Dead cell separating agent and method for producing cells using the same

Abstract

To provide a dead cell separating agent that enables selective and simple removal of dead cells present in a eukaryotic cell culture liquid, as well as a method for producing eukaryotic cells using the separating agent.SOLUTION: The foregoing problem is solved by the dead cell separating agent comprising an insoluble carrier and Tim4 (T-cell immunoglobulin and mucin domain 4) immobilized on the insoluble carrier.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a separating agent for dead cells and a method for producing cells using the same. In particular, the present invention relates to the separating agent having immobilized thereon a protein capable of specifically binding to dead cells, and a method for producing cells using the same. [Background technology]

[0002] In recent years, eukaryotic cells, including mammalian and insect cells, have been used for a variety of purposes in several industrial fields. For example, in the field of biopharmaceuticals, protein drugs, such as monoclonal antibodies, hematopoietic stimulating factors, and blood coagulation factors, as well as gene drugs, such as adeno-associated viruses and lentiviruses, are produced using genetically modified eukaryotic cells capable of expressing the proteins or viral vectors. Cellular medicines, in which eukaryotic cells themselves are administered to patients as pharmaceuticals, have also been commercialized. For example, a therapy is performed in which a patient's own blood cells are grown outside the body, a therapeutic gene is introduced using genetic engineering technology, and the cells are then activated and returned to the patient.

[0003] Eukaryotic cells are broadly classified into suspension cells and adhesive cells based on their growth pattern, but suspension cells are often used for the production of substances such as protein medicines and gene medicines because of their ease of scale-up.Furthermore, suspension cells are often used for cell medicine applications, as blood cells are naturally suspension cells.

[0004] When producing eukaryotic cells for the above-mentioned purposes, they are cultured in a liquid medium containing various nutrients suitable for the growth of the cells. As an example of culturing suspension cells, a liquid medium is added to a culture vessel called a bioreactor, and the eukaryotic cells to be cultured are then added. The cells are cultured in suspension by stirring while maintaining the medium temperature in the reactor at a temperature suitable for cell growth.

[0005] The culture medium containing cells after culture (hereinafter also referred to as "cell culture medium") often contains a certain number of dead cells (dead cells) in addition to living cells (viable cells) (hereinafter the ratio of live cells to the total cells in the cell culture medium will be referred to as "cell viability"). When the goal is substance production, only living cells contribute to substance production. Therefore, a high viability in the culture medium is preferable as it increases the efficiency of substance production. Furthermore, in cell medicine applications, only living cells exert their medicinal effects when administered to a patient, so a higher viability is preferable.

[0006] One method to increase the survival rate of eukaryotic cells in a culture medium is to discard a certain amount of the culture medium (the "bleeding method"). While this method is useful because it is easy to perform, it has the drawback of resulting in the loss of some valuable live cells because it does not distinguish between live and dead cells.

[0007] Another method for increasing the viability of eukaryotic cells contained in a culture medium is to selectively separate and remove dead cells contained in the culture medium. Patent Document 1 discloses an example of such a method, which utilizes the property of dextran to bind more strongly to dead cells to separate and remove dead cells from a cell population. However, because dextran also binds to live cells, there is a problem in that not only dead cells but also live cells are simultaneously removed. Patent Document 2 discloses a method for more selectively removing dead cells, which utilizes the property of annexin V to bind to phosphatidylserine, a phospholipid exposed on the surface of dead cells, in a calcium ion-dependent manner to separate dead cells by flow cytometry. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Special Publication No. 2012-513748 [Patent Document 2] Special Publication No. 9-505894 Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a separation agent that can selectively and easily remove dead cells contained in a eukaryotic cell culture medium, and a method for producing eukaryotic cells using the separation agent. [Means for solving the problem]

[0010] Taking the above points into consideration, the inventors conducted extensive research and discovered that a separation agent comprising an insoluble carrier and a specific protein immobilized on the insoluble carrier that can specifically bind to phosphatidylserine, a phospholipid exposed on the surface of dead cells, can selectively and easily remove dead cells contained in eukaryotic cell culture medium, thereby completing the present invention.

[0011] That is, the present invention includes the following aspects [1] to [4].

[0012] [1] A separation agent for separating live cells from dead cells in a culture medium of eukaryotic cells, comprising an insoluble carrier and Tim4 (T-cell immunoglobulin and mucin domain 4) immobilized on the insoluble carrier.

[0013] [2] A method for producing eukaryotic cells, comprising the steps of culturing eukaryotic cells, treating the culture medium after the culturing step with the separation agent described in [1], and separating dead cells.

[0014] [3] The manufacturing method described in [2] above, wherein the step of separating dead cells includes a step of forming a complex between the separation agent and dead cells, and a step of separating the complex from the culture medium and recovering live cells.

[0015] [4] The method for producing a cell culture medium according to [2] or [3], wherein the calcium ion concentration in the culture medium is 0.5 mmol / L or more and 2 mmol / L or less in the step of culturing the eukaryotic cells.

[0016] [5] The separation agent according to [1], wherein the eukaryotic cells are suspension cells.

[0017] [6] A method for producing a cell culture medium according to any one of [2] to [4], wherein the eukaryotic cells are suspension cells. [Effects of the Invention]

[0018] The present invention is characterized by using a separation agent for dead cells contained in a eukaryotic cell culture medium, the separation agent comprising an insoluble carrier and Tim4 (T-cell immunoglobulin and mucin domain 4) immobilized on the insoluble carrier. The separation agent can selectively and easily remove dead cells contained in the culture medium, thereby obtaining a eukaryotic cell culture medium with improved cell viability. In particular, the separation agent of the present invention is useful in the production of suspension cells, where selective removal of dead cells is considered difficult, because it can selectively and easily remove dead cells. DETAILED DESCRIPTION OF THE INVENTION

[0019] Hereinafter, the embodiments of the present invention will be described in detail, but the present invention is not intended to be limited to the following contents. The present invention can be practiced with appropriate modifications within the scope of its spirit.

[0020] The origin of the eukaryotic cells in the present invention is not particularly limited, and examples include mammals such as humans, monkeys, dogs, cats, rabbits, rats, nude mice, mice, guinea pigs, pigs, sheep, Chinese hamsters, and cows, and insects such as armyworm moths. Specific examples of cells include cultured cell lines such as Chinese hamster ovary (CHO) cells, MDCK cells derived from canine kidney, HEK293 cells derived from human fetal kidney, and Sf9 cells derived from armyworm moth; epithelial cells and endothelial cells that constitute various tissues and organs in the body; skeletal muscle cells that exhibit contractile properties, smooth muscle cells, cardiac muscle cells; neuronal cells that constitute the nervous system; glial cells; fibroblasts; blood cells (e.g., erythrocytes, platelets, leukocytes, hematopoietic stem cells, lymphoid stem cells, granulocytic stem cells, monocytes, megakaryocytes, lymphocytes, eosinophils, basophils, and neutrophils); macrophages, T cells, and dendritic cells that are involved in the body's immunity; hepatic parenchymal cells, non-hepatic parenchymal cells, and adipocytes that are involved in the body's metabolism; and cells with the ability to differentiate, such as induced pluripotent stem (iPS) cells, embryonic stem (ES) cells, and embryonic germ cells (ES) cells. These include various stem cells such as germ (EG) cells, embryonic cancer (EC) cells, mesenchymal stem cells, liver stem cells, pancreatic stem cells, skin stem cells, muscle stem cells, and germline stem cells, as well as progenitor cells of various tissues, and cells induced to differentiate from these.

[0021] The eukaryotic cells of the present invention may be suspension cells or adhesive cells. Adherent cells are cells that adhere to a solid surface and grow. Suspension cells are cells that grow suspended in a culture medium without requiring an adhesive surface. However, suspension cells are preferred from the viewpoint of ease of scale-up of production.

[0022] The cell density of the eukaryotic cell culture medium (referred to herein as "cultured eukaryotic cells in the medium"; the same applies hereinafter) from which dead cells are separated using the separation agent of the present invention is not particularly limited as long as it is within the range of cell densities obtained by culturing under conditions commonly used in the art. For example, when the eukaryotic cells are CHO cells, the cell density is 1.0 × 10 4 cells / mL or more 1.0×109 cells / mL or less is preferable, and 1.0 × 10 5 cells / mL or more 1.0×10 8 cells / mL or less is more preferable.

[0023] Examples of dead cells that can be separated using the separation agent of the present invention include cells undergoing necrosis or apoptosis. Necrosis is cell death that occurs when cells are physically or chemically damaged due to a sudden change in the culture environment. On the other hand, apoptosis, also known as programmed cell death, is genetically controlled cell death. Trypan blue exclusion can be used to distinguish between live and dead cells, but apoptotic cells may not be detected by trypan blue exclusion because their cell membrane structure is relatively well preserved.

[0024] Methods for separating dead cells from eukaryotic cell cultures rely on the physical properties that differ from those of live cells. For example, dead cells have a slightly lower density than live cells due to damaged cell membranes, making them suitable for separation by density differential techniques. One such method uses a continuous centrifuge, such as Thermo Fisher's counterflow centrifuge, the Rotea system. Another method utilizes the phenomenon of dead cells becoming slightly smaller than live cells to separate dead cells using an inertial microfluidic cell sorter (T. Kwon et al., Lab Chip, 2018, 18, 2826). However, in the culture of CHO cells used in the production of biopharmaceuticals, such as antibody drugs, it is said that more than 80% of dead cells generated in bioreactors are apoptotic (J. Goswami et al., Biotechnol Bioeng. 1999 Mar 20;62(6):632-40). Apoptotic cells retain their cell membrane structure relatively well, and their density is not significantly different from that of viable cells, making them difficult to separate and remove by centrifugation. Similarly, apoptotic cells are not necessarily smaller than viable cells, making them difficult to separate using inertial microfluidic cell sorting devices. However, apoptotic cells, like necrotic cells, exhibit phosphatidylserine on their cell membrane surface, making them easy to separate using separation agents immobilized with a ligand that specifically binds to phosphatidylserine.

[0025] The separating agent of the present invention is characterized by the use of Tim4 (T-cell immunoglobulin and mucin domain 4) as a ligand for dead cell separation, which is immobilized on an insoluble carrier. Tim4 is a type I transmembrane protein that can specifically bind to phosphatidylserine (PS), a phospholipid exposed on the surface of dead cells that have undergone necrosis or apoptosis (M. Miyanishi et al., Nature, 2007, Nov. 15, 450(7168):435-9). PS is a phospholipid that is rarely exposed on the surface of living cells.

[0026] The Tim4 used as the ligand may contain at least an IgV-like domain (Ig-like V-type domain), which is a PS-binding domain. For example, when human-derived or mouse-derived Tim4 is used as the ligand of the separation agent of the present invention, a polypeptide containing the full-length sequence (e.g., the full-length amino acid sequence of UniProt No. Q96H15 for human Tim4 or the full-length amino acid sequence of UniProt No. Q6U7R4 for mouse Tim4) may be used, or a polypeptide containing at least the IgV-like domain sequence (e.g., the sequence from glutamic acid at position 25 to asparagine at position 126 of the amino acid sequence of UniProt No. Q96H15 for human Tim4 or the sequence from alanine at position 23 to arginine at position 128 of the amino acid sequence of UniProt No. Q6U7R4 for mouse Tim4) may be used. Furthermore, as long as the protein has PS-binding ability, the amino acid sequence may undergo one or more of the following substitutions, deletions, insertions, and additions (hereinafter collectively referred to as "mutations") of one or more amino acid residues at one or more positions.

[0027] As used herein, "one or several" refers to any of the following, although it varies depending on the position of the amino acid substitution in the three-dimensional structure of Tim4 and the type of amino acid residue. Examples of such substitutions include 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, and 1. Furthermore, "substitution of one or several amino acid residues" as used herein includes not only amino acid substitutions at the specific positions described above, but also conservative substitutions between amino acids with similar physical and / or chemical properties. It is generally known to those skilled in the art that conservative substitutions maintain protein function between substituted and unsubstituted proteins. Examples of conservative substitutions include substitutions between glycine and alanine, serine and threonine, or glutamic acid and aspartic acid (Protein Structure and Function, Medical Science International, 9, 2005). Furthermore, "mutations of one or several amino acid residues" also include naturally occurring mutations (mutants or variants) based on differences in Tim4 origin or species.

[0028] The presence of calcium ions is thought to be necessary for Tim4 to specifically bind to PS exposed on the surface of dead cells (C. Santiago et al., Immunity, 2007, Dec. 27(6):941-51). Eukaryotic cell culture media contain a certain concentration of calcium ions as a medium component. For example, commonly used eukaryotic cell media such as RPMI 1640 contain 0.9 mmol / L of calcium ions, DMEM contain 1.8 mmol / L of calcium ions, and BalanCD CHO Growth A (Fujifilm-Irvine Scientific) contain 1 mmol / L of calcium ions. The specific binding of Tim4 to the PS is thought to be sufficient if the medium contains calcium ions at a concentration comparable to that found in these media (e.g., between 0.5 mmol / L and 2.0 mmol / L). The calcium ion concentration in the medium can be measured by ICP emission spectroscopy.

[0029] The insoluble carrier constituting the separating agent of the present invention is not limited to any particular material, so long as it is capable of immobilizing Tim4 and is insoluble in culture medium or buffer solution. Examples include polystyrene, polyethylene, polymethyl methacrylate, polyethylene terephthalate, polycarbonate, cellulose, cyclodextrin, acrylamide, alginate, dextran, gelatin, glass, ceramics, or a mixture of two or more of these materials. The insoluble carrier may also contain a magnetic material. Furthermore, the insoluble carrier may be a material that has a lower specific gravity than water and floats on the water surface, or a material that has a higher specific gravity than water and sinks in water. The shape and size of the insoluble carrier are also not particularly limited, as long as they are usable in the eukaryotic cell production process. Specifically, the insoluble carrier may be a spherical or oval sphere having a diameter of 0.1 μm to 1000 μm, a plate having a length and width of 1 mm to 10 mm, a nonwoven fabric having a length and width of 1 mm to 10 mm, or a rod having a length of 1 mm to 10 mm, or may be an insoluble carrier of an irregular shape, and the inner wall of a sealed container or a cylindrical container may also be considered to be an insoluble carrier.

[0030] The aforementioned immobilization of Tim4 to an insoluble support may be physical immobilization using hydrophobic bonds, or chemical immobilization using covalent or ionic bonds. Alternatively, the ligand may be modified with biotin and the insoluble support with avidin, and then immobilization may be performed via biotin-avidin interaction. Alternatively, the ligand may be modified with a tag peptide containing histidine, and the insoluble support may be loaded with a metal ion (e.g., nickel (II) ion), and then immobilization may be performed via peptide-metal ion interaction. By using the separation agent of the present invention, eukaryotic cells can be produced with a high survival rate. (a) culturing eukaryotic cells; (b) a step of reacting the culture solution obtained after the culturing step with the separating agent of the present invention; (c) separating dead cells contained in the culture medium; Each step will be described in detail below.

[0031] (a) Eukaryotic cell culture process When this step is carried out using a cell culture vessel (bioreactor), the vessel is not particularly limited as long as it is a vessel capable of culturing and growing cells by stirring the culture medium. Bioreactors can be broadly divided into suspension culture bioreactors, in which planktonic cells themselves or carriers with adherent cells attached are cultured in a suspended state, and fixed-bed bioreactors, in which adherent cells are cultured attached to a solid surface or entrapped in a gel. Examples of suspension culture bioreactors include those equipped with an internal impeller that stirs the culture medium by rotating the impeller, and those without an impeller that stir the culture medium by shaking the bioreactor itself. Fixed-bed culture bioreactors are bioreactors that use fixed carriers such as hollow fibers or nonwoven fabrics and stir only the liquid components. Either type may be used.

[0032] This process can be carried out using either batch culture or continuous culture. Batch culture also includes its variant, fed-batch culture. Perfusion culture is an example of continuous culture. Perfusion culture is a culture method in which medium is continuously supplied to a cell-containing culture system, while an equal amount of cell-free culture supernatant is continuously removed from the system, maintaining steady concentrations of nutrients, dissolved oxygen, and accumulated metabolic products. Because the growing eukaryotic cells are cultured within the system, if the culture environment can be properly maintained, higher cell densities can be achieved compared to other culture methods, enabling the production of target substances over long periods of time, which is expected to improve productivity.

[0033] The medium used to culture eukaryotic cells in this step is not particularly limited as long as it allows the cells to grow. Examples of such media include Minimum Essential Media (MEM), αMEM, Dulbecco's MEM (DMEM), Eagle's MEM (EMEM), Glasgow's MEM (GMEM), DMEM / Ham's F-12, Ham's F-12, Ham's F-10, Medium 199, and RPMI 1640. When the eukaryotic cells are CHO cells, commercially available CHO cell media such as BalanCD CHO Growth A (Fujifilm-Irvine Scientific) and EX-CELL Advanced CHO Fed-batch Medium (Sigma-Aldrich) may be used. Furthermore, the aforementioned media may contain amino acid additives, serum, or antibiotics. The type of serum is also not particularly limited. Examples include fetal bovine serum (FBS), bovine calf serum, adult bovine serum, horse serum, sheep serum, goat serum, pig serum, chicken serum, rabbit serum, and human serum. Among these, FBS is commonly used due to its ease of availability. There are no particular restrictions on the concentration of serum added to the medium, but for cost-effectiveness, it is generally used at a concentration of 20% (v / v) or less. Serum-free media may also be used that do not contain raw or unpurified serum but contain purified blood-derived components or animal tissue-derived components (e.g., growth factors).

[0034] As used herein, the term "culture medium" may include eukaryotic cells and a medium. In addition to the eukaryotic cells and a medium, the term "culture medium" may also include secretions secreted by the eukaryotic cells.

[0035] (b) A step of reacting the culture medium with the separating agent of the present invention. The method for reacting the separating agent of the present invention with the culture medium (eukaryotic cell culture medium) after step (a) may be selected appropriately depending on the shape of the separating agent of the present invention. For example, if the separating agent of the present invention is spherical, ellipsoidal, or flat, the separating agent of the present invention may be dispersed in the culture medium while stirring, or the culture medium may be passed through a column packed with the separating agent of the present invention. Furthermore, for example, if the separating agent of the present invention is rod-shaped, it may be immersed in the culture medium.

[0036] The separation agent of the present invention, which has been treated in the manner described above to bind dead cells contained in the culture medium, may be left standing or dispersed in the culture medium as is, but it is preferable to separate it from the culture medium, as this prevents the dead cells from being released from the separation agent of the present invention.

[0037] (c) Separating dead cells As with the contact method described above, the method for separating the separating agent of the present invention to which dead cells are bound from the eukaryotic cell culture medium may be appropriately selected depending on the shape of the separating agent of the present invention. For example, if the separating agent of the present invention is spherical, oval, flat, or nonwoven fabric-like and dispersed in the culture medium, the separating agent of the present invention may be separated by filtration from the culture medium. Furthermore, if the separating agent (insoluble carrier) of the present invention contains a magnetic material, the separating agent of the present invention may be separated by magnetic attraction using a magnet. Furthermore, if the separating agent (insoluble carrier) of the present invention has a lower specific gravity than water and floats to the water surface, it may be allowed to stand after contact with the eukaryotic cell culture medium, and the separating agent of the present invention may be separated after gathering at the surface of the culture medium. Furthermore, if the separating agent of the present invention is rod-shaped and contacted with the eukaryotic cell culture medium by immersion, the separating agent of the present invention may be simply removed from the cell culture medium.

[0038] Furthermore, when separation of dead cells contained in eukaryotic cell culture medium is carried out using a column packed with the separation agent of the present invention, this is preferable because passing the liquid through the column allows contact with the culture medium and separation from the culture medium to be carried out simultaneously.

[0039] The step of separating dead cells may also include the steps of forming a complex between the separating agent and the dead cells, separating the complex from the culture medium, and recovering the living cells.

[0040] The separation agent of the present invention separated from the eukaryotic cell culture medium may be discarded together with the bound dead cells, or may be reused after a regeneration step, which can be carried out by removing the dead cells bound to the separation agent of the present invention, for example, by washing with an alkali or acid, or by removing calcium ions with a chelating agent.

[0041] Use of the separation agent of the present invention improves the proportion of live cells (cell viability) contained in a eukaryotic cell culture medium. As used herein, "improving cell viability" may mean that the proportion of live cells contained in a eukaryotic cell culture medium is improved before and after the step of separating dead cells using the separation agent of the present invention. In other words, it may mean that the proportion of live cells contained in a eukaryotic cell culture medium is improved after the step of separating dead cells compared to before.

[0042] Cell viability can be measured, for example, by staining with Trypan blue, which stains the cytoplasm of dead cells blue, to determine whether the cells are viable or dead, followed by counting the total number of cells and the number of live cells. Cell counting can be performed using an automated cell counter. Note that in the early stages of apoptosis, the cell membrane remains intact, making Trypan blue staining ineffective. Another method for counting apoptotic cells is to use a combination of fluorescently labeled Annexin V and propidium iodide (PI) to distinguish between apoptotic and necrotic cells, and then calculate viability using flow cytometry. [Example]

[0043] The present invention will be described in more detail below using examples and comparative examples, but the present invention is not limited to these examples. Note that the reference examples do not constitute the present invention. Unless otherwise specified, commercially available reagents were used.

[0044] Example 1 Preparation of an insoluble carrier onto which Tim4 (T-cell immunoglobulin and mucin domain 4) is immobilized By adding commercially available magnetic particles Dynabeads M-280 Streptavidin (Thermo Fisher Scientific) to a commercially available biotinylated mouse Tim4 / human Fc chimera solution (Adipogen Life Sciences), Tim4 was immobilized to the particles via biotin-avidin binding, producing an insoluble carrier on which Tim4 was immobilized.

[0045] Reference Example 1: Removal of dead cells using Tim4-immobilized insoluble carrier (buffer system) (1) Preparation of cell culture medium (1-1) Chinese hamster ovary (CHO) cells (DG44 strain) were cultured in a 250 mL shake flask containing 20 mL of animal-derived component-free growth medium (BalanCD CHO Growth A: Fujifilm Wako Pure Chemical Industries, Ltd.) supplemented with L-glutamine. The cells were cultured in suspension at 130 rpm in a TAITEC NR-3 shaker at 37°C and 8.0% CO2 in an incubator. The culture medium was then subjected to agitation at 130 rpm in a TAITEC NR-3 shaker to obtain a logarithmic growth phase cell culture (hereafter referred to as "viable cell culture medium"). The viability of the cells in the viable cell culture medium was measured by trypan blue staining using a Vi-cell XR (Beckman Coulter) viability analyzer. The cell viability was determined to be >95%.

[0046] The calcium ion concentration in BalanCD CHO Growth A was measured using an ICP emission spectrometer (Agilent Technologies) and was found to be 1 mmol / L.

[0047] (1-2) During the culture of (1-1), 10 mL of culture medium containing cells in the logarithmic growth phase was taken, and the apoptosis-inducing reagent cycloheximide was added to a final concentration of 100 μmol / L. After culturing for 72 hours, a cell culture medium containing a large number of dead cells was obtained (hereafter, this culture medium is referred to as the "dead cell culture medium"). 0.5 mL of this dead cell culture medium was taken, and the cells were precipitated by centrifugation. After aspirating the supernatant, the cells were resuspended in 0.5 mL of phosphate-buffered saline (PBS). Fluorescently labeled Annexin V and propidium iodide (PI) (both from Nacalai Tesque) were added to stain apoptotic and necrotic cells, confirming the occurrence of apoptosis (and the simultaneous formation of dead cells).

[0048] (1-3) The viable cell culture medium obtained in (1-1) and the dead cell culture medium obtained in (1-2) were mixed at a ratio of 3:1, and 2 mL of the mixture was centrifuged at 100 × g for 2 minutes, and the supernatant was aspirated off. The pellet was then suspended in 2 mL of phosphate buffered saline (PBS) to prepare a cell suspension (hereinafter referred to as "viable cell suspension") to be used in the dead cell removal test described below (cell density: 1 × 10 6 cells / mL).

[0049] (2) Dead cell removal test (2-1) After 0.6 mL of the viable cell suspension obtained in (1) was placed in each of two test tubes, an aqueous calcium chloride solution was added to each test tube so that the calcium ion concentration became 1 mmol / L.

[0050] (2-2) In one of the test tubes, 1.2 × 10 6 After adding 100 pieces (corresponding to twice the number of cells present in the test tube), the test tubes, including the one to which the insoluble carrier had not been added, were reacted at room temperature for 60 minutes while mixing by inversion.

[0051] (2-3) After the reaction, the test tube to which the Tim4-immobilized insoluble carrier had been added was separated and removed from the suspension of live and dead cells using a magnet.

[0052] The viability of in vitro cell populations was measured by trypan blue staining using a Vi-cell XR live / dead cell autoanalyzer (Beckman Coulter). The addition of Tim4-immobilized insoluble carriers resulted in a viability of 96.6%, compared to 84.0% without the insoluble carrier. These results demonstrate that the Tim4-immobilized insoluble carriers can remove dead cells from viable / dead cell suspensions.

[0053] Example 2: Removal of dead cells using Tim4-immobilized insoluble carrier (culture medium system) In Reference Example 1, dead cells were removed in a system in which cells were suspended in a buffer solution. In this Example, however, there was a large amount of impurities, and we investigated whether dead cells could be similarly removed in a cell culture medium closer to the embodiment.

[0054] (1) The viable cell culture medium obtained in Reference Example 1 (1-1) and the dead cell culture medium obtained in Reference Example 1 (1-2) were mixed in a ratio of 3:1, and the cell density was 1 × 10 6 The cell culture medium was diluted with the medium used for cell culture to give a concentration of 1000 cells / mL, and the cell culture medium (hereinafter also referred to as "viable and dead cell culture medium") to be used in the dead cell removal test described below was prepared.

[0055] (2) Dead cells in the culture medium were removed in the same manner as in Reference Example 1(2), except that calcium chloride solution was not added to the test tube (because the medium used for cell culture contained 1 mmol / L of calcium ions).

[0056] When the viability of a cell population in a test tube was measured, the viability was 96.4% when Tim4-immobilized insoluble carriers were added, which was higher than the 83.4% viability without the insoluble carrier. These results demonstrate that Tim4-immobilized insoluble carriers can also remove dead cells contained in cell culture media.

[0057] Reference Example 2: Removal of dead cells using an insoluble carrier immobilized with Annexin V (buffer system) (1) Annexin V BACS Microbubbles included in the Akadeum Life Sciences Dead Cell Removal Microbubble Kit (Cat. No. 11510-211) were used as an insoluble carrier onto which annexin V was immobilized. The carrier is made of hollow glass and floats on the water surface.

[0058] (2) 0.6 mL of the viable and dead cell suspension obtained in Reference Example 1(1) was placed in each of two test tubes, and then an aqueous calcium chloride solution was added to each test tube so that the calcium ion concentration was 1 mmol / L.

[0059] (3) In one of the test tubes, 1.2 × 10 insoluble carrier immobilized with annexin V was added. 6 After adding 100 pieces (corresponding to twice the number of cells present in the test tube), the test tubes, including the one to which the insoluble carrier had not been added, were reacted at room temperature for 60 minutes while mixing by inversion.

[0060] (4) After the reaction, the mixture was centrifuged at 100×g for 1 minute, and the floating insoluble carrier was separated and removed by suction.

[0061] When the viability of a cell population in vitro was measured, the viability was 95.4% when an insoluble carrier immobilized with annexin V was added, which was higher than the 82.1% viability without the insoluble carrier. These results demonstrate that the insoluble carrier immobilized with annexin V, like the insoluble carrier immobilized with Tim4, can remove dead cells from a suspension of live and dead cells.

[0062] Comparative Example 1: Removal of dead cells using an insoluble carrier immobilized with Annexin V (culture medium system) (1) 0.6 mL of the viable cell culture medium prepared in Example 2(1) was placed in each of two test tubes, and then 1.2 × 10 insoluble carriers with immobilized annexin V were added to only one of the test tubes. 6(corresponding to twice the number of cells present in the test tube) was added, and the test tubes, including the one to which the insoluble carrier had not been added, were reacted for 60 minutes at room temperature while being mixed by inversion.

[0063] (2) After the reaction, the mixture was centrifuged at 100×g for 1 minute, and the floating insoluble carrier was separated and removed by suction.

[0064] When the viability of the cell population in vitro was measured, the viability was 85.5% when an insoluble carrier immobilized with annexin V was added, which was equivalent to the 86.3% viability without the insoluble carrier. These results demonstrate that it was difficult to remove dead cells contained in the cell culture medium using an insoluble carrier immobilized with annexin V.

[0065] Table 1 shows a comparison of the survival rate measurement results in Reference Examples 1 and 2, Example 2, and Comparative Example 1.

[0066] [Table 1]

[0067] Comparative Example 2: Dead cell removal using an insoluble carrier immobilized with Annexin V (examination of culture medium system and salt addition) In Comparative Example 1, it was difficult to remove dead cells contained in the cell culture medium using an insoluble carrier with immobilized annexin V. We investigated whether the dead cell removal rate could be improved by adding calcium ions to the culture medium.

[0068] Specifically, 0.6 mL of the viable cell culture medium prepared in Example 2(1) was placed in each of two test tubes, and calcium chloride aqueous solution was added to each test tube so that the calcium ion concentration was 2 mmol / L, 3 mmol / L, 5 mmol / L, 8 mmol / L, 11 mmol / L, 21 mmol / L, or 31 mmol / L (all concentrations taking into account the calcium ion (1 mmol / L) contained in the culture medium). The viability of the cell population in the test tube was measured in the same manner as in Comparative Example 1.

[0069] The results are shown in Table 2. Compared with the viability without the addition of annexin V-immobilized insoluble carrier, the addition of annexin V-immobilized insoluble carrier improved the viability in systems containing 3 mmol / L or 5 mmol / L calcium ions. However, the cell viability itself was 88.2% (calcium ions: 3 mmol / L) and 90.0% (5 mmol / L), which was inferior to the 96.4% obtained with the addition of Tim4-immobilized insoluble carrier. Furthermore, in systems containing calcium ions of 8 mmol / L or more, the cytotoxicity of high concentrations of calcium ions caused the cells to divide into numerous small bodies, making it impossible to measure the viability.

[0070] [Table 2]

[0071] Example 3: Removal of dead cells using Tim4-immobilized insoluble carriers (examination of culture medium and low calcium ion concentration) In Example 2, dead cells were removed using a cell culture medium containing 1 mmol / L of calcium ions. We investigated whether similar dead cell removal could be achieved using a cell culture medium with a lower calcium ion concentration.

[0072] (1) Preparation of cell culture medium (1-1) Chinese hamster ovary (CHO) cells (DG44 strain) were cultured in a 250 mL shake flask containing 20 mL of animal-derived component-free growth medium (EX-CELL Advanced CHO Fed-batch Medium: Sigma-Aldrich) supplemented with L-glutamine. The cells were cultured in suspension at 130 rpm in a TAITEC NR-3 shaker at 37°C and 8.0% CO2 in an incubator. The cell viability in the culture medium was determined by trypan blue staining using a Vi-cell XR (Beckman Coulter) autoanalyzer, and was found to be over 95%.

[0073] The calcium ion concentration in the EX-CELL Advanced CHO Fed-batch Medium was measured using an ICP emission spectrometer (Agilent Technologies) and was found to be 0.67 mmol / L. Therefore, the culture medium obtained here will be referred to as the "low-calcium viable cell culture medium."

[0074] (1-2) During the culture of (1-1), 10 mL of the culture medium containing cells in the logarithmic growth phase was taken, and the apoptosis-inducing reagent cycloheximide was added to a final concentration of 100 μmol / L. After culturing for 72 hours, a cell culture medium containing a large number of dead cells was obtained (hereafter, this culture medium is referred to as the "low-calcium-concentration dead-cell culture medium"). 0.5 mL of the low-calcium-concentration dead-cell culture medium was taken, and the cells were precipitated by centrifugation. After aspirating the supernatant, the cells were resuspended in 0.5 mL of phosphate-buffered saline (PBS). Fluorescently labeled Annexin V and propidium iodide (PI) (both from Nacalai Tesque) were added to stain apoptotic and necrotic cells, confirming the occurrence of apoptosis (and the simultaneous formation of dead cells).

[0075] (1-3) The low-calcium-concentration live cell culture medium obtained in (1-1) and the low-calcium-concentration dead cell culture medium obtained in (1-2) were mixed in a ratio of 3:1, and the cell density was adjusted to 1 × 10 6 The cell culture medium was diluted with the medium used for cell culture to give a concentration of 1000 cells / mL to prepare the cell culture medium (hereinafter also referred to as "low calcium concentration viable cell culture medium") to be used in the dead cell removal test described below.

[0076] (2) Dead cell removal test (2-1) 0.6 mL of the low calcium concentration viable / dead cell suspension obtained in (1) was placed in each of two test tubes, and then 1.2 × 10 6 After adding 100 pieces (corresponding to twice the number of cells present in the test tube), the test tubes, including the one to which the insoluble carrier had not been added, were reacted at room temperature for 60 minutes while mixing by inversion.

[0077] (2-2) After the reaction, the test tube to which the Tim4-immobilized insoluble carrier had been added was separated and removed from the low calcium concentration viable cell suspension using a magnet.

[0078] When the viability of a cell population in vitro was measured, the addition of Tim4-immobilized insoluble carriers resulted in a viability of 98.4%, compared to 82.5% without the insoluble carrier (a 15.9% improvement in viability). These results demonstrate that Tim4-immobilized insoluble carriers can also remove dead cells contained in cell culture media with low calcium ion concentrations.

[0079] Comparative Example 3: Dead cell removal using an insoluble carrier immobilized with Annexin V (culture medium system, low calcium ion concentration study) 0.6 mL of the low calcium concentration viable cell culture medium prepared in Example 3(1) was placed in each of two test tubes, and then 1.2 × 10 Annexin V-immobilized insoluble magnetic carriers prepared according to the instructions included with the EasySep Dead Cell Removal (Annexin V) Kit (manufactured by STEMCELL Technologies) were added to only one of the test tubes. 6 (corresponding to twice the number of cells present in the test tube) were added, and the reaction was carried out at room temperature for 60 minutes while mixing by inversion, including the test tube to which the insoluble magnetic carriers had not been added.

[0080] (2) After the reaction, the test tube to which the insoluble magnetic carriers immobilized with Annexin V had been added was separated and removed from the low calcium concentration viable and dead cell suspension using a magnet.

[0081] When the viability of a cell population in vitro was measured, the viability was 88.4% when Annexin V-immobilized insoluble magnetic carriers were added, which was an improvement of 2.9% compared to the 85.5% viability without the addition of the insoluble magnetic carriers. However, this improvement was inferior to the 15.9% improvement achieved with the addition of Tim4-immobilized insoluble carriers.

[0082] Table 3

Claims

1. A separation agent for separating live cells from dead cells in a culture medium of eukaryotic cells, comprising an insoluble carrier and Tim4 (T-cell immunoglobulin and mucin domain 4) immobilized on the insoluble carrier.

2. Culturing eukaryotic cells; a step of reacting the culture solution after the culturing step with the separating agent according to claim 1; and separating dead cells.

3. The method according to claim 2 , wherein the step of separating dead cells comprises the steps of forming a complex between the separation agent and dead cells, and separating the complex from the culture medium to recover live cells.

4. 4. The method according to claim 2, wherein the calcium ion concentration in the culture medium is 0.5 mmol / L or more and 2 mmol / L or less in the step of culturing the eukaryotic cells.

5. The separation agent according to claim 1 , wherein the eukaryotic cells are suspension cells.

6. The method of claim 2 or 3, wherein the eukaryotic cells are suspension cells.

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