JPH0690750A - Cultured cancer tissue and its production - Google Patents

Abstract

PURPOSE:To produce a cultured cancer tissue capable of sensitively measuring the effect of an anticancer agent in chemotherapy or radiotherapy, thermotherapy, etc., in physiotherapy on the cancer tissue in the living body in vitro by allowing cancer cells and normal cells to coexist and giving a duct structure to the cancer tissue. CONSTITUTION:Normal cells are cultured on a culture medium containing a temperature-sensitive polymer at a temperature of >=LCST so as to cover the surface of the culture medium with the normal cells. Thereby, a normal cell sheet is formed and cancer cells are inoculated and bonded to the normal cell sheet. The temperature is subsequently lowered to <=LCST and the normal cell sheet containing the cancer cells bonded thereto is released from the surface of the culture medium and allowed to float in the culture solution. This floating cell sheet is cultured on a cell-nonadhesive culture medium, thus producing the objective cultured cancer tissue. LCST is preferably 0 to 80 deg.C, further preferably 10 to 45 deg.C.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cultured cancer tissue containing cancer cells and normal cells and having a glandular duct structure that closely resembles the cancer tissue in a living body, and a method for producing the same.

Since the cultured cancer tissue of the present invention is very similar in shape and function to the in vivo cancer tissue, the cultured cancer tissue of the present invention can be used for chemotherapy or radiation treatment using an anticancer agent.
It is possible to measure the effect of physical therapy such as hyperthermia on cancer tissues in vivo with high sensitivity in vitro.

[0003]

2. Description of the Related Art As a method which has been conventionally developed as a method for producing a cancer tissue using a cell culture technique,
1) The most common method is to perform a flat culture of cancer cells on the surface of a culture substrate; 2) A method of embedding the cancer cells in a gel such as collagen or agar and performing a three-dimensional culture (eg, Enaki Jun flat, tissue Culture studies, 4 , 76, 1985); 3) A method of forming a cancer cell aggregate by utilizing the aggregability of cancer cells by spinner culture (Sutherland, R. et al.
M., et al., J. Nat. Cancer Inst., 46 , 113,
1971); and a method of forming cancer cell aggregates by culturing cancer cells on a substrate surface coated with non-cell-adhesive agar and the like, and the cancer cells spontaneously aggregate and desorb from the agar surface (Yuhas, J. M. et al., Cancer Res.,
37 , 3639, 1977); and the like.

On the other hand, in order to apply a cultured cancer tissue obtained by using cancer cells obtained from a living cancer tissue to the measurement of drug sensitivity such as an anti-cancer drug or sensitivity such as radiation or heat,
It is an important requirement that the cultured cancer tissue be qualitatively and morphologically similar to the living cancer tissue. This is because drug susceptibility and the like depend not only on the characteristics peculiar to cancer cells but also on the distribution of normal cells around cancer cells, that is, on the environment such as structures such as interstitial tissue and blood vessels.

When the cultured cancer tissue obtained by the above-mentioned conventional method is viewed from the viewpoint of the similarity to such in vivo cancer tissue, in the case of 1) flat culture, the obtained tissue is an artificial substrate. Since it is lined by, it can only take a planar structure, which is very different from the three-dimensional cancer tissue in vivo. Therefore, a cancer cell that forms a ductal structure in a living body becomes cobblestone-like by the flat culture method, and it is completely impossible to form a higher-order structure similar to that in a living body. In the case of the in-gel culture of 2), unlike the above-mentioned flat culture, it is possible to form a three-dimensional structure similar to that in the living body. However, in this culture method, it is difficult to supply nutrients and remove waste products from the outside of the gel to the cells inside the gel, and cancer cells can be efficiently proliferated like a flat culture to obtain a desired cancer. The major drawback is that it is very difficult to form tissue. The method of spontaneously forming an aggregate of cancer cells by utilizing the aggregability of cancer cells by spinner culture or culture on agar in 3) is to form a solid substance relatively similar to the in vivo cancer tissue. Suitable for However, in these methods, the above-mentioned cancer cell aggregate is formed by accident, and the number of cancer cells to be aggregated, that is, the size of the cancer cell aggregate can be controlled, and further, the aggregate having a desired size. It has the serious drawback that it is very difficult to manufacture in large quantities. Further, in this method, it is possible in principle to produce a mixed aggregate of cancer cells and normal cells, but as described above, the aggregate is formed by accident, and in general cancer cells Since the cohesiveness of the same cells is stronger than that of the normal cells, it is very difficult to control the mixing ratio of the cancer cells and normal cells in the aggregate to a desired value.

[0006] On the other hand, it was also reported that mixed agglomerates composed of HeLa cells as a model of cancer cells and fibroblasts as normal cells were formed by the agar culture method and used for the measurement of radiation sensitivity and drug sensitivity. Takeshi Sasaki, Journal of National Diseases, 55 , 515, 1988. However, it is very difficult to control the mixing ratio of HeLa cells and fibroblasts by this method, and almost no fibroblasts are found in the aggregates obtained by this method. No high-order structure peculiar to the cancer tissue is recognized in the. In particular, controlling the mixing ratio of cancer cells and normal cells to a desired value is an important elemental technique for clearly measuring the difference in drug sensitivity or radiation sensitivity between cancer cells and normal cells.

As outlined above, it is very difficult to produce a cultured cancer tissue having a glandular structure similar to that of an in vivo cancer tissue and formed of cancer cells and normal cells by the conventional culture technique. It was Therefore, the determination of drug sensitivity such as anti-cancer drug or the effect of radiation, hyperthermia, etc. obtained by using the conventional cultured cancer tissue has a serious defect that there is a large gap from that for actual cancer tissue. Was there.

[0008]

The object of the present invention is to detect drug sensitivity of actual cancer tissue (which was difficult when the cultured cancer tissue obtained by the above-mentioned conventional cell culture technique was used). It is an object of the present invention to provide a cultured cancer tissue that is morphologically and functionally very similar to a cancer tissue in a living body and a method for producing the same so that the effects of radiation and the like can be measured.

[0009]

According to the present invention, there is provided a cultured cancer tissue containing cancer cells and normal cells and having a ductal structure. Further, according to the present invention, there is provided a cultured cancer tissue containing cancer cells and normal cells, wherein the cancer cells and normal cells are substantially mixed. Further, according to the present invention, the LCST of the temperature-sensitive polymer compound is provided on a culture substrate containing at least the temperature-sensitive polymer compound.
After culturing the normal cells at a higher temperature until the normal cells cover the surface of the base material to form a normal cell sheet on the surface of the base material; after seeding and adhering cancer cells on the normal cell sheet and adhering By lowering the temperature below LCST,
The normal cell sheet to which the cancer cells adhere is completely desorbed from the surface of the substrate to form a cell sheet that floats in the culture medium; the floating cell sheet is cultured on a cell-nonadhesive substrate A method for producing a cultured cancer tissue is provided.
Here, in the cultured cancer tissue of the present invention, "the cancer cells and the normal cells are substantially mixed" means that, as described later, at the time of culturing for 3 hours after detachment from the culture substrate, the ratio described below. The ratio (A / A ₀ ) of A (the ratio of the number of normal cells / the number of cancer cells after culture) and the ratio A ₀ (the ratio of the number of normal cells / the number of cancer cells at the time of seeding the cancer cells) is A / A ₀ ≧ Say it is 0.05. On the other hand, according to the study by the present inventor, A / A ₀ <0.01 was obtained in the conventional cultured cancer tissue composed of cancer cells and normal cells. In other words,
In a conventional cultured cancer tissue composed of cancer cells and normal cells, the cancer cells and normal cells were not substantially mixed.

The cultured cancer tissue of the present invention will be described in detail below. (Gland duct structure) In the present invention, the presence or absence of the gland duct structure is preferably determined as follows. Immobilize the cultured cancer tissue obtained by culturing the cell sheet from the culture substrate for at least 1 day or more, and collect the obtained tissue section (thickness: about 4 to 6 μm, about the center of the cultured tissue) Is preferred), and when a glandular structure satisfying both of the following conditions is observed when observed under an optical microscope (400 times) with hematoxylin and eosin staining, it is determined in the present invention that "there is a glandular structure". . At least one ductal structure satisfying the following conditions should be present in the 500 μm × 500 μm field of view of the microscope. The average value (R ₀ ) of the major axis and the minor axis of the lumen of the ductal structure is 0.5 μm ≦ R ₀ ≦ 100 μm.

(Cancer cells) In the present invention, cancer cells (or tumor cells) refer to normal cells that have undergone malignant transformation, and more specifically, G-Band karyotype analysis (MEDrets, et al. , Proc. Natl. Acad. Sci., US
A., 68 , 2073 (1971)) refers to animal cells that show chromosomal abnormalities. Cancer cells generally have contact inhib
In the present invention, the presence or absence of contact inhibition can also be used as an indicator of the presence or absence of malignant transformation of cells in the present invention. The presence or absence of contact inhibition can be determined by determining whether or not the cells further grow when they are in a confluent state by monolayer culture (Imahori, Yamakawa, “Biochemistry Dictionary (2nd Edition)”, Tokyo Kagaku Dojin (1990), pages 640 and 808). The cancer cell preferably used in the present invention is a cancer cell that gives a ductal structure in vivo (that is, a cancer cell having a ductal structure-forming property). The cancer cells are preferably cells that give a ductal structure when cultured (in vitro) with human fibroblasts under defined conditions.

(Determination of gland duct structure forming ability in vitro) Collagen and poly-N-isopropylacrylamide (P
A 1: 1 mixture of NIPAAm) gives a dish with a thickness of about 2 μm. On the other hand, human dermal fibroblast (cell concentration: approximately 2 × 10 ⁵ cells / ml) dispersion 2
After incubating ml at 37 ° C, pour into the dish (coated with collagen / PNIPAAm mixture) preincubated at 37 ° C, and incubate at 37 ° C in air /
After culturing for 5 days in a 5% carbon dioxide gas incubator, the bottom surface of the dish is completely covered with fibroblasts. Next, sample cells (cells to be judged, cell concentration: about 2 x 10
⁵ cells / ml) 0.5 ml of the dispersion was kept warm at 37 ° C., and then injected into the fibroblast proliferation dish at 37 ° C.
The cells are left in an air / 5% carbon dioxide incubator for about 2 hours to completely attach the cancer cells on the fibroblast sheet. Then, the dish is taken out of the incubator and left at room temperature to peel off the fibroblast sheet having the cancer cells attached thereto from the bottom surface of the dish and float in the culture medium. If necessary, the sheet is washed with phosphate buffer 2-3 times, and then transferred to a cell non-adhesive dish (agar-coated dish, 35 mmφ),
Incubate at 37 ° C in an air / 5% carbon dioxide incubator for 7 days. The lumps obtained by culturing are fixed with 10% formalin buffer and paraffin-embedded tissue sections are prepared. The lump sections are stained with hematoxylin and eosin and used as a microscope sample. Using this sample, the presence or absence of the ductal structure inside the lump is judged by an optical microscope according to the above-mentioned criteria. The above-mentioned in vitro test method is used only for the determination of "cancer cells preferably used" in the present invention, and the conditions such as the above-mentioned culture conditions do not limit the scope of the present invention.

In the present invention, it is preferable to use, as the cancer cell, a cancer cell that forms the above-described gland duct structure, and more specifically, for example, gastric cancer or colon cancer that forms the gland duct structure in vivo. , Pancreatic cancer, bile cancer, bile duct cancer, liver cancer,
Cancer cells such as breast cancer, lung cancer and esophageal cancer are preferably used.

(Normal cell) In the present invention, "normal cell" means an animal cell other than the above-mentioned cancer cell. That is,
An animal cell that has not undergone malignant transformation. In the present invention, as normal cells,
It is particularly preferable to use fibroblasts which are generally present in the living body and are relatively easy to collect. In the present invention, if necessary, two or more types of cells may be used as normal cells.
As described above, by using two or more kinds of normal cells, it may be possible to more appropriately approximate the structure around the cancer in vivo.

(Ratio of number of normal cells / number of cancer cells) In the cultured cancer tissue of the present invention, the composition ratio of the cancer cells and the normal cells in the tissue (cultured tissue) at a predetermined time point described later is A
(Number of normal cells / number of cancer cells), and the composition ratio of the cancer cells to the normal cells at the time of seeding the cancer cells on the normal cells is A ₀
When defined as (the number of sheet-like normal cells / the number of seeded cancer cells), it is preferable that A ≧ 0.05. The composition ratio at the time of seeding the cancer cells and at a predetermined time after the culture is preferably 0.5 ≦ A / A ₀ ≦ 1.5.
As described above, in the conventional cultured cancer tissue obtained from normal cells and cancer cells, the normal cells are not effectively cultured, and only the cancer cells are substantially cultured. According to the study of A.
It is estimated that the value of A ₀ was less than 0.01. That is, it could not be said that normal cells and cancer cells were cultured in a “substantially mixed state” in the conventional culture tissue. On the other hand, in the cultured cancer tissue of the present invention, since normal cells and cancer cells are substantially mixed,
The value of A / A ₀ is 0.05 or more as described above. In other words, the value of A / A ₀ above 0.05 is an index that the normal cells and the cancer cells are “substantially mixed” in the cultured cancer tissue of the present invention.

(Measurement of A ₀ and A) In the present invention,
For the value of A ₀ , for example, the number of normal cells (N) at the time of seeding was measured using a phase contrast microscope, and the number of cancer cells (Nc) was measured using a phase contrast microscope to obtain N / Nc = A ₀ . Just calculate. On the other hand, the value of A can be obtained by, for example, fixing a cultured cancer tissue with formalin or the like to prepare a tissue section, staining it with hematoxylin / eosin, and utilizing a staining property or a morphological change under a microscope. A may be calculated by counting the number of normal cells and the number of cancer cells in the cultured cancer tissue. In addition, a sample (cultured cancer tissue 3 hours after detachment from the culture substrate) was immersed in a phosphate buffer containing 0.25% trypsin and 1 mmol of EDTA for about 30 minutes to form normal cells forming the culture tissue. And the number of cancer cells in the mixed dispersion obtained by separately separating the cells into cancer cells and cancer cells, and measuring the number of normal cells using a morphological difference under a phase contrast microscope. It is also possible to calculate the number / cancer cell number ratio (A). (A and A ₀ when a temperature-sensitive polymer is used) More specifically, for example, when culturing using a temperature-sensitive polymer described later in the present invention, A ₀ is performed as follows. And it is preferable to calculate A. A ₀ : The number of normal cells (N) put on a culture substrate having a certain area is measured under a phase contrast microscope. Next, the number of seeded cancer cells (Nc) was calculated by measuring the number of cells in the concentration of the cancer cell dispersion liquid seeded on the cell sheet, and N /
Calculate Nc = A ₀ . A: Cancer cells were seeded on a normal cell sheet that had been put on a culture substrate, the cancer cells were allowed to adhere to the surface of the sheet, and then the temperature was lowered below the LCST described below, After detaching the cell sheet from the surface of the substrate and completely suspending it in the culture medium, the cell sheet was transferred onto a cell-non-adhesive substrate and cultured for 3 hours after detaching the cell sheet. Obtain cultured cancer tissue. Next, the cultured cancer tissue obtained as described above was treated with 0.25% trypsin,
The cells are immersed in a phosphate buffer containing 1 mmol of EDTA for about 30 minutes to separate the cultured cancer tissue into normal cells and cancer cells. The number of normal cells and the number of cancer cells in the obtained mixed dispersion liquid of both cells are respectively measured under a phase contrast microscope by utilizing the difference in morphology, and A is calculated. As another method of calculating A, as in the above, the cancer tissue that has been cultured for 3 hours after being detached from the culture substrate is fixed with formalin or the like to prepare a tissue section, which is then stained with hematoxylin / eosin and examined under a microscope. It is also possible to use the staining property or morphological change to measure the number of normal cells and the number of cancer cells in the cultured cancer tissue, and calculate A.

(Mixing ratio of normal cells / cancer cells) In the cultured cancer tissue of the present invention, the mixing ratio of the number of normal cells (after culturing) / the number of cancer cells (B) measured in the same manner as the above-mentioned ratio A.
Is preferably 0.01 or more, more preferably 0.05 or more (the value of this “mixing ratio B” at a specific time point 3 hours after peeling from the culture substrate corresponds to the above-mentioned ratio A. ).
In the cultured cancer tissue of the present invention, the value of B may change with time. Therefore, the preferable value of B is at the time of actually using the cultured cancer tissue of the present invention (for example,
It suffices that the cultured cancer tissue is filled at the time of subjecting it to a drug resistance test or the like, and it does not necessarily have to be within the range of B at the time of shipping as a product.

(Temperature-Sensitive Polymer) The method for producing the cultured cancer tissue of the present invention is not particularly limited, but a temperature-sensitive polymer is preferably used in the production.
It is preferable for effectively preventing unnecessary damage to cells.
Here, "temperature-sensitive polymer compound" means LCST (Lo
wer Critical Solution Temperature). LCST is a transition temperature of hydration and dehydration of a temperature-sensitive polymer compound (for example, Hanskins,
M., et al., J. Macromol. Sci.-Chem., A2 (8), 1441 (1968)
See). In the present invention, the LCST of the temperature-sensitive polymer compound is 0 to 80 ° C (further 10 to 45 ° C).
C.) is preferred. This LCST is, for example,
It can be measured as follows.

The temperature-sensitive polymer is dissolved in a predetermined aqueous solution (for example, a culture solution), and the melting point measuring device is used for about 3 minutes per minute.
When the temperature is raised at a rate of ° C, the temperature at which turbidity first occurs is visually determined, and this temperature is referred to as LST (this LCS
For details of T, see T. Takezawa et al., Bio / Technolo.
gy, 8 , 854 (1990)). The temperature-sensitive polymer compound preferably used in the present invention is LCST
It is in the solid state at higher temperatures and becomes reversibly water soluble by lowering the temperature below the LCST. LC
Since the temperature-sensitive polymer compound is in a solid state at a temperature higher than ST, cells can grow while using the polymer compound as a scaffold. On the other hand, when detachment of the proliferated cells is required, if the temperature is set lower than LCST, the temperature-sensitive polymer compound becomes water-soluble, so that the binding between cells is not impaired, and The proliferating cells can be detached.

The temperature-sensitive polymer compound that can be preferably used in the present invention includes poly N-substituted acrylamide derivatives, poly N-substituted methacrylamide derivatives and their copolymers, polyvinyl methyl ether,
Polyethylene oxide, etherified methyl cellulose
And polyvinyl alcohol partial oxides. Particularly preferred are poly N-substituted acrylamide derivatives or poly N-substituted methacrylamide derivatives or copolymers thereof, polyvinyl methyl ether, polyvinyl alcohol partial oxides. Preferred polymer compounds are listed below in order of increasing LCST. Poly-N
-Acroylpiperidine; poly-N-n-propylmethacrylamide; poly-N-isopropylacrylamide; poly-N, N-diethylacrylamide; poly-N
-Isopropylmethacrylamide; poly-N-cyclopropylacrylamide; poly-N-acryloylpyrrolidine; poly-N, N-ethylmethylacrylamide;
Poly-N-cyclopropylmethacrylamide; poly-
N-ethyl acrylamide

The above polymers may be used alone or may be copolymerized with other monomers. As a monomer to be copolymerized, either a hydrophilic monomer or a hydrophobic monomer can be used. Generally, copolymerization with a hydrophilic monomer increases LCST, and copolymerization with a hydrophobic monomer decreases LCST.
Therefore, by selecting these, the desired LCS
A polymer compound having T can be obtained. As the hydrophilic monomer, N-vinylpyrrolidone, vinylpyridine, acrylamide, methacrylamide, N-methylacrylamide, hydroxyethyl methacrylate,
Hydroxyethyl acrylate, hydroxymethyl methacrylate, hydroxymethyl acrylate, acrylic acid having an acidic group, methacrylic acid and salts thereof, vinyl sulfonic acid, still sulfonic acid, and N having a basic group. , N-dimethylaminoethyl methacrylate, N, N-diethylaminoethyl methacrylate
And N, N-dimethylaminopropyl acrylamide and salts thereof. Since the cell membrane is usually negatively charged, it is preferable to use a copolymer with a basic monomer in order to improve the adhesion of cells to the substrate due to electrostatic interaction. On the other hand, examples of the hydrophobic monomer include acrylate derivatives and methacrylate derivatives such as ethyl acrylate, methyl methacrylate, and glycyl methacrylate, and N-substitution such as Nn-butyl methacrylamide. Examples thereof include alkylmethacrylamide derivatives, vinyl chloride, acrylonitrile, styrene, vinyl acetate and the like. The details of the above temperature-sensitive compound are described in Japanese Patent Application No. 2-49155.
Reference can be made to the Japanese patent application.

(Production Method of Cultured Cancer Tissue) Suitable Production Method of Cultured Cancer Tissue of the Present Invention (that is, Production Method of the Present Invention)
Will be described in detail below. In a preferred embodiment of the production method of the present invention, first, on a culture substrate containing the above-mentioned temperature-sensitive polymer compound (more preferably, a culture substrate containing a temperature-sensitive polymer compound and collagen), LCST (Lower Critical Solution Temperatur) of temperature sensitive polymer
e) higher temperature (preferably 1-40 ° C than LCST)
Normal cells are cultivated at high temperature, more preferably at 3 to 20 ° C. higher) to form a monolayer sheet of normal cells on the surface of the substrate. In the culture substrate used in the present invention,
For 1 part by weight of the temperature-sensitive polymer compound,
It is preferable to use 0.1 to 3 parts of gen (more preferably 0.5 to 2 parts). Next, cancer cells are seeded and adhered on the cell sheet. After the cancer cells are completely attached to the surface of the normal cell sheet, the temperature of the culture system is lower than LCST (preferably 1 to 40 ° C lower than LCST, more preferably 3 to 20 ° C lower). As a result, the temperature-sensitive polymer compound coated on the surface of the culture substrate is dissolved in the culture medium, and the normal cell sheet with the cancer cells attached thereto is peeled off from the surface of the substrate. The cultured cancer tissue of the present invention can be preferably produced by transferring the normal cell sheet to which the cancer cells are attached onto a cell-non-adhesive substrate (such as agar) and subjecting it to suspension culture.

One of the important features of the present invention is, as already mentioned, that the cells are released and released from a flat culture system on a substrate which is extremely unsuitable for morphogenesis similar to in vivo. , Induces the expression of the original function of the cell,
It is to enable the construction of a form very similar to that in the living body. Furthermore, in the embodiment using the temperature-sensitive polymer of the present invention, it is not necessary to use a conventional cell detachment agent such as a protease or a calcium chelating agent for releasing and releasing the cells from the substrate. Therefore, the cells do not fall apart one by one, and at the same time, damage to various receptors existing on the cell surface is significantly suppressed. As a result, it is an important feature of the present invention that functional differentiation of cells is further induced. Further, the mixing ratio of the cancer cells and the normal cells in the cultured cancer tissue of the present invention is, for example, by adjusting the number of both cells when the cancer cells are seeded on the normal cell sheet in the above-mentioned manufacturing process. The fact that it can be easily controlled is also an important feature of the present invention.

On the other hand, instead of using the culture substrate containing the temperature-sensitive polymer compound described above, the normal cell sheet with cancer cells attached thereto is mechanically peeled off from the substrate surface by a cell scraper or the like. Although it is possible to produce a cultured cancer tissue of, in terms of cytotoxicity,
The former method using a temperature-sensitive polymer compound is more preferable. The cultured cancer tissue of the present invention is usually composed of so-called multicellular spheroids and usually has a relatively large cell mass, so that it is not suitable for cryopreservation.
Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is determined by the description of the claims and is not limited by the following Examples.

[0025]

[Examples] Cancerous pleural effusions of a cholangiocarcinoma patient were aseptically collected, the red blood cell fraction and the lymphocyte layer were removed by a specific gravity centrifugation method, and the cellular components were adjusted to 37 using RPMI1640 as a medium.
Culturing was performed in a 5% carbon dioxide gas incubator at 0 ° C. Although floating cells were found to be mixed in the early stage of culture, cholangiocarcinoma cells could be isolated and proliferated by repeated passages. On the other hand, as normal cells, human dermal fibroblasts described below were used. A typical temperature-sensitive polymer, poly-N-isopropylacrylamide (PNIPA
Am) was dissolved in distilled water to prepare a 0.5 W / V% aqueous solution, which was sterilized by filtration with a sterilizing filter. The polymer aqueous solution and 0.5 W / V% aqueous solution of bovine dermal pepsin-solubilized type I collagen were mixed in equal amounts to prepare PNIPAAm.
A mixed solution of collagen and collagen was prepared. L of this mixed solution
As a result of measuring CST by changing turbidity, it was about 32 ° C. Next, 400 μl of the above mixed solution was added to a commercially available plastic dish for tissue culture (Falcon, 35 mm
φ) was injected into the bottom surface and aseptically air-dried at room temperature in a clean bench. By the method described above, a dish was obtained in which the bottom surface of the dish was coated with a 1: 1 mixture of collagen and PNIPAAm in a thickness of about 2 μm. Human dermal fibroblasts (cell concentration: about 2 × 10 ⁵ cells / ml) as a medium in DMEM medium (containing 10% fetal calf serum)
2 ml of the liquid dispersed in the above was kept warm at 37 ° C, and then the above (collagen / PNIPAAm
Pour into the dish (mixed coated),
The cells were cultured at 37 ° C in an air / 5% carbon dioxide gas incubator for 5 days. By this culture, the bottom surface of the dish was completely covered with fibroblasts.

Next, the above cholangiocarcinoma cells (cell concentration: about 2)
X 10 ⁵ cells / ml) in a DMEM medium similar to the above, 0.5 ml of the solution was kept warm at 37 ° C and then injected into the fibroblast proliferation dish obtained as described above, and air / 5% at 37 ° C. Leave it in the carbon dioxide incubator for about 2 hours,
The cancer cells were completely attached on the fibroblast sheet.
Then remove the dish from the incubator,
The fibroblast sheet attached with the cancer cells was peeled from the bottom of the dish by leaving it at room temperature. When the detachment process of the cell sheet was observed with a phase-contrast microscope, it was confirmed that the cell sheet was gradually detached from the dish end toward the central part, and the detached sheet wrapped around the upper surface while wrapping the cancer cells attached thereto. Further, in the process of peeling the sheet, no desorption of cancer cells from the fibroblast sheet was observed.

Approximately 10 minutes after being taken out from the incubator at 37 ° C., the cell sheet was completely detached from the bottom surface of the dish and floated in the culture medium (DMEM medium similar to the above). PNIPAAm lysed from the suspended cell sheet
And, in order to remove collagen, the sheet was washed with phosphate buffer 2-3 times, and then transferred onto a cell non-adhesive dish (agar-coated dish 35 mmφ), and air at 37 ° C./5% carbon dioxide gas. Cultured in an incubator. The cell sheet changed from a sheet-like shape to a lump-like shape in the process of suspension culture in a cell non-adhesive dish. On the 7th day of culture, the lumps were fixed with 10% formalin buffer to prepare paraffin-embedded tissue sections. When hematoxylin-eosin staining was performed on the agglomerate section, a clear ductal structure was observed inside the agglomerate (FIG. 1, corresponding color photograph is shown in “Reference Photo 1”). In FIG. 1, as shown in Reference Photo 1 above, the formation of a clear ductal structure is recognized.

On the other hand, the above-mentioned block of the lump was treated with anti-Carbohydrate A.
ntigen19-9 (CA 19-9, manufactured by France CIS) and anti-Carcino embryonic Antigen (CEA, manufactured by American DAKO) were each stained by an immunostaining method using a primary antibody to detect cancer cells inside the lump. Only the cytoplasm was clearly stained (Figs. 2 and 3, and corresponding color photographs are shown in "Reference Photo 2" and "Reference Photo 3", respectively). In these FIGS. 2 and 3, surprisingly, the ductal structure was mainly formed by cancer cells positive for immunostaining. FIG. 4 (a) shows a hematoxylin-eosin stained image of the primary tumor tissue of the cancer patient (corresponding color photograph is shown in “Reference photograph 4”).
The ductal structure as shown in (b) was very similar to that formed in the cultured cancer tissue of the present invention in FIG. Further, by analogy with the fact that high concentrations of CA19-9 and CEA were detected in the serum of the cancer patient, the present cholangiocarcinoma tissue showed CA.
Considered to be positive for 19-9 and CEA,
Also in this respect, it was revealed that the cultured cancer tissue of the present invention shown in FIGS. 2 and 3 closely resembles the primary tumor tissue.

[0029]

EFFECTS OF THE INVENTION Since the cultured cancer tissue of the present invention is composed of cancer cells and normal cells and has a ductal structure, it has a morphology and function very similar to that of the cancer-originating tissue from which the cancer cells are derived. There is. Therefore, drug sensitivity of anticancer drugs or radiation,
It can be widely applied to a method for highly sensitively measuring therapeutic effects such as heat, and its technical value is extremely high.

[Brief description of drawings]

FIG. 1 is a hematoxylin / eosin stained image (magnification: 650 times) of an example of the cultured cancer tissue of the present invention consisting of cholangiocarcinoma cells and normal fibroblasts, which is a color photograph corresponding to FIG. As shown in ", the formation of a clear ductal structure is observed.

FIG. 2 is an immunostaining image (magnification: 650 times) with an anti-CA19-9 antibody of an example of the cultured cancer tissue of the present invention consisting of cholangiocarcinoma cells and normal fibroblasts, in which only the cancer cells are stained, As shown in the “reference photograph 2” which is a color photograph corresponding to FIG. 2, it can be seen that the ductal structure is mainly formed by cancer cells.

FIG. 3 is an immunostaining image (magnification: 400 times) with an anti-CEA antibody of an example of the cultured cancer tissue of the present invention consisting of cholangiocarcinoma cells and normal fibroblasts, in which only the cancer cells are stained,
As shown in “reference photograph 3” which is a color photograph corresponding to FIG. 3, it can be seen that the ductal structure is mainly formed by cancer cells.

FIG. 4 is a hematoxylin-eosin stained image of the primary tumor tissue of the cholangiocarcinoma (FIG. 4 (a), magnification: 400 times).
As shown in Fig. 4 (b) schematically showing (a) and "Reference Photo 4" corresponding to Fig. 4 (a) above, a ductal structure is recognized.

[Explanation of symbols]

 1 wall 2 lumen

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[Procedure amendment]

[Submission date] July 9, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Brief description of the drawing

[Correction method] Change

[Correction content]

[Brief description of drawings]

FIG. 1 is a hematoxylin / eosin-stained image (magnification: 650 times) of an example of the cultured cancer tissue of the present invention composed of cholangiocarcinoma cells and normal fibroblasts, showing formation of a ductal structure.

FIG. 2 is an immunostaining image (magnification: 650 times) with an anti-CA19-9 antibody of an example of the cultured cancer tissue of the present invention consisting of cholangiocarcinoma cells and normal fibroblasts, in which only the cancer cells are stained, It can be seen that the ductal structure is mainly formed by cancer cells.

FIG. 3 is an immunostaining image (magnification: 400 times) with an anti-CEA antibody of an example of the cultured cancer tissue of the present invention consisting of cholangiocarcinoma cells and normal fibroblasts, in which only the cancer cells are stained,
It can be seen that the duct structure is mainly formed by cancer cells.

FIG. 4 is a hematoxylin-eosin stained image of the primary tumor tissue of the cholangiocarcinoma (FIG. 4 (a), magnification: 400 times), which is shown in FIG. 4 (b) schematically showing FIG. 4 (a). As such, a ductal structure is recognized.

[Explanation of symbols] 1 wall 2 lumen

Claims (6)

[Claims] 1. A cultured cancer tissue comprising cancer cells and normal cells and having a ductal structure. 2. A cultured cancer tissue, comprising cancer cells and normal cells, wherein the cancer cells and normal cells are substantially mixed. 3. The cancer cell is gastric cancer, colon cancer, pancreatic cancer,
The cultured cancer tissue according to claim 1 or 2, which is a cancer cell having a ductal structure-forming property selected from the group consisting of biliary carcinoma, cholangiocarcinoma, liver cancer, breast cancer, lung cancer and esophageal cancer. 4. The cultured cancer tissue according to claim 1, wherein the normal cells are fibroblasts. 5. On a culture substrate containing at least a temperature-sensitive polymer compound, normal cells are cultured at a temperature higher than the LCST of the temperature-sensitive polymer compound until the normal cells cover the surface of the substrate. To form a normal cell sheet on the surface of the base material; by seeding and adhering cancer cells on the normal cell sheet and then lowering the temperature to a temperature lower than the LCST, the normal cell sheet to which the cancer cells adhere is formed. A method for producing a cultured cancer tissue, which comprises completely desorbing from a substrate surface to form a cell sheet suspended in a culture solution; culturing the suspended cell sheet on a cell non-adhesive substrate. 6. The method for producing a cultured cancer tissue according to claim 5, wherein the culture substrate is a substrate containing a temperature-sensitive polymer compound and collagen.