JPH0731470A - Method for proliferating cancer cell - Google Patents

Abstract

PURPOSE:To selectively proliferate only a cancer cell by culturing the cancer cell containing a normal cell present therein and stopping or suppressing the extension or proliferation thereof without killing the normal cell. CONSTITUTION:A cancer cell containing a normal cell present therein is cultured on the surface of a culturing substrate substantially composed of a synthetic polymer and a cell growth factor to selectively proliferate the cancer cell. The culturing substrate is prepared by blending collagen and a temperature- sensitive polymer having 32 deg.C lower critical solution temperature (LCST) with water and drying the resultant coating solution at 1/6.7 weight ratio of the temperature-sensitive polymer to the growth factor at 28 deg.C.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for growing cancer cells.

[0002]

2. Description of the Related Art It is difficult to remove only cancer cells from a living body for culturing the cancer cells, and it is common that normal cells are mixed with the cancer cells. The taken out cells are cultured in vitro to increase the number of cells and used for inspections, tests, or for in vitro cultures. In these cases, the normal cells mixed with the cancer cells hinder the examination of the growth rate of the cancer cells. This is because the degree of proliferation is measured by the increase in the number of cells, the formation of colonies, the number of cells, etc., so that the normal cells mixed and proliferated give a large error to the measured value.

[0003] In order to remove normal cells, conventionally, they are naturally eliminated by soft agar culture or serum-free culture, physically removed, treated with an enzyme such as trypsin, or immunologically fractionated. The method of doing was suggested. These methods kill the normal cells or separate the cancer cells from the normal cells, and do not culture and selectively grow the cancer cells mixed with the normal cells.

[0004]

[Problems to be Solved by the Invention] Proliferation of cancer cells may be promoted by the growth factor (or growth factor) produced by normal cells of the stromal system. It was considered to culture. Since normal cells have adhesion dependence (or anchorage dependence), a culture substrate for adhering cells is necessary. But the cells
When cultured by adhering to a normal culture substrate such as a corona discharge treated polystyrene incubator, normal cells proliferate preferentially over cancer cells and spread over the entire surface of the culture substrate, and cancer cells are entangled and separated from the substrate. Therefore, it interferes with the culture of cancer cells.

[0005] It is not sufficient to simply increase the number of cells in the culture of cells, and it is important that the cells are grown while exhibiting the characteristics of the cells as in the case of growth in vivo. Unlike conventional cancer cell cultures, the present invention stops or suppresses the growth of normal cells mixed with cancer cells without killing them, and allows cancer cells to selectively and easily grow while exhibiting their characteristics. It is an object to provide a culture method.

[0006]

In order to solve the above-mentioned problems, the present invention cultivates cancer cells in which normal cells are mixed on a culture substrate surface consisting essentially of a synthetic polymer and a cell growth factor, Provided is a cancer cell growth method for selectively growing cancer cells. In order to stop or suppress the growth of normal cells such as fibroblasts and to grow cancer cells, it is necessary to understand the difference between the growth of cancer cells and normal cells. Considering the difference in growth between cancer cells and normal cells, cancer cells, when compared to normal cells, could not recognize their surroundings, lost contact inhibition, and proliferated, resulting in disordered cell arrangement (criscross or crossover). It also causes cell array) and overlapping growth (piled up growth). Normal cells are regularly arranged and stop growing when they hit the wall or boundary of the culture substrate, and do not cause overlapping growth. That is, since normal cells have normal external recognition and recognize the change in the surface property of the culture substrate, when they are recognized as such, they can stop proliferation unlike cancer cells.
The present invention is characterized by focusing on such a difference between a cancer cell and a normal cell, finding a condition that causes a difference in recognition between the two, and the culture substrate is provided with this condition. .

The adhesion of cells to a culture substrate becomes better as the surface property of the substrate becomes more hydrophilic, and conversely decreases when it becomes hydrophobic. When the surface of the base material is uniformly hydrophilic as a whole, the cancer cells proliferate in a plane unlike in the living body, and when the normal cells coexist, the normal cells preferentially proliferate. Examples of the growth factor used in the present invention include extracellular matrix represented by collagen, laminin, fibronectin, vitronectin, proteoglycan, glycosaminoglycan, gelatin, lectin, adhesive oligopeptide and the like.

The synthetic polymer used in the present invention is for imparting non-adhesiveness (or hydrophobicity) of the above-mentioned cells to the surface of the culture substrate to suppress or stop the spreading or proliferation of normal cells, It is preferably not toxic to killing cancer cells or normal cells. Preferred synthetic macromolecules are, for example, thermosensitive macromolecules and macromolecules having hydrophobicity such as polystyrene, and a solid mixed with a growth factor is produced by drying a liquid mixed with the growth factor in water. Those that do are preferred. LCST (Lower
Critical Solution Temperature: Lower critical solution temperature,
Or, if the temperature rises above the transition temperature of hydration and dehydration of the temperature-sensitive polymer, it becomes hydrophobic and becomes water-insoluble, and if cooled to a temperature below that, it shows hydrophilicity (water solubility). have. As the temperature-sensitive polymer, for example,
Examples thereof include poly-N-substituted acrylamide derivatives, poly-N-substituted methacrylamide derivatives, copolymers thereof, polyvinyl methyl ether, and polyvinyl alcohol partial acetic acid, which preferably have an LCST lower than the culture temperature. .

The culture substrate used in the present invention is not limited in shape, and may be layers, particles, plates, fibers, flakes, sponges, microbeads, etc., and may be an independent solid or coated on a support substrate. It may be a membrane. The supporting substrate is, for example, polystyrene or polyester that has been rendered hydrophilic by corona discharge treatment, or another solid having a hydrophilic surface such as an incubator. The coating film may be formed on all or part of the surface of the support substrate. When the coating film is formed on a part of the surface of the supporting substrate, for example, in the form of one or a plurality of spots, the seeded normal cells form lumps and are difficult to distinguish from the proliferated cancer cells. This is preferable because it can prevent the phenomenon.

The culture substrate is prepared, for example, by drying a liquid obtained by mixing a cell growth factor and a synthetic polymer in water. The liquid may be an aqueous solution of growth factor and synthetic polymer. The coating film can be formed by applying the liquid to a supporting substrate and drying. The liquid comprises a synthetic polymer in a weight ratio of more than 1 to the growth factor, preferably more than 5, more preferably 13.3 /.
Included in a weight ratio of 1 or less. When the synthetic polymer is contained at a ratio of 1 or less with respect to the growth factor, the normal cells grow preferentially over the cancer cells on the surface of the substrate, so that the cancer cells cannot be selectively grown. On the other hand, when the amount exceeds the upper limit of the above range, the surface of the base material becomes non-adhesive as a whole, and the cells may not adhere to the culture base material and may not be cultured.

The culture substrate can be produced, for example, by the following or the method, but the production method is not particularly limited. A coating solution containing a synthetic polymer in an amount of 0.125 w / v% or more, preferably 0.25 to 1.5 w / v%, and a weight ratio of the synthetic polymer to a growth factor satisfying the above-mentioned conditions is 25 ° C. or more, It is preferably dried at 27 to 29 ° C. Thus, a liquid containing a temperature-sensitive polymer at a low concentration is used at 25 ° C.
If it is dried at a temperature below 1, a substrate on which cancer cells can be selectively cultured cannot be obtained. If the polymer concentration is less than 0.125 w / v%, a substrate capable of selectively culturing cancer cells cannot be obtained no matter how the drying temperature is set.

0.75 w / v% or more of synthetic polymer,
A coating solution containing 0.9 to 2.0 w / v% of the synthetic polymer and having a weight ratio of the synthetic polymer to the growth factor satisfying the above-mentioned condition is lower than LCST, preferably 10 to 10.
Dry at 25 ° C. If the polymer is less than 0.75 w / v%, it is necessary to make the culture substrate by the above-mentioned method. If the high molecular weight is too high, the cells may not adhere to the substrate, so the above upper limit is preferred. When dried at a temperature higher than LCST, the temperature-sensitive polymer may agglomerate and peel from the incubator, resulting in non-uniform surface coating.

The type of cancer cells cultured in the present invention is not limited, and various cancer cells are possible. This point is different from the conventional method. The normal cells mixed with the cancer cells may be fibroblasts, and there is no limitation. This point is also different from the conventional method. The method of the present invention can be performed in the same manner as a conventional adherent culture method except that the above-mentioned culture substrate is used as an object to which cells are adhered. The liquid medium used for the culture and the culture conditions may be appropriately set depending on the cells to be cultured.

When the method of the present invention is applied to a cancer cell cytotoxicity test, the drug to be brought into contact with cancer cells is an anticancer drug or the like. The cancer cells may be brought into contact with the drug and then adhered to the culture substrate to be cultured, or the cancer cells adhered to the substrate may be cultured in the presence of the drug. The same or different agents may be contacted before and after adhesion to the substrate. Using the method of the present invention, it is possible to distinguish between cancer cells and normal cells. For example, when a culture substrate is partially formed on the surface of an incubator, which is a supporting substrate, in a spot shape and cultured, cancer cells grow on the entire surface of the culture substrate, but normal cells grow on the spot-shaped portion. Can be identified by this because it does not spread and stops growing.

The method of the present invention is a method of culturing primary cancer cells which are easily contaminated with normal cells, a method of using clinical specimens or transformed cells as an index of the canceration ability or malignancy of cells by focus formation, and isolation. Cancer cells, anticancer drug (anticancer drug) susceptibility test, research on enzymes related to cancer cells, cell growth factors, extracellular matrix, tumor markers, and commercialization of oncogenes, tumor suppressor genes and their products It is also useful for research and commercialization.

The cancer cells cultured by the method of the present invention selectively lyse one or both of a growth factor and a synthetic polymer (including a case of decomposing and lysing).
By doing so, it is possible to recover.

[0017]

According to the present invention, cancer cells mixed with normal cells are cultured on the surface of a culture substrate that is substantially composed of synthetic polymers and cell growth factors, so that the cancer cells cause cross cell arrangement or overlapping proliferation. . Moreover, even when culturing cancer cells mixed with normal cells, normal cells do not spread or stop or are suppressed from being proliferated by the synthetic polymer, whereas cancer cells contain the synthetic polymer. Also grows. Therefore, cancer cells mixed with normal cells can be selectively proliferated.

[0018]

EXAMPLES Examples of the present invention and comparative examples outside the scope of the present invention will be shown below, but the present invention is not limited to the following examples. The synthetic polymer aqueous solutions prepared in the following production examples were used in the following examples and comparative examples. (Production Example of Synthetic Polymer Aqueous Solution) 50 g of N-isopropylacrylamide was dissolved in 500 ml of benzene to prepare 2,2 ′.
Using 0.2 g of azobisisobutyronitrile (AIBN) as a polymerization initiator, polymerization was carried out at 60 ° C. for 12 hours in a nitrogen stream under stirring to give poly-N-isopropylacrylamide (hereinafter, referred to as “PNIPAAm”). Produced). Since this polymer precipitates in benzene, it is decanted and the precipitate is removed from tetrahydrofuran (TH
F) and dissolved in diethyl ether for purification by precipitation. The LCST (measured by the turbidity method) of the obtained 1.0 w / v% aqueous solution of PNIPAAm was about 32 ° C.
This aqueous solution was sterilized in an autoclave, re-dissolved at a temperature lower than LCST, and then 0.25, 0.5, 1.0,
2.0 and 4.0 w / v% aqueous solutions were obtained.

(Example 1A) 0.3 w / v% collagen aqueous solution (Cell matrix type I-C manufactured by Nitta Gelatin Co., Pepsin-solubilized type I collagen solution)
And 1.0 w / v% PNIPAAm aqueous solution were mixed in equal amounts, and stirred at 4 ° C. for 1 day with a stirrer to prepare a coating solution. This coating solution is PNIP
AAm 0.5w / v%, collagen 0.15w / v
Included at a concentration of%. Keep the coating solution warm at 28 ° C,
0.5 ml was poured into a plastic dish for cell culture (25010, manufactured by Iwaki Glass Co., Ltd.) which was kept at the same temperature, and the entire surface of the dish was wetted with the coating solution. Then, the excess liquid was immediately sucked and removed to remove the dish. It was placed in a constant temperature bath at 28 ° C. overnight and dried. The dried dish has collagen and PNIPAAm on the surface 1:
Having a weight ratio of 3.4. The dried dish was kept warm on a warm plate (DC-MP100DM, Kitasato Supply Co., Ltd.) at 37 ° C.
Dulbecco's modified Eagle medium (DME,
4x1 for Nissui Pharmaceutical Co., Ltd. containing 10% fetal bovine serum)
0 ⁵ at a ratio of cells / ml were seeded cell suspension 1ml was suspended colon carcinoma line (DLD-1). The dish containing the cell suspension was placed in an air / 5% carbon dioxide gas incubator and cultured at 37 ° C. for 4 days. The appearance of the dish surface after 4 days of culture was observed with a phase contrast microscope. The microscope image is shown in FIG. The above operation was performed under aseptic conditions.

(Comparative Example 1A) The procedure of Example 1A was repeated except that the coating solution was kept at 10 ° C. and the dish was kept at 10 ° C. in the same manner as in Example 1 A, except that the dish was dried in a constant temperature bath at 10 ° C. . The appearance of the dish surface after 4 days of culture was observed with a phase contrast microscope. The microscope image is shown in FIG. The above operation was performed under aseptic conditions.

(Example 1B) Example 1B except that the coating solution was kept at 30 to 34 ° C. and the dish was kept at 30 to 34 ° C., and the coating solution was dried in a constant temperature bath at 30 to 34 ° C. The operation of 1A was repeated. The appearance of the dish surface after 4 days of culture was observed with a phase contrast microscope. The microscope image is shown in FIG. The above operation was performed under aseptic conditions.

In each of FIGS. 1 to 3, A is the horizontal length of the screen 6
The total length is 64 μm and the total length is 430 μm. B is the horizontal total length of 2666.
The vertical total length is 1727 μm. 1-3,
The black granular image with white outline is a cancer cell. In Examples 1A and 1B, as seen in FIGS. 1 and 2, individual cells do not extend and are spherically rounded. In Comparative Example 1A, as shown in FIG. 3, individual cells are not rounded but extend in a polygonal shape. This phenomenon is flat cell spreading like commercial incubators. In addition, Examples 1A and 1B are also comparative examples 1.
A also showed overlapping growth characteristic of cancer cells.

(Example 1C) In Example 1A, the concentration of PNIPAAm in the coating solution was 1.0 w / v%.
The procedure of Example 1A was repeated, except that the drying was performed at 10 ° C. When the appearance of the dish surface after 4 days of culture was observed with a phase contrast microscope, the same results as in Example 1A were obtained. The above operation was performed under aseptic conditions.

(Example 1D) In Example 1A, the concentration of PNIPAAm in the coating solution was 2.0 w / v%.
The procedure of Example 1A was repeated, except that the drying was performed at 10 ° C. When the appearance of the dish surface after 4 days of culture was observed with a phase contrast microscope, the same results as in Example 1A were obtained. The above operation was performed under aseptic conditions.

From the results of the above Examples 1A to 1D and Comparative Example 1A, when the temperature-sensitive polymer was used as the synthetic polymer, the surface property of the culture substrate on the cells changed depending on the drying temperature of the coating solution, and the cells spread. It is understood that it causes changes in sex and growth form. (Examples 2A to 2C and Comparative Examples 2A to 2C) In Example 1A, the drying temperature of the coating solution was set to the temperature shown in Table 1, and the cancer cells isolated from the living body instead of the cancer cell line (see Table 1). The procedure of Example 1A was repeated except that the surgical material containing) was used. The appearance of the dish surface after 6 days of culture was observed with a phase contrast microscope. Figure 4
9 to 9 show the microscope images. The above operation was performed under aseptic conditions.

[0026]

[Table 1] ─────────────────────────────── Drying temperature [℃] Types of cancer cells Microscopic image ──── ─────────────────────────── Example 2A 28-30 Lung Cancer FIG. 4 Example 2B 28-30 Rectal Cancer FIG. 5 Example 2C 28 -30 Lung cancer Fig. 6 Comparative example 2A 10 Lung cancer Fig. 7 Comparative example 2B 10 Rectal cancer Fig. 8 Comparative example 2C 10 Lung cancer Fig. 9 ───────────────────────── ──────── Figures 4 to 9 show a total screen length of 664 μm and a vertical length of 430 μm.
Is. In FIGS. 4 to 6, the island-shaped region is a cancer cell colony, and a part of the colony is thick due to proliferation (black image).
And no fibroblasts are seen. In contrast,
7 to 9, fibroblasts (black elongated images) and cancer cells that are irregularly arranged and proliferate in a dome shape coexist. In FIG. 7, a region occupying a hook shape from the upper left to the lower center is a normal cell, and the other regions are cancer cells. In FIG. 8, the region protruding from the lower left in a peninsular shape is a normal cell, and the other regions are cancer cells. In FIG. 9, the upper right round region and the lower center round region are cancer cells, and the other regions are normal cells. In the examples, it was observed that only three cancer cells proliferated and fibroblasts did not proliferate in all three specimens, and it can be seen that the culture substrate of these examples can be used for selective culturing of cancer cells.

(Example 3A) 0.3 w / v% collagen
Aqueous solution (Product cell matrix of Nitta Gelatin Co., Ltd.
・ Type I-C, pepsin-solubilized type I collagen solution)
And 2.0 w / v% PNIPAAm aqueous solution mixed in equal amounts
Stir the resulting mixture with a stirrer at 4 ° C for 1 day.
Solution was made. This coating solution is PNIP
AAm 1.0w / v%, collagen 0.15w / v
Included at a concentration of%. Keep the coating solution warm at 10 ° C,
12 wells for cell culture kept at the same temperature
(MS-80120, Sumitomo Bakelite Co., Ltd.
The whole surface was dipped in 0.25 ml / well
Immediately after that, the excess liquid is sucked out, and it is kept in a constant temperature bath at 10 ° C for 24 hours.
Dried for hours. Place the dried plate on the inside of the well.
Contains collagen and PNIPAAm in a weight ratio of 1: 6.7.
Has a layer. Warm plate with dried plate
37 on (DC-MP100DM, Kitasato Supply)
Incubate at 37 ° C, keep each plate well at 37 ° C
Dulbecco's modified Eagle medium (DME, Nissui Pharmaceutical Co., Ltd.
4 × 10 per 10% fetal calf serum (made by Shiki company)^Fivecell/
Cell suspension in which lung cancer cell line (QG-56) was suspended at a ratio of ml
Add the suspension to a cell count of 5 x 10 ^FourTo be individual
Sowed. The plate containing this cell suspension is air / 5
Incubate in CO2 incubator for 5 days at 37 ℃
did. Using a phase-contrast microscope to observe the condition of the inner surface of the well after 5 days of culture
I observed. The microscope image is shown in A of FIG. The above
The crop was performed under aseptic conditions.

On the other hand, in the above operation, the same operation was repeated except that a fibroblast cell line (HFL-1, normal cell) was used instead of the lung cancer cell line. The state of the inner surface of the well after 5 days of culture was observed with a phase contrast microscope. The microscope image is shown to B of FIG. The above operation was performed under aseptic conditions. (Comparative Example 3A) In Example 3A, a 1.0 w / v% PNIPAAm aqueous solution was used, and a coating solution containing PNIPAAm at a concentration of 0.5 w / v% and collagen at a concentration of 0.15 w / v% was used. Example 3A except for that
Was repeated. The state of the inner surface of the well after 5 days of culture was observed with a phase contrast microscope. The microscope image is shown in A of FIG. The above operation was performed under aseptic conditions.

On the other hand, in the above operation, the same operation was repeated except that a fibroblast cell line (HFL-1, normal cell) was used instead of the lung cancer cell line. The state of the inner surface of the well after 5 days of culture was observed with a phase contrast microscope. The microscope image is shown to B of FIG. The above operation was performed under aseptic conditions. (Example 3B) The procedure of Example 3A except that a cell group in which HFL-1 and QG-56 were uniformly mixed at a cell number ratio of 4: 1 was used in place of the lung cancer cell line in Example 3A. Was repeated. The state of the inner surface of the well after 5 days of culture was observed with a phase contrast microscope. The microscope image is shown in FIG. The above operation was performed under aseptic conditions.

(Comparative Example 3B) Comparative Example 3B except that a cell group in which HFL-1 and QG-56 were uniformly mixed at a cell number ratio of 4: 1 was used in place of the lung cancer cell line in Comparative Example 3A. The operation of 3A was repeated. The state of the inner surface of the well after 5 days of culture was observed with a phase contrast microscope. The microscope image is shown in FIG. The above operation was performed under aseptic conditions.

In FIGS. 10 to 13, the total horizontal length of the screen is 2666 μm.
The vertical total length is 1727 μm. 10 to 13,
Black granular images are cancer cells, and black elongated images are fibroblasts. In Comparative Example 3A, as shown in FIG. 11, the cancer cells and fibroblasts proliferated flatly on the entire incubator in the same manner as when directly seeded in the commercially available incubator.
In particular, fibroblasts show a dense, parallel, regular arrangement (the cells are arranged to flow). On the other hand, in Example 3A, cancer cells were observed to overlap and grow (pile-up, one of the characteristics of cancer cells) (see FIG. 10B), and the overlap growth spreads to the entire incubator, but fibroblast The cells hardly grew (see FIG. 10A).

In Example 3B, as shown in FIG. 12, a large number of cancer cells aggregate and proliferate in a lump as shown in the center of the figure, whereas fibroblasts have a very small amount of base material. It adheres to but has hardly grown. Therefore, according to the present invention, it is possible to selectively grow cancer cells even when the cancer cells coexist with normal cells. On the other hand, in Comparative Example 3B, as shown in FIG. 13, fibroblasts preferentially proliferate on the entire culture substrate, and cancer cells planarly spread and partially proliferate.

In the above-mentioned Examples and Comparative Examples, cell lines were used for culturing in order to clearly show the effect of the culturing method of the present invention. Next, Examples and Comparative Examples in which primary culture was actually carried out Indicates. Example 4A In this example, surgical material was used on rectal tissue isolated from a living body. In this material, cancer cells and normal cells coexist. Dulbecco's modified Eagle medium (DME, manufactured by Nissui Pharmaceutical Co., Ltd., 1
The above material was dispersed by enzyme treatment at a rate of 4 × 10 ⁵ cells / ml in 0% fetal bovine serum) to obtain a cell suspension. The procedure of Example 3A was repeated except that this cell suspension was used in Example 3A. The appearance of the dish surface after 5 days of culture was observed with a phase contrast microscope. The microscope image is shown in FIG. The above operation was performed under aseptic conditions.

(Control 4A) In Example 4A, 0.15 w of collagen was used without PNIPAAm.
The procedure of Example 4A was repeated except that a coating solution containing a concentration of / v% was made. The appearance of the dish surface after 5 days of culture was observed with a phase contrast microscope. The microscope image is shown in FIG. The above operation was performed under aseptic conditions.

Comparative Example 4A Example 4A was repeated except that a coating solution containing no collagen and PNIPAAm at a concentration of 0.5 w / v% was prepared.
The operation of A was repeated. However, it could not be cultured because neither cancer cells nor fibroblasts adhered. The above operation was performed under aseptic conditions.

Example 4B The procedure of Example 4A was repeated except that the lung tissue surgical material separated from the living body was used. The appearance of the dish surface after 5 days of culture was observed with a phase contrast microscope. The microscope image is shown in FIG. The above operation was performed under aseptic conditions. (Control 4B) In Example 4B, PNIPA
The procedure of Example 4B was repeated except that a coating solution containing no Am and collagen at a concentration of 0.15 w / v% was prepared. The appearance of the dish surface after 5 days of culture was observed with a phase contrast microscope. The microscope image is shown in FIG. The above operation was performed under aseptic conditions.

(Example 4C) Using a surgical material of lung tissue excised from a living body, and drying at room temperature (2
The operation of Example 4A was repeated except that the drying was performed at 8 ° C ± 1 ° C. The appearance of the dish surface after 5 days of culture was observed with a phase contrast microscope. The microscope image is shown in FIG.
The above operation was performed under aseptic conditions.

(Control 4C) In Example 4C, 0.15 w of collagen was added without PNIPAAm.
The procedure of Example 4C was repeated except that a coating solution containing a concentration of / v% was made. The appearance of the dish surface after 5 days of culture was observed with a phase contrast microscope. The microscope image is shown in FIG. The above operation was performed under aseptic conditions.

14 to 19, the screen has a horizontal total length of 664 μm and a vertical total length of 430 μm. In FIGS. 14 and 16 to 19, images of black granular lumps and white large lumps that are white in places are cancer cell colonies, and black elongated images are fibroblasts. In FIG. 15, the large round region at the left end is a cancer cell. As shown in FIGS. 14, 16 and 18, according to the present invention, the cancer cells overlapped and proliferated to form colonies, whereas the fibroblasts adhered only slightly to the substrate, but proliferation was observed. However, the cancer cells were selectively cultured. On the other hand, as seen in FIGS. 15, 17 and 19, in the comparative example, the cancer cells (the circular region at the left end of FIG.
Fibroblasts proliferated preferentially around the black region (Figs. 17 and 19) and spread over the entire substrate.

(Comparative Example 5A) In Example 3A, 0.
PNIPA using 5w / v% PNIPAAm aqueous solution
Am 0.25w / v%, collagen 0.15w / v
The procedure of Example 3A was repeated except that a coating solution containing a concentration of 10% was made. The same operation was repeated except that a fibroblast cell line (HFL-1, normal cell) was used instead of the lung cancer cell line. After 5 days of culture, the DNA amount of cancer cells and fibroblasts was measured by the DNA fluorescence method. The above operation was performed under aseptic conditions.

(Examples 5A to 5F and Comparative Example 5B) In Comparative Example 5A, the coating solution PNIPAAm was used.
The operation of Comparative Example 5A was repeated except that the concentration or the drying temperature was changed as shown in Table 2. After 5 days of culture, DNA
The amount of DNA in cancer cells and fibroblasts was measured by the fluorescence method. The above operation was performed under aseptic conditions.

[0042]

[Table 2] ────────────────────────────────── Coating solution PNIPAAm concentration of coating solution Drying temperature [w / V%] [° C] ────────────────────────────────── Comparative Example 5A 0.25 10 Comparative Example 5B 0.5 10 Example 5A 1.0 10 Example 5B 2.0 10 ─────────────────────────────── Example 5C 0.25 28 Example 5D 0.5 28 Example 5E 1.0 28 Example 5F 2.0 28 ──────────────────── ─────────────── (Control 5A) In Comparative Example 5A, PNIPA
The procedure of Comparative Example 5A was repeated except that a coating solution containing no Am and collagen at a concentration of 0.15 w / v% was prepared. The same operation was repeated except that a fibroblast cell line (HFL-1, normal cell) was used instead of the lung cancer cell line. After 5 days of culture, the DNA amount was measured by the DNA fluorescence method. The above operation was performed under aseptic conditions.

The proliferation rate of cancer cells and fibroblasts in the culture of Examples 5A to 5F and Comparative Examples 5A to 5B was calculated by the following formula:

[0044]

[Equation 1]

[0045]

[Equation 2] Sought by. A degree of proliferation of 100% means that the degree of proliferation by culturing on a collagen substrate is the same. The results are shown in FIGS.

As shown in Table 2 and FIGS. 20 to 21, as the temperature-sensitive polymer concentration was increased, the proliferation rate of normal cells was
The degree of proliferation was significantly lower than that of cancer cells, and was higher when the drying temperature was high. The growth rate of cancer cells is hardly affected by the drying temperature. (Experiment 1) A culture experiment of a fibroblast cell line was performed using each culture substrate used in Example 4A and Comparative Example 4A. The dried plate was warmed (DC-MP100DM,
4 x in Dulbecco's modified Eagle medium (DME, containing 10% fetal calf serum, manufactured by Nissui Pharmaceutical Co., Ltd.) kept at 37 ° C on Kitasato Supply Co., Ltd. and kept at 37 ° C in each well of this plate. A cell suspension in which the fibroblast cell line (NB-1) was suspended at a rate of 10 ⁵ cells / ml was seeded so that the number of cells in each well was 7 × 10 ⁴ . The plate containing the cell suspension was placed in an air / 5% carbon dioxide incubator and cultured at 37 ° C. for 6 days. At the start of culture, 2
After 4 days and 6 days, the cells adhered to the inner surface of the well were fixed with ethanol, and the proliferation degree was measured by the DNA fluorescence method. The above operation was performed under aseptic conditions.

Fibroblast cell lines were designated HFL-1 and MRC-
The above Experiment 1 was repeated, changing to 5, respectively. At the start of the culture, 2 days, 4 days, and 6 days later, the cells adhered to the inner surface of the well were fixed with ethanol, and the proliferation degree was measured by the DNA fluorescence method. The above operation was performed under aseptic conditions. The measurement results in Experiment 1 are shown in the graph of A of FIG. 22 for the example and in the graph of B of FIG. 22 for the comparative example.

As shown in FIG. 22, in the culture substrate of Comparative Example 4A, every fibroblast similarly proliferates, whereas in the culture substrate of Example 4A, the number of cells at the start of culture is almost the same. No increase or only a slight increase indicates that the proliferation of fibroblasts is stopped or suppressed. It was confirmed by microscopic observation that the fibroblasts were not dead.

(Examples 6A to 6L, Comparative Examples 6A to 6F and Controls 6A to 6C) In Example 3A, the cancer cell lines shown in Table 3 were used, or the PNIPAAm concentration of the coating solution or the drying temperature. The operation of Example 3A was repeated except that the values were set to the values shown in Table 3. After 5 days of culture, the DNA amount was measured by the DNA fluorescence method. The above operation was performed under aseptic conditions. ZR
-75-1 is a breast cancer strain, C-1 is a colon cancer strain, and A-549 is a lung cancer strain.

[0050]

[Table 3] ───────────────────────────────── Coating Coating Cancer cell line type PNI of solution PAAm of solution Concentration Drying temperature [w / v%] [° C] Example 6A 0.125 28 ZR-75-1 Comparative example 6A 0.25 10 ZR-75-1 Example 6B 0.25 28 ZR-75-1 Comparative example 6B 0.5 10 ZR-75-1 Example 6C 0.5 28 ZR-75-1 Example 6D 1.0 10 ZR-75-1 Control 6A 0 28 ZR-75-1 Example 6E 0.125 28 C-1 Comparative Example 6C 0.25 10 C-1 Example 6F 0.25 28 C-1 Comparative Example 6D 0.5 10 C-1 Example 6G 0.5 28 C-1 Example 6H 1.0 10 C-1 Control 6B 0 28 C-1 Example 6I 0 125 28 A-549 Comparative Example 6E 0.25 10 A-549 Example 6J 0.25 28 A-549 Comparative Example 6F 0.5 10 A-549 Example 6K 0.5 28 A-549 Example 6L 1. 0 10 A-549 Control 6C 0 28 A-549 ───────────────────────────────── Example 6A-6L The degree of proliferation of the cancer cells cultured in Comparative Examples 6A to 6F was determined according to the above mathematical expression 1, and the results are shown in FIG.

As shown in FIG. 23, according to the culture method of the present invention, various cancer cells can be proliferated at a proliferation rate almost equal to or slightly lower than that of the collagen-based culture. (Experiment 2) The coating solution used in Example 3A was dropped into a commercially available incubator in an amount of 1 microliter and 10 microliters respectively, and the incubator surface was partially coated with spots of the coating solution, and the temperature was raised to 10 ° C. When dried at each temperature of 28 ° C., the results shown in Table 4 were obtained.

[0052]

[Table 4] ─────────────────────────────────── Drying temperature [℃] Dropping amount [microliter] Properties of coating film ─────────────────────────────────── 10 1 No separation layer 10 10 No separation layer 28 1 With separation layer 28 10 With separation layer ──────────────────────────────────── Where there is a separation layer Means that the circular coating film has an opaque inner circle and its transparent outer ring (concentric circles), and that there is no separation layer means that the coating film has a transparent and uniform circular shape. .

Since cells adhere to the inner circle and cells do not adhere to the concentric outer ring (outer circle), the inner circle is made of collagen and the outer circle is made of synthetic polymer. I found out that. from these things,
A method is also conceivable in which cells are recovered by melting the inner circle with an enzyme that dissolves collagen, such as type I collagenase.

(Experiment 3) In the coating solution used in Experiment 2, the collagen concentration was 0.001 each.
5, 0.015, and 0.15 w / v% were set, dropped in the same manner as in Experiment 2, dried at 28 ° C. to form a separation layer, and an inner circle and an outer circle were obtained with a micrometer under a microscope. Was measured. The results are shown in Table 5.

[0055]

[Table 5] ─────────────────────────────────── Dropping amount Collagen concentration [w / v%] [ Microliter] 0.0015 0.015 0.15 ──────────────────────────────────── 1 Inner circle Diameter 300 μm 500 μm 700 μm 1 Outer circle diameter 2.60 mm 2.76 mm 2.38 mm 10 Inner circle diameter 500 μm 750 μm 1000 μm 10 Outer circle diameter 3.8 mm 3.7 mm 3.33 mm ────────────── ────────────────────── The results in Tables 4 and 5 indicate that the culture substrate used in the present invention is a growth factor capable of adhering cancer cells to normal cells. The main part is surrounded by the part that is mainly composed of synthetic polymer, and there is a boundary that suppresses or stops the growth of normal cells. It is.

(Examples 7A to 7C and Comparative Examples 7A to 7B)
The operation of Example 3A was repeated except that the PNIPAAm concentration was changed to the value shown in Table 6 in Example 3A. After 6 days of culture, the state of the inner surface of the well was observed with a phase contrast microscope, and the area of cells in the visual field was evaluated according to the following criteria. The above operation was performed under aseptic conditions. A: 80 to 100% O: 60 to 80% B: 30 to 60% X: 0 to 30% The results are shown in Table 6.

[0057]

[Table 6] ─────────────────────────────────── PNIPAAm concentration [w / v%] Cancer cells Normal Cell ─────────────────────────────────── Comparative Example 7A 0.25 ◎ ◎ Comparative Example 7B 0.5 ◎ ○ Example 7A 0.75 ○ △ Example 7B 1.0 ○ △ Example 7C 2.0 △ × ───────────────────────── ─────────── As seen in Table 6, when the drying temperature is 10 ° C., the cancer cells are selectively grown at a synthetic polymer concentration of 0.75 to 2.0 w / v%. be able to.

[0058]

INDUSTRIAL APPLICABILITY By the method of the present invention, cancer cell culture,
In particular, when primary cancer cell culture is performed, cancer cells proliferate while exhibiting their characteristics similar to in vivo growth, and the extension and proliferation of normal cells mixed with cancer cells are stopped or suppressed. According to this invention, there is no need to use a drug that kills normal cells or set such culture conditions. According to the present invention, using the same conditions and apparatus as those for general cell culture except for the culture substrate, the cancer cells can be selectively proliferated regardless of the type and amount of the cancer cells and normal cells coexisting therewith. You can

According to the present invention, it is possible to easily distinguish between cancer cells and normal cells, which has been difficult in the past, and to cultivate the cancer cells independently. Therefore, it is possible to perform experiments and observations of cancer cells alone. In addition, the establishment of cancer cells by isolation becomes easier.
If a cancer cell cytotoxicity test is performed using the method of the present invention, it is expected that the success rate of the culture test will be improved. In the conventional primary cancer cell culture method, the influence of contaminated normal cells is very large, so the test data was analyzed by subtracting the influence. On the other hand, in the cytotoxicity test data using the method of the present invention, since the above-mentioned influence is small, the data itself is more accurate, and accuracy is expected even if the above-mentioned influence is subtracted.

[Brief description of drawings]

FIG. 1 is a phase-contrast microscope image showing cancer cells cultured in Example 1A.

FIG. 2 is a phase-contrast microscope image showing cancer cells cultured in Example 1B.

FIG. 3 is a phase-contrast microscope image showing cancer cells cultured in Comparative Example 1A.

FIG. 4 is a phase-contrast microscope image showing the cancer cells cultured in Example 2A.

FIG. 5 is a phase-contrast microscope image showing the cancer cells cultured in Example 2B.

FIG. 6 is a phase-contrast microscope image showing the cancer cells cultured in Example 2C.

FIG. 7 is a phase-contrast microscope image showing cells cultured in Comparative Example 2A.

FIG. 8 is a phase-contrast microscope image showing cells cultured in Comparative Example 2B.

FIG. 9 is a phase-contrast microscope image showing cells cultured in Comparative Example 2C.

FIG. 10 is a phase-contrast microscope image showing cells cultured in Example 3A.

FIG. 11 is a phase-contrast microscope image showing cells cultured in Comparative Example 3A.

FIG. 12 is a phase-contrast microscope image showing cells cultured in Example 3B.

FIG. 13 is a phase-contrast microscope image showing cells cultured in Comparative Example 3B.

FIG. 14 is a phase-contrast microscope image showing the cancer cells cultured in Example 4A.

FIG. 15 is a phase-contrast microscope image showing cells cultured in control 4A.

FIG. 16 is a phase-contrast microscope image showing cancer cells cultured in Example 4B.

FIG. 17 is a phase-contrast microscope image showing cells cultured in control 4B.

FIG. 18 is a phase-contrast microscope image showing the cancer cells cultured in Example 4C.

FIG. 19 is a phase contrast microscope image showing cells cultured in Control 4C.

FIG. 20 is a graph showing the degree of proliferation of cancer cell lines and fibroblast cell lines against the synthetic high molecular weight of the coating solution.

FIG. 21 is a graph showing the degree of proliferation of cancer cell lines and fibroblast cell lines against the synthetic high molecular weight of the coating solution.

FIG. 22 is a graph showing the time-dependent proliferation of fibroblasts according to the number of culture days.

FIG. 23 is a graph showing changes in the growth rate of cancer cells depending on the concentration of the coating solution and the drying temperature.

[Explanation of symbols]

 QG-56 Cancer cell line HFL-1 Fibroblast cell line NB-1 Fibroblast cell line MRC-5 Fibroblast cell line ZR-75-1 Breast cancer cell line C-1 Colon cancer cell line A-549 Lung cancer cell line

Claims (5)

[Claims] 1. A method for growing cancer cells, which comprises culturing cancer cells mixed with normal cells on the surface of a culture substrate substantially consisting of a synthetic polymer and a cell growth factor to selectively grow the cancer cells. 2. The method according to claim 1, wherein the culture substrate is obtained by applying a coating solution prepared by mixing a synthetic polymer and a cell growth factor in water to a supporting substrate and drying the coating solution. 3. The coating solution contains 0.12 synthetic polymer.
The method according to claim 2, wherein the content is 5 w / v% or more and the drying is performed at a temperature of 25 ° C or more. 4. The coating liquid contains 0.75 synthetic polymer.
The method according to claim 2, comprising w / v% or more. 5. The method according to claim 1, wherein the synthetic polymer is a temperature-sensitive polymer.