WO2016125884A1

WO2016125884A1 - Method for creating cancer-carrying mammal model

Info

Publication number: WO2016125884A1
Application number: PCT/JP2016/053467
Authority: WO
Inventors: 北原　真樹; 泰斗西野
Original assignee: 日産化学工業株式会社
Priority date: 2015-02-06
Filing date: 2016-02-05
Publication date: 2016-08-11
Also published as: TW201636422A; JPWO2016125884A1

Abstract

The present invention provides a method for creating a cancer-carrying non-human mammal, the method comprising the following steps: (1) a step for suspending a culture of cancer cells in a medium composition comprising a structure with which cells or tissue can be suspended and cultured; and (2) a step for transfecting, into a non-human mammal, the cancer cells obtained by the culture in (1).

Description

How to create a tumor-bearing mammal model

The present invention relates to a method for producing a cancer-bearing mammal. More specifically, the present invention relates to a method for producing a cancer-bearing mammal, which comprises transferring tumor cells suspended in a specific medium composition to a non-human mammal.

The mouse tumor-bearing model has characteristics such as in vivo drug efficacy evaluation of anticancer drugs, cancer cells isolated from cancer tissue, or cancer cells created by genetic manipulation, etc. It is an experimental system in an important position for evaluation. In addition, establishment of a new human cancer cell line is performed by subculturing primary cancer cells prepared from patient cancer tissue in a cancer-bearing model. In the mouse tumor-bearing model of human tumors, human cells are transplanted into the body of the mouse, so that the transplanted human cancer cells are subjected to mouse immune attack. Therefore, an immunodeficient mouse is used as a host mouse. Nude mice are used as the most common immunodeficient mice. For cancer cells with low growth ability, it is necessary to increase the engraftment rate by transplanting them into mice together with an extracellular matrix that constructs a cancer microenvironment such as Matrigel, which is a basement membrane preparation. In

Non-Patent Documents

1, 2, 3, 4, 5, and 6, a cancer-bearing model was created by transplanting monolayer-cultured cancer cells in the presence of Matrigel, but the cancer tissue grew to about 100 mm ^3. To administer anticancer drugs. And early transplantation of A549 in 3x10 ⁶ cells high cancerous tissue formation and anticancer evaluation are possible, but 4-8 weeks transplantation 4x10 ^⁶ ~ 5x10 ⁶ cells in MCF7 cells, in JIMT-1 cells 7.5 × 10 ⁶ cells The transplantation requires 25 days of rearing, and in the evaluation of anticancer drugs with MCF-7 cells or SKOV3 cells, the cancer tissue grows from 100 mm ³ to 500 mm ³ (or 3 to 4 times before anticancer drug administration) 25 each It takes ~ 30 days (about 1 month) or 17 days. In addition, since Matrigel is derived from mouse cancer tissue, transplantation of human cancer cells in the presence of Matrigel results in a different environment from that in actual human cancer tissue, which is satisfactory from the viewpoint of extrapolation to humans. Not a system. On the other hand, when transplanting cancer cells with a low engraftment rate into nude mice without using Matrigel, in order to establish cancer-bearing mice that can be used practically for the evaluation of anticancer agents, a larger number of cells per animal and a longer period of time Therefore, there are problems in the accuracy of experimental results and the physical burden on the experimenter. For new cancer cells with low immune tolerance or with unknown immunological tolerance, or cancer cells with low growth ability, more severe immunodeficient mice, such as NOD-scid mice, are used to establish tumor-bearing mice. . However, since NOD-scid mice are vulnerable to stress and infection with pathogenic bacteria and require careful attention to breeding, NOD-scid mice are less versatile as a model animal for anticancer drug evaluation. Recently, cancer immunotherapy and antitumor immunity drugs have been developed, and therefore, development of a model using normal mice that are not immunodeficient is required.

Conventionally, cancer cells used for transplantation for creating a tumor-bearing model have been used after being cultured in a monolayer on a cell adhesion plate and then recovered by enzymatic treatment with trypsin. In addition, Matrigel is added to a cell dispersion obtained by enzymatic treatment of cancer tissue with collagenase or the like and used for transplantation. In these methods, anchorage-dependent growth serves as the backbone of cell growth, and inherently the characteristics of cancer cells based on anchorage-independent growth are changed in the living body. Furthermore, a large amount of cells and long-term breeding may be required for the growth of cancer tissue in vivo. Therefore, the conventional tumor-bearing mouse model is not satisfactory in terms of versatility as a model for anticancer drug evaluation, economic efficiency of experimental research, convenience, and extrapolation to clinical practice.

The present inventors have succeeded in developing a medium composition capable of culturing cells and tissues in an anchorage-independent manner while maintaining a floating state without substantially increasing the viscosity in a liquid medium. (Patent Documents 1 and 2).

WO2014 / 017513 A1 US2014 / 0106348 A1

An object of the present invention is to provide a method for producing a cancer-bearing mammal having high cancer cell engraftment and high versatility regardless of the presence or absence of immunodeficiency. Another object of the present invention is to provide a method for producing a cancer-bearing mammal excellent in clinical extrapolation and a method for screening an anticancer agent.

The present inventors, after substantially culturing cancer cells in a medium composition capable of culturing cells and tissues while maintaining a floating state without substantially increasing the viscosity in the liquid medium, It has been found that the engraftment rate and the growth ability in a living body are improved by transplanting this to a recipient mammal. As a result, it has become possible to produce a cancer-bearing mammal having a large tumor tissue with a small number of cancer cells in a short period of time. In addition, cancer cells that do not almost engraft unless transplanted with an extracellular matrix such as Matrigel, and cancer cells that do not form a tumor can be engrafted in vivo even when transplanted in the absence of extracellular matrix by subjecting them to the suspension culture. And the tumor was organized. Furthermore, by culturing in this liquid medium, it is possible to optimize the size of cell aggregates of cancer cells, and damage the cells without subjecting the cell aggregates to enzyme treatment such as trypsin. It was possible to recover the cell aggregate without transplantation, and it was possible to transplant it as a cell aggregate.
Based on these findings, further studies were made and the present invention was completed.

That is, the present invention is as follows:

[1] A method for producing a cancer-bearing non-human mammal, comprising the following steps:
(1) Suspension culture of cancer cells in a medium composition containing a structure capable of culturing cells or tissues in suspension; and (2) Non-human mammals cancer cells obtained by the culture of (1) Introduce to animals.
[2] The method according to [1], further comprising the following steps:
(3) Breeding non-human mammals to which cancer cells have been transferred, and forming tumor tissue from the transferred cancer cells.
[3] The method of [1] or [2], wherein in (2), the cancer cells suspended in the physiological aqueous composition containing the basement membrane preparation are transferred to a non-human mammal.
[4] The method according to [1] or [2], wherein in (2), cancer cells suspended in a physiological aqueous composition substantially free of a basement membrane preparation are transferred to a non-human mammal. Method.
[5] The method according to any one of [1] to [4], wherein the non-human mammal is an immunodeficient non-human mammal.
[6] The method according to [5], wherein the cancer cells are allogeneic or xenogeneic with respect to the non-human mammal to be transferred.
[7] The method according to [6], wherein the cancer cell is a human cancer cell.
[8] The method according to any one of [1] to [7], wherein the cancer cells have the ability to form solid tumors.
[9] The method according to any one of [1] to [8], wherein the structure comprises deacylated gellan gum.
[10] The method according to [9], wherein the concentration of the deacylated gellan gum in the medium composition is 0.005 to 0.3% (w / v).
[11] The method according to any one of [1] to [10], wherein the medium composition has a viscosity at 37 ° C. of 8 mPa · s or less.
[12] A screening method for an anticancer agent comprising the following steps:
(1) administering a test compound to a cancer-bearing non-human mammal prepared by the method according to any one of [1] to [11];
(2) Measuring the size of the tumor tissue in the cancer-bearing non-human mammal obtained in (1);
(3) To compare the size of the tumor tissue measured in (2) with the size of the tumor tissue of a control cancer-bearing non-human mammal not administered with the test compound.
[13] A method for promoting engraftment of cancer cells in non-human mammals, comprising suspension-culturing cancer cells in a medium composition containing a structure capable of culturing cells or tissues in suspension. .

The present invention provides a cancer-bearing mammal model that is excellent in versatility, economy, convenience, and / or clinical extrapolation. According to the present invention, since the engraftment rate and the growth ability in a living body at the time of in vivo transplantation of cancer cells are improved, a cancer-bearing mammal having a tumor tissue can be created in a shorter period of time with a smaller number of cancer cells. Is possible. In addition, cancer cells that hardly engraft unless transplanted with an extracellular matrix such as Matrigel can be engrafted in vivo under the absence of the extracellular matrix to form a tumor. It is. Further, a medium composition capable of culturing cells and tissues while maintaining a floating state without substantially increasing the viscosity in the liquid medium, and a cell aggregate cultured in the medium containing the composition are: Since it is considered to provide a culture environment that approximates the cancer tissue in an actual living body, it can be expected to reproduce a tumor tissue that approximates the actual clinical case. It is also possible to create a cancer-bearing animal model using non-immune-deficient animals. The cancer-bearing mammal model created by the method of the present invention is useful for cancer characteristic evaluation, individual cancer diagnosis, anticancer agent evaluation and the like.

The cancer tissue formation by the transplanted HCT116 cell is shown. The vertical axis shows the tumor volume. Attach-2D: adhesion culture. Low attach: Cultured on non-cell-adhesive plates in deacylated gellan gum-free medium. FP001-3D: Suspension culture in a medium containing deacylated gellan gum. Matrigel: transplanted in the presence of Matrigel. Medium: transplanted in the absence of Matrigel. The cancer tissue formation by the transplanted HCT116 cell is shown. The vertical axis shows the tumor volume. Attach-2D: adhesion culture. Low attach: Cultured on non-cell-adhesive plates in deacylated gellan gum-free medium. FP001-3D: Suspension culture in a medium containing deacylated gellan gum. The cancer tissue formation by the transplanted HCT116 cell is shown. The vertical axis shows the tumor volume. (FBS): transplanted using FBS-containing medium. (-): Transplanted using FBS-free medium. Two-dimensional: adherent culture. FP001: Suspension culture in a medium containing deacylated gellan gum. Anticancer drug effect on cancer tissue formation when HCT116 cells dispersed in a single cell after suspension culture in a medium containing deacylated gellan gum and transplanted in the absence of Matrigel. The vertical axis shows tumor volume (left panel) and tumor tissue weight (right panel). Comparison of cancer tissue formation between spheroid transplantation and single cell transplantation. HCT116 cells suspended in suspension in a medium containing deacylated gellan gum were transplanted in the absence of Matrigel. The vertical axis shows the tumor volume. The effect of Trametinib on cancer tissue formation when HCT116 cells suspended in suspension in a medium containing deacylated gellan gum were transplanted in the absence of Matrigel in the form of spheroids. The vertical axis shows tumor volume (left panel) and tumor tissue weight (right panel). Cancer tissue formation when SKOV3 cells dispersed in single cells after suspension culture in a medium containing deacylated gellan gum were transplanted in the absence of Matrigel. 2D: Adhesive culture. FP001: Suspension culture in a medium containing deacylated gellan gum. The vertical axis shows the tumor volume. The effect of an anticancer agent on cancer tissue formation when SKOV3 cells dispersed in a single cell after suspension culture in a medium containing deacylated gellan gum and transplanted in the absence of Matrigel. The vertical axis shows tumor volume (left panel) and tumor tissue weight (right panel). Comparison of cancer tissue formation between spheroid transplantation and single cell transplantation. SKOV3 cells suspended in suspension in a medium containing deacylated gellan gum were transplanted in the absence of Matrigel. The vertical axis shows the tumor volume. The effect of MK2206 on cancer tissue formation when SKOV3 cells suspended in suspension in a medium containing deacylated gellan gum are transplanted in the absence of matrigel in the state of spheroids. The vertical axis shows tumor volume (left panel) and tumor tissue weight (right panel). The effect of Gefitinib on cancer tissue formation when MCF7 cells suspended in suspension in a medium containing deacylated gellan gum were transplanted in the absence of Matrigel in the state of spheroids. Cancer tissue formation when LS123 cells suspended in suspension in a medium containing deacylated gellan gum are transplanted in the presence of matrigel (matrigel) or in the absence of matrigel (-). 2D: Adhesive culture. FP3D: suspension culture in a medium containing deacylated gellan gum. The vertical axis shows the tumor volume.

(1) Medium composition used in the present invention The present invention relates to a non-cancer-bearing composition comprising suspension culture of cancer cells using a medium composition capable of culturing cells and tissues while maintaining a floating state. Provide human mammal production technology. The medium composition makes it possible to culture cancer cells to be evaluated and tissues containing the same while maintaining a floating state. The medium composition can be prepared as described in WO2014 / 017513 A1 and US2014 / 0106348 A1. Hereinafter, the culture medium composition may be referred to as culture medium composition I.

A cell is the most basic unit constituting an animal, and has, as its elements, a cytoplasm and various organelles inside a cell membrane.

The cancer cells used in the present invention are mammalian cancer cells. Examples of mammals include rodents such as mice, rats, hamsters, and guinea pigs, rabbit eyes such as rabbits, ungulates such as pigs, cows, goats, horses, and sheep, cats such as dogs and cats, and humans Primates such as monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans and chimpanzees. The mammal is preferably a rodent (such as a mouse) or a primate, more preferably a human.

Examples of cancer include, but are not limited to, stomach cancer, esophageal cancer, colon cancer, colon cancer, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell cancer, basal cell cancer, Adenocarcinoma, bone marrow cancer, renal cell cancer, ureter cancer, liver cancer, bile duct cancer, cervical cancer, endometrial cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, epithelial cancer, craniopharyngeal cancer, Laryngeal cancer, tongue cancer, fibrosarcoma, mucosal sarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, hemangiosarcoma, lymphangiosarcoma, lymphatic endothelial sarcoma, synovial, mesothelioma, Ewing tumor, smooth Myoma, rhabdomyosarcoma, seminoma, Wilms tumor, glioma, astrocytoma, myeloblast, meningioma, melanoma, neuroblastoma, medulloblastoma, retinoblastoma, malignant Examples include tissues such as lymphoma and blood derived from cancer patients. The cancer cell may be a cancer cell (primary culture) isolated from a cancer tissue collected from a cancer patient with an enzyme or the like, or may be a established cancer cell line. Examples of cancer cell lines include, but are not limited to, human breast cancer cell lines HBC-4, BSY-1, BSY-2, MCF-7, MCF-7 / ADR RES, HS578T, MDA- MB-231, MDA-MB-435, MDA-N, BT-549, T47D, HeLa as human cervical cancer cell line, A549 as human lung cancer cell line, EKVX, HOP-62, HOP-92, NCI-H23, NCI-H226, NCI-H322M, NCI-H460, NCI-H522, DMS273, DMS114, Caco-2, COLO-205, HCC-2998, HCT-15, HCT-116, HT-29 as human colon cancer cell lines KM-12, SW-620, WiDr, human prostate cancer cell line DU-145, PC-3, LNCaP, human central nervous system cancer cell line U251, SF-295, SF-539, SF-268, SNB- 75, SNB-78, SNB-19, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, SK-OV-3, IGROV-1 as human ovarian cancer cell lines, RXF- as human renal cancer cell lines 631L, ACHN, UO-31, SN-12C, A498, CAKI-1, RXF-393L, 786-0, TK-10, AGS, MKN45, MKN28, St-4, MKN-1, MKN as human gastric cancer cell lines -7, MKN-74, skin cancer cell line LOX-IMVI, LO X, MALME-3M, SK-MEL-2, SK-MEL-5, SK-MEL-28, UACC-62, UACC-257, M14, CCRF-CRM, K562, MOLT-4, HL- as leukemia cell lines 60TB, RPMI8226, SR, UT7 / TPO, Jurkat, A431 as a human epithelioid cancer cell line, A375 as a human melanoma cell line, HOS, MNMG, MNNG / HOS as a human osteosarcoma cell line, BxPC3 as a human pancreatic cancer cell line, COLO-357HPAC, MIAPaCa-2, Panc-1, and LS123 as human colon cancer cell lines. Examples of cell lines include, but are not limited to, HEK293 (human embryonic kidney cells), MDCK, MDBK, BHK, C-33A, AE-1, 3D9, Ns0 / 1, NIH3T3, PC12, S2 , Sf9, Sf21, High® Five (registered trademark), Vero, and the like.

The floating of cells (or cell aggregates) and / or tissues in the present invention means a state in which cells (or cell aggregates) and / or tissues can contact the bottom surface but do not adhere to the culture vessel (non-adhesion). ). Furthermore, in the present invention, when cells (or cell aggregates) and / or tissues are grown, differentiated or maintained, external pressure or vibration to the liquid medium composition, shaking in the composition, rotation operation, etc. A state in which cells (or cell aggregates) and / or tissues are uniformly dispersed in the liquid medium composition and are in a floating state without being accompanied by “floating standing”. Cultivation) and / or tissue culture is referred to as “floating stationary culture”. In addition, the period that can be floated in “floating standing” includes 5 minutes or more, 1 hour or more, 24 hours or more, 48 hours or more, 7 days or more, etc. It is not limited to.

The medium composition used in the present invention is a suspension of cells (or cell aggregates) and / or tissues in at least one point in a temperature range (for example, 0 to 40 ° C.) in which cells and tissues can be maintained and cultured. Is possible. The medium composition used in the present invention is preferably capable of allowing cells (or cell aggregates) and / or tissues to float in suspension at least at one point in the temperature range of 25 to 37 ° C., and most preferably at 37 ° C. .

Whether or not the suspension can be suspended is determined by, for example, dispersing the cells to be cultured at a concentration of 2 × 10 ⁴ cells / ml uniformly in the medium composition to be evaluated and injecting 10 ml into a 15 ml conical tube. By standing at 37 ° C for at least 5 minutes or more (eg, 1 hour or more, 24 hours or more, 48 hours or more, 7 days or more), and observing whether or not the floating state of the cells is maintained Can be evaluated. If more than 70% of all cells are floating, it can be concluded that the floating state was maintained. Instead of cells, polystyrene beads (Size 500-600 μm, manufactured by Polysciences Inc.) may be substituted for evaluation.

The medium composition used in the present invention is a composition containing a structure and a medium in which cells (or cell aggregates) or tissues can be suspended and cultured (preferably capable of floating stationary culture).
The medium composition used in the present invention is preferably a composition capable of recovering cells (or cell aggregates) or tissues from the medium composition after replacement of the medium composition at the time of culture and after completion of the culture. More preferably, the composition does not require any temperature change, chemical treatment, enzyme treatment, or shearing force when cells (or cell aggregates) or tissues are collected from the medium composition.
The structure in the present invention is formed from a specific compound and exhibits the effect of uniformly floating cells (or cell aggregates) and / or tissues. More specifically, it includes those in which polymer compounds are aggregated via ions, or in which polymer compounds form a three-dimensional network. In addition, it is known that polysaccharides form microgels via metal ions (for example, JP-A-2004-129596), and the structure of the present invention includes the microgel as one embodiment.
Moreover, as a thing with which the high molecular compound aggregated via ion, the film-like structure is mentioned as one aspect | mode.
The size of the structure in the present invention is preferably one that passes through a filter having a pore diameter of 0.2 μm to 200 μm when filtered through a filter. The lower limit of the pore diameter is more preferably more than 1 μm, and more preferably more than 5 μm in consideration of stably floating cells (or cell aggregates) or tissues. The upper limit of the pore diameter is more preferably 100 μm or less, and even more preferably 70 μm or less considering the size of cells (or cell aggregates) or tissue.
The specific compound in the present invention forms an amorphous structure when the specific compound is mixed with a liquid medium, the structure is uniformly dispersed in the liquid, and substantially increases the viscosity of the liquid. It has the effect of substantially retaining the cells and / or tissues and preventing their sedimentation. “Does not substantially increase the viscosity of the liquid” means that the viscosity of the liquid does not exceed 8 mPa · s. In this case, the viscosity of the liquid (that is, the viscosity of the medium composition used in the present invention) is 8 mPa · s or less, preferably 4 mPa · s or less, more preferably 2 mPa · s or less at 37 ° C. is there. Furthermore, the structure is formed in a liquid medium, and the effect of causing cells (or cell aggregates) and / or tissue to float uniformly (preferably to float and stand still) without substantially increasing the viscosity of the liquid. As long as it shows, the chemical structure, molecular weight, physical properties, etc. of the specific compound are not limited at all.
The viscosity of the liquid containing the structure can be measured, for example, by the method described in the examples described later. Specifically, E type viscometer under the condition of 37 ° C (Toki Sangyo Co., Ltd., TV-22 type viscometer, model: TVE-22L, cone rotor: standard rotor 1 ° 34´ × R24, rotation speed 100rpm) Can be measured.

The medium composition used in the present invention can achieve improvement in the engraftment rate and viability in the living body when transplanted to a recipient mammal by suspension culture of cancer cells in the medium composition. As long as the viscosity of the medium composition used in the present invention is substantially increased, it does not prevent the viscosity of the medium composition from being increased by the specific compound (the viscosity of the liquid exceeds 8 mPa · s). (Viscosity does not exceed 8 mPa · s). The viscosity of the medium composition used in the present invention is 8 mPa · s or less, preferably 4 mPa · s or less, more preferably 2 mPa · s or less at 37 ° C.

Examples of the specific compound used in the present invention include, but are not particularly limited to, a polymer compound, preferably a polymer compound having an anionic functional group.
Examples of the anionic functional group include a carboxy group, a sulfo group, a phosphate group, and salts thereof, and a carboxy group or a salt thereof is preferable.
As the polymer compound used in the present invention, one having one or more selected from the group of anionic functional groups can be used.
Preferable specific examples of the polymer compound used in the present invention include, but are not particularly limited to, a polysaccharide obtained by polymerizing 10 or more monosaccharides (for example, triose, tetrose, pentose, hexose, heptose, etc.). More preferably, an acidic polysaccharide having an anionic functional group is used. The acidic polysaccharide here is not particularly limited as long as it has an anionic functional group in its structure. For example, a polysaccharide having uronic acid (for example, glucuronic acid, iduronic acid, galacturonic acid, mannuronic acid) is used. , Polysaccharides having a sulfate group or phosphate group in a part of the structure, or polysaccharides having both structures, not only polysaccharides obtained from nature, but also polysaccharides and genes produced by microorganisms Also included are engineeringly produced polysaccharides or polysaccharides artificially synthesized using enzymes. More specifically, hyaluronic acid, gellan gum, deacylated gellan gum (hereinafter sometimes referred to as DAG), rhamzan gum, diyutan gum, xanthan gum, carrageenan, xanthan gum, hexuronic acid, fucoidan, pectin, pectinic acid, pectinic acid, Examples include those composed of one or more from the group consisting of heparan sulfate, heparin, heparitin sulfate, kerato sulfate, chondroitin sulfate, dermatan sulfate, rhamnan sulfate and salts thereof. The polysaccharide is preferably hyaluronic acid, DAG, Dieutan gum, xanthan gum, carrageenan or a salt thereof, and can float cells or tissues with a low concentration, and facilitate recovery of the cells or tissues. In view of the above, DAG is most preferable.
Examples of the salt herein include salts of alkali metals such as lithium, sodium and potassium, salts of alkaline earth metals such as calcium, barium and magnesium, and salts of aluminum, zinc, copper, iron, ammonium, organic bases and amino acids. Is mentioned.
The weight average molecular weight of these polymer compounds (polysaccharides and the like) is preferably 10,000 to 50,000,000, more preferably 100,000 to 20,000,000, still more preferably 1,000, 000 to 10,000,000. For example, the molecular weight can be measured in pullulan conversion by gel permeation chromatography (GPC).
Furthermore, phosphorylated DAG can be used. The phosphorylation can be performed by a known method.

In the present invention, a plurality of (preferably two) polysaccharides may be used in combination. The type of combination of polysaccharides is that the above-mentioned structure is formed in a liquid medium, and the cells (or cell aggregates) and / or tissues are uniformly suspended without substantially increasing the viscosity of the liquid medium (preferably Is not particularly limited as long as it can be left floating), but preferably the combination contains at least DAG or a salt thereof. That is, suitable polysaccharide combinations include DAG or a salt thereof, and polysaccharides other than DAG or a salt thereof (eg, xanthan gum, alginic acid, carrageenan, diutan gum, methylcellulose, locust bean gum or a salt thereof). It is. Specific polysaccharide combinations include DAG and Ramzan gum, DAG and Dieutan gum, DAG and Xanthan gum, DAG and Carrageenan, DAG and Xanthan gum, DAG and locust bean gum, DAG and κ-carrageenan, DAG and sodium alginate, DAG And methyl cellulose, but are not limited thereto.

More preferable specific examples of the specific compound used in the present invention include hyaluronic acid, deacylated gellan gum, diyutan gum, carrageenan and xanthan gum, and salts thereof. Considering the ease of tissue recovery, the most preferable example is deacylated gellan gum or a salt thereof.

The deacylated gellan gum in the present invention is a linear chain composed of four sugar molecules of 1-3 linked glucose, 1-4 bonded glucuronic acid, 1-4 bonded glucose and 1-4 bonded rhamnose. In the following general formula (I), R ₁ and R ₂ are both hydrogen atoms, and n is a polysaccharide represented by an integer of 2 or more. However, R ₁ may contain a glyceryl group and R ₂ may contain an acetyl group, but the content of acetyl group and glyceryl group is preferably 10% or less, more preferably 1% or less.
The structure in the present invention can take various forms depending on the specific compound. Deacylated gellan gum will be described in the case of deacylated gellan gum. When deacylated gellan gum is mixed with a liquid medium, metal ions (for example, calcium Ions) and form an amorphous structure via the metal ions, and the cells (or cell aggregates) and / or tissues are suspended. The viscosity of the medium composition used in the present invention prepared from deacylated gellan gum is 8 mPa · s or less, preferably 4 mPa · s or less, and is easy to recover cells (or cell aggregates) or tissues. Considering this point, it is more preferably 2 mPa · s or less.

The specific compound in the present invention can be obtained by a chemical synthesis method, but when the compound is a natural product, it can be extracted from various plants, animals, and microorganisms containing the compound using conventional techniques. It is preferable to obtain by separation and purification. In the extraction, the compound can be extracted efficiently by using water or supercritical gas. For example, as a method for producing gellan gum, the produced microorganism is cultured in a fermentation medium, and the mucosa produced outside the cells is collected by a normal purification method, and after drying, pulverization, etc., it is powdered. Good. Further, in the case of deacylated gellan gum, it may be recovered after subjecting it to alkali treatment when recovering the mucosa and deacylating the glyceryl group and acetyl group bonded to the 1-3 bonded glucose residue. Purification methods include, for example, liquid-liquid extraction, fractional precipitation, crystallization, various ion exchange chromatography, gel filtration chromatography using Sephadex LH-20, etc., adsorption chromatography using activated carbon, silica gel, or thin layer It is possible to purify by removing impurities by using adsorption / desorption treatment of an active substance by chromatography or high-performance liquid chromatography using a reverse phase column alone or in combination in any order and repeatedly. Examples of gellan gum producing microorganisms include, but are not limited to, Sphingomonas elodea and microorganisms modified from the genes of the microorganisms.
In the case of deacylated gellan gum, commercially available products such as “KELCOGEL (registered trademark of CPE Kelco) CG-LA” manufactured by Sanki Co., Ltd., “Kelcogel (CPP) manufactured by Saneigen FFI Co., Ltd.・ Registered trademark of Kelco) ”or the like. In addition, “Kelcogel (registered trademark of CPI Kelco) HT” manufactured by San-Ei Gen FFI Co., Ltd. can be used as native gellan gum.

The concentration of the specific compound in the medium depends on the type of the specific compound, and the specific compound forms the above-described structure in the liquid medium (preferably without substantially increasing the viscosity of the liquid medium). It can be set as appropriate as long as cells (or cell aggregates) and / or tissues can be floated uniformly (preferably allowed to stand still), but usually 0.0005% to 1.0% (w / v), Preferably, it may be 0.001% to 0.4% (w / v), more preferably 0.005% to 0.1% (w / v), and still more preferably 0.005% to 0.05% (w / v). For example, in the case of deacylated gellan gum, 0.001% to 1.0% (w / v), preferably 0.003% to 0.5% (w / v), more preferably 0.005% to 0.3% (w / v), more preferably It may be added to 0.01% to 0.05% (w / v), most preferably 0.01% to 0.03% (w / v) medium. In the case of xanthan gum, 0.001% to 5.0% (w / v), preferably 0.01% to 1.0% (w / v), more preferably 0.05% to 0.5% (w / v), most preferably 0.1% to 0.2%. % (W / v) medium may be added. In the case of a mixed system of κ-carrageenan and locust bean gum, the sum of both compounds is 0.001% to 5.0% (w / v), preferably 0.005% to 1.0% (w / v), more preferably 0.01% to 0.1%. (W / v), most preferably 0.03% to 0.05% (w / v) may be added to the medium. In the case of native gellan gum, it may be added in a medium of 0.05% to 1.0% (w / v), preferably 0.05% to 0.1% (w / v).

When the polysaccharide is used in combination of a plurality of types (preferably two types), the concentration of the polysaccharide is such that the combination of the polysaccharide forms the above-described structure in the liquid medium (preferably, the liquid medium It can be appropriately set within a range in which cells (or cell aggregates) and / or tissues can be suspended in a uniform manner (preferably allowed to stand still) without substantially increasing the viscosity. For example, when a combination of DAG or a salt thereof and a polysaccharide other than DAG or a salt thereof is used, the concentration of DAG or a salt thereof is 0.005 to 0.02% (w / v), preferably 0.01 to 0.02% (w / v v) is exemplified, and the concentration of polysaccharides other than DAG or a salt thereof is 0.0001 to 0.4% (w / v), preferably 0.005 to 0.4% (w / v), more preferably 0.1 to 0.4% (w / v) is exemplified. The following are examples of specific combinations of concentration ranges.
DAG or a salt thereof: 0.005 to 0.02% (preferably 0.01 to 0.02%) (w / v)
Polysaccharide xanthan gum other than DAG: 0.1-0.4% (w / v)
Sodium alginate: 0.0001 to 0.4% (w / v) (preferably 0.1 to 0.4% (w / v))
Native gellan gum: 0.0001-0.4% (w / v)
Locust bean gum: 0.1-0.4% (w / v)
Methyl cellulose: 0.1 to 0.4% (w / v) (preferably 0.2 to 0.4% (w / v))
Carrageenan: 0.05-0.1% (w / v)
Dieutan gum: 0.05-0.1% (w / v)

The concentration can be calculated by the following formula.
Concentration [% (w / v)] = weight of specific compound (g) / volume of medium composition (ml) × 100

The compound can be further changed into another derivative by a chemical synthesis method, and the derivative thus obtained can also be used effectively in the present invention. Specifically, in the case of deacylated gellan gum, the hydroxyl group corresponding to R ₁ and / or R ₂ of the compound represented by the general formula (I) is a C _1-3 alkoxy group, a C _1-3 alkylsulfonyl group, Derivatives substituted with monosaccharide residues such as glucose or fructose, oligosaccharide residues such as sucrose and lactose, and amino acid residues such as glycine and arginine can also be used in the present invention. Alternatively, the compound can be crosslinked using a crosslinker such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC).

The specific compound or salt thereof used in the present invention can exist in any crystal form depending on the production conditions, and can exist as any hydrate. These crystal forms, hydrates, and mixtures thereof. Are also included within the scope of the present invention. Moreover, although it may exist as a solvate containing organic solvents, such as acetone, ethanol, and tetrahydrofuran, all of these forms are contained in the scope of the present invention.

The specific compound used in the present invention exists in the form of a tautomer, geometric isomer, tautomer or mixture of geometric isomers, or a mixture thereof formed by intra-ring or exocyclic isomerization. Also good. Regardless of whether the compound of the present invention is produced by isomerization, when it has an asymmetric center, it may exist in the form of a resolved optical isomer or a mixture containing them in an arbitrary ratio.

The medium composition used in the present invention may contain metal ions such as divalent metal ions (calcium ions, magnesium ions, zinc ions, iron ions, copper ions, etc.), and preferably contains calcium ions. . The metal ions can be used in combination of two or more, such as calcium ions and magnesium ions, calcium ions and zinc ions, calcium ions and iron ions, calcium ions and copper ions. Those skilled in the art can appropriately determine the combination. In one embodiment, a metal ion (eg, calcium ion) is contained in the medium composition, whereby the polymer compound is aggregated via the metal ion, and the polymer compound forms a three-dimensional network (for example, The structure of the present invention is formed by the polysaccharide forming a microgel via metal ions (eg, calcium ions). The concentration of the metal ion is such that the specific compound forms the above-described structure in the liquid medium, and preferably (without substantially increasing the viscosity of the liquid medium) cells (or cell aggregates) and / or tissue. It can be set as appropriate as long as it can be uniformly suspended (preferably allowed to float and stand). The metal ion concentration is 0.1 mM to 300 mM, preferably 0.5 mM to 100 mM, but is not limited thereto. The metal ions may be mixed with the medium, or a salt solution may be separately prepared and added to the medium. Further, the medium composition used in the present invention may contain an extracellular matrix, an adhesion molecule, etc. described later.

The structure composed of a specific compound used in the present invention contains the structure of the specific compound when the cell (or cell aggregate) and / or tissue are cultured in vitro. It shows the effect of floating in a liquid (preferably the effect of floating and standing). Due to the floating effect, it is possible to increase the number of cells (or cell aggregates) and / or the number of tissues per fixed volume as compared with monolayer culture. In addition, when a conventional suspension culture method involves a rotation or shaking operation, a shearing force acts on cells (or cell aggregates) and / or tissues, so that the proliferation rate and recovery rate of cells and / or tissues are low, or Cell function may be impaired. However, by using the medium composition containing the structure of the specific compound of the present invention, cells (or cell aggregates) and / or tissue can be uniformly dispersed without performing operations such as shaking. Target cells (or cell aggregates) and / or tissues can be obtained easily and in large quantities without loss of cell function. In addition, when cells (or cell aggregates) and / or tissues are suspended and cultured in a medium containing a conventional gel substrate, it is difficult to observe or recover the cells (or cell aggregates) and / or tissues. In some cases, the function may be impaired. However, by using a medium composition containing the structure of the specific compound of the present invention, cells (or cell aggregates) and / or tissues are cultured in suspension, and observed and recovered without losing their state and function. be able to. Moreover, the culture medium containing the conventional gel base material has a high viscosity, and replacement | exchange of a culture medium may be difficult. However, since the medium composition containing the structure of the specific compound of the present invention has a low viscosity, the medium can be easily replaced using a pipette, a pump, or the like.

When culturing cells (or cell aggregates) and / or tissues using the specific compound in the present invention, the medium used for culturing cells (or cell aggregates) and / or tissues and the specific compound are mixed. Thus, a medium composition can be prepared. The classification according to the composition of the medium includes a natural medium, a semi-synthetic medium, and a synthetic medium, and the classification according to the shape includes a semi-solid medium, a liquid medium, and a powder medium (hereinafter sometimes referred to as a powder medium). When cells (or cell aggregates) and / or tissues are derived from animals, any medium can be used as long as it is a medium used for culturing animal cells. Examples of such media include Dulbecco's Modified Eagle Medium (Dulbecco's Modified Eagles's Medium; DMEM), Ham F12 Medium (Ham's Nutrient Mixture F12), DMEM / F12 Medium, McCoy's 5A medium, les' MEMs Medium (E Minimum Essential Medium (EMEM), αMEM medium (alpha Modified Eagles's Minimum Essential Medium; αMEM), MEM medium (Minimum Essential Medium), RPMI1640 medium, Iscove's modified Dulbecco's medium, Iscove's Modified Dulbecco's Medium medium; , IPL41 medium, Fischer's medium, StemPro34 (manufactured by Invitrogen), X-VIVO-10 (manufactured by Cambridge), X-VIVO-15 (manufactured by Cambridge), HPGM (manufactured by Cambridge), StemSpan-H3000 (Stem Cell Technology) ), StemSpanSFEM (manufactured by Stem Cell Technology), Stemline II (Sigma Aldrich) QBSF-60 (Quality Biological), StemProhESCSFM (Invitrogen), Essential8 (registered trademark) medium (Gibco), mTeSR1 or 2 medium (Stem Cell Technology), Repro FF or Repro FF2 ( Reprocell), PSGro hESC / iPSC medium (System Bioscience), NutriStem (registered trademark) medium (Biological Industries), CSTI-7 medium (Cell Science Laboratories), MesenPRO RS medium (Gibco) MF-Medium (registered trademark) mesenchymal stem cell growth medium (Toyobo Co., Ltd.), Sf-900II (Invitrogen), Opti-Pro (Invitrogen), and the like.

The medium used for culturing cancer cells can contain a cell adhesion factor in the medium. Examples thereof include matrigel, collagen gel, gelatin, poly-L-lysine, poly-D-lysine, laminin, and fibronectin. These cell adhesion factors can be added in combination of two or more. Furthermore, thickeners such as guar gum, propylene glycol alginate, locust bean gum, gum arabic, tara gum, tamarind gum and methylcellulose can be further mixed with the medium used for culturing cancer cell spheres.

Those skilled in the art may freely add sodium, potassium, calcium, magnesium, phosphorus, chlorine, various amino acids, various vitamins, antibiotics, serum, fatty acids, sugars, and the like to the above medium according to the purpose. In the case of animal-derived cells (or cell aggregates) and / or tissue culture, those skilled in the art can add one or more other chemical components or biological components in combination according to the purpose.
Components added to animal-derived cells (or cell aggregates) and / or tissue culture media include fetal bovine serum, human serum, horse serum, insulin, transferrin, lactoferrin, cholesterol, ethanolamine, sodium selenite, Monothioglycerol, 2-mercaptoethanol, bovine serum albumin, sodium pyruvate, polyethylene glycol, various vitamins, various amino acids, agar, agarose, collagen, methylcellulose, various cytokines, various hormones, various growth factors, various extracellular matrices and various Examples include cell adhesion molecules.

Examples of antibiotics added to the medium include sulfa drugs, penicillin, pheneticillin, methicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, nafcillin, ampicillin, penicillin, amoxicillin, cyclacillin, carbenicillin, ticarcillin, piperacillin, piperacillin, Mecuzurocillin, mecillinam, andinocillin, cephalosporin and its derivatives, oxophosphoric acid, amifloxacin, temafloxacin, nalidixic acid, pyromido acid, ciprofloxane, sinoxacin, norfloxacin, perfloxacin, rosoxacin, ofloxacin, enoxacin, pipexamic acid, sulbactam acid, sulbactam acid, sulbactam acid, sulbactam acid , Β-bromopenicillanic acid, β-chloropenicillanic acid, 6-acetylmethylene-penicillanic acid Cefoxazole, sultampicillin, adinocillin and sulbactam formaldehyde and foodlate esters, tazobactam, aztreonam, sulfazetin, isosulfazetin, nocardicin, m-carboxyphenyl, methyl phenylacetamidophosphonate, chlortetracycline, oxytetracycline, tetracycline, deme Examples include clocycline, doxycycline, metacycline, and minocycline.

When the specific compound in the present invention is added to the above medium, the specific compound is first dissolved or dispersed in an appropriate solvent (this is referred to as a medium additive). Thereafter, the concentration of the specific compound in the medium, as detailed above (preferably without substantially increasing the viscosity of the liquid medium), the cells (or cell aggregates) and / or tissue are uniformly distributed. Concentration at which it can be floated (preferably suspended), for example 0.0005% to 1.0% (w / v), preferably 0.001% to 0.4% (w / v), more preferably 0.005% to 0.1% (w / v), more preferably 0.005% to 0.05% (w / v), the medium additive may be added to the medium. For example, in the case of deacylated gellan gum, 0.001% to 1.0% (w / v), preferably 0.003% to 0.5% (w / v), more preferably 0.005% to 0.3% (w / v), most preferably 0.01% to 0.05% (w / v) medium may be added. In the case of xanthan gum, 0.001% to 5.0% (w / v), preferably 0.01% to 1.0% (w / v), more preferably 0.05% to 0.5% (w / v), most preferably 0.1% to 0.2%. % (W / v) medium may be added. In the case of a mixed system of κ-carrageenan and locust bean gum, the sum of both compounds is 0.001% to 5.0% (w / v), preferably 0.005% to 1.0% (w / v), more preferably 0.01% to 0.1%. Most preferably, it may be added to a 0.03% to 0.05% (w / v) medium. In the case of a mixed system of deacylated gellan gum and diyutan gum, the total of both compounds should be added to the medium between 0.001% and 1.0% (w / v), most preferably 0.005% to 0.01% (w / v). It ’s fine. In the case of a mixed system of deacylated gellan gum and methylcellulose, the total amount of both compounds may be added to a medium of 0.001% to 1.0% (w / v), most preferably 0.005% to 0.2% (w / v). . In the case of a mixed system of deacylated gellan gum and locust bean gum, the total of both compounds should be added to the medium between 0.001% and 1.0% (w / v), most preferably between 0.01% and 0.1% (w / v). It ’s fine. In the case of a mixed system of deacylated gellan gum and sodium alginate, the total of both compounds can be added to a medium of 0.001% to 1.0% (w / v), most preferably 0.01% to 0.1% (w / v). good. In the case of a mixed system of deacylated gellan gum and xanthan gum, the total of both compounds may be added to a medium of 0.001% to 1.0% (w / v), most preferably 0.01% to 0.1% (w / v). . In the case of a mixed system of deacylated gellan gum and κ-carrageenan, the total of both compounds is added to the medium in a range of 0.001% to 1.0% (w / v), most preferably 0.01% to 0.1% (w / v). It ’s fine. The concentration can be calculated by the following formula.
Concentration [% (w / v)] = weight of specific compound (g) / volume of medium composition (ml) × 100

Here, examples of suitable solvents used for the medium additive include aqueous solvents such as water, dimethyl sulfoxide (DMSO), various alcohols such as methanol, ethanol, butanol, propanol, glycerin, propylene glycol, and butylene glycol. However, it is not limited to these. At this time, the concentration of the specific compound is 0.001% to 5.0% (w / v), preferably 0.01% to 1.0% (w / v), more preferably 0.1% to 0.6% (w / v). At that time, an additive that enhances the effect of the specific compound or lowers the concentration at the time of use can be further added. Examples of such additives include one or more polysaccharides such as guar gum, propylene glycol alginate, locust bean gum, gum arabic, tara gum, tamarind gum, methylcellulose, carboxymethylcellulose, agarose, tamarind seed gum, pullulan, etc. be able to. In addition, the specific compound can be used by immobilizing on the surface of the carrier or supporting the specific compound inside the carrier. The specific compound can be in any shape at the time of provision or storage. The specific compound is bound to formulated solids such as tablets, pills, capsules, granules, liquids such as solutions or suspensions dissolved in appropriate solvents and solubilizers, or substrates or single substances. It can be in the state. Additives for formulation include antiseptics such as p-hydroxybenzoates; excipients such as lactose, glucose, sucrose and mannitol; lubricants such as magnesium stearate and talc; polyvinyl Examples include binders such as alcohol, hydroxypropyl cellulose, and gelatin; surfactants such as fatty acid esters; and plasticizers such as glycerin. These additives are not limited to those described above, and can be freely selected as long as they are available to those skilled in the art. In addition, the specific compound in the present invention may be sterilized as necessary. The sterilization method is not particularly limited, and examples thereof include radiation sterilization, ethylene oxide gas sterilization, autoclave sterilization, and filter sterilization. The material of the filter part when performing filter sterilization (hereinafter sometimes referred to as filter sterilization) is not particularly limited. For example, glass fiber, nylon, PES (polyethersulfone), hydrophilic PVDF (polyvinylidene fluoride), cellulose Examples thereof include mixed esters, cellulose acetate, and polytetrafluoroethylene. The pore size of the filter is not particularly limited, but is preferably 0.1 μm to 10 μm, more preferably 0.1 μm to 1 μm, and most preferably 0.1 μm to 0.5 μm. In these sterilization treatments, the specific compound may be in a solid state or a solution state.

By adding a solution or dispersion of the specific compound to the liquid medium according to the above preparation, the structure is formed in the liquid medium, and the medium composition used in the present invention can be obtained. The medium usually contains metal ions (eg, divalent metal ions such as calcium ions) at a concentration sufficient for polymer compounds to assemble via ions or to form a three-dimensional network. Therefore, the medium composition used in the present invention can be obtained only by adding the solution or dispersion of the specific compound of the present invention to the liquid medium. Alternatively, the medium may be added to a medium additive (a solution or dispersion of the specific compound). Furthermore, the medium composition used in the present invention can also be prepared by mixing a specific compound and medium components in an aqueous solvent (for example, water containing ion-exchanged water or ultrapure water). As a mode of mixing, (1) a liquid medium and a medium additive (solution) are mixed, (2) the polymer compound (solid such as a powder) is mixed in the liquid medium, (3) a medium additive ( Examples include, but are not limited to, mixing a powder medium with (solution), (4) mixing the powder medium and the above polymer compound (solid such as powder) with an aqueous solvent, and the like. In order to prevent non-uniform distribution of the specific compound in the medium composition used in the present invention, the embodiment of (1) or (4) or (1) or (3) is preferable.
When the specific compound is dissolved in a solvent (for example, an aqueous solvent such as water or a liquid medium), or when the specific compound and the powder medium are dissolved in the solvent, it is preferable to heat the mixed solution to promote dissolution. The heating temperature includes, for example, 80 ° C. to 130 ° C., preferably 100 ° C. to 125 ° C. (eg, 121 ° C.) that is sterilized by heating. After heating, the obtained solution of the specific compound is cooled to room temperature. By adding the above metal ions (eg, divalent metal ions such as calcium ions) to the solution (for example, by adding the solution to a liquid medium), the structure composed of the specific compound Is formed. Alternatively, the specific compound is heated (for example, 80 ° C. to 130 ° C.) when dissolved in a solvent (eg, water, an aqueous solvent such as a liquid medium) containing the above metal ions (eg, divalent metal ions such as calcium ions). The above structure composed of the specific compound can also be formed by cooling the resulting solution to room temperature at a temperature of 0 ° C., preferably 100 ° C. to 125 ° C. (eg, 121 ° C.).

Although the preparation method of the culture medium composition used for this invention is illustrated, this invention is not limited by this. A specific compound is added to ion exchange water or ultrapure water. And it stirs at the temperature (for example, 60 degreeC or more, 80 degreeC or more, 90 degreeC or more) which can melt | dissolve the said specific compound until it becomes a transparent state. After dissolution, the mixture is allowed to cool with stirring and sterilized (for example, autoclaved at 121 ° C. for 20 minutes). After returning to room temperature, the sterilized aqueous solution is added to the medium while stirring (for example, a homomixer) an arbitrary medium used for stationary culture, and mixed so as to be uniform with the medium. The method for mixing the aqueous solution and the medium is not particularly limited, and examples thereof include manual mixing such as pipetting, and mixing using equipment such as a magnetic stirrer, mechanical stirrer, homomixer, and homogenizer. Moreover, you may filter the culture medium composition used for this invention with a filter after mixing. The pore size of the filter used for the filtration treatment is 5 μm to 100 μm, preferably 5 μm to 70 μm, more preferably 10 μm to 70 μm.

Alternatively, the medium composition used in the present invention is prepared by mixing the powder medium and the polymer compound (solid such as powder) with an aqueous solvent and heating at the above temperature.

For example, when preparing a deacylated gellan gum, it is 0.1% to 1% (w / v), preferably 0.2% to 0.5% (w / v), more preferably 0.3% to 0.4% (w / v). Thus, deacylated gellan gum is added to ion-exchanged water or ultrapure water. In another aspect, when preparing deacylated gellan gum, 0.1% to 1% (w / v), preferably 0.2% to 0.8% (w / v), more preferably 0.3% to 0.6% (w / v) Add deacylated gellan gum to ion-exchanged water or ultrapure water.
The temperature may be any temperature at which the deacylated gellan gum can be dissolved. For example, it is transparent by stirring at 60 ° C. or higher, preferably 80 ° C. or higher, more preferably 90 ° C. or higher (eg, 80 to 130 ° C.). Dissolve until ready. After dissolution, the mixture is allowed to cool with stirring, and autoclaved at 121 ° C. for 20 minutes, for example. After returning to room temperature, for example, while stirring the DMEM / F12 medium with a homomixer or the like, this aqueous solution is added to the medium so as to have a desired final concentration (for example, when the final concentration is 0.015%, a 0.3% aqueous solution: The ratio of the medium is 1:19). The method for mixing the aqueous solution and the medium is not particularly limited, and examples thereof include manual mixing such as pipetting, and mixing using equipment such as a magnetic stirrer, mechanical stirrer, homomixer, and homogenizer. Moreover, you may filter the culture medium composition used for this invention with a filter after mixing. The pore size of the filter used for the filtration treatment is 5 μm to 100 μm, preferably 5 μm to 70 μm, more preferably 10 μm to 70 μm.

Preferred embodiments of the medium composition used in the present invention and the production method thereof will be described below.
The medium composition used in the present invention is preferably a medium composition that can be cultured by suspending cells or tissues, and the viscosity of the medium composition is 8 mPa · s or less (under 37 ° C.), And a medium composition comprising deacylated gellan gum or a salt thereof. In one embodiment, the concentration of deacylated gellan gum or salt thereof in the medium composition is 0.01-0.05% (w / v). In one embodiment, the medium composition further contains a polysaccharide other than deacylated gellan gum or a salt thereof. In one embodiment, the medium composition contains a divalent metal ion (eg, calcium ion) at a concentration sufficient to form a structure in which the deacylated gellan gum can float and culture cells or tissues. The concentration is, for example, 0.1 mM to 300 mM, preferably 0.5 mM to 100 mM.

The medium composition can be produced by mixing deacylated gellan gum or a salt thereof and a medium. In one embodiment, the medium is a liquid medium. In one embodiment, the liquid medium contains a concentration of divalent metal ions (eg, calcium ions) sufficient to form a structure in which the deacylated gellan gum can be cultured by suspending cells or tissues. The concentration is, for example, 0.1 mM to 300 mM, preferably 0.5 mM to 100 mM. In one embodiment, the medium is mixed with deacylated gellan gum or salt thereof dissolved or dispersed in a solvent. In one embodiment, the deacylated gellan gum or salt thereof dissolved or dispersed in a solvent is in a sterile state. In one embodiment, sterilization is performed by autoclave sterilization. In another embodiment, sterilization is performed by filter sterilization. In one embodiment, filter sterilization is performed by passing through a 0.1-0.5 μm filter.

(2) Method for producing cancer-bearing non-human mammal The present invention provides a method for producing a cancer-bearing non-human mammal, comprising the following steps:
(1) Suspension culture of cancer cells in medium composition I; and (2) Transfer the cancer cells obtained by the culture of (1) to a non-human mammal.
By subjecting cancer cells to suspension culture in medium composition I, the survival rate and growth rate of cancer cells in vivo when transferred to a non-human mammal are improved. The present invention also includes a method for promoting the engraftment of such cancer cells in a non-human mammal.

The cancer cell used in the present invention is preferably a cancer cell having the ability to form a solid tumor when engrafted in vivo.

Cancer cells having the ability to form solid tumors include stomach cancer, esophageal cancer, colon cancer, colon cancer, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell cancer, basal cell cancer, adenocarcinoma, Renal cell cancer, ureteral cancer, liver cancer, bile duct cancer, cervical cancer, endometrial cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, epithelial cancer, craniopharyngeal cancer, laryngeal cancer, tongue cancer, Fibrosarcoma, mucosal sarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, lymphangiosarcoma, lymphatic endothelial sarcoma, synovial tumor, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma Cells such as, but not limited to, carcinoma, seminoma, Wilms tumor, glioma, astrocytoma, meningioma, melanoma, neuroblastoma, medulloblastoma, retinoblastoma . The cancer cells having the ability to form solid tumors are preferably epithelial cell-derived malignant tumors, ie carcinoma (carcinoma) cells (eg, colon carcinoma, breast cancer, ovarian carcinoma, colon carcinoma).

The cancer cells used in the present invention may be allogenic, allogenic, or xenogenic to the recipient non-human mammal to be transferred. In view of the effect of the present invention that improves the survival rate of cancer cells that are difficult to engraft in vivo, the cancer cells are allogeneic or xenogeneic with respect to the recipient non-human mammal to be transferred, more preferably Heterogeneous. In one embodiment, the cancer cell used in the present invention is a human cancer cell.

In step (1), the cancer cells are subjected to suspension culture (preferably stationary suspension culture) in the medium composition I.

In suspension culture of cancer cells, petri dishes, flasks, plastic bags, Teflon (registered trademark) bags, dishes, petri dishes, tissue culture dishes, multi dishes, micro plates, micro cells generally used for cell culture are used. It is possible to culture using a culture apparatus such as a well plate, a multi-plate, a multi-well plate, a chamber slide, a cell culture flask, a spinner flask, a tube, a tray, a culture bag, or a roller bottle. These culture substrates are desirably low cell adhesion. As a non-cell-adhesive culture container, the surface of the culture container is not artificially treated (for example, coating treatment with an extracellular matrix or the like) for the purpose of improving the adhesion with cells, A surface whose surface is artificially treated for the purpose of reducing adhesion to cells can be used.

The above-mentioned cancer cell culture automatically executes cell seeding, medium exchange, cell image acquisition, and culture cell recovery under closed control under mechanical control, while controlling pH, temperature, oxygen concentration, etc. It can also be carried out by a bioreactor capable of high-density culture or an automatic culture device.

The form and state of cancer cells to be cultured can be arbitrarily selected by those skilled in the art. Specific preferred examples thereof include, but are not particularly limited to, cancer cells dispersed in medium composition I in a single cell state, cancer cells adhered on a carrier surface, cancer cells as a carrier Examples include a state of being embedded inside, and a state in which a plurality of cancer cells are aggregated to form a cell aggregate (sphere (spheroid)). The cancer cells are preferably in a state of being dispersed in the medium composition I in a single cell state, or in a state in which a plurality of cells are aggregated to form a cell aggregate (sphere (spheroid)). The suspension culture (preferably suspension static culture) is performed in the suspension culture. That is, preferably, a carrier for adhering or embedding cancer cells is not used. Among these states, the state in which cell aggregates (spheres (spheroids)) are formed is because cell-cell interactions and cell structures close to the in vivo environment have been reconstructed, and cell functions are maintained for a long time. Can be cultured as it is, and the cells can be collected relatively easily. The sphere size is preferably 500 μm or less, and most preferably 50 to 100 μm, as an average value of the maximum diameter.

The method for forming a cell aggregate (sphere (spheroid)) is not particularly limited and can be appropriately selected by those skilled in the art. Examples thereof include a method using a container having a cell non-adhesive surface, a hanging drop method, a swirl culture method, a three-dimensional scaffold method, a centrifugation method, a method using aggregation by an electric field or a magnetic field, and the like.

Spheres can also be formed using the culture composition I. For example, a sphere can be obtained by collecting target cancer cells as single cells by cell-dispersing enzyme treatment, then uniformly dispersing them in the culture composition I, and allowing them to stand for 3 to 10 days for suspension culture. Prepared. The spheres prepared here can be collected by centrifugation or filtration.

When culturing cancer cells, separately prepared cancer cells may be added to the culture composition I and mixed so as to be uniformly dispersed. The mixing method in that case is not particularly limited, and examples thereof include manual mixing such as pipetting, and mixing using equipment such as a stirrer, a vortex mixer, a microplate mixer, and a shaker. After mixing, the obtained cancer cell suspension may be cultivated by standing, or the culture solution may be rotated, shaken or stirred as necessary. The number of rotations and frequency may be appropriately set according to the purpose of those skilled in the art. In addition, when it is necessary to change the medium composition during the period of stationary culture, the cancer cell and the medium composition are separated by centrifugation or filtration, and then the fresh medium composition I is added to the cells. What is necessary is just to add. Alternatively, after the cancer cells are appropriately concentrated by performing centrifugation or filtration treatment, the fresh medium composition I may be added to the concentrate.

In one embodiment, in the suspension culture of adherent cancer cells, for example, the cancer cells are collected from the subculture and dispersed into a single cell or a state close thereto. The cancer cells are dispersed using an appropriate cell dissociation solution. As the cell dissociation solution, for example, EDTA; proteolytic enzymes such as trypsin, collagenase IV, metalloprotease and the like can be used alone or in appropriate combination. The dispersed adherent cancer cells are suspended in the medium composition I, and this is subjected to suspension culture (preferably suspension stationary culture). In the culture, the cancer cells grow while floating in the medium composition I in a single cell state or in a sphere state. In this culture, it is preferable to use a cell non-adhesive incubator, but this is not a limitation.

The culture period of the cancer cells in the medium composition I is not particularly limited as long as it is sufficient to improve the engraftment of the cancer cells in the non-human mammal, but usually 1 day or more, preferably 3 More than 6 days, more preferably more than 6 days. The upper limit of the culture period is theoretically infinite, but from the viewpoint of avoiding changes in the characteristics of cancer cells, for example, it is preferably within 30 days, preferably within 10 days. .

The temperature at which the cancer cells are cultured is usually 25 to 39 ° C, preferably 37 ° C. The CO ₂ concentration is usually 4 to 10% by volume, preferably 5% by volume, in the culture atmosphere. The oxygen concentration is 15 to 50% by volume, preferably 20% by volume, in the culture atmosphere.

By such a culture operation, cancer cells with improved engraftment in vivo can be obtained.

Next, the cancer cells obtained by this culture are transferred to a non-human mammal.

The type of non-human mammal is not particularly limited, but examples include rodents such as mice, rats, hamsters, guinea pigs, rabbit eyes such as rabbits, ungulates such as pigs, cows, goats, horses, sheep, and dogs. And cats such as cats, primates such as monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans and chimpanzees. The mammal is preferably a rodent (mouse, rat, etc.).

In one aspect, the non-human mammal is preferably immunodeficient, but not limited thereto. By using an immunodeficient non-human mammal, it is possible to avoid rejection when allogeneic or xenogeneic cancer cells are transferred and to promote cancer cell engraftment. Examples of immunodeficient non-human mammals include nude mice, SCID mice, IL-2Rg KO mice, NOD / Shi mice, NOD / Shi-SCID mice, NOD / SCID mice, NOG mice, RAG2 KO mice, RAG2 IL- Examples include 2Rg dKO mice, but are not limited thereto. Non-immune deficient animals include C57BL / 6J mice, C3H / HeN mice, and the like.

In the present invention, cancer cells are subjected to suspension culture in the medium composition I, so that the survival rate and growth rate of cancer cells in a living body when transferred to a non-human mammal are improved. Without severe immunodeficient non-human animals (eg, NOD / SCID), immunodeficient non-humans whose degree of immunodeficiency is relatively mild even for cancer cells that could not form tumor tissue in vivo. Tumor tissue can be formed in vivo using animals (eg, nude mice). In addition, tumor cells that had previously been modeled for tumor-bearing mice in nude mice are transplanted into normal immune non-human animals (eg, BALB / c, C57BL / 6j) to form tumor tissue in vivo. It is also possible.

In transferring cancer cells into a non-human mammal, the cancer cells are usually suspended in a physiological aqueous composition, and the resulting suspension is transferred into the body of the non-human mammal. Cancer cells may be treated with a protein such as trypsin or a chelating agent such as EDTA and dispersed in a single cell, then suspended in a physiological aqueous composition and transferred into the body, or physiologically in the state of spheroids. It may be suspended in an aqueous composition and transferred into the body. The transfer in the spheroid state tends to enhance the formation of cancer tissue, while the transfer in the single cell state tends to be more sensitive to the anticancer agent. Physiological aqueous compositions include physiological aqueous solutions and physiological aqueous gels. Examples of the physiological aqueous solution include phosphate buffer, carbonate buffer, citrate buffer, Tris buffer, borate buffer physiological buffer, cell culture medium, and the like. Examples of aqueous gels include extracellular matrix constituents (proteoglycans such as aggrecan; glycosaminoglycans such as hyaluronic acid; protein fibers such as collagen and elastin; or a mixture thereof (eg, basement membrane preparation)), polysaccharides, etc. A gel containing (agarose etc.) can be mentioned. A basement membrane preparation has the function of controlling cell morphology, differentiation, proliferation, movement, functional expression, etc. when seeding and culturing desired cells of the same or different types having basement membrane-forming ability on it. Means the isolated basement membrane. In the basement membrane preparation, for example, cells having the ability to form a basement membrane that adhere to the support through the basement membrane are removed from the support using a solution or an alkaline solution having lipid-dissolving ability of the cells. Can be produced. An example of a basement membrane preparation is Matrigel (Corning product). By using the aqueous gel, dispersion of the transplanted cancer cells in the living body can be suppressed.

In one embodiment, cancer cells to be transplanted are suspended in a physiological aqueous composition (eg, aqueous gel) containing an extracellular matrix constituent factor. By using an extracellular matrix constituent factor, engraftment of the transplanted cancer cells in vivo is promoted.

The extracellular matrix component used in the present invention is preferably isolated. “Isolation” means that an operation to remove a target component or a factor other than cells has been performed, and the state existing in nature has been removed. The purity of “isolated protein X” (percentage of the weight of protein X in the total protein weight) is usually 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 99% or more, Most preferably 100%. Therefore, the “isolated extracellular matrix constituent factor” does not include an endogenous extracellular matrix constituent factor produced from a cancer cell to be transplanted and contained in the cancer cell or suspension. In one aspect, the invention includes providing a physiological aqueous composition (eg, aqueous gel) comprising an isolated extracellular matrix component.

When a physiological aqueous composition containing an extracellular matrix constituent factor is used, the concentration of the extracellular matrix constituent factor in the composition is not particularly limited as long as it promotes the engraftment of cancer cells to be transplanted, but usually 1 to 10 mg / ml, preferably about 2 to 4 mg / ml. When Matrigel (trade name, manufactured by Corning) is used as an extracellular matrix, it is preferably diluted to about 25 to 50% (v / v).

In one embodiment, cancer cells to be transplanted are suspended in a physiological aqueous composition that is substantially free of isolated extracellular matrix components. In the present invention, cancer cells are subjected to suspension culture in the medium composition I, so that the engraftment rate and growth rate of cancer cells in vivo when transferred to a non-human mammal are improved. Cancer cells that could not form tumor tissue in vivo unless suspended and transplanted in a physiological aqueous composition containing an outer matrix constituent factor are substantially free of the extracellular matrix constituent factor. Tumor tissue can be formed in vivo using the physiological aqueous composition.

“Substantially free of isolated extracellular matrix constituents” means that the concentration of isolated extracellular matrix constituents in a physiological aqueous composition promotes the engraftment of transplanted cancer cells Means below. More specifically, the concentration of the isolated extracellular matrix component in the “physiological aqueous composition substantially free of isolated extracellular matrix component” is usually 0.01% (w / v). Hereinafter, it is preferably 0.001% (w / v) or less, more preferably 0.0001% (w / v) or less, and most preferably 0% (w / v).

In addition, the “isolated physiological matrix composition substantially free of extracellular matrix constituents” includes endogenous extracellular cells produced from cancer cells to be transplanted and contained in the cancer cells and suspensions. Inclusion of matrix constituents is allowed. Further, the “isolated extracellular matrix constituent factor concentration” does not include the concentration of an endogenous extracellular matrix constituent factor produced from the cancer cell to be transplanted and contained in the cancer cell or suspension.

Physiological aqueous composition may or may not contain serum. From the viewpoint of forming a larger cancer tissue, it is preferable to use a physiological aqueous composition containing serum. When serum is included, the concentration is not particularly limited, but is usually 0.1 to 20 (v / v)%. On the other hand, from the viewpoint of avoiding mixing of undetermined components, it is preferable to use a physiological aqueous composition that does not contain serum. In the present invention, cancer cells are subjected to suspension culture in the medium composition I, so that the survival rate and growth rate of cancer cells in vivo when transferred to a non-human mammal are improved. For cancer cells that could not form a sufficiently large tumor tissue in vivo unless they were suspended and transplanted in a physiological aqueous composition containing It can be used to form tumor tissue in vivo.

The physiological aqueous composition used in the present invention includes the following embodiments.
Contains extracellular matrix constituents and serum.
-Contain substantially no isolated extracellular matrix constituents and serum.
Contains extracellular matrix constituents and no serum.
-Substantially free of isolated extracellular matrix constituents and free of serum.

When suspending cancer cells in a physiological aqueous composition, the cancer cell concentration in the suspension should be appropriately set within a range where tumor tissue can be formed when transferred into the recipient non-human mammal. However, it is usually about 0.01-1x10 ⁸ cells / ml.

The number of cancer cells to be transferred into the recipient non-human mammal can be appropriately set within a range in which tumor tissue can be formed in the non-human mammal, but usually 0.1 to 10 × 10 ⁶ cells / animal. Degree. In the present invention, by subjecting cancer cells to suspension culture in medium composition I, the engraftment rate and growth rate of cancer cells in vivo when transferred to a non-human mammal is improved. Tumor tissue of the same size can be formed with a smaller number of cells than when I is not used.

The site of cancer cell transfer is not particularly limited as long as a tumor tissue can be formed in the recipient non-human mammal, and may be an orthotopic transplant or an ectopic transplant. The site of cancer cell transfer in the case of ectopic transplantation is not particularly limited, and examples include subcutaneous, intradermal, intramuscular, intravenous, and intraperitoneal. Since the transplantation technique is simple and the formed tumor tissue can be easily observed with the naked eye, the cancer cells are preferably transferred subcutaneously.

After the transfer of cancer cells, the recipient non-human mammal may be bred usually under SPF or aseptic conditions as necessary. By breeding the recipient non-human mammal for a predetermined period, a tumor tissue is formed from the transferred cancer cells. Breeding conditions are not limited, but specific examples include temperature conditions of 20 to 26 ° C., humidity conditions of 30 to 70%, feeding and water supply conditions: free intake, lighting cycle: 12-hour light / dark cycle.

The breeding period is not particularly limited as long as a tumor tissue can be formed from the transferred cancer cells, but it is usually preferable to breed the recipient non-human mammal for 3 days or more, preferably 3 weeks or more after transplantation.

The cancer-bearing non-human mammal prepared by the method of the present invention is useful for screening of anticancer agents, evaluation of pharmaceuticals for treating or preventing cancer, and the like.

(3) Screening Method for Anticancer Agent The present invention provides a screening method for an anticancer agent comprising the following steps:
(1) administering a test substance to a cancer-bearing non-human mammal prepared by the method of the present invention;
(2) Measuring the size of the tumor tissue in the cancer-bearing non-human mammal obtained in (1);
(3) To compare the size of the tumor tissue measured in (2) with the size of the tumor tissue of a control cancer-bearing non-human mammal not administered with the test compound.

The test substance may be any known compound or new compound, such as a nucleic acid, carbohydrate, lipid, protein, peptide, low molecular organic compound, compound library prepared using combinatorial chemistry technology, random peptide Examples include natural components derived from libraries, random nucleic acid libraries, microorganisms, animals and plants, marine organisms, and the like. Moreover, the artificial polymer which carry | supported the said substance may be sufficient.

The administration form of the test substance includes, but is not limited to, an aqueous solution or suspension containing a medium such as methylcellulose (MC), carboxymethylcellulose (CMC), polyethylene glycol (PEG), or ethanol (EtOH). The administration route of the test substance includes oral, subcutaneous, intraperitoneal, intravenous, intraportal and the like, but is not limited thereto. The period during which the test substance is administered is a period sufficient to evaluate the effect, and during this period, the cancer-bearing non-human mammal is bred based on the laboratory animal ethics regulations of each research institute. The breeding period is usually 3 days or longer, preferably 10 days or longer, more preferably 21 days or longer.

After breeding, the size of the tumor tissue in the cancer-bearing non-human mammal administered with the test substance is measured. Methods for measuring tumor tissue size are well known to those skilled in the art.

Next, the size of the tumor tissue in the cancer-bearing non-human mammal administered with the test substance is compared with the size of the tumor tissue in the control cancer-bearing non-human mammal not administered with the test compound. The comparison of tumor tissue size can preferably be made based on the presence or absence of a statistically significant difference. In addition, the size of the tumor tissue of the control cancer-bearing non-human mammal to which the test substance was not added was measured in advance with respect to the measurement of the size of the tumor tissue in the cancer-bearing non-human mammal administered with the test substance. Although it may be a thing measured simultaneously, what was measured simultaneously is preferable from the viewpoint of the precision and reproducibility of experiment.

As a result of the comparison, a substance whose tumor tissue size has been reduced by the addition of the test substance can be selected as a candidate substance for an anticancer agent (particularly, a candidate substance for an anticancer agent effective against transferred cancer cells). .

The contents of all publications, including the patents and patent application specifications mentioned herein, are hereby incorporated by reference herein to the same extent as if all were expressly cited. .

Hereinafter, the present invention will be described in more detail by specifically describing analysis examples and test examples of the medium composition used in the present invention as examples, but the present invention is not limited thereto.

[Reference Example 1]
Production of Deacylated Gellan Gum-Containing Medium A medium composition used for suspension culture of cells was prepared according to the production method described in WO2014 / 017513A1. That is, deacylated gellan gum powder was dissolved in pure water so as to be 0.3% (w / v). Dissolution was carried out by boiling with stirring with a stirrer, and then the resulting deacylated gellan gum aqueous solution was returned to room temperature overnight and then sterilized with an autoclave. The above-mentioned 0.3% (w / v) deacylated gellan gum aqueous solution of 1/20 volume of the medium amount was vigorously injected into the recommended medium of each cell, and immediately stirred vigorously to mix and disperse.

[Test Example 1]
Cell culture HCT116 cells of human colon cancer cell line were cultured in a monolayer in McCoy's 5A medium containing 10% FBS. The cells were trypsinized and then suspended at 1-5 × 10 ⁷ cells / ml in McCoy's 5A medium containing 10% FBS. Add the cell suspension to McCoy's 5A medium containing 0.015% (w / v) deacylated gellan gum and 10% (v / v) FBS to 1 ~ 5x10 ⁵ cells / ml and suspend uniformly. did. The obtained cell suspension was seeded on a cell non-adherent culture plate or petri dish, and the cells were cultured in a stationary culture. The medium was changed once every 3 days and cultured for 6 days. In the medium exchange, the culture solution containing the cells was collected in a centrifuge tube, diluted 2-3 times with McCoy's 5A medium, and centrifuged at 1500 rpm for 5 minutes to collect 10% (v / v) cells in the lower layer. This cell-containing layer is resuspended in McCoy's 5A medium containing 0.015% (w / v) deacylated gellan gum and 10% (v / v) FBS at least twice the initial amount before dilution, and the cells are not attached. The cells were seeded on a culture plate or a petri dish, and the cells were subsequently subjected to suspension static culture.

[Test Example 2]
Cell Recovery after Culture The culture solution containing the HCT116 cells cultured in Test Example 1 was collected in a centrifuge tube and centrifuged at 500 rpm for 1 minute. The non-precipitated supernatant fraction was collected, diluted 2-3 times with McCoy's 5A medium or PBS, and centrifuged at 1500 rpm for 5 minutes to collect 10% (v / v) cells in the lower layer. Furthermore, it was suspended in 5 to 10 times the volume of the McCoy's 5A medium or PBS, and centrifuged again at 1500 rpm for 5 minutes to recover the precipitated cells. Cells were resuspended in a small amount of McCoy's 5A medium to obtain a sphere cell suspension. Add trypsin solution 1: 1 (v / v) to this cell solution, treat at 37 ° C for 5 minutes, and then add 10% (v / v) FBS-containing McCoy's 5A medium to a final FBS concentration of 1% (v / v). The mixture was centrifuged at 1000 rpm for 3 minutes, and the precipitated cells were suspended again in a small amount of McCoy's 5A medium to obtain a single cell suspension.

[Test Example 3]
Cell culture SKOV3 cells of a human ovarian cancer cell line were cultured in a single layer in McCoy's 5A medium containing 10% (v / v) FBS. After trypsinization, the cells were suspended in 15% (v / v) FBS-containing McCoy's 5A medium at 1 to 5 × 10 ⁷ cells / ml. The cell suspension was added to 0.01-5% (w / v) deacylated gellan gum and 15% (v / v) FBS-containing McCoy's 5A medium to 1 to 5 × 10 ⁵ cells / ml to suspend uniformly. . The obtained cell suspension was seeded on a cell non-adherent culture plate or petri dish, and the cells were cultured in a stationary culture. The medium was changed once every 3 days, and the cells were cultured for 6 days. In the medium exchange, the culture medium containing the cells was collected in a centrifuge tube, diluted 2-3 times with McCoy's 5A medium, and centrifuged at 1500 rpm for 5 minutes to collect 10% (v / v) cells in the lower layer. This cell-containing layer is resuspended in McCoy's 5A medium containing 0.015% (w / v) deacylated gellan gum and 15% (v / v) FBS at least twice the initial amount before dilution, and the cells are not attached. The cells were seeded on a culture plate or a petri dish, and the cells were subsequently subjected to suspension static culture.

[Test Example 4]
Cell Recovery after Culture The culture solution containing SKOV3 cells cultured in Test Example 3 was collected in a centrifuge tube and centrifuged at 500 rpm for 1 minute. The non-precipitated supernatant fraction was collected, diluted 2-3 times with McCoy's 5A medium or PBS, and centrifuged at 1500 rpm for 5 minutes to collect 10% (v / v) cells in the lower layer. Furthermore, it was suspended in 5 to 10 times the volume of the McCoy's 5A medium or PBS, and centrifuged again at 1500 rpm for 5 minutes to recover the precipitated cells. Cells were resuspended in a small amount of McCoy's 5A medium to obtain a sphere cell suspension. Add trypsin solution 1: 1 (v / v) to this cell solution, treat at 37 ° C for 5 minutes, and then add 10% (v / v) FBS-containing McCoy's 5A medium to a final FBS concentration of 1%. Centrifugation was performed at 1000 rpm for 3 minutes, and the precipitated cells were suspended again in a small amount of McCoy's 5A medium to obtain a single cell suspension.

[Test Example 5]
Cell Culture MCF7 cells of a human breast cancer cell line were cultured in a monolayer in an E-MEM medium containing 10% (v / v) FBS and 1% non-essential amino acid (NEAA). After trypsinization, the cells were suspended in E-MEM medium containing 10% (v / v) FBS and 1% (v / v) NEAA at 1 to 5 × 10 ⁷ cells / ml. The cell suspension is 1-5 x 10 ⁵ cells / ml in E-MEM medium containing 0.015% (w / v) deacylated gellan gum, 10% (v / v) FBS, and 1% (v / v) NEAA. Was added and suspended uniformly. The obtained cell suspension was seeded on a culture plate or a petri dish, and the cells were subjected to suspension static culture. The medium was changed once every 3 days, and the cells were cultured for 6 days. In the medium exchange, the culture medium containing the cells is collected in a centrifuge tube, diluted 2-5 times with 1% NEAA-containing E-MEM medium, centrifuged at 1500 rpm for 5 minutes, and 10% (v / v) cells in the lower layer. Was recovered. This cell-containing layer is more than twice the initial volume before dilution, 0.015% (w / v) deacylated gellan gum, 10% (v / v) FBS, and 1% (v / v) NEAA containing E- The cells were resuspended in MEM medium and seeded on a culture plate or a petri dish, and the cells were subsequently subjected to suspension static culture.

[Test Example 6]
Cell Recovery after Culture The culture solution containing MCF7 cells cultured in Test Example 5 was collected in a centrifuge tube and centrifuged at 500 rpm for 1 minute. Collect the non-precipitated supernatant fraction, dilute 2 to 3 times with 1% (v / v) NEAA-containing E-MEM medium or PBS, and centrifuge at 1500 rpm for 5 minutes to obtain 10% (v / v) of the lower layer. ) Cells were collected. Further, the cells were suspended in E-MEM medium or PBS containing 1% (v / v) NEAA containing 5 to 10 times the volume of the residue, and centrifuged again at 1500 rpm for 5 minutes to recover the precipitated cells. The cells were resuspended in a small amount of E-MEM medium containing 1% NEAA to obtain a sphere cell suspension. Add trypsin solution 1: 1 (v / v) to this cell solution, treat at 37 ° C for 5 minutes, and then add 10% (v / v) FBS-containing 1% (v / v) NEAA-containing E-MEM medium to FBS. Add to a final concentration of 1%, centrifuge at 1000 rpm for 3 minutes, and resuspend the precipitated cells in a small amount of 1% (v / v) NEAA-containing E-MEM medium to obtain a single cell suspension. Obtained.

[Test Example 7]
Cell Culture Human colon cancer cell line LS123 cells were cultured in a monolayer in E-MEM medium containing 10% (v / v) FBS and 1% non-essential amino acid (NEAA). The cells were treated with trypsin and suspended in E-MEM medium containing 10% (v / v) FBS and 1% (v / v) NEAA at 1 to 5 × 10 ⁷ cells / ml. The cell suspension is made up to 1-5x10 ⁵ cells / ml in E-MEM medium containing 0.015% (w / v) deacylated gellan gum, 10% (v / v) FBS, and 1% (v / v) NEAA. Was added and suspended uniformly. The obtained cell suspension was seeded on a culture plate or a petri dish, and the cells were subjected to suspension static culture. The medium was changed once every 3 days, and the cells were cultured for 6 days. In the medium exchange, the culture medium containing the cells is collected in a centrifuge tube, diluted 2 to 5 times with 1% (v / v) NEAA-containing E-MEM medium, centrifuged at 1500 rpm for 5 minutes, and 10% (v / v) cells were collected. This cell-containing layer is resuspended in E-MEM medium containing 0.015% (w / v) deacylated gellan gum, 10% (v / v) FBS, and 1% NEAA at least twice the initial volume before dilution. It became turbid and seeded on a culture plate or petri dish, and the cells were subsequently cultured in a stationary culture.

[Test Example 8]
Cell Recovery after Culture The culture solution containing the LS123 cells cultured in Test Example 7 was collected in a centrifuge tube and centrifuged at 500 rpm for 1 minute. Collect the non-precipitated supernatant fraction, dilute 2-3 times with 1% NEAA-containing E-MEM medium or PBS, and centrifuge at 1500 rpm for 5 minutes to collect 10% (v / v) cells in the lower layer did. Further, the cells were suspended in E-MEM medium or PBS containing 1% (v / v) NEAA containing 5 to 10 times the volume of the residue, and centrifuged again at 1500 rpm for 5 minutes to recover the precipitated cells. The cells were resuspended in a small amount of E-MEM medium containing 1% NEAA to obtain a sphere cell suspension. Add trypsin solution 1: 1 (v / v) to this cell solution, treat at 37 ° C for 5 minutes, and then add 10% (v / v) FBS-containing 1% (v / v) NEAA-containing E-MEM medium to FBS. Add to a final concentration of 1% (v / v), centrifuge at 1000 rpm for 3 minutes, and resuspend the settled cells in a small amount of 1% (v / v) NEAA-containing E-MEM medium A cell suspension was obtained.

[Test Example 9]
Preparation of a tumor-bearing model Each cancer cell obtained in Test Examples 2, 4, 6 and 8 is medium containing 10% FBS or not containing FBS so as to be 0.5 to 5 × 10 ⁶ cells / animal / 150 μL (or 200 μL). It was diluted with McCoy's 5A medium or E-MEM medium containing 1% NEAA. In Matrigel co-transplantation, the cell concentration was doubled as described above, and the cell suspension and Matrigel were mixed 1: 1 (v: v).
Seven week old female BALB / c-nu / nu (nude) mice were purchased from CLEA Japan. Under isoflurane anesthesia, each cancer cell was injected subcutaneously into the shoulder of the animal. After cell injection, food and water were ingested freely and reared for 4 to 5 weeks in groups. The cancer tissue was observed once a week and the size (outside dimension) was measured. The volume of the cancer tissue was calculated by the following formula. In addition, cancer tissues 4 to 5 weeks later were removed and weighed.
Volume V = 4π a b c / 3 (a = width, b = length, c = height)

[Test Example 10]
Judgment of anticancer drug effect in cancer-bearing model The size of the cancer tissue of the mouse prepared in Test Example 9 was measured once every 2 to 3 days from the first week of transplantation, and the individual with a cancer tissue volume of 200 to 300 mm ³ Administration of test substance or solvent was started. Administration was continued for a period from the start of administration until sufficient growth of cancer tissue in the control group (control group) was achieved. Meanwhile, the size of the cancer tissue was measured once every 2-3 days.

The results of a cancer bearing model creation test and an anticancer drug effect determination test are shown below.
FIG. 1 and FIG. 2 show the results of transplantation of HCT116 cells dispersed in a single cell after suspension culture in a medium containing deacylated gellan gum in the presence and absence of Matrigel. When transplanted in the presence of matrigel, non-adherent suspension cultured cells in a medium containing deacylated gellan gum were approximately 110% of non-adherent precipitated cells (cells in non-acylated gellan gum-free medium, cell-free). About 210% of the cancer cells were cultured and were capable of forming cancerous tissue. On the other hand, in the case of transplantation in the absence of matrigel, cells cultured in suspension in a medium containing deacylated gellan gum were found to have prominent cancer tissue from the 2nd week of transplantation, and the volume of the cancer tissue at 4th week was an adherent monolayer culture cell. About 1000% of non-adherent sedimentation cultured cells, and high cancer cell engraftment and cancer tissue forming ability. Furthermore, FIG. 3 shows the result of transplantation in the absence of Matrigel in a medium not containing FBS. Similarly, cells cultured in suspension in a medium containing deacylated gellan gum showed higher engraftment and cancer organization in mice than adherent monolayer cultured cells.

FIG. 4 shows the anticancer drug effect on cancer tissue formation when HCT116 cells dispersed in a single cell after suspension culture in a medium containing deacylated gellan gum and transplanted in the absence of Matrigel. The growth of cancer tissues formed by transplanting cells cultured in suspension in a medium containing deacylated gellan gum is approximately 74% (cancer tissue volume) with the administration of paclitaxel (7 mg / kg / day, 3day / week), Trametinib ( Administration of 3 mg / kg / day, 3 day / week) suppressed about 49% (cancer tissue volume). In addition, the same suppression was observed in cancer weight.

FIG. 5 shows a comparison of spheroid transplantation and single cell transplantation in the absence of matrigel in HCT116 cells suspended in suspension in a medium containing deacylated gellan gum. Spheroid transplantation showed about 150% higher engraftment and cancer tissue formation than single cell transplantation with the same number of cells.

FIG. 6 shows the effect of an anticancer agent on cancer tissue formation when HCT116 cells suspended in suspension in a medium containing deacylated gellan gum were transplanted in the absence of Matrigel in the form of spheroids. Cancer tissue formation was suppressed by about 35% by administration of Trametinib (3mg / kg / day, 3day / week). The degree of this suppression tended to be comparable or weaker than single cell transplantation.

FIG. 7 shows the results of cancer tissue formation when SKOV3 cells dispersed in single cells after suspension culture in a medium containing deacylated gellan gum were transplanted in the absence of Matrigel. In the case of transplantation of 6.8 × 10 ⁵ cells, in the absence of Matrigel, cancer engraftment and cancer formation were extremely low in adherent monolayer cultured cells, and evaluation was difficult even at 5 weeks. On the other hand, in the cells cultured in suspension in a medium containing deacylated gellan gum, remarkable cancer tissues were confirmed from the third week of transplantation, and 685 mm ³ of cancer tissues were formed at the fifth week. In the transplantation of 1x10 ⁶ cells, the formation of marked cancer tissue was observed in the adherent monolayer cultured cells from the 4th week, but in the cells cultured in suspension in a medium containing deacylated gellan gum, the cells were more prominent than the 1st week of transplantation. Formation of cancerous tissue was observed, and the volume of the cancerous tissue at 5 weeks was about 490% compared to the adherent monolayer cultured cells.

FIG. 8 shows the anticancer drug effect on cancer tissue formation when SKOV3 cells dispersed in a single cell after suspension culture in a medium containing deacylated gellan gum and transplanted in the absence of Matrigel. Proliferation of cancer tissue formed by transplanting SKOV3 cells suspended in suspension in a medium containing deacylated gellan gum was suppressed by about 69% (cancer tissue volume) by administration of MK2206 (120 mg / kg / day, 3day / week). It was. In addition, the same suppression was observed in cancer weight.

FIG. 9 shows a comparison between spheroid transplantation and single cell transplantation in the absence of matrigel of SKOV3 cells suspended in suspension in a medium containing deacylated gellan gum. Spheroid transplantation showed comparable engraftment and cancer tissue formation compared to single cell transplantation of the same number of cells.

FIG. 10 shows the effect of an anticancer agent on cancer tissue formation when SKOV3 cells suspended in suspension in a medium containing deacylated gellan gum were transplanted in the absence of matrigel in the form of spheroids. Cancer tissue formation was suppressed by about 57% (cancer tissue volume) by the administration of MK2206 (120 mg / kg / day, 3 day / week). In addition, the same suppression was observed in cancer weight.

When MCF7 cells suspended in suspension in a medium containing deacylated gellan gum were transplanted to nude mice in the absence of Matrigel in the presence of spheroids, 5 x 10 ⁶ cells were transplanted, and 333 ± 37 mm ³ cancer in 6 of 18 mice at 3 weeks after transplantation Tissue formation was confirmed. On the other hand, when the same number of monolayer cultured cells were transplanted in the absence of Matrigel, 0 out of 6 animals were confirmed to have cancerous tissue formation at 3 weeks after transplantation. Further, FIG. 11 shows the effect of an anticancer agent against cancer formed when MCF7 cells suspended in a deacylated gellan gum-containing medium were transplanted in a spheroid state. Gefitinib (100mg / kg / day, 3day / week) suppressed the enlargement of cancer tissue.

[Test Example 11]
Cell Recovery after Culture The culture solution containing the LS123 cells cultured in Test Example 7 was collected in a centrifuge tube and centrifuged at 500 rpm for 1 minute. Collect the non-precipitated supernatant fraction, dilute 2-3 times with 1% (v / v) NEAA-containing E-MEM medium or PBS, and centrifuge at 1500 rpm for 5 minutes to obtain 10% (v / v) of the lower layer. ) Cells were collected. Furthermore, it was suspended in 5% to 10 times the volume of 1% (v / v) NEAA-containing E-MEM medium or PBS, and centrifuged again at 1500 rpm for 5 minutes to recover the precipitated cells. The cells were resuspended in a small amount of 1% (v / v) NEAA-containing E-MEM medium to obtain a sphere cell suspension.

[Test Example 12]
Creation of a tumor-bearing model The E-MEM medium containing 1% NEAA so that the cancer cells obtained in Test Example 11 are ⁶ × 10 ⁶ cells / animal / 200 μL in 2D culture and 3 × 10 ⁶ cells / animal / 250 μL in DAG suspension culture. Diluted with In Matrigel co-transplantation, the cell suspension and Matrigel were mixed 1: 1 (v: v) for 2D culture and 2.5: 1 (v: v) for DAG suspension culture.
Seven week old female BALB / c-nu / nu (nude) mice were purchased from CLEA Japan. Under isoflurane anesthesia, 200 μL of each cancer cell suspension under the shoulder of the animal (2D cultured cells: ⁶ × 10 ⁶ cells or 3 × 10 ⁶ cells in the presence of Matrigel) or 250 μL (DAG suspension cultured cells: 3 × 10 ⁶ cells) or 350 μL (DAG suspension culture cells: 3 × 10 ⁶ cells (in the presence of Matrigel)) was injected. After cell injection, food and water were ingested freely and reared for 4 weeks in group breeding. The cancer tissue was observed once a week and the size (outside dimension) was measured. The volume of the cancer tissue was calculated according to the formula described in Test Example 9. The results are shown in FIG.
In the transplantation in the absence of Matrigel, both 2D cultured cells and DAG suspension cultured cells were observed to have tumors like cancer tissues one week after transplantation. The size of DAG suspension cultured cells was almost double even though the number of cells was half that of 2D cultured cells. The tumor disappeared completely in the 2D cultured cells after the 2nd week, but maintained in the DAG suspension cultured cells until the 4th week although it was reduced. In the transplantation in the presence of Matrigel, compared to the transplantation in the absence of Matrigel, the tumor formed in the 2D cultured cells was small and disappeared after the second week, and the formation of the tumor was not confirmed in the DAG suspension cultured cells.

The present invention provides a cancer-bearing mammal model that is excellent in versatility, economy, convenience, and / or clinical extrapolation. According to the present invention, the engraftment rate and the growth ability of cancer cells in vivo are improved, so that it is possible to create a cancer-bearing mammal having a large tumor tissue with a small number of cancer cells in a short period of time. It becomes. In addition, it is possible to engraft cancer cells that do not engraft unless they are transplanted with an extracellular matrix such as Matrigel, and that do not form a tumor, in vivo, under the condition that there is no extracellular matrix. . In addition, a medium composition capable of culturing cells or cell aggregates or tissues while maintaining a floating state without substantially increasing the viscosity in a liquid medium, and the cells or cells obtained by the culture Aggregates are thought to provide a culture environment that approximates the cancer tissue in an actual living body. It is also possible to create a cancer-bearing animal model using non-immune-deficient animals. Therefore, it is possible to expect a tumor tissue that is more similar to an actual clinical case. The cancer-bearing mammal model created by the method of the present invention is useful for cancer characteristic evaluation, individual cancer diagnosis, anticancer agent evaluation and the like.

The contents of all publications, including patents, patent application specifications, and scientific literature mentioned herein, are hereby incorporated by reference herein to the same extent as if all were expressed. Is.

This application is based on Japanese Patent Application 2015-022585 (filing date: February 6, 2015) and Japanese Patent Application 2015-067781 (filing date: March 28, 2015) filed in Japan. All of which are included in this specification.

Claims

A method for producing a cancer-bearing non-human mammal comprising the following steps:
(1) Suspension culture of cancer cells in a medium composition containing a structure capable of culturing cells or tissues in suspension; and (2) Non-human mammals cancer cells obtained by the culture of (1) Introduce to animals.
The method of claim 1, further comprising the following steps:
(3) Breeding non-human mammals to which cancer cells have been transferred, and forming tumor tissue from the transferred cancer cells.
3. The method according to claim 1 or 2, wherein in (2), cancer cells suspended in a physiological aqueous composition containing a basement membrane preparation are transferred to a non-human mammal.
3. The method according to claim 1 or 2, wherein in (2), cancer cells suspended in a physiological aqueous composition substantially free of a basement membrane preparation are transferred to a non-human mammal.
The method according to any one of claims 1 to 4, wherein the non-human mammal is an immunodeficient non-human mammal.
6. The method according to claim 5, wherein the cancer cells are allogeneic or xenogeneic with respect to the non-human mammal to be transferred.
The method according to claim 6, wherein the cancer cell is a human cancer cell.
The method according to any one of claims 1 to 7, wherein the cancer cells have the ability to form solid tumors.
The method according to any one of claims 1 to 8, wherein the structure comprises deacylated gellan gum.
10. The method according to claim 9, wherein the concentration of the deacylated gellan gum in the medium composition is 0.005 to 0.3% (w / v).
The method according to any one of claims 1 to 10, wherein the viscosity of the medium composition at 37 ° C is 8 mPa · s or less.
A screening method for an anticancer agent, comprising the following steps:
(1) administering a test compound to a cancer-bearing non-human mammal prepared by the method according to any one of claims 1 to 11;
(2) Measuring the size of the tumor tissue in the cancer-bearing non-human mammal obtained in (1);
(3) To compare the size of the tumor tissue measured in (2) with the size of the tumor tissue of a control cancer-bearing non-human mammal not administered with the test compound.
A method of promoting engraftment of cancer cells in a non-human mammal, comprising suspension-culturing cancer cells in a medium composition containing a structure capable of culturing cells or tissues in suspension.