the well is relatively low, it can be determined that the competition with the added anti-membrane protein antibody has intensified due to the large amount of antibody produced. It is possible and quantitative evaluation can be performed.

Hereinafter, the present invention will be described in more detail with reference to examples of the present invention, but the technical scope of the present invention is not limited thereto.

As an example of the present invention, an incubator in which a protein binding site and a temperature-responsive site were immobilized was prepared, and selective separation and recovery of antibody-producing cells were tested.

As the protein binding site, a non-peptide mimetic represented by the following chemical formula was used. The atomic group shown in the following chemical formula mimics the antibody binding ability of protein A. In addition, ε-polylysine was used as the temperature-responsive site. As the temperature-responsive site, a hydrophilic site made of polyethylene glycol was used as a side chain.

The incubator was specifically prepared according to the following procedure. First, 1-ethyl-3-carbodiimide (WSC), N-hydroxysuccinimide (NHS) and carboxylic acid-terminated polyethylene glycol (PEG) were dissolved in pure water. The solution was then cooled to 4 ° C. and polylysine was added with stirring. As polylysine, a mixture in which various molecular weights were mixed was added. Then, it was stirred at room temperature for 2 hours to obtain a PEG-modified polylysine to which polyethylene glycol was bonded as a side chain.

Next, the synthesized PEG-modified polylysine was immobilized on a polystyrene resin plate. Specifically, a resin plate in which 1-ethyl-3-carbodiimide (WSC) and N-hydroxysuccinimide (NHS) are added to an aqueous solution of PEG-modified polylysine at a concentration of 1 mol%, and the surface is pre-modified with an amino group. "SUMILLON carbodiimide" (manufactured by Sumitomo Bakelite Co., Ltd.) was immersed. Then, after reacting at room temperature for 2 hours, it was washed 3 times with pure water to obtain a resin plate on which PEG-modified polylysine was immobilized.

Next, the non-peptide mimetic was bound to the PEG-modified polylysine immobilized on the resin plate. Specifically, the resin plate was immersed in a 2% glutaraldehyde solution diluted with a carbonate buffer solution and allowed to stand at 37 ° C. for 2 hours for reaction. The resin plate after the reaction was then washed 3 times with phosphate buffer (PBS). Then, the resin plate was immersed in a non-peptide mimetic solution previously adjusted to a concentration of 0.1% by mass with a phosphate buffer solution (PBS) having a pH of 7.4, and PEG modification to which the non-peptide mimetic was bound. An incubator on which polylysine was immobilized was obtained.

Next, the selective separation and recovery of antibody-producing cells were tested using the prepared incubator. Two types of CRL-1606 cells and CHO-S cells were used as candidate cells. These cell lines have a known ability to produce antibodies in advance. CRL-1606 cells are mouse hybridomas conserved in ATCC (American Type Culture Collection), and are floating cells that secrete anti-fibronectin antibody extracellularly. In addition, CHO-S cells are cell lines that do not have the ability to produce antibodies by genetically modifying CHO-K1 cells, which are adherent cells, into floating cells.

First, each of CRL-1606 cells and CHO-S cells was cultured in an incubator on which PEG-modified polylysine bound to non-peptide mimetics was immobilized. A predetermined serum-free medium was used for culturing CRL-1606 cells, and a CD-CHO medium (manufactured by Invitrogen) was used for culturing CHO-S cells, and the cells were grown in a 5% CO ₂ atmosphere at 37 ° C. Subsequently, an anti-CD44 antibody and an acid were aseptically added to each culture solution to lower the temperature of the culture solution and adjust the pH to 6.5. Then, after reacting at room temperature for 1 hour, each culture solution was recovered from the incubator.

As a result, CRL-1606 cells having an antibody-producing ability were recovered from the culture medium in which CRL-1606 cells were cultured. On the other hand, the cells were not recovered from the culture medium in which the CHO-S cells were cultured, and the CHO-S cells having no antibody-producing ability remained attached to the incubator. That is, in an incubator in which a protein binding site and a temperature-responsive site are immobilized, candidate cells are cultured, and then the temperature-responsive site is phase-translated to selectively separate and recover antibody-producing cells in a single tank. It was confirmed that it can be performed by a simple process.

1 Inner wall (support)
2 Protein binding site 3 Temperature responsive site 4 Hydrophilic site 9 Base body 10 well 100 Cell separator 101 Operation room 102 Heat block (temperature control means)
103 Pipette device (sowing means)
104 Suction nozzle (collection means)
105 Culture solution recovery tank 106 Supply system 107 Culture room 108 Gas exchange device 109 Temperature control device 111 Cell suspension 112 Medium reagent 113 Anti-membrane protein antibody reagent 114 pH adjustment reagent 120a Bottom wall 120b Side wall A1 Anti-membrane protein antibody Ab Antibody C Candidate cell P incubator P10 membrane protein

Claims (2)

A cell separator that selectively separates and collects antibody-producing cells from a plurality of candidate cells.
An operation unit that can install an incubator with multiple wells,
A seeding means for seeding candidate cells in a culture medium held in each of the plurality of wells, and
A temperature control means for controlling the temperature of the culture solution and
Reagent addition means for adding an anti-membrane protein antibody to each of the culture broths held in the plurality of wells, and
A collection means for collecting the culture solution held in each of the plurality of wells is provided.
On the inner wall surface of the well of the incubator, a protein binding site composed of a molecule capable of binding to an antibody and a three-dimensional structure reversibly changed by a temperature change across the phase transition temperature, the protein binding site and the antibody It is bound to a temperature-responsive site consisting of a molecular chain that changes the binding affinity of
In the seeding means, candidate cells expressing a membrane protein are seeded in each of the plurality of wells.
The reagent addition means adds an anti-membrane protein antibody capable of binding to the membrane protein to the culture medium in which the seeded candidate cells are cultured.
The temperature control means changes the temperature of the culture solution held in each of the plurality of wells from the culture temperature of the candidate cells to a temperature exceeding the phase transition temperature of the temperature-responsive site.
The recovery means collects the culture solution held in each of the plurality of wells, and among the seeded candidate cells, the antibody non-antibody that binds to the protein binding site via the anti-membrane protein antibody. A cell separation apparatus characterized in that antibody-producing cells that are not bound to the protein binding site and are suspended in a culture solution are selectively separated and collected while leaving the producing cells. A cell separation method that selectively separates and collects antibody-producing cells from a group of candidate cells.
A temperature consisting of a protein binding site consisting of a molecule capable of binding to an antibody and a molecular chain consisting of a molecular chain whose three-dimensional structure is reversibly changed by a temperature change across the phase transition temperature to change the binding affinity between the protein binding site and the antibody. Candidate cells expressing the membrane protein were seeded in each of the plurality of wells in which the responsive site is bound to the inner wall surface.
The seeded candidate cells were cultured and
An anti-membrane protein antibody capable of binding to the membrane protein is added to the culture solution held in each of the plurality of wells, and the liquid temperature of the culture solution held in each of the plurality of wells is set as the candidate. The membrane protein expressed by the candidate cell is bound to the anti-membrane protein antibody by changing from the cell culture temperature to a temperature exceeding the phase transition temperature of the temperature-responsive site, and the protein binding site is combined with the protein binding site. The anti-membrane protein antibody, the protein binding site, and the antibody produced by the candidate cell are bound to each other.
The culture solution held in each of the plurality of wells is collected, and among the seeded candidate cells, antibody-producing cells that are bound to the protein binding site via the anti-membrane protein antibody are left. , A cell separation method characterized by selectively separating and recovering antibody-producing cells that are not bound to the protein binding site and are suspended in a culture solution.

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