JP2012165719A - Method for constructing three-dimensional carcinoma tissue model under pseudo-microgravity environment - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently constructing a homogeneous three-dimensional carcinoma tissue model.SOLUTION: The method for constructing the three-dimensional carcinoma tissue model includes culturing a carcinoma cell under a pseudo-microgravity environment. Preferably, the carcinoma cell to be seeded is an established cell line or a primary culture cell.

Description

  The present invention relates to a method for constructing a three-dimensional cancer tissue model in a pseudo microgravity environment. In particular, the present invention relates to a method for simultaneously and homogeneously constructing a large number of three-dimensional cancer tissue models using a large number of cell scaffold materials seeded with cancer cells.

  In conventional cancer cell efficacy screening, cancer cells are seeded and cultured on a general-purpose monolayer cell culture dish, and then a culture solution in which a drug for examining the effect is added is added. It has been evaluated whether it causes apoptosis. However, in recent years, it has been suggested that a cancer tissue model obtained by two-dimensional culture may not exhibit the same drug response as in vivo. In a three-dimensional assembly of cancer cells (that is, a three-dimensional cancer tissue model), it is considered that it is difficult to reach the inside due to physical space problems, although it is possible to reach the surface of the administered anticancer drug, It is considered that the reactivity to the administered drug differs between the two-dimensional (monolayer culture) cancer tissue model and the three-dimensional cancer tissue model.

  From such a background, evaluation using a three-dimensional cancer tissue model closer to an in vivo state is desired. As one solution, a technique has been devised in which cancer cells are seeded and cultured on a special surface having irregularities and the like to produce and evaluate spheroids (Non-patent Document 1).

  This method is advantageous in that a three-dimensional cancer tissue model can be constructed, but it is difficult to make the shape and size of the constructed spheroid tissue constant. Therefore, when the three-dimensional cancer tissue model is used in drug efficacy screening, data variation cannot be avoided, and it is very difficult to select a drug with efficacy from a large number of drug libraries.

  Evaluation of drug efficacy of cancer cells by monolayer culture and spheroid culture has the above-mentioned problems, and a technique capable of efficiently constructing a homogeneous three-dimensional tissue model is demanded in this field.

The pseudo-microgravity environment means a simulated microgravity environment artificially created by imitating the microgravity environment in outer space. Today, various bioreactors that generate a pseudo microgravity environment have been developed (for example, RWV (Rating-Wall Vessel: Patent Document 1), RCCS (Rotary Cell Culture System ^™ : Synthecon Incorporated), 3D Clinostat), and cells. (Patent Literature 2).

US Pat. No. 5,002,890 WO2005 / 056072

Watanabe M.M. et al, Biological behavior of prosthetic cancer cells in 3D culture systems. 2008 Jan; 128 (1): 37-44. Review. Japanese

  An object of the present invention is to provide a method for efficiently and simultaneously constructing a large number of homogeneous three-dimensional cancer tissue models.

  As a result of intensive studies to solve the above problems, the present inventors have found that a homogeneous three-dimensional cancer tissue model can be constructed by three-dimensional tissue culture in a pseudo microgravity environment. Furthermore, it was found that a large number of homogeneous three-dimensional cancer tissue models can be constructed by seeding a large number of scaffold materials with the same number of cancer cells and rotating them simultaneously in a pseudo-microgravity environment. The present invention is based on these findings.

The present invention has the following features.
[1] A method for constructing a three-dimensional cancer tissue model, comprising culturing cancer cells in a pseudo-microgravity environment.
[2] The construction method of [1], wherein the cancer cells to be seeded are established cell lines or primary cultured cells.
[3] The method includes the steps of seeding cancer cells on a plurality of homogeneous cell scaffold materials, and culturing the cancer cells seeded on the cell scaffold materials in a pseudo microgravity environment, [1] or [2] How to build.
[4] The construction method according to [3], wherein the cell scaffold material includes collagen, a polymer, or a mixture thereof.
[5] The construction of [4], wherein the collagen is selected from the group consisting of type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, and mixtures thereof. Method.
[6] The polymer contains one or more substances selected from the group consisting of polycaprolactone, polylactic acid, polyglycolic acid, polyalginic acid, and poly (D, L-lactic acid), [4] How to build.
[7] The construction method according to any of [4] to [6], wherein the seeding of the cancer cells on the cell scaffold material is performed by immersing the cell scaffold material in a suspension of the cancer cells.
[8] The construction method according to [7], wherein the step of immersing the cell scaffold material in a suspension of cancer cells is performed under reduced pressure.
[9] The construction method according to any one of [1] to [8], wherein the pseudo microgravity environment is an environment that gives the object gravity equivalent to 1/100 to 1/10 of the earth's gravity on a time average.
[10] Using a uniaxial rotating bioreactor or a biaxial rotating bioreactor that realizes a pseudo microgravity environment on the ground by offsetting the gravity of the earth by the stress generated by the rotation. The construction method according to any one of [1] to [9] realized.
[11] The construction method according to [10], wherein the uniaxial rotating bioreactor is an RWV (Rotating Wall Vessel) bioreactor.
[12] The construction method according to [10], wherein the biaxial bioreactor is a clinostat.
[13] The construction according to any one of [1] to [12], further comprising a step of subjecting a three-dimensional cancer tissue model obtained by culturing in a pseudo microgravity environment to monolayer culture or three-dimensional culture. Method.

  According to the present invention, a homogeneous three-dimensional cancer tissue model can be efficiently constructed. According to the present invention, a large number of homogeneous three-dimensional cancer tissue models can be efficiently constructed by using a scaffold material. By using a large number of homogeneous three-dimensional cancer tissue models, it is possible to perform drug screening such as anticancer drugs and drug efficacy evaluation with high accuracy.

FIG. 1 is a photograph showing a hematoxylin-eosin stained image of an MG63 three-dimensional cancer tissue model prepared by RWV rotation culture without using a scaffold material. FIG. 2 is a graph showing changes over time in the amount of DNA produced by exposure to doxorubicin in an MG63 three-dimensional cancer tissue model produced by RWV rotation culture. FIG. 3 is a graph showing changes over time in the amount of DNA produced by exposure to doxorubicin in an MG63 cancer tissue model prepared by planar culture. FIG. 4 is a graph showing a comparison of the amount of DNA generated by exposure to doxorubicin in a cancer tissue model of MG63 produced by flat culture (Flat) or RWV rotation culture (3D) (left 24 hours, right 48 hours).

  The present invention relates to a method for constructing a three-dimensional cancer tissue model, which comprises culturing cancer cells in a pseudo microgravity environment.

  The three-dimensional cancer tissue model produced by the method of the present invention can be used for drug efficacy screening and drug efficacy evaluation of anticancer agents and the like.

  The “three-dimensional cancer tissue model” means a three-dimensional aggregate (three-dimensional aggregate) of cancer cells obtained by three-dimensional culture of cancer cells. In the three-dimensional assembly, cancer cells can grow three-dimensionally, and by cell culture (two-dimensional cell culture) that extends two-dimensionally so that the cells stick on a cell culture plate or the like. Compared to the obtained tissue model, it is possible to realize a culture environment closer to that in the living body.

  In the present invention, various cancer cells can be used without any particular limitation. Preferably, it is a cancer cell derived from solid cancer. Solid cancer means cancer excluding blood cancer (such as leukemia), for example, stomach cancer, esophageal cancer, colon cancer, colon cancer, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, Basal cell cancer, adenocarcinoma, bone marrow cancer, renal cell cancer, ureter cancer, liver cancer, bile duct cancer, cervical cancer, endometrial cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, epithelial cancer, Craniopharyngeal cancer, laryngeal cancer, tongue cancer, fibrosarcoma, mucosal sarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, hemangiosarcoma, lymphangiosarcoma, lymphatic endothelial sarcoma, synovial tumor, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, seminoma, Wilms tumor, glioma, astrocytoma, myeloblastoma, meningioma, melanoma, neuroblastoma, medulloblastoma, retinoblast Examples include, but are not limited to, cell tumors. The cancer cell may be an established cell line or a primary cultured cell.

The cancer cells may be seeded on a cell scaffold material (scaffold).
By using a cell scaffold material, a plurality of three-dimensional cancer tissue models can be constructed simultaneously under the same conditions, and a plurality of homogeneous three-dimensional cancer tissue models can be produced. In contrast, when the cell scaffold material is not used, the cells seeded in the medium aggregate in one in the rotation culture described in detail below, compared with the case where the cell scaffold material is used. Large three-dimensional cancer tissue models can be manufactured.

  Here, “homogeneous” means that there is little or very little property difference between each constructed three-dimensional cancer tissue model, for example, for stimuli given identically under the same conditions. On the other hand, it means that each three-dimensional cancer tissue model shows the same reaction.

  “Cell scaffold material” means a material in which various cell functions such as cell adhesion, proliferation, differentiation, activation, migration, migration, and morphological change are expressed and promoted by cancer cells coming into contact with the material. . Examples of such a material include, but are not limited to, hydroxyapatite, β-TCP (tricalcium phosphate), ceramics such as α-TCP, collagen, polymer, and the like. Preferably, the cell scaffold material comprises collagen, a polymer, or a mixture thereof. Here, “collagen” is any of type I to type VII collagen, or a mixture thereof. The collagen used may be commercially available, extracted and purified from animal tissue, or purified after recombinant expression. The “polymer” includes one or more of polycaprolactone, polylactic acid, polyglycolic acid, polyalginic acid, and poly (D, L-lactic acid). The above list of materials is exemplary and not limiting.

  The cell scaffold material may be composed of any one of the above substances, or may be composed of a complex containing a plurality of kinds.

  The form of the cell scaffold material is not particularly limited, and can be a sponge, a mesh, a non-woven cloth shaped article, or the like, but is preferably porous so that cells can be uniformly seeded. “Porosity” is a structure in which an infinite number of pores (voids) of several μm to several 100 μm exist, and in the present invention, the porosity (ratio of the volume of the void portion relative to the total volume of the cell scaffold material) ) Is 40 to 90%, more preferably 60 to 90%. If the porosity is too low, cell penetration becomes insufficient, and if the porosity is too high, the strength of the structure cannot be maintained.

  The shape of the cell scaffold material is not particularly limited, and any shape such as a spherical shape, a disk shape, a particle shape, or a block shape can be used. The size of the cell scaffold material is not particularly limited, and for example, the length (diameter and the like) of the longest portion can be appropriately selected from the range of 0.2 mm to 10 mm.

  As the cell scaffold material, a commercially available material (not particularly limited, for example, Terdermis (Olympus Terumo Biomaterial Co., Ltd.)) can be used.

  The cell scaffold materials used for the culture are preferably all the same in material and size in order to ensure the homogeneity of the three-dimensional cancer tissue model to be constructed.

  The number of cell scaffold materials used for the culture is preferably adjusted as appropriate according to the volume of the bioreactor used, the number of desired three-dimensional cancer tissue models, and the like. Usually, a single three-dimensional cancer tissue model is constructed from one cell scaffold material seeded with cancer cells.

  The seeding of the cancer cells on the cell scaffold material can be performed using a known method, and the suspension obtained by suspending the cancer cells in a liquid such as a buffer solution, physiological saline, a culture solution, or a collagen solution. It can be carried out by immersing the cell scaffold material in a liquid, or by injecting the suspension into the cell scaffold material. Moreover, you may seed | inoculate on drawing pressure or pressurization conditions as needed. The number of cells to be seeded (seeding density) can be adjusted by the cell concentration and the injection amount of the suspension, and is preferably adjusted as appropriate according to the characteristics of the type of cells used and the scaffold material.

  The cell scaffold material seeded with cancer cells is preferably subjected to static culture for 1 to 24 hours before culturing in a pseudo-microgravity environment to adhere the cancer cells to the cell scaffold material.

  In the present invention, the “pseudo microgravity environment” means a simulated microgravity environment artificially created by imitating the microgravity environment in outer space. Such a pseudo microgravity environment is realized, for example, by offsetting the earth's gravity by the stress generated by the rotation. That is, since the rotating object receives a force represented by the vector sum of the earth's gravity and stress, its size and direction change with time. Therefore, on a time-average basis, the object acts only with a gravity much smaller than the earth's gravity (g), and a “pseudo-microgravity environment” that closely resembles outer space is realized.

  The “pseudo microgravity environment” needs to be an environment in which cells can proliferate and differentiate in a uniformly dispersed state without sedimentation, and aggregate in three dimensions to form a tissue mass. In other words, it is desirable to adjust the rotational speed to synchronize with the sedimentation speed of the seeded cells to minimize the effect of the Earth's gravity on the cells. Specifically, the microgravity applied to the cultured cells is desirably about 1/100 to 1/10 times the earth's gravity (g) on a time average.

In the present invention, a rotating bioreactor is preferably used in order to realize a pseudo microgravity environment. Examples of such bioreactors include RWV (Rating-Wall Vessel: US Pat. No. 5,002,890), RCCS (Rotary Cell Culture System ^™ : Synthecon Incorporated), 3D clinostat, and JP-A-8-173143. No. 9, JP-A-9-37767, JP-A-2002-45173, and the like can be used. Among them, RWV and RCCS are excellent in that they have a gas exchange function.

  Among these bioreactors, there are a uniaxial rotating type and a multi-axial rotating type having two or more axes. In the present invention, it is preferable to use a uniaxial rotating type bioreactor. In multi-axis rotation type (for example, 2-axis type clinostat etc.), shear stress (shear stress) cannot be minimized, and the sample itself rotates, so that it is in the vessel like a single axis bioreactor. This is because the state where the sample floats softly cannot be reproduced. This fluffy state is important to obtain a large three-dimensional tissue mass even in the absence of special scaffold materials.

  The RWV used in the embodiments of the present invention is a single-shaft rotary bioreactor with a gas exchange function developed by the National Aeronautics and Space Administration (NASA). The RWV is filled with a culture solution in a horizontal cylindrical bioreactor and seeded with cells, and then cultured while rotating along the horizontal axis direction of the cylinder. Within the bioreactor, a “microgravity environment” is realized that is substantially smaller than the Earth's gravity due to the stress of rotation. In this pseudo-microgravity environment, the cells are uniformly suspended in the culture solution, cultured for a required time under a minimum shear stress, and aggregate to form a tissue mass. In the case of culture using the scaffold material, the cells spread uniformly throughout the scaffold material and grow well.

  The rotation speed of the rotary bioreactor is appropriately adjusted so that the cell mass or tissue mass in the bioreactor floats in a substantially constant position without rotating. In order to maintain the above state, the rotational speed of the rotary bioreactor can be appropriately changed over time during the culture period.

  The preferred rotational speed when using RWV is appropriately set according to the diameter of the vessel and the size and mass of the tissue mass. For example, if a vessel with a diameter of 10 cm is used, it is 8 to 8 when no scaffold material is used. When using a scaffold material, about 24 rpm, it is desirable that it is about 12-15 rpm. When culturing at such a rotational speed, the gravity acting on the cells in the vessel is substantially about 1/10 to 1/100 of the ground gravity (1 g).

  As the medium, a medium usually used for culturing cancer cells, such as a MEM medium, an α-MEM medium, and a DMEM medium, can be appropriately selected according to the characteristics of the cells. In addition, antibiotics such as FBS (manufactured by Sigma) and Antibiotic-Antilytic (manufactured by GIBCO BRL) may be added to these media.

It is desirable to culture the cells under conditions of 3 to 10% CO ₂ and 30 to 40 ° C., particularly 5% CO ₂ and 37 ° C. The culture period is not particularly limited, but is at least 4 days, preferably 6 to 28 days.

  According to the method of the present invention, a large number of homogeneous three-dimensional cancer tissue models can be constructed simultaneously. Since the three-dimensional cancer tissue model is homogeneous, variation in reactivity to the same compound can be reduced or avoided, and a compound having efficacy such as anticancer activity can be screened from a large number of drug libraries. It can be said that it is extremely useful.

  Compound screening and drug efficacy evaluation using the three-dimensional cancer tissue model can be performed using a conventionally known general technique. For example, a three-dimensional cancer tissue model and a candidate compound are brought into contact with each other, and a compound showing a desired reaction in the three-dimensional cancer tissue model can be selected and identified. For example, in the case of screening a compound having an anticancer activity, a 3D cancer tissue model is brought into contact with a candidate compound, and a compound that causes growth inhibition and / or apoptosis in the 3D cancer tissue model is selected and identified. Growth inhibition and / or detection of apoptosis can be performed using a conventionally known general technique.

  In compound screening and / or drug efficacy evaluation, the three-dimensional cancer tissue model may be cultured in a monolayer culture or in a three-dimensional culture. In this case, the “monolayer culture” means that a part of cells constituting the three-dimensional cancer tissue model may adhere to the surface of the culture dish, and the three-dimensional cancer tissue model is a part of the three-dimensional structure or It means culture in a state where the whole is maintained. In this case, the “three-dimensional culture” is preferably performed using a clinostat. By using these culture methods, operations such as screening of compounds can be simplified.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the technical scope of the present invention is not limited to these examples.

Example 1: Construction of a three-dimensional cancer tissue model by culturing without using a cell scaffold material MG63 (human myeloma-derived cell line) was cultured in a culture solution (DMEM (high glucose) (WAKO) + 10% FBS (Sigma) + antibiotics−. After monolayer culture with antimycotics (Gibco, BRL)), they were detached and collected using trypsin. The collected cells were suspended in 50 cc of a culture solution having the same composition (cell concentration 2 × 10 ⁶ cells / ml), and rotational culture was performed using an RWV bioreactor (manufactured by Synthecon).

Rotational culture using the RWV bioreactor was performed for 3 weeks under the conditions of a rotational speed of 8 to 24 rpm, 37 ° C., and 5% CO ₂ using a vessel having a diameter of 10 cm (capacity 50 cc). The number of rotations was frequently adjusted so that the cell mass was visually floating. The culture medium was exchanged once every 3 days and the bubbles collected in the culture vessel were removed.

  After culturing, the obtained cell mass was fixed with microwaves using 4% paraformaldehyde and 0.1% glutaraldehyde, dehydrated according to a conventional method, and embedded in paraffin. Sections were prepared with a thickness of 5 μm, stained with hematoxylin and eosin according to a conventional method, and observed with a microscope.

  The results of hematoxylin and eosin staining are shown in FIG. As shown in FIG. 1, a large-sized (about 1.5 cm diameter) homogeneous three-dimensional cancer tissue model could be constructed.

Example 2: Construction of a large number of three-dimensional cancer tissue models using cell scaffold materials Cell scaffold materials produced collagen sponges (Terdermis (graft for dermal defect: collagen monolayer type: Olympus Terumo Biomaterials Co., Ltd.)) The ones (150 pieces) cut out by the inspection punch (3 mmφ) were used.

MG63 (Established cell line derived from human bone marrow species) was cultured in a single layer in a culture solution (DMEM (high glucose) (WAKO) + 10% FBS (Sigma) + antibiotics-antimycotics (Gibco, BRL)), and then detached using trypsin.・ Recovered. The collected cells were suspended in the same medium (cell concentration: 1 × 10 ⁶ cells / ml), 150 collagen sponges were immersed in the obtained suspension, and cells were seeded on the collagen sponge under reduced pressure. Then, after static culture for 5 hours, 150 scaffold materials / cancer cell composites were poured into a culture vessel (for 250 cc) of the RWV bioreactor together with a fresh culture solution (same composition), and rotational culture was performed. The rotation speed was initially 12 rpm, and the rotation speed was visually adjusted so that the scaffold material / cancer cell composite floated softly in the culture medium. After culturing for 6 days (rotational speed was adjusted in the range of 12 to 15 rpm), a large number of homogeneous three-dimensional cancer tissue models could be constructed (a size of about 1.5 mm in diameter).

Example 3: Evaluation of efficacy of 3D cancer tissue model The 3D cancer tissue model obtained in Example 2 was transferred to a 96-well plate, and fresh culture solution (DMEM (high glucose) (WAKO) + 10% FBS ( (Sigma) + antibiotics-antimycotics (Gibco, BRL)) for 24 hours. Next, the medium was replaced with a medium (10 μg / ml, 1.0 μg / ml, 0.1 μg / ml, 0 μg / ml, respectively) supplemented with an anticancer drug (doxorubicin, Sigma), and cultured for 2 days. Sampling was performed every 12 hours (n = 6 samples for each anticancer agent composition) and stored at −80 ° C. For comparative experiments, MG63 was flat-cultured (5000 cells / well) in a 96-well plate to construct a two-dimensional cancer tissue model, treated with doxorubicin as above, and stored at −80 ° C.

DNA quantification: Picogreen assay PicoGreen (Quanti-T ^™ PicoGreen dsDNA Reagent and Kits P11396, Invitrogen) was used for DNA quantification of each cancer tissue model. A sample stored at −80 ° C. was naturally thawed, and a cell lysate (SCIVAX Spheroid Lysis Buffer: SLB) was added to each sample at 10% of the culture solution and homogenized at room temperature (Qiagen; TissueLyser II). After collecting the supernatant by centrifugation, the same amount of TE buffer solution was added, the picogreen solution was prepared according to the manufacturer's protocol, added to the sample solution and allowed to stand for 10 minutes, and then the amount of DNA was measured by performing fluorescence measurement (Molecular) Devices SPECTRA MAX GEMINI EM).

The quantification results of DNA in each cancer tissue model are shown in FIGS.
As shown in FIG. 2, the change in the amount of DNA due to doxorubicin exposure in the three-dimensional cancer tissue model obtained by RWV rotation culture was dependent on the concentration of doxorubicin. However, when the doxorubicin concentration is 0 μg / ml (Dox0) and 0.1 μg / ml, the influence on the DNA amount of the three-dimensional cancer tissue model is small, and when the doxorubicin concentration is 1 μg / ml and 10 μg / ml, the tertiary It was observed that the influence of the original cancer tissue model on the amount of DNA was relatively large.

  On the other hand, as shown in FIG. 3, the change in the amount of DNA due to exposure of doxorubicin in the two-dimensional cancer tissue model obtained by planar culture was also dependent on the concentration of doxorubicin. However, an inversely proportional relationship was observed between the concentration change of doxorubicin and the amount of DNA, which was clearly different from the results of the three-dimensional cancer tissue model in FIG.

  FIG. 4 is a bar graph showing the DNA amount of each cancer tissue model at 24 hours and 48 hours after exposure to doxorubicin. The amount of DNA in each cancer tissue model is indicated by a relative value where the expression level is 100 when the doxorubicin concentration is 0 μg / ml (Dox0). As is clear from this result, in the two-dimensional cancer tissue model (Flat) obtained by planar culture, there is an inversely proportional relationship between the concentration change of doxorubicin and the amount of DNA, whereas RWV In the three-dimensional cancer tissue model (3D) obtained by rotating culture, a drastically high effect was obtained when doxorubicin at a concentration of 1 μg / ml or more was used.

  These results indicate that the efficacy of the same drug varies greatly between a cancer tissue model obtained by two-dimensional culture and a cancer tissue model obtained by three-dimensional culture. In general, clinical reports have shown that the effects of anticancer agents are low in solid cancers. The reason is that the cancer cells have a three-dimensional tissue structure, so that the anticancer agent is less likely to infiltrate the inside of the tissue, and the reactivity to the anticancer agent is different from that in the two-dimensional culture depending on the three-dimensional tissue. It is mentioned. As described above, the data of the cancer tissue model obtained by the three-dimensional culture was clearly different from the data of the cancer tissue model obtained by the two-dimensional culture. This is considered to be a difference in invasiveness of the anticancer agent into the tissue of the cancer tissue model and / or a change in the reactivity of the three-dimensional tissue to the anticancer agent.

  The three-dimensional cancer tissue model obtained by the method of the present invention is considered to reflect the reactivity of solid cancer with anticancer agents, and is suitable for screening drugs that are truly effective in cancer tissues.

  According to the present invention, a homogeneous three-dimensional cancer tissue model can be efficiently constructed. For this reason, the present invention can provide a three-dimensional cancer tissue model useful for drug efficacy screening and drug efficacy evaluation such as anticancer drugs, and a great contribution is expected in the field of anticancer drug development.

Claims (13)

  A method for constructing a three-dimensional cancer tissue model, comprising culturing cancer cells in a pseudo-microgravity environment.   The construction method according to claim 1, wherein the cancer cells to be seeded are established cell lines or primary cultured cells.   The construction method according to claim 1, comprising the steps of seeding cancer cells on a plurality of homogeneous cell scaffold materials, and culturing the cancer cells seeded on the cell scaffold materials in a pseudo-microgravity environment. .   The construction method of claim 3, wherein the cell scaffold material comprises collagen, a polymer, or a mixture thereof.   The construction method according to claim 4, wherein the collagen is selected from the group consisting of type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, and a mixture thereof. .   5. The polymer of claim 4, wherein the polymer comprises one or more substances selected from the group consisting of polycaprolactone, polylactic acid, polyglycolic acid, polyalginic acid, and poly (D, L-lactic acid). Construction method.   The construction method according to any one of claims 4 to 6, wherein the seeding of the cancer cells on the cell scaffold material is performed by immersing the cell scaffold material in a suspension of the cancer cells.   The construction method according to claim 7, wherein the step of immersing the cell scaffold material in a suspension of cancer cells is performed under reduced pressure.   The construction method according to any one of claims 1 to 8, wherein the pseudo microgravity environment is an environment that gives an object gravity corresponding to 1/100 to 1/10 of the earth's gravity on a time average.   A pseudo microgravity environment is realized by using a single-axis rotating bioreactor or a biaxial rotating bioreactor that realizes a pseudo-microgravity environment on the ground by offsetting the gravity of the earth by the stress generated by the rotation. The construction method according to any one of claims 1 to 9.   The construction method according to claim 10, wherein the single-shaft rotating bioreactor is an RWV (Rotating Wall Vessel) bioreactor.   The construction method according to claim 10, wherein the biaxial bioreactor is a clinostat.   Furthermore, the construction method of any one of Claims 1-12 including the process of attaching | subjecting the three-dimensional cancer tissue model obtained by culture | cultivating in a pseudo microgravity environment to a monolayer culture or a three-dimensional culture. .

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