JP2011115106A - Cancer cell aggregate and method for preparing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new cancer cell aggregate that can accurately reflect the behavior of cancer cells in vivo and a method for preparing the same. <P>SOLUTION: There is disclosed: an aggregate formed by unicellularize a cancer tissue obtained from a cell mass or an individual originated from a cancer tissue and then cells in the unicellularized material are mutually aggregated into an aggregate consisting of three or more cells; or its cultured material, wherein a cancer cell aggregate retaining proliferating ability is prepared in vitro. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a cancer cell aggregate and a method for preparing the same. More specifically, the present invention relates to a cancer cell aggregate that can reconstruct cancer in vitro and retains the ability to grow.

  In recent years, as a result of various studies accumulated to overcome cancer, the results of treatment for early-stage cancer have improved dramatically. However, the treatment of advanced cancer remains difficult and cancer continues to be the leading cause of death among Japanese. According to the 2007 vital statistics by the Ministry of Health, Labor and Welfare, more than 340,000 people die from cancer each year.

  In cancer research so far, especially when examining the behavior in vitro, experiments using cancer cell lines established by subculture under conditions optimized for culture are the mainstream. Such cancer cell lines include human breast cancer cell lines (MDF7, NCI / ADR HS578T, MDA-MB-22231 / ATCC, MDA-MB-4335, MDA-N, BT-549, T-47D), human offspring Cervical cancer cell line (HeLa), human lung cancer cell lines (A549, EKVX, HOP-62, HOP-92, NCI-H23, NCI-H226, NCI-H322M, NCI-H460, NCI-H522) and human colon cancer cells Strains (Caco-2, COLO 205, HCC-2998, HCT-15, HCT-116, HT29, KM12, SW-620) human prostate cancer cell lines (DU-145, PC-3, LNCaP), etc. In fact, it is widely used for research.

In order to realize diagnosis and treatment for each cancer patient, primary culture of cancer cells is considered promising and research is ongoing. For example, a CD-DST method (Collagen gel droplet embedded drug sensitivity test) using primary cultured cells has been developed. This in vitro test method is a drug sensitivity test in which an isolated tissue or cell from a patient is embedded in a collagen gel droplet and verified by combining three-dimensional culture and image colorimetry (for example, non-patented) Reference 1). However, primary culture cells are difficult to handle because no culture method has been established.

As a result of research on cancer cells, cancer cells that make up cancer may be composed of multiple subpopulations, which are called “tumor progenitor cells” or “tumor stem cells”, but are small populations that are self-replicating. There are a number of reports that support the existence of subpopulations that are possible and that can be the source of the majority of cancer cells by differentiation (eg, Non-Patent Documents 2 and 3). Such stem cells can be obtained, for example, by separating a tumor extracted from a living body into single cells and sorting them, and some of them are said to show proliferative ability even in vitro ( Non-patent document 4). However, there is a negative report on the theory of explaining the origin of cancer by stem cells in this way (Non-Patent Document 5), and it does not go beyond the hypothesis.

Even in the current state of cancer research, there are many unknowns about cancer.

Takamura Y et al. (2002) Prediction of chemotherapeutic response by collagen gel droplet embedded culture-drug sensitivity test in human breast cancers. Int. J. Cancer, 98, 450-455 Vermeulen L et al. (2008) Single-cell cloning of colon cancer stem cells reveals a multi-lineage differentiation capacity. PNAS Vol.105 No.36 13427-13432 Ricci-Vitiani L et al. (2007) Identification and expansion of human colon-cancer-initiating cells. Nature Vol.445 111-115 Todaro M et al. (2007) Colon cancer stem cells dictate tumor growth and resist cell death by production of interleukin-4.Cell Stem Cell 1: 389-402 Shmelkov S V et al. (2008) CD133 expression is not restricted to stem cells, and both CD133 + and CD133- metastatic colon cancer cells initiate tumors. The Journal of Clinical Investigation Vol. 118 2111-2120

The purpose of the present invention is to analyze the cancer cells in vivo, to reproduce the behavior of cancer cells in vitro, and to accurately verify the effects in vivo in drug sensitivity tests or radiation sensitivity tests. It is to provide a novel cancer cell aggregate useful as a sample for research.

The present invention also provides a novel, useful sample as a sample for cancer analysis and treatment research that can be used for the preparation of a simple cancer animal model that has sufficient colonization in a small amount in transplantation into different animals. A cancer cell aggregate is provided.

The present inventors intend to conduct a therapeutic sensitivity test for individual cancer patients, and considering the possibility that the cell line that has been used as a research material for cancer research is different from patient cancer, As a result of intensive studies on the primary culture method of cancer cells as a research material in order to solve the above problems, a novel cancer cell aggregate and a method for preparing the same were found, and the present invention was completed.

  That is, the present invention relates to an aggregate formed by agglutination of cells in the single cell product into three or more cells after treating the cell mass derived from a cancer tissue into a single cell, a culture thereof, or an individual An aggregate formed by aggregating the cancer tissue obtained from the cell into a single cell and then aggregating the cells in the single cell product into 3 or more cells, or a culture thereof, Cancer cell aggregates, which can hold

In the present invention, after the cancer tissue-derived cell mass or the cancer tissue obtained from an individual is made into a single cell, the cells in the single cell product are 3 cells in the presence of a ROCK (Rho binding kinase) inhibitor. The present invention relates to an aggregate formed by aggregating more than one or a culture thereof, and a cancer cell aggregate capable of retaining a proliferation ability in vitro.

The ROCK inhibitor may be Y27632®.

The present invention also relates to a cancer cell aggregate comprising three or more cancer cell aggregates and having a substantially spherical shape or an elliptical spherical shape.

  The present invention also relates to a cancer cell aggregate comprising three or more cancer cell aggregates: and a basement membrane-like substance existing on the outer peripheral surface of the cancer cell aggregate, and having a substantially spherical shape or an elliptical spherical shape.

The cancer cell aggregate is preferably substantially free of cells other than cancer cells.

The basement membrane-like material can be laminin.

The cancer cell aggregate may have a diameter of 40 μm to 250 μm.

The cancer cell can be derived from an epithelial cancer cell.

The cancer cells can be derived from colon cancer, ovarian cancer, breast cancer, lung cancer, prostate cancer, kidney cancer, bladder cancer, pharyngeal cancer, pancreatic cancer.

The present invention also includes a step of enzyme-treating a cancer tissue-derived cell mass or a cancer tissue excised from a living body into a single cell; and a step of aggregating cells in the single cell product into three or more cells. A method for preparing a cancer cell aggregate.

The present invention also includes a step of enzyme-treating a cancer tissue-derived cell mass or a cancer tissue excised from a living body into a single cell; and cells in the single cell product in the presence of a ROCK inhibitor, The present invention relates to a method for preparing a cancer cell aggregate including a step of aggregating cells to 3 or more cells.

The method for preparing a cancer cell aggregate may further include a step of culturing the aggregated components for 3 hours or more.

The enzyme treatment may be a treatment using trypsin.

The ROCK inhibitor may be Y27632®.

The present invention also relates to a cancer cell aggregate obtained by the above preparation method.

  The cancer cell aggregate of the present invention exhibits behavior similar to that in vivo in vitro, and can reconstruct those exhibiting such behavior, and can retain proliferation ability over a certain period of time. . Such cancer cell aggregates can be used for amplification by culturing cancer cells, and can be widely and conveniently used in vitro for drug sensitivity tests or radiation sensitivity tests. Since it is excellent in tumor colonization with respect to heterogeneous animals, it can be used to create simple tumor-forming animals.

It is a figure which shows the cancer tissue origin cell cluster obtained from the mouse | mouth. It is a figure which shows the cancer tissue origin cell cluster obtained from various cancer tissues. It is a figure which shows the cancer tissue origin cell cluster obtained from the mouse islet tumor. It is a figure which shows the cancer cell aggregate obtained from the cancer tissue origin cell mass. It is a figure which shows the cancer cell aggregate derived from a human colon cancer operation specimen. It is a figure which shows the state after freeze-preserving and thaw | melting the cancer tissue origin cell mass after trypsin treatment. It is the figure which compared the collection | recovery rate of the cell after cryopreserving and thaw | melting cancer tissue origin cell mass by the presence or absence of a trypsin process. It is the figure which compared the proliferation ability after freezing preservation | save and thaw | melting the cancer tissue origin cell mass by the presence or absence of a trypsin process. It is a figure which shows the result of the drug sensitivity test by the doxorubicin in vitro using the cancer cell aggregate of this invention. It is a figure of the tumor tissue obtained by transplanting the cancer cell aggregate of the present invention to a mouse.

The cancer cell aggregate of the present invention is a cancer cell-derived cell mass or a cancer tissue obtained from an individual, and is converted into single cells, and then the individual cells in the single cell product are completely separated from each other or completely into individual cells. Formed by aggregation of several cells that have not been separated, or individual cells and some cells that have not been completely separated into individual cells, to aggregate to a total of 3 or more cells Aggregates or cultures thereof, such that they can retain their proliferative ability in vitro.

Here, “to make a cancer tissue-derived cell mass or a cancer tissue obtained from an individual into a single cell” means that at least a part of the cancer tissue-derived cell mass or the obtained cancer tissue is separated into single cells in vitro. It means to perform the processing. Thus, typically, after such treatment, there may be cells that have separated into individual single cells, but some cells may not be separated into individual cells, and Even in this case, it corresponds to “single cell” as used herein. In this case, those that are not separated until they are mixed include aggregates of up to 10 cells, preferably aggregates of 2 to 3 cells.

First, a cancer tissue-derived cell mass that can be a starting material for the cancer cell aggregate of the present invention will be described. The cancer tissue-derived cell mass is a novel cell mass found by the present inventors, and is itself a separated product obtained by separating and treating as a mass containing three or more cancer cells from a cancer tissue obtained from an individual. The culture may be such that it can retain its proliferative ability in vitro.

Here, the “separate separated from a cancer tissue obtained from an individual as a mass containing three or more cancer cells” is obtained by treating a cancer tissue obtained from a cancer generated in a living body. Or an isolate containing 3 or more, preferably 8 or more cancer cells. Such isolates do not include those that have been separated into single cells, nor do they include constructs that have been separated into single cells and then reconstituted. However, this separated product includes not only a product immediately after being separated from a living body but also a product that has been kept in physiological saline for a certain period of time, or a product that has been frozen or refrigerated.

“A culture of an isolate separated from a cancer tissue obtained from an individual as a mass containing three or more cancer cells” is obtained by treating a cancer tissue obtained from a cancer generated in vivo. In addition, it refers to those obtained by culturing in vitro an isolated product separated as a mass containing 3 or more cancer cells. The culture time is not particularly limited as long as it is present in the medium even for a short time. Such a culture often exhibits a substantially spherical shape or an elliptical sphere shape by culturing for a certain period, preferably 3 hours or more. The culture here includes a substantially spherical or elliptical spherical culture after elapse of a certain period of time, and an amorphous culture up to that. Further, an indefinite shape obtained by further dividing such a substantially spherical or elliptical spherical culture, and a substantially spherical or elliptical spherical product by further culture are also cultures referred to herein.

Such a cancer tissue-derived cell mass can retain its proliferative ability for a period of 10 days or more, preferably 13 days or more, more preferably 30 days or more by continuing the culture as it is. By performing mechanical division or forming the cancer cell aggregate of the present invention, the proliferation ability can be maintained substantially indefinitely.

The machine division can be performed by using a scalpel, a knife, scissors, an ophthalmic sword or the like. Alternatively, it can also be performed by attaching an injection needle to the syringe and repeating the suction and discharge of the cancer tissue-derived cell mass together with the culture solution. For example, a 1 ml syringe and a 27G needle are preferably used in the present invention, but are not limited thereto.

Here, the medium for culturing the cell mass derived from the cancer tissue of the present invention is not particularly limited, but an animal cell culture medium is preferably used. Particularly preferably, a serum-free medium for stem cell culture is used. Such a serum-free medium is not limited as long as it is used for culturing stem cells. The medium and conditions for culturing the cancer tissue-derived cell mass are the same as those for culturing the cancer cell aggregate.

The number of cancer cells constituting the cancer tissue-derived cell cluster is at least 3 or more, preferably 8 or more, more preferably 10 or more, still more preferably 20 or more, and most preferably 50 or more. When the cancer tissue-derived cell mass is a separated product, the number is preferably 1000 or less, more preferably about 500 or less. In the case of a culture after culturing the isolate, the number can be increased by culturing. However, even if it is a culture, it is preferably 10,000 or less, more preferably 5000 or less.

In the present invention, it is particularly derived from colon cancer tissue, ovarian cancer tissue, breast cancer tissue, lung cancer tissue, prostate cancer tissue, kidney cancer tissue, bladder cancer tissue, pharyngeal cancer tissue, or pancreatic cancer. Is particularly preferred, but not limited.

In the case of a cancer tissue-derived cell mass derived from colorectal cancer tissue, the contained cancer cells are not particularly limited, but may express CD133.

The cancer tissue-derived cell mass may alternatively include three or more cancer cell aggregates and have a substantially spherical shape or an elliptical spherical shape.

Although not limited, it may include a basement membrane-like substance existing on the outer peripheral surface of the cancer cell aggregate.

In the cancer tissue-derived cell mass of the present invention, a thin membranous basement membrane-like material is preferably formed on the order of several nanometers, preferably about 40 to 120 nm, although it is not limited.

The size of the cell mass derived from the cancer tissue of the present invention is not limited, and includes an irregular shape having a particle size or a volume average particle size of about 8 μm to 10 μm. Also included. The diameter is preferably 40 μm to 1000 μm, more preferably 40 μm to 250 μm, and still more preferably 80 μm to 200 μm.

The cancer tissue-derived cell mass of the present invention often has one or more sequences selected from the group consisting of a shelf-like array, a sheet-like array, a multilayered array, and a syncytial array, but is not particularly limited.

The cancer tissue-derived cell mass of the present invention is typically a step of subjecting a fragment of cancer tissue excised from a living body to an enzyme treatment; and a mass containing 3 or more cancer cells among the enzyme-treated products. Can be prepared by a method comprising the steps of:

Furthermore, although not limited, the cancer tissue-derived cell mass of the present invention can be prepared by a method including a step of culturing the components thus collected for 3 hours or more.

First, cancer tissue excised from a living body can be fragmented as it is, and first, it can be maintained in an animal cell culture medium before fragmentation. Such animal cell culture media include, but are not limited to, Dulbecco MEM (such as DMEM F12), Eagle MEM, RPMI, Ham's F12, Alpha MEM, Iskov modified Dulbecco and the like. At this time, suspension culture is preferably performed in a cell non-adhesive incubator.

It is also preferred that the cancer tissue be washed prior to stripping. Such washing includes, but is not limited to, acetate buffer (acetate + sodium acetate), phosphate buffer (phosphate + sodium phosphate), citrate buffer (citric acid + sodium citrate), boric acid A buffer solution such as a buffer solution, a tartrate buffer solution, a Tris buffer solution, or a phosphate buffered saline can be used. In the present invention, it is particularly preferable that the tissue can be washed in HBSS. The appropriate number of washings is 1 to 3 times.

The fragmentation can be performed by dividing the tissue after washing with a knife, scissors, a cutter (manual, automatic) or the like. The size and shape after the fragmentation are not particularly limited and can be performed randomly, but preferably 1 mm to 5 mm square, more preferably 1 mm to 2 mm square.

Next, the fragmented product thus obtained is subjected to an enzyme treatment. Such enzyme treatment can be treatment with one of collagenase, trypsin, papain, hyaluronidase, C. histolyticum neutral protease, thermolysin, and dispase, or a combination of two or more thereof. Enzymatic treatment conditions may include isotonic salt solutions buffered to physiologically acceptable pH, eg about 6-8, preferably about 7.2-7.6, eg, PBS or Hanks balanced salt solution, For example, at about 20-40 ° C., preferably about 25-39 ° C., for a time sufficient to degrade the connective tissue, for example about 1-180 minutes, preferably 30-150 minutes, a concentration sufficient for that, for example about It may be 0.0001-5% w / v, preferably about 0.001% -0.5% w / v.

Although not limited, the conditions for the enzyme treatment may be, for example, treatment with a mixed enzyme containing collagenase. More preferably, one or more proteases selected from the group consisting of C. histolyticum neutral protease, thermolysin, and dispase; and one or more collagenases selected from the group consisting of collagenase I, collagenase II, and collagenase IV Treatment with a mixed enzyme containing.

Such mixed enzymes include, but are not limited to, Liberase Blendzyme 1 (registered trademark) and the like.

Next, it is preferable to select and collect a mass containing three or more cancer cells from the enzyme-treated product thus obtained. The method of sorting and collecting is not particularly limited, and any method known to those skilled in the art for distributing the size can be used.

The size distribution method is not particularly limited as long as it is visual, fractionation using a phase-contrast microscope, or a sieve as a simple method, as long as it is a fractionation method based on particle diameters available to those skilled in the art. When using a sieve, it is preferable to collect components that pass through a sieve mesh size of 20 μm and do not pass through 500 μm. More preferably, components that pass through a sieve mesh size of 40 μm and do not pass through 250 μm are recovered.

Here, the mass containing three or more cancer cells to be selected is the cancer tissue-derived cell mass of the present invention, and has a certain range of sizes. The size within a certain range includes small particles having a volume average particle diameter of about 8 μm to 10 μm, but in the case of a nearly spherical shape, the diameter is 20 μm to 500 μm, preferably 30 μm to 400 μm, more preferably 40 μm to 250 μm, In the case of an elliptical shape, the major axis is 20 μm or more and 500 μm or less, preferably 30 μm or more and 400 μm or less, more preferably 40 μm or more and 250 μm or less, and in the case of an irregular shape, the volume average particle diameter is 20 μm or more and 500 μm or less, preferably 30 μm or more and 400 μm or less. More preferably, it is 40 μm or more and 250 μm or less. The volume average particle diameter can be measured by evaluating the particle size distribution and particle shape using a phase contrast microscope (IX70; manufactured by Olympus Corporation) with a CCD camera.

Both of the separation-treated product and its culture, which are thus selected and collected components, are cancer tissue-derived cell masses. The culture may be one in which the separated and collected components are present in the medium for a short time, for example, at least 3 hours or more, preferably 10 hours or more and 36 hours, more preferably 24 hours. By culturing for a period of ˜36 hours or longer, it may have a substantially spherical shape or a substantially elliptical spherical shape. The culture time may exceed 36 hours, and several days, 10 days or more, 13 days or more, or 30 days or more may have elapsed.

Cultivation can be performed as it is for a long time in the medium, but preferably, the ability to proliferate can be maintained substantially infinitely by performing mechanical division periodically during the culture.

Next, the cancer cell aggregate of the present invention will be described in detail. In the present invention, “cancerous tissue obtained” from an individual is acquired so that it can be handled in vitro for histological examination with an injection needle or endoscope in addition to cancerous tissue obtained by excision by surgery or the like. Refers to cancerous tissue.

“Aggregating to 3 or more cells” means individual cancer cells obtained from cancerous tissue generated in vivo or cancer tissue-derived cell masses found by the present inventors as single cells. It refers to a state in which a group of cells that have not been separated from each other or individual cells, or a combination thereof, gathered to contain at least three or more cells.

In the case of subjecting a cancer tissue derived from a cancer tissue-derived cell mass or a cancer tissue obtained from a living body to a single cell treatment, it is not limited, but includes an enzyme treatment of a cancer tissue obtained from an individual. .

The enzyme treatment may typically be treatment with trypsin, dispase, and optionally one of collagenase, papain, hyaluronidase, C. histolyticum neutral protease, and thermolysin, or combinations of two or more thereof. . Enzymatic treatment conditions may include isotonic salt solutions buffered to physiologically acceptable pH, eg about 6-8, preferably about 7.2-7.6, eg, PBS or Hanks balanced salt solution, For example, at about 20-40 ° C., preferably about 25-39 ° C., for a time sufficient to degrade the connective tissue, for example about 1-180 minutes, preferably 30-150 minutes, a concentration sufficient for that, for example about It may be 0.0001-5% w / v, preferably about 0.001% -0.5% w / v.

Without limitation, the enzyme treatment typically may be trypsin or dispase treatment alone.

Individual cells are usually obtained after the single cell treatment. However, cells that are not completely separated individually are also included.

Such cells may be aggregated as they are, but preferably, for example, a ROCK inhibitor is present and aggregated immediately after the single cell treatment.

ROCK is a Rho-associated coiled-coil kinase (ROCK: GenBank accession number: NM_005406) and is one of the main effector molecules of Rho GTPase and is known to control various physiological phenomena. (Also called Rho-binding kinase). As a ROCK inhibitor, Y27632 etc. are illustrated, for example. In addition, Fasudil (HA1077), H-1152, Wf-536 (these are all available from Wako Pure Chemical Industries, Ltd.), and derivatives thereof, as well as antisense nucleic acids, RNA interference-inducing nucleic acids for ROCK, and these Vector containing.

Prior to aggregation, the 96-well culture plate was used to treat the treatment separated into trypsin treatment (eg, but not limited to, 0.25% trypsin-EDTA, treatment at 37 ° C. for 5 minutes) to a single cell or a population of 10 cells or less. Seed at low density (eg, 500 / 0.32 cm ² , medium volume of about 0.15 ml). Immediately or after culturing for several days, the ROCK inhibitor can be added at a concentration of about 1 to 100 μM, preferably about 10 μM.

Such aggregates can be cultured in vitro. The culture time is not particularly limited as long as it is present in the medium even for a short time. Such a culture often exhibits a substantially spherical shape or an elliptical sphere shape by culturing for a certain period, preferably 3 hours or more. The culture here includes a substantially spherical or elliptical spherical culture after elapse of a certain period of time, and an amorphous culture up to that. Further, an indefinite shape obtained by further dividing such a substantially spherical or elliptical spherical culture, and a substantially spherical or elliptical spherical product by further culture are also cultures referred to herein.

In vitro, the cancer cell aggregate of the present invention “can retain growth ability” means at least 10 days or more, preferably 13 days under a cell culture condition of a temperature of 37 ° C. and a 5% CO ₂ incubator. As described above, it means that the growth ability can be maintained for a period of 30 days or more.

Such cancer cell agglomerates can maintain their proliferative ability for a period of 10 days or more, preferably 13 days or more, and more preferably 30 days or more by continuing the culture as they are. The ability to proliferate can be maintained substantially indefinitely by performing general division or by further unicellularization and aggregation.

The machine division can be performed by using a scalpel, a knife, scissors, an ophthalmic sword or the like. Alternatively, it can be performed by attaching an injection needle to the syringe and repeating the suction and discharge of the cancer cell aggregates together with the culture solution. For example, a 1 ml syringe and a 27G needle are preferably used in the present invention, but are not limited thereto.

Here, the medium for culturing the cancer cell aggregate of the present invention is not particularly limited, but an animal cell culture medium is preferably used. Particularly preferably, a serum-free medium for stem cell culture is used. Such a serum-free medium is not limited as long as it is used for culturing stem cells. A serum-free medium refers to a medium that does not contain unprepared or unpurified serum, and can be used after adding purified blood-derived components or animal tissue-derived components (for example, growth factors).

The serum-free medium of the present invention can be prepared using a medium used for animal cell culture as a basal medium. As the basal medium, for example, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, RPMI 1640 medium, Fischer's medium , And combinations thereof.

The serum cell aggregate can be cultured by adding a serum substitute to such a serum-free medium. Serum substitutes contain, for example, albumin, amino acids (eg, non-essential amino acids), transferrin, fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol or 3 ′ thiolglycerol, or equivalents thereof as appropriate. Can be.

In the culture method of the present invention, a commercially available serum substitute can also be used. Examples of such commercially available serum substitutes include knockout serum replacement (KSR), chemically-defined lipid concentrated fatty acid concentrate (manufactured by Gibco), and glutamax (manufactured by Gibco).

The medium for culturing the cancer cell aggregate of the present invention can also contain vitamins, growth factors, cytokines, antioxidants, pyruvate, buffers, inorganic salts and the like.

In particular, any serum-free medium such as a serum-free medium containing EGF and bFGF, such as a serum-free medium containing a serum substitute such as knockout serum replacement (KSR, manufactured by Invitrogen) and bFGF can be preferably used. . The content of serum substitute or EGF is preferably 10-30% w / v of the whole medium.

Such a medium is not limited, but a commercially available product includes a serum-free medium (Gibco) for STEMPRO human ES cells.

Incubators used for culturing cancer cell aggregates are not particularly limited as long as animal cells can be cultured. For example, flasks, tissue culture flasks, dishes, petri dishes, tissue culture dishes , Multi-dish, microplate, microwell plate, multiplate, multiwell plate, chamber slide, petri dish, tube, tray, culture bag, roller bottle.

The incubator is preferably non-cell-adhesive and is three-dimensionally cultured in the presence of a cell support substrate such as an extracellular matrix (ECM) in the medium. The cell support substrate can be intended for adhesion of cancer cell aggregates. Examples of such a cell supporting substrate include matrigel using an extracellular matrix, such as collagen gel, gelatin, poly-L-lysine, poly-D-lysine, laminin, and fibronectin. Such conditions are suitably used particularly when it is desired to grow the cancer cell aggregate of the present invention.

Other culture conditions can be appropriately set. For example, the culture temperature is not limited, but is preferably about 30 to 40 ° C. Most preferably, it is 37 degreeC. The CO ₂ concentration is, for example, about 1 to 10%, preferably about 2 to 5%.

The cancer cell aggregate of the present invention can be cultured in such a medium and culture conditions. Furthermore, the culturing of cancer cell aggregates may require co-culture with other cells due to their individual nature, or may require the presence of additional specialized supplements such as hormones.

Specifically, co-culture may be performed together with feeder cells. As feeder cells, stromal cells such as fetal fibroblasts can be used. Specifically, although not limited, NIH3T3 or the like is preferable.

Alternatively, for specific types of breast cancer, uterine cancer, and prostate cancer, it is preferable to culture in the presence of hormones. Specifically, estrogen for breast cancer, profesterone for uterine cancer, testosterone for prostate cancer, and the like, but not limited thereto, various hormones can be added to conveniently adjust the culture conditions. Furthermore, by investigating how the behavior of cancer cell aggregates after culturing changes due to the presence of such hormones, it is possible to determine the hormone dependence of cancer in the patient from which it is derived, and the effectiveness of antihormonal drug treatment May be predictable.

The cancer cell aggregate of the present invention can also be cultured in suspension culture. In suspension culture, cancer cell aggregates are cultured in a medium under conditions that are non-adherent to the incubator. Examples of such suspension culture include embryoid body culture method (Keller et al., Curr. Opin. Cell Biol. 7, 862-869 (1995)), and SFEB method (eg, Watanabe et al., Nature Neuroscience 8, 288- 296 (2005); International Publication No. 2005/123902). This is particularly preferred when it is desired to maintain the cancer cell aggregates rather than the growth.

The number of cancer cells constituting the cancer cell aggregate is at least 3 or more, preferably 8 or more, more preferably 10 or more, still more preferably 20 or more, and there is no particular upper limit in the number. When the cancer cell aggregate of the present invention is a separated product, the number is preferably 1000 or less, more preferably 500 or less. In the case of a culture after culturing the isolate, the number can be increased by culturing. However, even if it is a culture, it is preferably 10,000 or less, more preferably 5000 or less.

In the present invention, the term “cancer cell” is used in a commonly used meaning, and refers to a disordered cell-ordered cell such as unlimited division / proliferation and deviation from apoptosis in vivo. More specifically, it refers to a cell that has lost its cell growth control function or is extremely attenuated, and typically has acquired infinite growth ability with a frequency of 80% or more, many of which also have invasive metastatic ability. It often means that it is a cell that is positioned as a malignant neoplasm that leads to death, including humans, especially mammals.

In the present invention, the type of cancer tissue derived is not particularly limited, and lymphoma, blastoma, sarcoma, liposarcoma, neuroendocrine tumor, mesothelioma, schwannoma, meningioma occurring in animals including mammals Adenomas, melanomas, leukemias, lymphoid malignancies, and the like, and cancers that occur in mammalian epithelial cells are particularly preferable. Non-small cell lung cancer, hepatocellular carcinoma, biliary tract cancer, esophageal cancer, stomach cancer, colorectal cancer, pancreatic cancer, cervical cancer, ovarian cancer, endometrial cancer, bladder cancer, Examples include pharyngeal cancer, breast cancer, salivary gland cancer, renal cancer, prostate cancer, labial cancer, anal cancer, penile cancer, testicular cancer, thyroid cancer, and head and neck cancer. There are no particular restrictions on animals including mammals, but animals belonging to primates including monkeys and humans, animals belonging to rodents such as mice, squirrels and rats, animals belonging to rabbits, cats such as dogs and cats, etc. An animal belonging to the eye is exemplified.

Among them, in the present invention, in particular, from colon cancer tissue, ovarian cancer tissue, breast cancer tissue, lung cancer tissue, prostate cancer tissue, kidney cancer tissue, bladder cancer tissue, pharyngeal cancer tissue, or pancreatic cancer It is particularly preferred, but not limited.

In the case of a cancer cell aggregate derived from a colon cancer tissue, the cancer cells contained are not particularly limited, but may express CD133.

The cancer cell aggregate of the present invention may alternatively include three or more cancer cell aggregates and have a substantially spherical shape or an elliptical spherical shape.

Although not limited, it may include a basement membrane-like substance existing on the outer peripheral surface of the cancer cell aggregate.

Here, although not particularly limited, the cancer cells forming the aggregate may have one or more surface antigens selected from the group consisting of CD133, CD44, CD166, CD117, CD24, and ESA on the cell surface. Many. CD133, CD44, CD166, CD117, CD24, and ESA are generally surface antigens expressed on cells such as leukocytes such as lymphocytes, fibroblasts, epithelial cells, and tumor cells. These surface antigens are involved in various signal transductions in addition to their function as cell-cell and cell-matrix adhesion, but are also surface markers for various stem cells.

In the present invention, when a cell group “expresses” a surface antigen such as CD133, 80% or more, preferably 90% or more, more preferably substantially all of the cells present in the cell group are surface antigens. Indicates the state.

In the present specification, the “basement membrane-like substance” is not limited, but preferably contains at least one of collagen, laminin, nidogen, proteoglycan such as heparan sulfate proteoglycan, and glycoprotein such as fibronectin. Refers to a substance. In the present invention, a basement membrane-like material containing laminin is preferable.

Laminin is a high-molecular glycoprotein that constitutes the basement membrane. The functions of laminin are diverse and are involved in cell functions such as cell adhesion, signal transduction, proliferation of normal cells and cancer cells. Laminin has a structure in which each of three different subunits is linked by a disulfide bond, and 11 types are found depending on the different types of each subunit.

Among these, laminin 5 is usually produced only from epithelial cells, and is known as a component having an activity of promoting the adhesion of epithelial cells to the basement membrane and the motor function. This laminin 5 has a structure in which each one of α3 chain, β3 chain, and γ2 chain forms a complex. In particular, γ2 chain is considered to be unique to LN5 and is not included in other LN molecular species. Absent.

The cancer cell aggregate of the present invention may have a configuration in which the outer periphery of an aggregate of cancer cells is entirely wrapped in a film formed by such a basement membrane-like substance. Such morphology can be analyzed by observing cancer cell aggregates with an electron microscope, immunostaining of basement membrane components, or a combination of both.

The presence of laminin can be detected by, for example, contacting an antibody recognizing laminin, for example, a mouse laminin-derived rabbit antibody of Sigma-Aldrich with a cancer cell aggregate and measuring the antibody antigen reaction.

It is also possible to use a specific antibody that specifies the type of laminin. For example, the presence of laminin 5 can be detected by, for example, contacting an antibody reactive to the above-described unique γ2 chain or a fragment thereof with a cancer cell aggregate and measuring the reaction of the antibody. .

In the cancer cell aggregate of the present invention, a thin membrane-like basement membrane-like substance is preferably formed on the order of several μm, preferably about 40 to 120 nm, but is not limited.

The size of the cancer cell aggregate of the present invention is not limited, and includes those having an irregular shape having a particle size or a volume average particle size of about 8 μm to 10 μm, and those having a particle size of 1 mm or more that grows greatly after culturing. Is also included. The diameter is preferably 40 μm to 1000 μm, more preferably 40 μm to 250 μm, and still more preferably 80 μm to 200 μm.

The cancer cell aggregate of the present invention often has one or more sequences selected from the group consisting of a shelf-like array, a sheet-like array, a multilayered array, and a syncytial array, but is not particularly limited.

The cancer cell aggregate of the present invention typically includes a step of making a cancer tissue excised from a living body into a single cell; and a step of aggregating cells in the single cell product into three or more cells. It can be prepared by a method.

Furthermore, although not limited thereto, the cancer cell aggregate of the present invention can be prepared by a method including a step of culturing the aggregated component for 3 hours or more.

First, when the cancer cell aggregate of the present invention is obtained from a cancer tissue-derived cell mass, it is directly subjected to the enzyme treatment, but the cancer tissue removed from the living body is converted into a single cell by being subjected to the enzyme treatment as it is. On the other hand, it is preferable to make a fragment before the enzyme treatment. Prior to fragmentation, it can be maintained in animal cell culture media. Such animal cell culture media include, but are not limited to, Dulbecco MEM (such as DMEM F12), Eagle MEM, RPMI, Ham's F12, Alpha MEM, Iskov modified Dulbecco and the like. At this time, suspension culture is preferably performed in a cell non-adhesive incubator.

It is also preferred that the cancer tissue be washed prior to stripping. Such washing includes, but is not limited to, acetate buffer (acetate + sodium acetate), phosphate buffer (phosphate + sodium phosphate), citrate buffer (citric acid + sodium citrate), boric acid A buffer solution such as a buffer solution, a tartrate buffer solution, a Tris buffer solution, or a phosphate buffered saline can be used. In the present invention, it is particularly preferable that the tissue can be washed in HBSS. The appropriate number of washings is 1 to 3 times.

The fragmentation can be performed by dividing the tissue after washing with a knife, scissors, a cutter (manual, automatic) or the like. The size and shape after the fragmentation are not particularly limited and can be performed randomly, but preferably 1 mm to 5 mm square, more preferably 1 mm to 2 mm square.

Next, the fragmented product thus obtained is subjected to an enzyme treatment. Such enzyme treatment can be mainly trypsin treatment or dispase treatment as described above.

Next, the cells in the single cell product thus obtained are aggregated to 3 or more cells. Prior to aggregation, preferably a ROCK inhibitor can be quickly added to a single cell product.

Here, the aggregate containing 3 or more cancer cells obtained by aggregation is the cancer cell aggregate of the present invention and has a certain range of sizes. The size within a certain range includes small particles having a volume average particle diameter of about 8 μm to 10 μm, but in the case of a nearly spherical shape, the diameter is 20 μm to 500 μm, preferably 30 μm to 400 μm, more preferably 40 μm to 250 μm, In the case of an elliptical shape, the major axis is 20 μm or more and 500 μm or less, preferably 30 μm or more and 400 μm or less, more preferably 40 μm or more and 250 μm or less, and in the case of an irregular shape, the volume average particle diameter is 20 μm or more and 500 μm or less, preferably 30 μm or more and 400 μm or less. More preferably, it is 40 μm or more and 250 μm or less. The volume average particle diameter can be measured by evaluating the particle size distribution and particle shape using a phase contrast microscope (IX70; manufactured by Olympus Corporation) with a CCD camera.

Any of the thus obtained aggregates or cultures thereof is the cancer cell aggregate of the present invention. The culture may be one in which the separated and collected components are present in the medium for a short time, for example, at least 3 hours or more, preferably 10 hours or more and 36 hours, more preferably 24 hours. By culturing for a period of ˜36 hours or longer, it may have a substantially spherical shape or a substantially elliptical spherical shape. The culture time may exceed 36 hours, and several days, 10 days or more, 13 days or more, or 30 days or more may have elapsed.

Cultivation can be performed as it is for a long time in the medium, but preferably, the ability to proliferate can be maintained substantially infinitely by performing mechanical division periodically during the culture.

The thus obtained cancer cell aggregate of the present invention exhibits the same behavior as in vivo cancer tissue in vitro, can be stably cultured, and retains the proliferation ability. Therefore, it is useful, for example, for identifying the types of existing drugs to which the obtained tumor derived from cancer tissue is sensitive, or for confirming the sensitivity to radiation individually for each patient. For the drug or radiosensitivity, any known method can be used and is not limited. Drug sensitivity can be performed by measuring the proliferation rate of cancer cell aggregates in vitro. Such measurement includes, for example, visually observing the number of viable cells after addition of the test drug, several hours or several days later together with a control example, image analysis after photographing with a CCD camera, or included in each cell. Colorimetric measurement of the amount of protein by staining with a protein-binding dye (for example, sulforhodamine B) is included.

Such cancer cell aggregates are also useful for screening unknown drugs. Such unknown drug sensitivities can also be performed by measuring the proliferation rate of cancer cell aggregates in vitro or by determining cell viability. For the measurement of the growth rate, for example, the number of viable cells after several hours or days after the addition of the test drug is visually observed together with a control example, image analysis is performed after taking a CCD camera, or included in each cell. Colorimetric measurement as protein amount by staining with protein-binding dye sulforhodamine B, measurement of SD (Succinyl dehidrogenase) activity, and the like are included.

Test compound susceptibility measurement data for all human cultured cells, ie, the concentration that inhibits cell growth by 50% (GI ₅₀ ), the concentration that apparently inhibits cell growth (TGI), and the number of cells is reduced to 50% at the time of seeding. It is possible to perform information processing by calculating the concentration (LC ₅₀ ) and the like. The GI ₅₀ , TGI, and LC ₅₀ values are values specific to the cancer cell aggregate to be tested. The overall average GI ₅₀ , TGI, and LC ₅₀ values are obtained, the difference between this average value and the Log GI ₅₀ value in each individual cell is obtained, and they are converted into absolute values based on the average Log GI ₅₀ value and made positive or negative. Is written. The larger the positive value, the more sensitive the drug can be judged.

As a radiation sensitivity test using the cancer cell aggregate of the present invention, X-rays, γ-rays using a radioactive isotope of cobalt as a radiation source, electron beams are extracted by a particle accelerator accelerated by a linear accelerator, a cyclotron, etc. Known tests using heavy particle beams such as alpha rays alone or in combination with a radiosensitizer.

Furthermore, the cancer cell aggregate of the present invention has a high degree of colonization in transplantation into a heterologous animal even when, for example, the cancer cell aggregate is 10 micrometers or less (corresponding to 1000 cells or less) having a diameter of 100 micrometers. Therefore, the cancer cell aggregate of the present invention is useful for easy preparation of cancer model animals including mice, and treatment modes including more strict cancer tissue verification, drug sensitivity evaluation, or radiotherapy. Can be evaluated.

The cancer cell aggregate of the present invention can be stored frozen and can retain its growth ability under normal storage conditions.

  The cancer cell aggregate of the present invention can be cryopreserved in a culturable state in vitro and can be used for a wide range of applications. And it can be made to proliferate by culture | cultivation and the cancer cell proliferation from a trace amount specimen is enabled. Furthermore, the cancer cell aggregate of the present invention can be widely used for drug sensitivity tests or radiation sensitivity tests, and can be used for the production of simple tumor-forming animals. For this reason, the cancer cell aggregate of the present invention can bring about a dramatic improvement in anticancer agents and radiation therapy that are currently generally used in trial and error or cocktail therapy. In other words, before performing such therapy, cancer cell aggregates obtained from each patient can predict the effects of drugs and radiation therapy in advance, and only effective drugs can be administered to patients Become. Furthermore, since the cancer cell aggregate of the present invention may be of a size that can be collected with an injection needle, it can also be obtained from a patient before surgery, and can be obtained from an anticancer agent or radiation therapy with less burden on the patient. It is also possible to predict the effect.

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. In addition, all the parts and% in each example are based on weight. The culture conditions not specifically defined below are all under 37 ° C. and 5% CO ₂ incubator conditions. Centrifugation conditions are 4 ° C., 1000 rpm, and 5 minutes unless otherwise specified.

Example 1
(Preparation of cancer tissue-derived cell mass from mouse colon cancer transplanted tumor)
A mouse colon cancer transplanted tumor was prepared by the xenotransplantation method as follows.

First, a surgically removed specimen of a human tumor (colon cancer) is cut into approximately 2 mm cubes under aseptic operation. Next, a small incision of about 5 mm is made on the back of severely immunodeficient mice (nude mice, preferably NOD / SCID mice), and the subcutaneous tissue is exfoliated. After the prepared tumor piece is inserted subcutaneously, it is closed with a skin suture clip. Some transplanted tumors are observed as subcutaneous tumors after about 14 days to 3 months.

The obtained colon cancer mouse was bred under SPF (specific pathogen free) breeding conditions. When the tumor became 1 cm in size, the tumor was removed and 20 ml of DMEM (Gibco; 11965-092) + 1% Pen Strep ( Gibco; 15140-022) (both at a final concentration of 100 units / ml penicillin, 100 μg / ml) were collected in a 50 ml centrifuge tube (IWAKI; 2345-050).

Next, 20 ml HBSS (Gibco; 14025-092) was added, and the tumor was washed by inversion mixing. Next, 20 ml of new HBSS was added, and this operation was repeated twice, and the tumor tissue was transferred to a 10 cm tissue culture dish (tissue culture dish) (IWAKI; 3020-100). On this culture dish, the necrotic tissue was removed using a surgical knife.

The tumor pieces from which the necrotic tissue had been removed were transferred to a new 10 cm dish containing 30 ml of HBSS. Next, the tumor piece was cut into about 2 mm square using a surgical knife.

Tumor debris together with HBSS was transferred to a new 50 ml centrifuge tube, centrifuged, and the supernatant was discarded and washed with 20 ml HBSS by inversion mixing.

Centrifugation and washing were repeated. Thereafter, 20 ml of DMEM + 1% Pen Strep + 0.28 U / ml (final concentration) Blendzyme 1 (Roche; 11988417001) was added and mixed. This was transferred to a 100 ml Erlenmeyer flask and treated with Liberase Blendzyme 1 (Roche Diagnostics) for 2 hours while rotating the stirrer at low speed in a 37 ° C constant temperature bath.

  Next, the enzyme-treated product was collected in a 50 ml centrifuge tube, centrifuged, the supernatant was discarded, and 20 ml HBSS was added and mixed. The components that passed through the stainless steel mesh (500 μm) and passed through the filter were collected in a 50 ml centrifuge tube, and further subjected to centrifugation. Discard the supernatant, add 1 mg / ml DNaseI solution (Roche; 1284932) (100 mg of 10 mg / ml stock + 900 μl of PBS), mix, leave at 4 ° C for 5 minutes, add 20 ml HBSS, mix, and centrifuge Separation was performed and the supernatant was discarded. After mixing with 20 ml HBSS, it was sieved in steps of 500-250-100 μm and then passed through a 40 μm cell strainer (BD; 352340). A cell strainer was immersed in a 10 cm tissue culture dish (tissue culture dish) containing 30 ml of HBSS and gently shaken to remove single cells, small cell clusters of 40 μm or less, and debris. The cell strainer was transferred to another 10 cm tissue culture dish (tissue culture dish) containing 30 ml of HBSS, and the cell mass captured by the cell strainer was collected by pipetting.

  Further, the same centrifugation operation as described above was performed several times, and the resulting component was mixed with 4 ml StemPro hESC SFM (Gibco; A10007-01) + 8 ng / ml bFGF (Invitrogen; 13256-029) + 0.1 mM 2-mercapto Ethanol (Wako; 137-06862) + 1% PenStrep + 25 μg / ml Amphotericin B (Wako; 541-01961) was added and mixed, and transferred to a 6 cm non-treated dish (EIKEN CHEMICAL; AG2000).

This was cultured at 37 ° C. for 36 hours in a 5% CO ₂ incubator (Sanyo MCO-17AIC).

As a result, as shown in FIG. 1, it changes from an irregular shape to a well-formed spherical shape with the passage of time, is substantially spherical after at least 3 to 6 hours, and is perfectly spherical after 24 hours. A derived cell mass was obtained.

(Example 2)
(Preparation of cancer tissue-derived cell mass from human colorectal cancer surgical specimen)
A cancer tissue-derived cell mass was obtained in the same manner as in Example 1 except that a colorectal cancer surgical specimen was used. As a result, as shown in FIG. 2, a substantially spherical cancer tissue-derived cell cluster similar to that in FIG. 1 was obtained after at least 12 hours.

(Example 3)
(Preparation of cancer tissue-derived cell mass from human ovarian cancer surgical specimen)
A cancer tissue-derived cell mass was obtained in the same manner as in Example 2 except that the ovarian cancer surgical specimen was used. As a result, as shown in FIG. 2, a substantially spherical cancer tissue-derived cell cluster similar to that in FIG. 1 was obtained after at least 12 hours.

Example 4
(Preparation of cancer tissue-derived cell mass from human pancreatic cancer surgical specimen)
A cancer tissue-derived cell mass was obtained in the same manner as in Example 2 except that a pancreatic cancer surgical specimen was used. As a result, as shown in FIG. 2, a substantially spherical cancer tissue-derived cell cluster similar to that in FIG. 1 was obtained after at least 12 hours.

(Example 5)
(Preparation of cancer tissue-derived cell mass from human small cell carcinoma surgical specimen)
A cancer tissue-derived cell mass was obtained in the same manner as in Example 2 except that a small cell cancer surgical specimen which is a type of lung cancer was used. As a result, as shown in FIG. 2, a substantially spherical cancer tissue-derived cell cluster similar to that in FIG. 1 was obtained after at least 12 hours.

(Example 6)
(Preparation of cancer tissue-derived cell mass from human renal cancer surgical specimen)
A cancer tissue-derived cell mass was obtained in the same manner as in Example 2 except that a renal cancer surgical specimen was used. As a result, as shown in FIG. 2, a substantially spherical cancer tissue-derived cell cluster similar to that in FIG. 1 was obtained after at least 12 hours.

(Example 7)
(Preparation of cancer tissue-derived cell mass from human bladder cancer surgical specimen)
A cancer tissue-derived cell mass was obtained in the same manner as in Example 2 except that a bladder cancer surgical specimen was used. As a result, as shown in FIG. 2, a substantially spherical cancer tissue-derived cell cluster similar to that in FIG. 1 was obtained after at least 12 hours.

(Example 8)
(Preparation of cancer tissue-derived cell mass from human breast cancer surgical specimen)
A cancer tissue-derived cell mass was obtained in the same manner as in Example 2 except that a breast cancer surgical specimen was used. As a result, as shown in FIG. 2, a substantially spherical cancer tissue-derived cell cluster similar to that in FIG. 1 was obtained after at least 12 hours.

Example 9
(Preparation of cancer tissue-derived cell mass from human prostate cancer surgical specimen)
A tissue-derived cell mass was obtained in the same manner as in Example 2 except that a prostate cancer surgical specimen was used. To the culture medium, dihydrotestosterone (DHT) at a concentration of 10 ⁻⁸ mol / L was added and cultured in the same manner as in Example 1. As a result, as shown in FIG. 2, a substantially spherical cancer tissue-derived cell cluster similar to that in FIG. 1 was obtained after at least 12 hours.

(Example 10)
(Preparation of cancer tissue-derived cell mass from human pharyngeal cancer surgical specimen)
A cancer tissue-derived cell mass was obtained in the same manner as in Example 2 except that the pharyngeal cancer surgical specimen was used. As a result, as shown in FIG. 2, a substantially spherical cancer tissue-derived cell cluster similar to that in FIG. 1 was obtained after at least 12 hours.

(Example 11)
(Preparation of cancer tissue-derived cell mass from mouse islet tumor)
RipTag is a transgenic mouse in which SV40-T antigen is forcibly expressed under the control of the rat insulin promoter. Tumors develop in islets. A cancer tissue-derived cell mass was obtained in the same manner as in Example 2 except that the islet tumor of the RipTag mouse was used. As a result, a substantially spherical cancer tissue-derived cell mass similar to that shown in FIG. 1 was obtained after at least 12 hours (FIG. 3).

(Example 12)
(Preparation of cancer cell aggregates from cancer tissue-derived cell clusters)
Using the cancer tissue-derived cell mass obtained by the same method as in Example 2, the following treatment was performed. First, a collagen gel (Cell Matrix type IA: 5x DMEM: buffer for gel reconstitution = 7: 2: 1) 50 · L / well was spread on the center of a 24-well plate (untreated dish). The collagen gel was solidified by standing at 37 ° C. for 30 minutes. Cell masses derived from cancer tissue in suspension culture (100 per well) are collected in a 1.5 mL tube. This was centrifuged for about 5 seconds, and the supernatant was removed. The cancer tissue-derived cell mass was suspended in a collagen gel (30 μL per well), and 30 μL was placed on the pre-solidified gel. The mixture was allowed to stand at 37 ° C. for 30 minutes to solidify, and StemPro (EGF 50 ng / mL) 600 μL / well was added. The culture was performed for 10 days while changing the medium once every 2-3 days.
Next, the medium was replaced with 1 mL / well of DMEM (Gibco; 11965-092, containing collagenase IV 200 mg / mL) and cultured at 37 ° C. for about 5 hours.
After incubation, transfer to a 1.5 mL Eppendorf tube, centrifuge (approximately 5 seconds), remove the supernatant, add 1 mL of PBS to suspend, centrifuge (Chibitan, approximately 5 seconds), and remove the supernatant. Repeated twice. 1 mL of Trypsin / EDTA (0.05%) was added and suspended, and the mixture was allowed to stand at 37 ° C. for 8 minutes. After suspending several times, it was confirmed that there was no large mass like a cell mass derived from cancer tissue. This was transferred to a 15 mL tube and suspended by adding 2 mL of DMEM (Gibco; 11965-092).
Next, the suspension was centrifuged (1000 rpm, 5 minutes), and the supernatant was removed. It was suspended in 2 mL StemPro (EGF 50 ng / mL, Y-27632 10 μM) and transferred to a φ35 mm non-treated dish (Iwaki: 1000-035). This was cultured overnight at 37 ° C.
After 12 hours, formation of a cell mass derived from a cancer tissue having a diameter of about 40 μm was confirmed. The medium was replaced with StemPro (EGF 50 ng / mL).

As a result, as shown in FIG. 4, after 4 days, a perfectly arranged spherical cancer cell aggregate was obtained.

(Example 13)
(Preparation of cancer cell aggregates from human colon cancer surgical specimens)
A cancer cell aggregate was obtained in the same manner as in Example 12 except that a colorectal cancer surgical specimen was used. As a result, as shown in FIG. 5, a substantially spherical cancer cell aggregate similar to FIG. 1 was obtained after at least 12 hours.

(Example 14)
Cell preservation of the cancer tissue-derived cell mass obtained by the same method as in Example 2 was performed. The cancer tissue-derived cell mass was treated with trypsin in the same manner as in Example 12 to obtain a single cell. The cryopreservation solution used was Cell Banker 1 (Juji Field) with Y-27632 added.

What was made into a single cell and cryopreserved for 10 days was then reconstituted for a short time in a 37 ° C. water bath. This was suspended in PBS, further centrifuged at 1000 rpm at 4 ° C., and the supernatant was discarded. The obtained precipitate was suspended in StemPro (manufactured by Invitro) and cultured. As shown in FIG. 6, the state of the cells at 24 hours after thawing was good, and the cancer tissue-derived cell mass was reformed after thawing.

Next, the recovery rate of cryopreserved cancer tissue-derived cell mass was examined. 1,000 cancer tissue-derived cell masses were frozen and thawed 7 days later. The recovery rates were compared when the cancer tissue-derived cell mass was made into a single cell and cryopreserved, and when the cancer tissue-derived cell mass was cryopreserved as it was. Suspension culture was performed, and after 2 days, spherical cell clusters having a diameter of 40 μm or more were counted as cancer tissue-derived cell clusters. As a result, the recovery rate of the cancer tissue-derived cell mass was significantly better when the cancer tissue-derived cell mass was made into a single cell and cryopreserved than when the cancer tissue-derived cell mass was cryopreserved (FIG. 7). In the case of single cells, about 9,000 cancer tissue-derived cell masses could be recovered from 1,000 cancer tissue-derived cell masses.

The proliferation ability of cryopreserved cancer tissue-derived cell mass was examined. The cancer tissue-derived cell mass was three-dimensionally cultured, and the proliferation ability of the cancer tissue-derived cell mass was observed according to its size. The cancer tissue-derived cell mass was made into single cells and stored frozen. This was thawed to re-form a cancer tissue-derived cell mass, and then cultured in collagen gel (Nitta Gelatin: CellMatrix type I-A) for 6 days. A control was obtained by similarly culturing a cell mass derived from cancer tissue before cryopreservation. The cell mass derived from cancer tissue after cryopreservation is almost the same as the cell mass derived from cancer tissue before cryopreservation, the diameter is about 3 times, and it retains the same proliferation ability after cryopreservation as before cryopreservation. (Fig. 8).

Evaluation items in Examples and the like were measured as follows.

<Identification of surface antigen>

The cancer cell aggregate obtained in Example 1 was dispersed into single cells using trypsin / EDTA. These cells were reacted with a surface antigen-specific antibody labeled with fluorescence, and then analyzed by flow cytometry.

<Confirmation of basement membrane-like material>
The cancer cell aggregate obtained in Example 12 was cultured for 3 days in 1 cc of a serum-free medium (Gibco) for STEMPRO human ES cells under the conditions of 37 ° C. and 5% CO ₂ incubator. This was fixed in formalin, embedded in paraffin, sliced and stained with anti-laminin antibody (Sigma-Aldrich, mouse laminin-derived rabbit antibody) according to the manufacturer's instructions. Laminin antigenicity was observed in the cytoplasm of cells near the periphery. Thus, it was found that the cancer cell aggregate of the present invention was surrounded by laminin around the cancer cell aggregate. On the other hand, the expression of laminin could not be confirmed 24 hours after the surgical specimen treatment.

<Detection of hypoxia>
Example of detection of hypoxia using pimonidazole The nitroimidazole compound, pimonidazole, has the property of forming adducts with proteins and nucleic acids in the absence of oxygen. The hypoxic region of the tissue treated with pimonidazole under hypoxia can be recognized using an antibody that specifically recognizes pimonidazole. In a cancer tissue, a hypoxic region appears when it is separated from a blood vessel by about 100 micrometers, but the cancer cell aggregate obtained in Example 12 is a hypoxic region with an internal part of about 100 micrometers from the outer edge as a boundary. Cell death was observed.

<Evaluation of in vitro proliferation ability>
The proliferation ability of cancer cell aggregates in vitro was verified as follows. The cancer cell aggregate obtained in Example 12 was treated with collagen gel (CellMatrix type IA (Nitta Gelatin): 5x DMEM (Gibco; 12100-038): buffer for gel reconstitution (50 mM NaOH, 260 mM NaHCO3, 200 mM HEPES) = 7. : 2: 1) was embedded in 10 × pieces, and cultured in 1 cc of a serum-free medium (Gibco) for STEMPRO human ES cells under the conditions of 37 ° C. and 5% CO ₂ incubator. The state of the cells was regularly observed, and the size was measured with a phase contrast microscope (40 times magnification) equipped with a CCD camera. As a result, the growth ability could be maintained for at least 13 days without mechanical resolution. Furthermore, when mechanical division was performed on the 13th day, it was confirmed that the proliferation ability was maintained for at least 13 days. The mechanical division was performed by dividing a cancer cell aggregate having a diameter of 500 micrometers into four with an ophthalmic pointed knife.

<Confirmation of cell number>
In the same manner as in Example 12, 100 to 250 μm cancer cell aggregates were treated with 0.25% trypsin and 2.6 mM EDTA for 3 minutes, and mechanically degraded by pipetting approximately 30 times. The solution was diluted and dispensed so that one cell would enter one well of a 96-well culture plate. For cell clusters that were not unicellularized, the number of constituent cells was counted and recorded. Thereafter, culture (conditions same as above) was performed, and the increase in the number of cells in each well was recorded, and the culture was observed for 30 days. As a result, it was confirmed that if there were three cells, the cell mass could grow.

<Drug sensitivity test>
Doxorubicin is known to exert an antitumor effect by inserting between the base pairs of tumor cell DNA, inhibiting the DNA polymerase, RNA polymerase, and topoisomerase II reactions and suppressing the biosynthesis of both DNA and RNA. Was used to conduct a drug sensitivity test using the sample of Example 12. In the test, cancer cell aggregates were treated with collagen gel (CellMatrix type IA (Nitta Gelatin): 5x DMEM (Gibco; 12100-038): Gel reconstitution buffer (50 mM NaOH, 260 mM NaHCO ₃ , 200 mM HEPES) = 7: 2: Each of the cells was embedded in 10) in 1), and cultured in 1 cc of a serum-free medium (Gibco) for STEMPRO human ES cells under the culture conditions of a temperature of 37 ° C. and a 5% CO ₂ incubator. Furthermore, doxorubicin was applied at a concentration of 0.1 μM, 1 μM, and 10 μM, and the state on the eighth day of culture was comparatively evaluated. The result is shown in FIG. About the increase rate regarding the area of a cancer cell aggregate, the increase rate regarding the area in the medicine non-application culture was relatively described as 1. In FIG. 9, cancer cell proliferation on the 8th day of culture was suppressed depending on the concentration of doxorubicin, and it was proved that the cancer cell aggregate of the present invention was useful in the drug sensitivity test.

<Transplantation test to different animals>
Ten cancer cell aggregates having a diameter of about 100 micrometers obtained in Example 12 and cultured for 3 days according to the present invention were suspended in Matrigel (BD) and administered subcutaneously to the back of NOD-SCID mice. Tumor formation was evaluated by measuring tumor size over time. As a result, remarkable tumor formation was observed in the individual mouse transplanted with the cancer cell aggregate of Example 2 of the present invention, and it was confirmed that the cancer cell aggregate of the present invention has a high tumor forming ability. When this tissue was analyzed, it was found that a similar tissue type was obtained between a tumor formed by transplanting into a mouse and a tumor existing in the living body (FIG. 10).

Claims (16)

It is an aggregate formed by cancer cells derived from a cancer tissue or a cancer tissue obtained from an individual, and an aggregate formed by aggregating cells in the single cell product into three or more cells or a culture thereof. And
Can retain proliferative ability in vitro,
Cancer cell aggregate. After the cancer tissue-derived cell mass or the cancer tissue obtained from an individual is made into a single cell, it is formed by aggregation of the single cells to 3 or more cells in the presence of a ROCK (Rho binding kinase) inhibitor. Agglomerates or cultures thereof,
Can retain proliferative ability in vitro,
Cancer cell aggregate. The cancer cell aggregate according to claim 2, wherein the ROCK inhibitor is Y27632 (registered trademark). A cancer cell aggregate containing three or more cancer cell aggregates and having a substantially spherical shape or an elliptical spherical shape. A cancer cell aggregate comprising three or more cancer cell aggregates: and a basement membrane-like substance existing on the outer peripheral surface of the cancer cell aggregate, and having a substantially spherical shape or an elliptical spherical shape. The cancer cell aggregate according to any one of claims 1 to 5, which contains substantially no cells other than cancer cells. The cancer cell aggregate according to claim 5, wherein the basement membrane-like substance is laminin. The cancer cell aggregate according to any one of claims 1 to 7, which has a diameter of 40 µm to 250 µm. The cancer cell aggregate according to any one of claims 1 to 8, wherein the cancer cells are derived from epithelial cancer cells. The cancer cell aggregate according to claim 9, wherein the cancer cells are derived from colon cancer, ovarian cancer, breast cancer, lung cancer, prostate cancer, kidney cancer, bladder cancer, pharyngeal cancer, pancreatic cancer. A cancer cell comprising a step of enzyme-treating a cancer tissue-derived cell mass or a cancer tissue excised from a living body into a single cell; and a step of aggregating cells in the single cell product into three or more cells A method for preparing agglomerates. A step of enzymizing a cancer tissue-derived cell mass or a cancer tissue excised from a living body into a single cell; and cells in the single cell product in the presence of a ROCK inhibitor, the number of cells is 3 or more. The method for preparing a cancer cell aggregate according to claim 11, comprising a step of agglutinating the cells. Furthermore, the preparation method of the cancer cell aggregate of Claim 11 or 12 including the process of culturing the said aggregated component for 3 hours or more. The method for preparing a cancer cell aggregate according to any one of claims 11 to 13, wherein the enzyme treatment is treatment using trypsin. The method for preparing a cancer cell aggregate according to any one of claims 12 to 14, wherein the ROCK inhibitor is Y27632 (registered trademark). A cancer cell aggregate obtained by the preparation method according to any one of claims 11 to 15.