Low tissue factor activity cells and method for producing same

Abstract

To provide a cultured cell with low tissue factor (TF) activity.SOLUTION: The present invention relates to cell that is obtained by culturing on a culture substrate showing a lower limit critical solution temperature (LCST), cooling the culture substrate to a temperature equal to or lower than the lower limit critical solution temperature LCST and then peeling and collecting.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to cells with low tissue factor activity and a method for producing the same. [Background technology]

[0002] Cell therapy is a method of treating a disease using one's own cells or cells from another person, and has been attracting attention as a new treatment for diseases that have been said to be difficult to treat, such as graft-versus-host disease (GVHD), myocardial infarction, and spinal cord injury. Cells used in cell therapy are cultured in an in vitro environment using a culture substrate such as a petri dish, and the cultured cells are collected as a cell suspension, and the concentration is adjusted with physiological saline or the like to prepare a formulation, which is then intravenously injected into a patient. On the other hand, since a highly concentrated cell suspension is administered into a blood vessel, there is a concern about the formation of a blood clot after administration. In response to this, Patent Document 1 discloses that by culturing mesenchymal stem cells in a serum-free medium, the expression of tissue factor (TF) in mesenchymal stem cells can be reduced to 60% or less compared to mesenchymal stem cells obtained under conventional culture conditions. However, the effect of suppressing the expression of tissue factor (TF) in mesenchymal stem cells is not sufficient, and cells with a lower expression of tissue factor (TF) in mesenchymal stem cells are desired. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] WO2018 / 159432 issue Summary of the Invention [Problem to be solved by the invention]

[0004] The objective of this patent is to provide a cultured cell with low TF activity and a method for producing the same. [Means for solving the problem]

[0005] In view of the above, the present inventors conducted extensive research and discovered that tissue factor (TF) activity is suppressed in cells cultured on a culture vessel exhibiting a lower critical solution temperature (LCST), and then the culture vessel is cooled to a temperature below the LCST to detach and recover cells, thereby completing the present invention.

[0006] That is, the present invention includes the following aspects. <1> A cultured cell that has been cultured in vitro and then detached from a culture ware by cooling treatment, and is characterized in that the tissue factor (TF) activity is 40% or less than that of a cultured cell that has been cultured in vitro and then detached using a proteolytic enzyme. <2> The cell is a mesenchymal stem cell. <1> The cell according to claim 1. <3> The mesenchymal stem cells are derived from human bone marrow. <2> The cell according to claim 1. <4> A method for producing cells, characterized in that the cultured cells are detached from a culture vessel by cooling treatment after in vitro culture, and have a TF activity level of 40% or less compared to cultured cells detached using a proteolytic enzyme after in vitro culture. <5> <1> ~ <3> A cell recovery method comprising culturing any one of the cells described above on a culture vessel exhibiting a lower critical solubility temperature (LCST) and then recovering the cells by a cooling treatment. <6> <5> A cell recovery method, comprising the step of: providing a culture vessel exhibiting the above-described lower critical solution temperature (LCST) in which a temperature-responsive block copolymer is coated on a support material. Effect of the Invention

[0007] By adopting the configuration of the present invention, it is possible to obtain cultured cells having a lower tissue factor (TF) activity value than cultured cells detached using a protease in the same detachment time as when the protease is used. Therefore, when the cultured cells obtained by adopting the configuration of the present invention are intravenously injected into a patient, it is possible to reduce the risk of thrombus formation in the patient after administration. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] Hereinafter, the embodiments of the present invention will be described in detail, but the present invention is not limited to the following contents. The present invention can be carried out by appropriately modifying it within the scope of the spirit of the present invention.

[0009] The cells used in the present invention are not particularly limited as long as they can be cultured in vitro on a culture vessel. For example, in addition to various cultured cell lines such as Chinese hamster ovary-derived CHO cells, mouse connective tissue L929 cells, human fetal lung-derived normal diploid fibroblasts (TIG-3 cells), human fetal kidney-derived cells (HEK293 cells), and human cervical cancer-derived HeLa cells, for example, epithelial cells and endothelial cells that constitute each tissue and organ in the body, skeletal muscle cells that exhibit contractility, smooth muscle cells, cardiac muscle cells, neuron cells that constitute the nervous system, glial cells, and fibroblasts, hepatic parenchymal cells, non-hepatic parenchymal cells, and adipocytes that are involved in the metabolism of the body, and cells with differentiation potential such as induced pluripotent stem (iPS) cells, embryonic stem (ES) cells, embryonic germ (EG) cells, embryonic carcinoma (EC) cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, skin stem cells, muscle stem cells, and germ stem cells, or precursor cells of each tissue, and further cells induced to differentiate from them, etc., are particularly preferably used.

[0010] Mesenchymal stem cells refer to a population of stem cells and their precursor cells that can differentiate into all or some of the mesenchymal cells, such as chondrocytes, osteoblasts, and adipocytes. The origin of mesenchymal stem cells is not particularly limited, but includes tissue origins such as bone marrow, fat, dental pulp, umbilical cord blood, placenta, and synovium, and pluripotent stem cells such as ES cells and iPS cells, with bone marrow origin being particularly preferred. The origin of mesenchymal stem cells is also not particularly limited, but includes human, animal, and plant origin, with human origin being particularly preferred.

[0011] Tissue factor (TF) is a membrane-associated glycoprotein that forms a complex with factor VIIa. The factor VIIa / TF complex is known to initiate the blood clotting process. Once formed, the factor VIIa / TF complex activates a series of specific enzymes involved in the extrinsic and intrinsic pathways of the blood clotting cascade, ultimately resulting in the generation of thrombin, fibrin production, platelet activation, and clot formation, leading to thrombus formation.

[0012] Tissue factor (TF) activity (also referred to as "TF activity") refers to the activation reaction of factor VIIa, which is the initiating factor of the blood coagulation cascade, and it is known that the lower this activation reaction is, the more clot formation is suppressed.

[0013] The cells of the present invention may have a TF activity level of 40% or less of that of conventional cultured cells, preferably 35% or less, and more preferably 30% or less to 0%.

[0014] The method for measuring TF activity may be a method generally used in the art, but from the viewpoints of ease of operation, quantitativeness, detection sensitivity, etc., a TF activity colorimetric assay in which the activation of factor X by factor VIIa / TF complex to factor Xa is directly measured using a chromogenic substrate for factor Xa is preferred. A commercially available assay kit for measuring TF activity may also be used. In the case of the TF activity colorimetric assay, an example of the commercially available assay kit is the Human Tissue Factor Chromogenic Activity Assay Kit (manufactured by Assaypro, LLC). In the case of the TF activity colorimetric assay, the TF activity can be calculated, for example, by measuring a sample with known TF activity and using a calibration curve obtained.

[0015] Protease refers to enzymes such as trypsin, accutase, collagenase, natural protease, chymotrypsin, elastase, papain, pronase, or recombinant forms thereof. Cultured cells detached using proteases are cultured cells detached and collected using these proteases by methods commonly used in the art.

[0016] The shape of the culture equipment is not particularly limited, and examples thereof include dishes, flasks, microwell plates, culture bags, tubes, trays, culture tanks, etc. The materials of these equipment are also not particularly limited, and examples thereof include glass, polypropylene, polystyrene, stainless steel, and combinations thereof.

[0017] The surface of the culture vessel may be further coated with an extracellular matrix. The type of extracellular matrix is ​​not particularly limited, and may be, for example, collagen, atelocollagen, hyaluronic acid, elastin, proteoglycan, glycosaminoglycan, fibronectin, laminin, vitronectin, gelatin, or matrigel containing laminin, collagen IV, heparan sulfate proteoglycan, entactin / nidogen 1, 2, etc. as the main component, or may be one type of these or a combination of two or more types. Also, it may be a segment of these extracellular matrices.

[0018] The lower critical solution temperature (LCST) is the temperature below which a polymer dissolves in water to form a transparent solution, but above which it becomes insoluble and cloudy, or precipitates and undergoes phase separation. In the present specification, the presence of an LCST may be referred to as having temperature responsiveness. The LCST is preferably in the range of 20°C to 40°C, more preferably in the range of 25°C to 35°C. Examples of repeating units exhibiting LCST and their LCSTs with respect to water include N-isopropylacrylamide (LCST = 32°C), Nn-propylmethacrylamide (LCST = 22°C), N-tetrahydrofurfurylacrylamide (LCST = 28°C), N-ethoxyethylacrylamide (LCST = 35°C), N,N-diethylacrylamide (LCST = 32°C), Nn-propylmethacrylamide (LCST = 28°C), N-tetrahydrofurfurylmethacrylamide (LCST = 35°C), N-methyl-N-isopropylacrylamide (LCST = 23°C), and N-methyl-Nn-propylacrylamide (LCST = 20°C). The temperature at which the LCST appears varies depending on the concentration of the aqueous solution, but N-isopropylacrylamide is preferred because it has low concentration dependency of LCST appearance. The repeating units used in the culture vessel exhibiting LCST in the present invention may be one type only, or two or more types may be combined. In addition, if the copolymer has temperature responsiveness, it may contain a different repeating unit in addition to the repeating unit exhibiting the LCST. The LCST of a copolymer containing different repeating units may differ from the LCST of a polymer consisting of a single repeating unit, and the LCST of the copolymer is measured by actual measurement.

[0019] There is no particular limitation on the method of producing a cultureware exhibiting an LCST. For example, a method of polymerizing a repeating unit exhibiting an LCST by electron beam irradiation to coat the surface of the vessel, and a method of coating the vessel with a membrane by applying a surface treatment agent in which a polymer containing a repeating unit exhibiting an LCST is dissolved in a solvent to the vessel. The polymer is not particularly limited, but a method of synthesizing the polymer using a living radical polymerization technique described in "Radical Polymerization Handbook" pp. 161-225 (2010) published by NTS Co., Ltd. can be used. Cells cultured and grown using a cultureware generally need to be treated with a protease such as trypsin to be peeled off and collected from the vessel surface, but by using a cultureware exhibiting an LCST, the adhesive force of the vessel surface is weakened by the sol transition of the temperature-responsive polymer due to the temperature drop in the surrounding environment, and the cells can be peeled off and collected without using a protease.

[0020] When using a polymer membrane containing a repeating unit exhibiting LCST, it is preferable that the polymer component constituting the membrane contains a repeating unit for fixing the membrane to a substrate in addition to the repeating unit exhibiting LCST. Examples of such a repeating unit include styrene and its derivatives, 2-methoxyethyl acrylate, n-propyl acrylate, n-propyl methacrylate, n-butyl acrylate, and n-butyl methacrylate, and a block copolymer containing a repeating unit of 2-methoxyethyl acrylate, a repeating unit of n-butyl acrylate, and a repeating unit of N-isopropylacrylamide is particularly preferable.

[0021] The composition of the medium used in the present invention is not particularly limited as long as the cells adhere and grow, and is composed of a basal medium and serum, and may contain an antibiotic. The type of basal medium is not particularly limited, and for example, MEM, αMEM, DMEM, EMEM, GMEM, DMEM / Ham'sF-12, Ham'sF-12, Ham'sF-10, Medium199, RPMI1640, etc. can be used. The type of serum is not particularly limited, and for example, fetal bovine serum (FBS), calf serum, adult bovine serum, horse serum, sheep serum, goat serum, pig serum, chicken serum, rabbit serum, and human serum are used, but FBS is generally used because of its ease of availability. The serum concentration in the medium is not particularly limited. For cost-effectiveness, it is generally used at a concentration of 20 vol% or less, but it may be at a concentration of more than 20 vol%. Alternatively, the medium may be a serum-free medium that does not contain any untreated or unpurified serum, but contains purified blood-derived components or animal tissue-derived components (such as growth factors).

[0022] Cell culture is performed at a temperature higher than the LCST of the temperature-responsive polymer coating the surface of the cultureware. When human-derived cells are used, the cell culture is preferably performed at temperatures close to body temperature in order to obtain high culture efficiency, more preferably in the temperature range of 35 to 39°C, and even more preferably in the temperature range of 36 to 38°C.

[0023] There is no particular limitation on the cell culture density as long as the cells adhere and proliferate. In the case of mesenchymal stem cells, for example, 1.0×10 1 ~1.0×10 5 cells / cm 2 is preferred, and 1.0×10 2 ~1.0×10 4 cells / cm 2 Other culture conditions are not particularly limited, and the culture may be performed under conditions generally used in the art.

[0024] Methods for detaching and recovering cells from cultureware that exhibits an LCST include, for example, changing the ambient temperature to a temperature lower than the LCST of the cultureware and then waiting for the cells to detach naturally, locally impacting the cells by tapping or shaking, using a cell scraper, and pipetting. Of these, the pipetting method is preferred in order to minimize damage to the cells and shorten the time required for recovery.

[0025] The temperature at which the cultureware is cooled is preferably 1°C or more lower than the LCST. In order to reduce damage caused by cooling, the temperature at which the cultureware is cooled is preferably 0°C to a temperature 1°C lower than the LCST, more preferably 3°C to a temperature 3°C lower than the LCST, and even more preferably 4°C to a temperature 8°C lower than the LCST. Methods for lowering the temperature of the cultureware (also called "cooling treatment") include replacing with a cooled liquid and storing in a cold place, but replacing with a cooled liquid is preferable in order to shorten the time required for cooling. There is no particular limitation on the cooled liquid, and it can be selected according to the purpose, such as culture solution, other medium solution, isotonic solution, etc. In addition, in order to reduce damage to cells, the cooling time is preferably 60 minutes or less, more preferably 30 minutes or less, particularly preferably 20 minutes or less, and most preferably 10 minutes or less. EXAMPLES

[0026] Examples of the present invention are described below, but the present invention is not limited to these examples. Unless otherwise specified, commercially available reagents were used. <Block copolymer composition> Proton nuclear magnetic resonance spectroscopy ( 1 H-NMR spectroscopy was used to determine the molecular weight. <Molecular weight and molecular weight distribution of block copolymer> Weight average molecular weight (Mw), number average molecular weight (Mn) and molecular weight distribution (Mw / Mn) were measured by gel permeation chromatography (GPC). The GPC apparatus used was a Tosoh Corporation HLC-8320GPC, and two Tosoh Corporation TSKgel SuperAWM-H columns were used. The column temperature was set to 40°C, and the eluent was 10 mM sodium trifluoroacetate containing 10 mM sodium trifluoroacetate. The measurement sample was prepared at 1.0 mg / mL and measured. The molecular weight calibration curve was made using polymethylmethacrylate (Polymer Laboratories) with known molecular weight. <Synthesis example of block copolymer> 0.650 g (5 mmol) of 2-methoxyethyl acrylate (MEA) was added to a 200 mL two-neck flask, followed by 31.8 mg (100 μmol) of cyanomethyl dodecyl trithiocarbonate, 1.6 mg (10 μmol) of azobisisobutyronitrile, and 10 mL of tert-butyl alcohol. After replacing with argon gas, the mixture was heated and stirred at 62° C. for 24 hours.

[0027] After the first heating and stirring, 3.845 g (30 mmol) of n-butyl acrylate (BA) was added, followed by 1.6 mg (10 μmol) of azobisisobutyronitrile and 5 mL of tert-butyl alcohol. After replacing with argon gas, the mixture was heated and stirred at 62° C. for 24 hours.

[0028] After the second heating and stirring, 7.355 g (65 mmol) of N-isopropylacrylamide (IPAAm LCST = 32°C) was added to the above, and 1.6 mg (10 µmol) of azobisisobutyronitrile and 85 mL of tert-butyl alcohol were further added. After replacing with argon gas, the mixture was heated and stirred at 62°C for 24 hours.

[0029] After the third heating and stirring, the reaction solution was purified by reprecipitation with water and dried under reduced pressure to obtain a yellow solid. The obtained yellow solid was dissolved in chloroform, and the chloroform phase was recovered using a separatory funnel. The recovered chloroform phase was concentrated using an evaporator and purified by reprecipitation with heptane. The precipitate was recovered by filtration and dried under reduced pressure to obtain 8.295 g of block copolymer poly(MEA-BA-IPAAm). The composition of the obtained block copolymer was MEA:BA:IPAAm = 5:30:65 (mol%), and Mn was 11.8 × 10 4 , Mw / Mn was 1.45, and LCST was 28°C. <Preparation of surface treatment agent> Add 19.910 g of 2-methoxyethanol to 90 mg of block copolymer and stir. All of the components were dissolved to prepare a 0.45 wt % surface treatment agent for the block copolymer. <Preparation of temperature-responsive cultureware> A temperature-responsive cultureware coated with a block copolymer was prepared by adding 300 μL of the surface treatment agent to the center of an IWAKI tissue culture dish (φ10 cm) and spin-coating it with a spin coater (Mikasa, product name MS-B200) at a rotation speed of 3,000 rpm for 60 seconds. The amount of the block copolymer coated on the surface of this device was measured by total reflection Fourier transform infrared spectroscopy (ATR / FT-IR) and found to be 0.55 μg / cm for the temperature-responsive block. 2 It was.

[0030] Example 1 Bone marrow-derived human mesenchymal stem cells (Lonza Japan, Product Code: PT-2501, Lot Number: 0000603525) were cultured at 1.0 × 10 5 The cells were seeded per dish and cultured at 37°C and 5% CO2 concentration. The culture medium used was Dulbecco-Voigt modified Eagle's minimum essential medium (10 vol% FBS / DMEM) containing 10 vol% fetal bovine serum (Colombia).

[0031] After culturing for 7 days, the culture medium was removed, and fresh culture medium cooled to 4°C was added and cooled at room temperature for 10 minutes. After 10 minutes, the culture medium was pipetted using a pipetter so that the culture medium was applied to the entire culture surface of the culture equipment, and the culture medium was recovered together with the cells. The recovered culture medium was centrifuged under conditions of 160 rcf, 25°C, and 5 minutes, after which the supernatant was removed and 500 μL of PBS(-) was added to suspend the cells. 10 μL of the obtained cell suspension was added to a slide for measuring cell count (manufactured by Thermo Fisher Scientific Co., Ltd., product name: Countess (registered trademark) Cell Counting Chamber Slid) and counted using an automatic cell counter (The The cell number was measured using Thermo Fisher Scientific Co., Ltd. (trade name: Countess (registered trademark) II). After measuring the cell number, the mixture was centrifuged again at 160rcf, 25°C, and 5 minutes, the supernatant was removed, and 200μL of 1% Triton X-100 / Tris-HCl (pH 8.0) was added and suspended. After standing on ice for 15 minutes, the mixture was centrifuged at 14,000rcf, 4°C, and 20 minutes to obtain a protein solution. The concentration of the obtained protein solution was measured using a protein concentration measurement kit (manufactured by Thermo Fisher Scientific Co., Ltd., trade name: Pierce BCA Protein Assay Kit). After measuring the concentration, the TF activity of the protein solution was measured using a human TF measurement kit (manufactured by Assaypro, LLC, trade name: Human Tissue Factor Chromogenic Activity Assay Kit) to measure the change in absorbance at a wavelength of 405nm due to a color-developing substrate, and calculated from a calibration curve prepared from a TF sample with known activity. As a result, the value of TF activity per total protein amount was 1.4 pM / μg, which was 30% of the value in Comparative Example 1 where culture was performed by the conventional culture method.

[0032] Example 2 TF activity was measured in the same manner as in Example 1, except that human adipose-derived stem cells (manufactured by Lonza Japan, Product Code: PT-5006) were used. As a result, the TF activity value per total protein amount was 2.9 pM / μg, which was 39% of the value in Comparative Example 2, where the cells were cultured by the conventional culture method.

[0033] Example 3 TF activity was measured in the same manner as in Example 1, except that normal diploid fibroblasts derived from human fetal lung were cultured for 5 days. As a result, the TF activity value per total protein amount was 0.5 pM / μg, which was 36% of the value in Comparative Example 3, where the cells were cultured by the conventional culture method.

[0034] Example 4 After culturing for 7 days, the culture medium was removed, new culture medium at 25°C was added, and TF activity was measured in the same manner as in Example 1, except that the culture medium was cooled at room temperature for 20 minutes. As a result, the TF activity value per total protein amount was 1.5 pM / μg, which was 33% of the value in Comparative Example 1, where culture was performed by the conventional culture method.

[0035] Comparative Example 1 When detaching and recovering mesenchymal stem cells from an IWAKI tissue culture dish (φ10 cm), instead of adding culture medium cooled to 4°C, TF activity was measured in the same manner as in Example 1, except that after washing with PBS, 1 mL of a protease (Thermo Fisher Scientific, product name: Trypsin-EDTA (0.25%)) was added, the cells were left to stand at 37°C for 10 minutes, medium was added, and the cells were recovered. As a result, the value of TF activity per total protein amount was 4.6 pM / μg.

[0036] Comparative Example 2 TF activity was measured in the same manner as in Comparative Example 1, except that human adipose-derived stem cells (manufactured by Lonza Japan, Product Code: PT-5006) were used. As a result, the value of TF activity per total protein amount was 7.4 pM / μg.

[0037] Comparative Example 3 TF activity was measured in the same manner as in Comparative Example 1, except that normal diploid fibroblasts derived from human fetal lung were cultured for 5 days. As a result, the value of TF activity per total protein amount was 1.4 pM / μg.

[0038] [Table 1]

[0039]

Table 2

[0040]

Table 3

Claims

1. A method for recovering cultured cells detached from a culture vessel by cooling treatment after in vitro culture, comprising the steps of: A method for recovering cells, the recovered cultured cells having a tissue factor (TF) activity value of 40% or less compared to cultured cells detached using a protease, comprising: The cultured cells are any one of bone marrow-derived human mesenchymal stem cells, human adipose-derived stem cells, and human fetal lung-derived normal diploid fibroblast cells, After culturing on a culture vessel exhibiting a lower critical solution temperature (LCST) in which a temperature-responsive block copolymer is coated on a support material, A cell recovery method, comprising recovering cells by a cooling treatment, which is a treatment for changing the temperature to a temperature lower than the LCST.

2. The method according to claim 1, wherein the temperature-responsive block copolymer is a temperature-responsive block copolymer comprising a repeating unit of 2-methoxyethyl acrylate, a repeating unit of n-butyl acrylate, and a repeating unit of N-isopropylacrylamide.

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