JPH0576355A - First group of cultured cancerous cell and its production - Google Patents

Abstract

PURPOSE: To obtain a primary culture cancer cell cluster by forming a normal cell layer on a cell culture substrate at a temperature higher than the LCST of a temperature-sensitive polymeric compound, scattering cancer cells on the layer and lowering the temperature to effect the desorption of the normal cells.

CONSTITUTION: A normal cell is cultured on a cell culture substrate containing a temperature-sensitive polymeric compound and a cell adhesion and proliferation factor at a temperature higher than the LCST of the above polymeric compound until the surface of the substrate is completely covered with the normal cells to form a normal cell layer on the substrate, cancer cells directly collected from a patient are scattered and bonded to the cell layer, the temperature is lowered to a level lower than the LCST of the above polymeric compound to effect the complete desorption of the normal cell layer holding the bonded cancer cells from the substrate surface to form a cell sheet floating in a culture liquid, and the sheet is cultured on the above substrate to obtain the objective primary culture cancer cell.

COPYRIGHT: (C)1993,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a primary cultured cancer cell aggregate which contains cancer cells and normal cells collected from a patient and has a desired size and a desired composition ratio of the cancer cells and normal cells. The manufacturing method is related.

The primary cultured cancer cell aggregate of the present invention is obtained by primary culturing the cancer cells obtained by separating from the cancer tissue extracted from the cancer patient by the method of the present invention. By using the primary cultured cancer cell aggregate of the present invention, it is possible to set the conditions of chemotherapy such as the type, dose, and administration period of the most effective anticancer agent for cancer of the cancer patient or the conditions of physical therapy such as radiation and heat. It is possible in vitro. In addition, it is possible to simultaneously measure the effect of the above-mentioned cancer treatment conditions on normal cells due to the presence of normal cells at the same time.

[0003]

2. Description of the Related Art Attempts have been made to culture cancer cells in vitro for the purpose of drug sensitivity test of anticancer agents or determination of effects of physical therapy such as radiation and heat.

At present, almost all established cancer cells can be cultivated and propagated by ordinary flat culture. However, most of the established cancer cells lose contact with other cells in the cancer tissue during the process of establishment, and thus lose many of the features of the primary cancer cells (Fraslin, J. et al., EMB
OJ (1985) 4: 2487).

Therefore, it is necessary to use cancer cells having the characteristics of cancer tissue in the drug sensitivity test of anticancer agents and the like, rather than using established cancer cells that have lost the characteristics of cancer tissue. Flat culture of cancer cells is very difficult and has hardly succeeded to date. As one of the causes, it is considered that normal cells mixed in cancer tissue, especially fibroblasts, show strong proliferative property in flat culture and inhibit the proliferation of primary cancer cells. Another major cause is that it is very difficult to collect cancer cells from a cancer tissue without mixing fibroblasts, and at the same time, it is difficult to distinguish both cells. Therefore, a culture system has been developed in which fibroblasts are less likely to grow even in flat culture. That is, Bal derived from a mouse having the ability to inhibit contact of fibroblasts
This is a method of culturing and proliferating primary cancer cells on a monolayer culture (cell mat) of b / 3T3 cells (Kenji Miki et al., Tissue Culture Study, (1991) 9:35). In addition, a method for inhibiting the growth of normal cells in lung cancer tissue and specifically proliferating only lung cancer cells on a monolayer culture of C3H10T1 / 2 cell line has been developed. (Klein, JC et al., Cancer Res. (1987) 47: 325.
1). On the other hand, even if the proliferation of mixed fibroblasts is suppressed by the above method, the planar culture system is greatly different from the environment of the cancer cells in the living body. Cancer cells exist in a three-dimensional construct in the living body, and a three-dimensional culture system is required to culture and proliferate while maintaining the characteristics of the cancer cells. Have been found to be excellent for maintaining the properties of cancer cells for a long time (Miller, BE, et al, Cancer Res. (1985) 45: 420.
0). A method of culturing cancer cells using collagen gel as a matrix for a three-dimensional culture system (Enonami Junpei, Tissue Culture (1983) 9: 397), a method of culturing cancer cells in double soft agar medium (N, Tanigawa , et al, Cancer Res, (1982) 42:21
59) and so on. However, it is very difficult to maintain the characteristics of cancer cells for a long time even by these gel culture methods.

On the other hand, a method has been developed in which cancer cells are suspension-cultured on a non-cell-adhesive substrate such as agarose to spontaneously aggregate cancer cells to form cancer cell aggregates and perform three-dimensional culture. (J. Calsson. Et al., Sphero
ids in Cancer Research (1984), 1, edited by Acker, H.
et al., Springer-Verlag). Since the cancer cell aggregates produced by this method are morphologically similar to solid cancers in vivo, the cancer cell aggregates are formed using established cancer cells and the drug sensitivity of the anticancer drug is used as a model of solid cancers. , Used to evaluate radiation sensitivity (RMSutherland, Science
(1988) 240: 177).

[0007] Attempts have also been made to form cancer cell aggregates from cancer cells collected from patients using this method (J.
L. Darling, et al., Cell Biology International Report
s (1983) 7:23, EKRofstad, Br.J. Cancer (1986) 54:56.
9). Attempts were made to produce cell aggregates of cancer cells directly collected from individual patients, but the success rate was significantly lower than that of the established cancer cells. The reason for this is that the method naturally forms aggregates by utilizing the aggregating properties of cancer cells, and therefore the aggregating properties differ remarkably depending on the cancer cell type and cannot be applied to all cancer cells. In particular, it is considered that the agglutinability of cancer cells directly collected from a patient has a remarkably wider distribution than that of cell lines. Another problem with this method is that aggregates are formed by spontaneous aggregation, and the size and yield of aggregates are significantly affected by the size of the agglutinability of cancer cells. It is very difficult to produce a large amount with good reproducibility, and it has been very difficult to quantitatively measure the drug sensitivity or radiosensitivity of an anticancer agent using these cancer cell aggregates.

On the other hand, using this method, the established cancer cell line Hela
Attempts have been made to prepare mixed cell aggregates of cells and fibroblasts (Sasaki, T., Cancer Res. (1984) 34:21). It has been recognized that co-culturing fibroblasts improves the ability of cancer cells with low aggregation to form aggregates. However, in the co-culture system of this method, the agglutinability of established cell lines was remarkably higher than that of fibroblasts, and substantially only the cancer cells formed the obtained mixed cell aggregate. . Therefore, as long as this method is used, it is difficult to control the composition ratio of cancer cells and fibroblasts in a mixed cell aggregate and to achieve a high yield.

As outlined above, it is difficult to culture and proliferate cancer cells directly collected from a patient by conventional culture techniques. Furthermore, it is almost impossible to prepare a primary cancer cell aggregate in which normal cells are quantitatively mixed in order to measure the effect on normal cells, which is a measure of side effects of chemotherapy or physical therapy on the living body. Furthermore, it is extremely difficult to control the size of cancer cell aggregates and achieve a high yield, which are important conditions for quantitatively measuring drug sensitivity, radiation sensitivity and the like. The conventional cancer cell aggregates and the method for producing the same have the above-mentioned serious drawbacks.

[0010]

The object of the present invention was difficult to obtain by the above-mentioned conventional cell culture techniques. 1) It is possible to measure drug sensitivity, radiation sensitivity, etc. of cancer tissue of individual patients. In order to measure the effect on normal cells, which is a measure of the side effects of chemotherapy with drugs, physical therapy with radiation, etc. To provide a primary culture cancer cell aggregate in which normal cells are quantitatively mixed in 3), and to further enable quantitative measurement of drug sensitivity, radiation sensitivity, etc. with good reproducibility It is to control the composition of cancer cells and normal cells therein, control the size of cell aggregates, and achieve high yield.

[0011]

[Means for Solving the Problems] The above object is to provide a primary culture characterized by containing cancer cells and normal cells directly collected from a patient and having a desired size and a desired composition ratio of cancer cells and normal cells. It has been achieved by the present invention, which provides a cancer cell population. Furthermore, the normal cells cover the surface of the base material with the temperature-sensitive polymer compound on the culture substrate containing the cell adhesion / growth factor at a temperature higher than the LCST of the temperature-sensitive polymer compound. Culture, form a normal cell layer on the surface of the base material, and inoculate and adhere cancer cells directly collected from a cancer patient on the normal cell layer;
By lowering the temperature to a temperature lower than ST, the normal cell layer 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, and then the floating cell sheet is It was achieved by the present invention which provides a method of culturing on a non-adhesive substrate.

The primary cultured cancer cell aggregate of the present invention will be described in detail below.

In the present invention, the term "cancer cells directly collected from a patient" means that the cancer cells of a patient, which are judged based on the ubiquity, size and deep staining of cell nuclei or the presence or absence of a ductal structure, are freed from the cancer tissue of the patient. Cancer cells.

In the present invention, the "normal cell" means an animal cell other than the above cancer cell. That is, it refers to an animal cell that has not undergone malignant transformation.
In the present invention, it is particularly preferable to use, as normal cells, fibroblasts that 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. In this way, by using two or more types of normal cells, in vivo
In some cases, a more suitable approximation of the structure around the cancer tissue can be made possible.

The size of the primary cultured cancer cell aggregate and the composition of the cancer cells and normal cells are controlled as follows.

A mixture of collagen and poly-N-isopropylacrylamide (PNIPAAm) was inoculated with fibroblasts kept at a temperature above LCST (for example, 37 ° C.) in a dish coated on the bottom surface, and the mixture was heated at a temperature above LCST. Incubate until the cells are confluent to completely cover the bottom of the dish.

Next, a temperature above LCST (for example, 37
A cancer cell dispersion directly collected from a patient kept warm at (.degree. C.) is seeded on the confluent fibroblast monolayer to completely adhere the cancer cells on the fibroblast monolayer. Then, the temperature of the dish is lower than LCST (for example, room temperature:
The fibroblast sheet to which the cancer cells are attached is peeled from the bottom of the dish by controlling the temperature to about 25 ° C.) and suspended in the culture medium. If necessary, add 2 sheets of the sheet with phosphate buffer.
After washing ~ 3 times, transfer to a cell non-adhesive dish and culture.

In the above steps, the size and cell composition of the cell aggregate are determined by the number of normal cells such as fibroblasts and the number of cancer cells in the cell aggregate. The number of fibroblasts is calculated as the product of the area of the bottom of the culture dish and the cell density of the confluent cell monolayer. The cell density of a confluent cell monolayer can be easily and accurately calculated by a known means such as a phase contrast microscope before seeding cancer cells. Cell density in confluent cell monolayers depends on cell type,
For example, in the case of fibroblasts, the cell density in a confluent state is 6-8 × 10 ⁴ cells / cm ² . Therefore, the number of fibroblasts in the cell aggregate is controlled only by the culture area of the dish. On the other hand, the number of cancer cells in the cell aggregate is, for example, the method of directly calculating the number of cancer cells attached to the fibroblasts by a phase contrast microscope or the number of unattached cancer cells floating in the culture solution. It can be measured by a method of calculating and subtracting from the number of cancer cells at the time of seeding.
Therefore, the number of cancer cells in the cell aggregate can be controlled by the seeding density on the confluent fibroblast monolayer.
However, in the case of the cancer cells, unlike the case of normal cells such as fibroblasts, the number of cancer cells may change with time because the cancer cells may proliferate in the cell aggregate.

Therefore, the size and the cell composition of the controllable cell aggregate in the present invention are the initial ones of the cell aggregate.

The method for producing the cultured cancer tissue of the present invention is not particularly limited, but regarding the production, use of a temperature-sensitive polymer compound effectively prevents unnecessary damage to cells. preferable.

Here, the "temperature-sensitive polymer compound" means
A polymer compound having an LCST (Lower Critical Solution Temperature) (eg, Heskins, M., et al., J.
See Macromol, Sci-Chem., A2 8,1441 (1968)).

In the present invention, the temperature-sensitive polymer compound has an LCST of 0 to 80 ° C. (further 10 to 4).
0 ° C.) is preferable. This LCST can be measured as follows, for example.

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 ℃, the temperature at which turbidity first occurs is visually observed and this temperature is referred to as LCST (this LCST
For more information on T. Takezawa et al., Bio / Technology,
8, 654 (1990)).

The temperature-sensitive polymer compound preferably used in the present invention is in a solid state at a temperature higher than LCST, and becomes reversibly water-soluble by lowering the temperature lower than LCST. Since the temperature-sensitive polymer compound is in a solid state at a temperature higher than LCST, cells can grow while using the polymer compound as a scaffold.

On the other hand, when exfoliation of the proliferated cells is required, if the temperature is made lower than LCST, the temperature-sensitive polymer compound becomes water-soluble, so that the bond between cells is not impaired. Proliferating cells can be easily detached.

Examples of the temperature-sensitive polymer compound that can be preferably used in the present invention include poly N-substituted acrylamide derivatives, poly N-substituted methacrylamide derivatives and their copolymers, polyvinyl methyl ether,
Examples thereof include polyethylene oxide, etherified methyl cellulose, and polyvinyl alcohol partial acetic acid. Particularly preferred are poly N-substituted acrylamide derivatives, poly N-substituted methacrylamide derivatives and their copolymers, polyvinyl methyl ether, and polyvinyl alcohol partial acetic acid.

The preferred polymer compounds are listed below in order of increasing LCST.

Poly-N-acryloylpiperidine; poly-N-n-propylmethacrylamide; poly-N-isopropylacrylamide; poly-N, N-diethylacrylamide; poly-N-isopropylmethacrylamide; poly-N-cyclopropylacrylamide; poly-N- Acryloylpyrrolidine; poly-N, N-ethylmethyl acrylamide; poly-N-cyclopropyl methacrylamide; poly-N-ethyl acrylamide Even if the above polymer is obtained by polymerizing a single monomer, other polymer It may be a copolymer with the body. The monomer to be copolymerized may be either a hydrophilic monomer or a hydrophobic monomer. Generally, LCST increases when copolymerized with a hydrophilic monomer and LST increases when copolymerized with a hydrophobic monomer.
CST goes down. Therefore, a polymer compound having a desired LCST can be obtained by appropriately selecting these.

Examples of the hydrophilic monomer include N-vinylpyrrolidone, vinylpyridine, acrylamide, methacrylamide, N-methylacrylamide, hydroxyethylmethacrylate, hydroxyethylacrylate, hydroxymethylmethacrylate, hydroxymethylacrylate and acidic groups. Having acrylic acid, methacrylic acid and salts thereof, vinyl sulfonic acid, stil sulfonic acid, etc., and basic N, N-dimethylaminoethyl methacrylate, N, N-diethylaminoethyl methacrylate, N, N-dimethylaminopropyl Examples thereof include, but are not limited to, acrylamide and salts thereof.

Since the cell membrane is usually negatively charged,
In order to improve the adhesion of cells to the substrate due to electrostatic interaction, it is preferable to use a copolymer with a basic monomer.

On the other hand, as the hydrophobic monomer, acrylate and methacrylate derivatives such as ethyl acrylate, methyl methacrylate and glycidyl methacrylate, N-substituted alkyl methacrylamide derivatives such as Nn-butyl methacrylamide, vinyl chloride,
Examples include, but are not limited to, acrylonitrile, styrene, vinyl acetate, and the like.

The temperature-sensitive polymer compound used in the present invention preferably has a molecular weight of 1.0 × 10 ⁵ or more. More preferably, it is 1.0 × 10 ⁶ or more. The molecular weight referred to in the present invention refers to the average molecular weight obtained from the viscosity. For example,
Regarding poly-N-isopropylacrylamide, the relational expression between the average molecular weight and the limiting concentration is represented by the following formula.

[0033]

[Formula 1] On the other hand, the above-mentioned temperature-sensitive polymer compound constituting the culture substrate in the present invention has a poor affinity with cells, and cells adhere and grow on the culture substrate consisting only of the temperature-sensitive polymer compound. Can not do it. Therefore, in order to improve cell adhesion and proliferation of the culture substrate, the following cell adhesion / growth factors were used in combination.

Examples of the cell adhesion / culture factor in the present invention include collagen, fibronectin, vitronectin, laminin, proteoglycan, glycosaminoglycan, gelatin, lectin, mussel-derived adhesion protein, and adhesive oligopeptide.

The cell culture substrate used in the present invention is typically produced by coating a temperature-sensitive polymer compound having an LCST lower than the cell culture temperature and a cell adhesion / growth factor on a support. can do.

As such an adhesive substrate, an aqueous solution of a mixture of a temperature-sensitive polymer compound and a cell adhesion / growth factor is LCS.
It is obtained by coating all or part of the support at a temperature lower than T and drying the coating layer. Also, adhesion of temperature-sensitive polymer compound and cells
The culture substrate of the present invention can also be obtained by a step of immersing the support in an aqueous solution of a mixture of growth factors at a temperature of LCST or lower and drying.

Further, the culture substrate used in the present invention can also be prepared by forming a layer of a temperature-sensitive polymer on the surface of a support and then forming a layer of cell adhesion / growth factor on the layer. can get.

The mixing ratio of the temperature-sensitive polymer compound used in the present invention and the cell adhesion / growth factor is typically 1 :.
It is 0.1 to 1: 3, and this value changes depending on the type of cell adhesion / growth factor and cell type used.

The thickness of the coating layer after drying is 0.2 μm.
m or more, more preferably 1.0 μm or more. When the thickness of the coating layer is 0.2 μm or less, the desorption property of cells is remarkably deteriorated, so that it takes a very long time to desorb the cells, and as a result, the function of the cells becomes unstable.

The support used in the present invention is preferably transparent or translucent, and glass or plastic is preferable. Typical plastics include polyethylene, polycarbonate, polymethylmethacrylate, polypropylene, polyvinyl chloride, polyacrylonitrile, polytetrafluoroethylene and polydimethylsiloxane. The form of the support is not particularly limited, and it may be used in the form of a dish, plate, film, sheet or the like.

A preferred method for producing the primary cultured cancer cell aggregate of the present invention (that is, the production method of the present invention) is described in detail below.

In a preferred embodiment of the production method of the present invention, first, a temperature higher than the LCST of the temperature-sensitive polymer compound is first applied to the culture substrate containing the temperature-sensitive polymer compound and the cell adhesion / growth factor. (Preferably LCST
The normal cells are cultured at a temperature higher by 1 to 40 ° C., more preferably a temperature higher than 3 to 20 ° C.) to form a monolayer sheet of normal cells on the surface of the substrate.

Next, the cancer cells directly collected from the patient 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 adjusted to L
When the temperature is lower than CST (preferably 1-40 ° C lower than LCST, more preferably 3-20 ° C lower), the temperature-sensitive polymer compound coated on the surface of the culture substrate is dissolved in the culture medium. The normal cell sheet to which the cancer cells adhere is separated from the surface of the base material. The primary cultured cancer cell aggregate of the present invention can be suitably produced by transferring the normal cell sheet to which the cancer cells are attached onto a cell-non-adhesive substrate and subjecting to suspension culture.

One of the important features of the present invention is, as described above, that the cancer cells are released and released from a flat culture system which is extremely unsuitable for culturing the cancer cells directly collected from the patient. The purpose is to carry out primary culture while maintaining the original function that it has. Furthermore, in the embodiment using the temperature-sensitive polymer of the present invention, since the use of a conventional cell releasing agent, a protease or a calcium chelating agent, is not necessary for release and release of cells from the substrate,
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 also an important feature of the present invention that the function of cells is more favorably maintained.

An important feature of the present invention is that normal cells can coexist at the same time as cancer cells collected from a patient, the efficacy of anticancer drugs and the like can be evaluated, and at the same time side effects on normal cells such as anticancer drugs can be evaluated under the same conditions. It is something that can be evaluated.

Further, the mixing ratio of the cancer cells and the normal cells in the primary cultured cancer cell aggregate of the present invention is determined by, for example, the number of both cells when seeding the cancer cells on the normal cell sheet in the above-mentioned manufacturing process. It is an important feature of the present invention that it can be easily controlled by preparing it.

On the other hand, instead of using the above-mentioned culture substrate containing the temperature-sensitive polymer compound, a normal cell sheet having 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 the primary culture cancer cell aggregate of, the method using the former temperature-sensitive polymer compound is more preferable from the viewpoint of cell damage.

[0048]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is determined by the scope of the claims and is not limited by the following examples. . (Example 1) A tumor mass (0.5 cm x 0.5 cm x 0.3 cm) aseptically collected from a gastric cancer patient (TK, female, 38 years old)
Penicillin 100 U / ml, gentamicin 50 μg / m
l, kanamycin 100 μg / ml, fungizone 0.5 μg /
After dipping in Hanks' solution containing ml for about 30 minutes, the tumor mass was put in a petri dish and finely cut (1 to ³ mm ³ ) with scissors.
Then, the cancer tissue section was treated with 0.01% hyaluronidase, 0.1%.
It was digested in RPMI-1640 culture medium (TIL) containing 004% DNA, 0.1% collagenase (type V) at 37 ° C. for about 60 minutes while stirring with a stirrer.
The digested liquid was filtered using a 40 μm mesh, and then specific gravity centrifugation was performed with Ficoll at 2000 rpm for 20 minutes to remove blood components and collect cancer cells. The cancer cells were dispersed in a medium DMEM solution at a concentration of about 2.0 × 10 ⁴ cells / ml and about 1.0 × 10 ⁵ cells / ml.

Poly-N-isopropylacrylamide (PNIPAAm), which is a typical temperature-sensitive polymer compound, was dissolved in distilled water to prepare a 0.5 W / V% aqueous solution, which was sterilized by filtration with a sterilizing filter. A 0.5 W / V% aqueous solution of bovine dermal pepsin-solubilized type I collagen was mixed in equal amounts with the polymer compound aqueous solution to prepare a mixed solution of PNIPAAm and collagen. About 160 μl and about 80 of the mixed solution
μl for each 12 well dish culture wear (dish bottom area: about 3.8 cm ² , Iwaki Glass Co., Ltd.)
And a 4-well multi-dish (bottom area of dish: about 1.9 cm ² , manufactured by Nippon Intermed Co., Ltd.), and then aseptically air dried at room temperature in a clean bench. Collagen and P on the bottom of the dish by the above method
A dish was obtained which was coated with a 1: 1 mixture of NIPAAm to a thickness of about 2 μm.

A human dermal-derived fibroblast DMEM medium (containing 10% fetal calf serum) dispersion was kept at 37 ° C., and then the collagen / pre-heated at 37 ° C.
The PNIPAAm mixture was poured into a coated dish. The seeding concentration of the fibroblasts was about 4 × 10 ⁴ cells / c
It was m ² . Fibroblast culture was carried out at 37 ° C. in an air / 5% carbon dioxide incubator for 5 days, and the bottom of the dish was completely covered with fibroblasts by this culture. Next, a DMEM dispersion of the above-mentioned cancer cells (cell concentration: about 2.0 × 10 ⁴ cells / ml. 1.0 × 10 ⁵ cells / ml)
After incubating 0.5 ml of the cells at 37 ° C, the cells were injected into the fibroblast proliferation dish obtained above and left for 17 hours at 37 ° C in an air / 5% carbon dioxide gas incubator to allow the above-mentioned cancer cells to become confluent fibroblasts alone. Deposited on the layer. Then, the dish was taken out of the incubator at 37 ° C., and cooled to 4 ° C. to completely detach the fibroblast monolayer having the cancer cells attached thereto from the bottom surface of the dish.
When observing the cell monolayer detachment process with a phase microscope, the cell layer is detached gradually from the dish edge toward the central portion,
It was confirmed that the peeled sheet wraps around the upper surface while wrapping the cancer cells attached thereto. Further, in the process of peeling the sheet, almost no detachment of cancer cells from the fibroblast layer was observed. The peeling process was completed in about 5 minutes, and the cell sheet was completely peeled from the bottom surface of the dish and floated in the culture medium. PNIPAAm lysed from the suspended cell sheet
And to remove collagen, wash the sheet 2-3 times with phosphate buffer and then transfer onto a non-cell-adhesive dish (agar-coated dish).
The cells were cultured at 7 ° C in an air / 5% carbon dioxide gas 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 second day of suspension culture, the cultured cancer cell aggregates were fixed with 10% formalin buffer, and paraffin-embedded tissue sections were prepared.
The cancer cell aggregate section was stained with hematoxylin and eosin, and the composition ratio of cancer cells to fibroblasts and the size of the aggregate were measured under a microscope. The results are shown in Table 1. The aggregate was subjected to suspension culture for 21 days, fixed in the same manner, tissue sections were prepared, and hematoxylin staining was performed. The results are shown in FIG.

For comparison, a hematoxylin / eosin stained image of the tumor mass of the cancer patient (TK) is shown in FIG. Surprisingly, the cancer cell image in the cultured cancer cell aggregate (indicated by an arrow in FIG. 1) and the cancer cell image of the patient (indicated by an arrow in FIG. 2) were very similar. As can be seen from Table 1, the composition ratio of the cancer cells in the cultured cancer cell aggregate to the fibroblasts is the bottom area of the culture dish, that is, the density of confluent fibroblasts (6 to 8 × 10 ⁴ cells / cm ² ). It could be controlled very well by the number of seeded cancer cells. Further, it was found that the size of the cancer cell aggregate depends on the total number of fibroblasts and cancer cells, and it was possible to control the size with high accuracy by the method of the present invention.

[0052]

[Table 1] (Comparative Example 1) 2 ml of a DMEM (containing 10% fetal calf serum) dispersion liquid (cell concentration: about 1.0 × 10 ⁵ cells / ml) of a cancer cell prepared by the same method as in Example 1 was used as a commercial product. As a result of injecting into a tissue culture dish (Falcon # 3001, 35 mmΦ) and culturing at 37 ° C. in an air / 5% carbon dioxide gas incubator, adhesion and culturing of cancer cells were not observed at all. (Example 2) cancer patients (U.N. woman, 63 years old) 1 in peritoneal fluid 3 _liter. 5 liter primary pancreatic-derived ascites metastases cells collected by centrifuging the DMEM (10
% Fetal bovine serum).

The human dermis-derived fibroblasts used in Example 1 were seeded on the 12-well dish culture wear coated with the collagen / PNIPAAm mixture used in Example 1 and incubated at 37 ° C. in an air / 5% carbon dioxide gas incubator. After culturing for 5 days, a confluent fibroblast monolayer was obtained. Then, a DMEM dispersion liquid of the above-mentioned pancreatic cancer-derived ascites metastatic cancer cells kept at 37 ° C. (cell concentration: about 8 × 10 5
0.5 cell ( ⁴ cells / ml) was seeded on the fibroblast layer in a confluent state and left standing in a 37 ° C. air / 5% carbon dioxide gas incubator for 24 hours. After 24 hours, it was confirmed by phase contrast microscopy that most of the cancer cells adhered to the confluent fibroblast monolayer, and the dish was taken out from the 37 ° C incubator and cooled to 4 ° C.

As a result of observation with a phase-contrast microscope, the fibroblast monolayer in a confluent state in which the cancer cells adhered to the upper surface was peeled off so as to wrap around the cancer cells adhering from the periphery of the dish toward the center, and about 5 minutes later. The cell sheet completely separated from the bottom of the dish and floated in the culture solution. Almost no detachment of the cancer cells from the fibroblast sheet was observed during this detachment process. 2 to remove dissolved PNIPAAm and collagen from the exfoliated cell sheet
After washing with a phosphate buffer solution once, the cells were transferred to the cell non-adhesive dish used in Example 1 and subjected to suspension culture at 37 ° C. in an air / 5% carbon dioxide gas incubator. It changed into a massive cell aggregate. After 21 days of suspension culture, the cancer cell aggregates were fixed with 10% formalin buffer and paraffin-embedded tissue sections were prepared.

When the tissue section was stained with hematoxylin / eosin and the cancer cell image was observed by a phase contrast microscope,
It was very similar to the image of cancer cells in the pancreatic primary lesion of the cancer patient. Comparative Example 2 2 ml of a DMEM dispersion liquid (cell concentration: about 8 × 10 ⁴ cells / ml) of ascites metastatic cancer cells derived from the primary pancreatic cancer used in Example 2 was added to a commercially available tissue culture dish (Falcon # 3001, 35 mmΦ) and cultured at 37 ° C. in an air / 5% carbon dioxide gas incubator. As a result, adhesion and growth of the cancer cells were not observed at all.

[0056]

INDUSTRIAL APPLICABILITY The primary cultured cancer cell aggregate of the present invention has cancer cells and normal cells directly collected from a patient's cancer tissue, and the composition ratio and size of the cancer cells and normal cells are precisely controlled. Therefore, by using the cancer cell aggregate of the present invention, it is possible to set the most effective cancer treatment method for a patient who has collected the cancer cells, and the technical value thereof is extremely high.

[Brief description of drawings]

FIG. 1 is a hematoxylin-stained image after 21 days of culture of an example of a primary cultured cancer cell aggregate of the present invention, which comprises cancer cells collected from a gastric cancer patient and fibroblasts derived from human dermis, and the arrow in the figure indicates cancer. Shows cells.

[Fig. 2] Hematoxylin of the primary lesion of gastric cancer shown in Fig. 1.
It is an eosin stained image, and the arrow in the figure indicates a cancer cell.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yuichi Mori 1642-21 B-1-21 Kamariya-cho, Kanazawa-ku, Yokohama, Kanagawa Prefecture B-4 (72) Inventor Toshiaki Takezawa 31-15, Higashi Naruse, Isehara City, Kanagawa 34-302

Claims (2)

[Claims] 1. A primary culture cancer cell aggregate comprising cancer cells and normal cells directly collected from a patient and having a desired size and a desired composition ratio of the cancer cells and the normal cells. . 2. On a cell culture substrate containing a temperature-sensitive polymer compound and a cell adhesion / growth factor, normal cells are added to the cell culture substrate at a temperature higher than the LCST of the temperature-sensitive polymer compound. By culturing until the material surface is covered, a normal cell layer is formed on the cell culture substrate, and the cancer cells directly collected from the patient are seeded and adhered onto the normal cell layer to adhere to the temperature-sensitive material. By lowering the temperature below the LCST of the molecular compound, the normal cell layer to which the cells are attached is completely desorbed from the surface of the substrate to form a cell sheet that floats in the culture medium, and the floating cell sheet is A method for producing a primary cultured cancer cell, comprising the steps of culturing on a non-cell-adhesive substrate.