Human hepatoma cell sheet and method for culturing human hepatoma cells

Abstract

To provide a model liver cell highly expressing BSEP (Bile Salt Export Pump) by using an ordinary liver cancer cell strain.SOLUTION: In a human liver cancer cell sheet of the present invention, PLC / PRF / 5 cells are present in a gap of a porous body, and the PLC / PRF / 5 cells expressing bile acid transporter BSEP are unevenly distributed on one principal surface side. A culture method of a human liver cancer cell of the present invention uses the porous body as a cell culture carrier, and the human liver cancer cell is a PLC / PRF / 5 cell.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a human hepatocellular carcinoma cell sheet and a method for culturing human hepatocellular carcinoma cells.

Background Art

[0002] One of the side effects of drugs is Drug-Induced Liver Injury (DILI). It is known that the earliest symptom of DILI is cholestatic liver toxicity (liver toxicity caused by inhibited bile excretion from hepatocytes that produce bile; for example, it occurs when bile excretion via the bile acid transporter BSEP (Bile Salt Export Pump) in hepatocytes is inhibited). That is, if cholestatic liver toxicity can be evaluated and predicted in advance in the early stage of the development of pharmaceuticals and the like, it is thought that the development of drugs with reduced occurrence of the side effect DILI will be promoted.

[0003] On the other hand, in vitro evaluation of cholestatic liver toxicity requires model hepatocytes that highly express BSEP, which is the evaluation target molecule. Currently, the most mainstream model cells are normal human hepatocytes, but these have problems such as inability to obtain reproducible results due to individual differences among donor providers and variations between production lots, inability to perform long-term culture, and high cost. Novel cell sources have also emerged, such as hepatocytes derived from chimeric mice transplanted with human hepatocytes and the normal hepatocyte-derived immortalized cell line HepaRG (registered trademark; Non-Patent Document 1), but the problem of high cost of the cells and dedicated special media has not been solved.

[0004] For this reason, there has been a demand for the development of model hepatocytes using general hepatocellular carcinoma cell lines that are inexpensive, simple, highly reproducible, and capable of long-term culture. However, many hepatocellular carcinoma cell lines have reduced or lost liver function, and it is difficult to achieve high BSEP expression under conventional culture conditions.

Prior Art Literature

Non-Patent Literature

[0005] [Non-Patent Document 1] Gripon P et al. Infection of a human hepatoma cell line by hepatitis B virus. Proc Natl Acad Sci US A., 99, 15655-15660 (2002) [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] The object of the present invention is to provide a model hepatocyte that highly expresses BSEP using a common liver cancer cell line. [Means for solving the problem]

[0007] This invention relates to the following inventions: [1] A human hepatocellular carcinoma cell sheet in which PLC / PRF / 5 cells are present in the voids of a porous material, The PLC / PRF / 5 cells expressing the bile acid transporter BSEP are predominantly located on one of the main surfaces. Human liver cancer cell sheet. [2] Human liver cancer cell sheet from [1], used to evaluate bile excretion function. [3] A method for culturing human hepatocellular carcinoma cells using a porous material as a cell culture carrier, The human liver cancer cells are characterized by being PLC / PRF / 5 cells. A method for culturing human liver cancer cells. [4] Step 1: A step of preparing two types of culture vessels having wells (culture vessel (A) and culture vessel (B)), a porous body, PLC / PRF / 5 cells, and a culture medium for the PLC / PRF / 5 cells. Step 2: A step of filling the wells with the culture medium until all of the porous bodies placed on the bottom surface of the wells of the culture vessel (A) are submerged. Step 3: A step of seeding the PLC / PRF / 5 cells onto the main surface of the porous body, which is placed on the bottom surface of the well, on the side opposite to the bottom surface of the well. Step 4: A step of culturing the PLC / PRF / 5 cells on the porous body in the well. Step 5: A step of filling the wells of the culture vessel (B) with the culture medium so that all of the porous material is immersed in the wells in the subsequent steps. Step 6: Remove the porous body from the well of the culture vessel (A) along with the cultured PLC / PRF / 5 cells. Step 7: The step of placing the porous body removed in Step 6 onto the bottom surface of the well of the culture vessel (B) such that the side of the porous body on which the PLC / PRF / 5 cells were seeded is opposite to the bottom surface of the well. Step 8: A step of culturing the PLC / PRF / 5 cells in the wells of the culture vessel (B), It is equipped with, A method for culturing human liver cancer cells according to [4], characterized in that the area on which the porous material can be installed on the bottom surface of the well of culture vessel (B) is larger than the area on which the porous material can be installed on the bottom surface of the well of culture vessel (A). [5] The method for culturing human liver cancer cells according to [4], characterized in that the culture period of the PLC / PRF / 5 cells in step 8 is longer than 5 days. [Effects of the Invention]

[0008] The human liver cancer cell sheet and human liver cancer cell culture method of the present invention can provide model liver cells that highly express BSEP. [Brief explanation of the drawing]

[0009] [Figure 1] This graph was created by plotting the fluorescence intensity of PLC / PRF / 5 cell nuclei and BSEP in the thickness direction of the human liver cancer cell sheet prepared in Example 6. [Figure 2] This graph was created by plotting the ratio of BSEP fluorescence intensity to PLC / PRF / 5 cell nucleus fluorescence intensity in the thickness direction of the human liver cancer cell sheet prepared in Example 6. [Modes for carrying out the invention]

[0010] (Human hepatocellular carcinoma cell sheet of the present invention) In the human hepatocellular carcinoma cell sheet of the present invention, PLC / PRF / 5 cells are present in the voids of a porous body, and the PLC / PRF / 5 cells expressing the bile acid transporter BSEP are unevenly distributed on one main surface side.

[0011] The porous body used as a cell culture carrier in the present invention is not particularly limited as long as it is a sheet-like structure having a three-dimensional space (voids) capable of inducing proliferation and differentiation of cells. Examples thereof include fiber sheets, more specifically woven fabrics, knitted fabrics, fiber webs, nonwoven fabrics, porous films, foams, and the like. Fiber webs or nonwoven fabrics are preferably used because they are close to in vivo structures where cells actually exist.

[0012] When a fiber sheet is used as the porous body, the fibers constituting the fiber sheet are not limited as long as they can form a fiber sheet that can be used as a carrier for inducing cell proliferation and differentiation. For example, either organic fibers or inorganic fibers, or both organic fibers and inorganic fibers can be employed.

[0013] As constituent components of organic fibers, for example, polyamide polymer, polyacrylonitrile, polylactic acid, poly(lactic-co-glycolic acid) (PLGA), polycaprolactone, polybutylene succinate, or proteins (e.g., gelatin, collagen), water-soluble polymers (e.g., polyvinyl alcohol, polyethylene glycol, poly(N-vinylpyrrolidone), Nafion), general-purpose polymers (e.g., polystyrene, poly(methyl methacrylate) (PMMA)), etc., can be used alone or in combination.

[0014] As the constituent components of the inorganic fiber, for example, SiO₂, Al₂O₃, B₂O₃, TiO₂, ZrO₂, CeO₂, FeO, Fe₃O₄, Fe₂O₃, VO₂, V₂O₅, SnO₂, CdO, LiO₂, WO₃, Nb₂O₅, Ta₂O₅, In₂O₃, GeO₂, PbTi₄O₉, LiNbO₃, BaTiO₃, PbZrO₃, KTaO₃, Li₂B₄O₇, NiFe₂O₄, SrTiO₃, etc. can be mentioned. The inorganic fiber may be composed of one of these oxides or two or more of these oxides.

[0015] An exemplary culture method mentioned in the present invention includes a step of moving the porous body together with PLC / PRF / 5 cells from a first culture vessel (culture vessel (A)) to a second culture vessel (culture vessel (B)), therefore, a fiber sheet exhibiting sufficient strength and shape retention is preferable. For example, a fiber sheet in which fibers are bonded or fused to each other is preferable, and an inorganic fiber sheet having rigidity in the component itself is more preferable, specifically, a silica fiber nonwoven fabric with inter-fiber bonding can be mentioned.

[0016] When the fiber sheet is composed of two or more types of fibers with different compositions, it may be a fiber sheet in which various fibers are mixed, or it may be a fiber sheet provided with fiber layers having different compositions from each other. Further, it may be a fiber sheet in which the aforementioned organic fibers and inorganic fibers are mixed, or a fiber sheet provided with an organic fiber layer and an inorganic fiber layer.

[0017] In the present invention, PLC / PRF / 5 cells are used as human hepatoma cells. PLC / PRF / 5 cells are a cell line derived from human hepatoma, also called Alexander cells, and are known to express the bile salt export pump (BSEP), which is involved in bile excretion (Tomonari T et al. Oncotarget. 2016; 7:7207-7215.). They are available from various cell banks (e.g., ATCC, JCRB Cell Bank), and can be cultured, for example, in RPMI 1640 medium or DMEM medium containing 10% by volume fetal bovine serum at 37°C under conditions of 5% carbon dioxide concentration.

[0018] In the human liver cancer cell sheet of the present invention, PLC / PRF / 5 cells expressing BSEP are predominantly located on one main surface. In this specification, "biased towards one main surface" means that the distribution of BSEP expression is biased towards one main surface compared to the distribution of PLC / PRF / 5 cells in the human liver cancer cell sheet according to the present invention. Specifically, it is possible to determine whether a human liver cancer cell sheet has BSEP-expressing PLC / PRF / 5 cells biased towards one main surface by the following method.

[0019] (Judgment method 1) (1) Stain the human liver cancer cell sheet to stain the cell nuclei and BSEP. At this time, staining should be performed so that the color development of the stained cell nuclei (e.g., red fluorescence) and the color development of the stained BSEP (e.g., green fluorescence) are different. (2) The stained human liver cancer cell sheet is cut in the thickness direction (a direction perpendicular to the main surface) from one main surface to the other, and a cross-sectional photograph of the human liver cancer cell sheet is taken with the stained cell nuclei fluorescing. Note that by using a device that can observe the inside of the specimen, such as a confocal laser microscope, a cross-sectional photograph can also be obtained without actually cutting the sheet. Next, a cross-sectional photograph of the human liver cancer cell sheet is taken with the stained BSEP fluorescing in the same cross-section. (3) A new cross-sectional image is created by superimposing the two captured cross-sectional images, and the areas where the fluorescence of the stained cell nuclei and the fluorescence of the stained BSEP overlap and produce different fluorescence colors (for example, areas where red and green overlap to produce yellow fluorescence) are visually confirmed. If no areas with different fluorescence colors are found, it can be determined that there are no human liver cancer cells expressing BSEP in the human liver cancer cell sheet. Furthermore, the more areas with different fluorescence colors there are, the more BSEP is expressed in the human liver cancer cell sheet. In the aforementioned new cross-sectional photograph, if there are more areas with different fluorescent colors on one main surface side of the human liver cancer cell sheet than on the other main surface side, it can be determined that the cells expressing BSEP (such as PLC / PRF / 5 cells) in the human liver cancer cell sheet are predominantly located on the other main surface side.

[0020] Furthermore, if it is necessary to more accurately determine the distribution of BSEP-expressing PLC / PRF / 5 cells in a human hepatoma cell sheet, the following method can be used to determine whether or not the BSEP-expressing PLC / PRF / 5 cells are predominantly distributed on one side of the human hepatoma cell sheet.

[0021] (Judgment method 2) (1) Stain the human liver cancer cell sheet to stain the cell nuclei and BSEP. At this time, staining should be performed so that the color development of the stained cell nuclei (e.g., red fluorescence) and the color development of the stained BSEP (e.g., green fluorescence) are different. (2) The stained human liver cancer cell sheet is cut at 0.5 μm intervals in the thickness direction (direction perpendicular to the main surface) from one main surface to the other main surface, so as to be parallel to one of the main surfaces, thereby obtaining multiple thin films of the human liver cancer cell sheet. Note that by using a device that can observe the inside of the specimen, such as a confocal laser microscope, it is also possible to obtain main surface photographs of each thin film described later without actually cutting the sheet. (3) Take a main surface photograph of each thin film at a magnification of 400x with the stained cell nuclei fluoresced. Then, take a main surface photograph of each thin film with the stained BSEP fluoresced. (4) For all photographs taken, determine the fluorescence value c of the cell nuclei and the fluorescence value b of the BSEP in each photograph. Then, the smallest fluorescence value c of each cell nucleus is set as the reference value (=1), and the relative value of the fluorescence value c of the cell nuclei in each photograph with respect to this reference value is treated as the fluorescence amount of the cell nuclei in each photograph. Similarly, the smallest fluorescence value b of each BSEP is set as the reference value (=1), and the relative value of the fluorescence value b of the BSEP in each photograph with respect to this reference value is treated as the fluorescence amount of the BSEP in each photograph. (5) A graph is created by plotting the quantified fluorescence amount of the cell nucleus and the fluorescence amount of BSEP in each thin film on a coordinate system where the X axis is the length (thickness, in μm) from one main surface of the human liver cancer cell sheet in the thickness direction, and the Y axis is the fluorescence amount (for example, Figure 1 created in the example described below). (6) Plot the length (thickness, in μm) from one main surface of the human liver cancer cell sheet in the thickness direction on the X axis, and the ratio of the fluorescence amount of BSEP to the fluorescence amount of cell nuclei in each thin film, as determined in item (5) above, on the Y axis to create a graph (for example, Figure 2 created in the example described below). In the graph created in this way (5), the fluorescence intensity plot of the cell nucleus can be considered as the distribution of the number of cells in the sheet thickness direction (e.g., the number of PLC / PRF / 5 cells), and the fluorescence intensity plot of BSEP can be considered as the expression level of BSEP expressed in cells present in the sheet thickness direction (e.g., PLC / PRF / 5 cells). Furthermore, in the graph of the coordinate axes created in item (6) above, if the value of the Y axis increases significantly as you move toward one end of the X axis, it can be determined that in the human liver cancer cell sheet, cells expressing BSEP (such as PLC / PRF / 5 cells) are predominantly located on the other major surface side rather than the other major surface side.

[0022] The cell density within the sheet and the cellular state other than BSEP expression are not particularly limited as long as the cells are in a healthy state when the sheet is used. However, it is preferable that the cells completely fill the main surface direction of the sheet, leaving no gaps, so that bile acid transport evaluation can be performed with the sheet as a boundary. Furthermore, it is preferable that the entire sheet functions as a membrane. Specifically, it is preferable that proteins necessary for the formation of tight junctions (such as E-cadherin and ZO-1) are expressed between cells, and that the sheet has transepithelial electrical resistance (TEER). It is also preferable that the uptake transporter NTCP (sodium taurocholate cotransporting polypeptide), which functions in conjunction with the bile acid excretion transporter BSEP, is present on the main surface side other than the main surface side where BSEP is eccentrically distributed.

[0023] The presence or absence of BSEP expression can be confirmed, for example, by immunostaining using an anti-BSEP antibody (primary antibody) and a secondary antibody labeled with a fluorescent dye, as shown in the examples described later. In this case, by using a staining agent that can stain all cells regardless of whether BSEP is expressed (e.g., a nuclear staining agent), cells expressing BSEP will show positive results in both immunostaining and cell staining (e.g., nuclear staining), while cells that do not express BSEP will show positive results in cell staining only.

[0024] In the embodiment described later, when the sheet was placed on a petri dish, PLC / PRF / 5 cells expressing BSEP were concentrated on the main surface side of the sheet (the side that does not come into contact with the bottom of the petri dish), while PLC / PRF / 5 cells that do not express BSEP were concentrated on the main surface side of the sheet (the side that comes into contact with the bottom of the petri dish). In other words, in the embodiments described later, we were able to provide a human liver cancer cell sheet in which PLC / PRF / 5 cells expressing BSEP are predominantly located on one main surface.

[0025] This structure (i.e., a structure that shows eccentricity regarding BSEP expression) is capable of reproducing bile uptake (function of the side where BSEP is not expressed) and excretion (function of the side where BSEP is expressed). By using the human liver cancer cell sheet of the present invention, it becomes possible to evaluate bile uptake and excretion, and it is expected that this will promote the development of drugs that reduce the occurrence of drug-induced liver injury, one of the side effects of drugs.

[0026] The uses of the human liver cancer cell sheet of the present invention are not particularly limited, but for example, it can be used to cover the interface that divides a culture space into two, and to evaluate the transport of substances between the two spaces. Inserts (e.g., Transwell (Corning), Cell Culture Insert (Thermo Fisher Scientific)) are known as tools for dividing the wells of a culture vessel into two spaces, inner and outer, and this human liver cancer cell sheet can be used by placing it in the part of these inserts where the membrane is installed. If the side of this human liver cancer cell sheet where BSEP is unevenly distributed faces inward into the insert, and a culture medium containing bile acids is added to the outside of the insert and a normal culture medium is added to the inside of the insert, it is thought that the bile acids will be transported from the outside to the inside at a constant rate via the BSEP of this sheet. As an example of drug evaluation, a method can be used in which a test substance is added to the inner and outer culture media and the change in the transport rate of bile acids from the outside to the inside is measured. If the measurement results in a decrease in the transport rate of bile acids, it can be evaluated that the test substance may inhibit the function of BSEP and cause cholestatic toxicity.

[0027] (Method for culturing human liver cancer cells according to the present invention) In the present invention's method for culturing human liver cancer cells, PLC / PRF / 5 cells are used as the human liver cancer cells, and a porous material is used as the cell culture carrier. Furthermore, in a preferred embodiment of the human liver cancer cell culture method of the present invention, Step 1: A step of preparing two types of culture vessels having wells (culture vessel (A) and culture vessel (B)), a porous material, PLC / PRF / 5 cells, and a culture medium for the PLC / PRF / 5 cells. Step 2: A step of filling the wells with the culture medium until all of the porous bodies placed on the bottom surface of the wells of the culture vessel (A) are submerged. Step 3: A step of seeding the PLC / PRF / 5 cells onto the main surface of the porous body, which is placed on the bottom surface of the well, on the side opposite to the bottom surface of the well. Step 4: A step of culturing the PLC / PRF / 5 cells on the porous body in the well. Step 5: A step of filling the wells of the culture vessel (B) with the culture medium so that all of the porous material is immersed in the wells in the subsequent steps. Step 6: Remove the porous body from the well of the culture vessel (A) along with the cultured PLC / PRF / 5 cells. Step 7: The step of placing the porous body removed in Step 6 onto the bottom surface of the well of the culture vessel (B) such that the side of the porous body on which the PLC / PRF / 5 cells were seeded is opposite to the bottom surface of the well. Step 8: A step of culturing the PLC / PRF / 5 cells in the wells of the culture vessel (B). We will implement this.

[0028] In step 1, two types of well plates having wells (culture vessel (A) and culture vessel (B)), a porous material, PLC / PRF / 5 cells, and a culture medium for the PLC / PRF / 5 cells are prepared.

[0029] The culture vessels (A) and (B) prepared in step 1 are not particularly limited, as long as the area where the porous material can be installed on the well bottom surface of culture vessel (A) (Sa) is larger than the area of ​​the main surface (Sp) of the porous material, and the area where the porous material can be installed on the well bottom surface of culture vessel (B) (Sb) is larger than Sa. Culture vessels commonly used for cell culture can be appropriately selected from, for example, 24-well plates, 12-well plates, 8-well plates, 6-well plates, 4-well plates, petri dishes, glass-bottom dishes, etc.

[0030] The ratio of Sa to Sp (Sa / Sp) is not particularly limited as long as it is greater than 1, but it is preferable that it be close to 1 so that the seeded cells can be efficiently arranged and adhere to one main surface of the porous body. Therefore, it is more preferable that it is 2 or less, and even more preferable that it is 1.5 or less. Furthermore, the ratio of Sb to Sa (Sb / Sa) is not particularly limited as long as it is greater than 1, but is preferably 5 or more, more preferably 10 or more, and even more preferably 20 or more. The reason why BSEP is highly expressed and unevenly distributed in relation to BSEP expression by the culture method of the present invention is not yet clear, but according to the inventors' speculation, it is thought that this is because the area on which the porous material can be installed on the bottom surface of the well is increased, causing it to move to an environment rich in culture medium. While there is no specific upper limit to the Sb / Sa ratio, in practice, it is typically 50 or less due to constraints such as the placement of culture vessels and the cost of culture media.

[0031] The porous material and PLC / PRF / 5 cells prepared in step 1 can be described in the same way as above in the description of (the human liver cancer cell sheet of the present invention). For PLC / PRF / 5 cells, for example, RPMI1640 medium or DMEM medium containing 10% fetal bovine serum by volume can be used as the culture medium.

[0032] In step 2, the culture medium is filled into the wells until all of the porous bodies placed on the bottom surface of the wells of the culture vessel (A) are submerged. In step 2, the amount of culture medium to fill the wells of the culture vessel (A) is not particularly limited as long as it is enough to immerse all of the porous bodies placed on the bottom surface of the wells. However, if the culture medium is too deep, the distance between the porous bodies and the gas phase will increase, which may result in poor oxygen supply. Therefore, it is typically preferable that the culture medium is 1 to 10 mm deep from the bottom surface of the wells.

[0033] In step 3, the PLC / PRF / 5 cells are seeded on the main surface of the porous body, which is placed on the bottom surface of the wells of the culture vessel (A), on the side opposite to the bottom surface of the wells. The number of PLC / PRF / 5 cells to be seeded per unit area of ​​the bottom surface of the wells can be appropriately determined according to, for example, the size of the wells and the porous body and the number of culture days, for example, 1.0 × 10 4 ~3.0×10 5 cells / cm 2 That is the case.

[0034] In step 4, PLC / PRF / 5 cells are cultured on a porous material in the wells of culture vessel (A) (hereinafter referred to as primary culture), and it is preferable that the PLC / PRF / 5 cells are adhered to the porous material at this time. PLC / PRF / 5 cells can be cultured at 37°C and 5% carbon dioxide concentration using, for example, RPMI1640 medium containing 10% fetal bovine serum by volume or DMEM medium. The culture period can be adjusted, but if culture is continued in culture vessel (A), unwanted cells that spilled from the porous material during seeding will also proliferate, accelerating the deterioration of the culture medium and potentially adversely affecting the cells on the porous material. Therefore, it is preferable that the culture period be the time from cell adhesion to the porous material until the deterioration of the culture medium begins. Specifically, it is preferable that this period is from 4 hours after seeding, which is the time required for cell adhesion, until the color of the indicator used to determine deterioration in the culture medium begins to change (e.g., phenol red, which changes from reddish-purple to yellow due to the accumulation of acidic waste products from the cells). Furthermore, as long as it is possible to maintain a state where the culture medium does not deteriorate through replacement, the primary culture period can be extended by replacing the culture medium as needed.

[0035] In step 5, the culture medium is filled into the wells of the culture vessel (B) so that all of the porous material will be immersed in the subsequent steps. In step 5, the amount of culture medium to fill the wells of the culture vessel (B) is not particularly limited, as is the same as in step 2, as long as it is enough to immerse all of the porous bodies placed on the bottom surface of the wells, but typically it is preferably 1 to 10 mm above the bottom surface of the wells.

[0036] In step 6, the porous body is removed from the well of the culture vessel (A) along with the cultured PLC / PRF / 5 cells. In the culture method of the present invention, it is preferable to use an inorganic fiber sheet in which fibers are bonded together to exhibit sufficient strength and shape retention as the porous body.

[0037] In step 7, the porous body removed in step 6 is placed on the bottom surface of the well of the culture vessel (B) such that the side of the porous body on which the PLC / PRF / 5 cells are seeded is opposite to the bottom surface of the well. Furthermore, in step 8, the PLC / PRF / 5 cells are cultured in the wells of the culture vessel (B) (hereinafter referred to as secondary culture).

[0038] The transfer of the porous material from culture vessel (A) to culture vessel (B) in steps 6 and 7 shall be carried out in such a manner that the state of the porous material transferred to culture vessel (B) remains unchanged from the state of the porous material placed in culture vessel (A) (i.e., the relationship between the upper and lower surfaces of the porous material remains unchanged).

[0039] The secondary culture can be carried out in the same manner as the primary culture in step 4, except that the area where the porous material can be installed on the bottom surface of the wells of culture vessel (B) (Sb) is larger than the area where the porous material can be installed on the bottom surface of the wells of culture vessel (A) (i.e., the amount of culture medium is increased).

[0040] While there are no particular limitations on the number of days for secondary culture, it is preferable to culture for at least two days to increase BSEP expression, and more preferably for more than five days to ensure a skewed distribution of PLC / PRF / 5 cells in the porous medium in terms of BSEP expression. As an upper limit, although BSEP expression increases with the number of culture days, it is practically limited to 20 days or less to avoid unnecessarily prolonging the time required to obtain a human liver cancer cell sheet and to prevent problems such as internal necrosis due to excessive cell proliferation. During secondary culture, it is preferable to change the culture medium as needed while carefully monitoring the color change of the indicator used to determine deterioration in the culture medium.

[0041] According to the culture method of the present invention, since PLC / PRF / 5 cells are used as human hepatocytes, it is possible to provide a human hepatocyte sheet carrying human hepatocytes that highly express BSEP. For example, as shown in Comparative Examples 2 and 3 described later, when HepG2 cells, a human hepatocyte-derived cell line, were used, BSEP was not expressed.

[0042] Furthermore, the culture method of the present invention makes it possible to achieve three-dimensional culture that mimics the biological environment by using a porous material as a cell culture carrier, thereby providing a human hepatocellular carcinoma cell sheet that carries more human hepatocellular carcinoma cells expressing BSEP. For example, as shown in Comparative Example 1 described later, even when using PLC / PRF / 5 cells, BSEP expression could not be confirmed when seeded directly into wells without using a porous material.

[0043] Furthermore, according to a preferred embodiment of the culture method of the present invention that performs both primary and secondary culture, it is possible to provide human hepatocellular carcinoma cell sheets carrying human hepatocellular carcinoma cells that express more BSEP and exhibit eccentric distribution of BSEP expression in a shorter period of time compared to the culture method of the present invention that performs only primary culture (for example, Examples 1 and 2 described later). [Examples]

[0044] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0045] Preparation of human liver cancer cell sheets In Examples 1-6 and Comparative Examples 1-3 described below, unless otherwise specified, human liver cancer cell sheets were prepared according to the following culture procedure. A 24-well plate (diameter of circular wells: 15.6 mm) filled with RPMI1640 medium (250 μL / well) containing 10% fetal bovine serum by volume was prepared, and a circular silica fiber nonwoven fabric (Cellbed (diameter: 13 mm, average fiber diameter: approx. 1 μm, thickness: 250 μm, registered trademark); manufactured by Nippon Vilene Co., Ltd.) was immersed in it and placed on the bottom surface of the wells as a porous cell culture carrier, with 3.0 × 10⁶ cells per well placed on top of it. 5 Human hepatocellular carcinoma-derived cell line PLC / PRF / 5 (JCRB cell bank, cell number IFO50069, culture lot 10252013) was seeded in a suspension medium at a cell / 250 μL concentration. Primary culture was performed for 2, 7, or 14 days at 37°C with a 5% carbon dioxide concentration. For a 2-day culture, no medium changes were performed during the culture period. For a 7-day culture, all 500 μL / well of medium was replaced with fresh medium on days 2, 4, and 6 after seeding. For a 14-day culture, all 500 μL / well of medium was replaced with fresh medium on days 2 and 4 after seeding, and then all 500 μL / well of medium was replaced with fresh medium daily from day 6 onward.

[0046] After primary culture for the prescribed number of days, the silica fiber nonwoven fabric was transferred to a 100 mm petri dish filled with RPMI1640 medium (10 mL) containing 10% fetal bovine serum by volume, and secondary culture was performed for the prescribed number of days to prepare human liver cancer cell sheets. After transferring to the 100 mm petri dish, the culture medium was completely replaced every three days.

[0047] Examples 1 and 2 In this example, human liver cancer cell sheets were prepared by performing only primary culture in a 24-well plate lined with silica fiber nonwoven fabric. Primary culture was performed for 7 days in Example 1 and for 14 days in Example 2.

[0048] Examples 3-6 In this example, human liver cancer cell sheets were prepared by performing primary culture for two days in a 24-well plate lined with silica fiber nonwoven fabric, then transferring the silica fiber nonwoven fabric along with the cells to a 100 mm petri dish for secondary culture. Secondary culture was performed for 2 days in Example 3, 5 days in Example 4, 8 days in Example 5, and 12 days in Example 6.

[0049] Comparative Example 1 In this comparative example, a single-layer human hepatoma cell sheet consisting solely of the human hepatoma-derived cell line PLC / PRF / 5 was prepared by culturing the cells under single-layer culture conditions using a flat-bottomed culture vessel without using silica fiber nonwoven fabric. For subsequent fluorescence observation, a glass-bottomed dish that does not exhibit autofluorescence was used. Furthermore, since cell detachment occurs if the cells become too densely packed under single-layer culture conditions, the cell seeding density and other conditions were adjusted so that a perfectly gapless single-layer cell sheet was formed when the culture period was set to the same 7 days as in Example 1. Specifically, 2.0 × 10⁶ cells were seeded in a glass-bottomed dish with a circular, flat bottom and a diameter of 35 mm. 4 Cells were seeded in a cell suspension at a concentration of 2 mL / cell and cultured for 7 days at 37°C with a 5% carbon dioxide concentration. The same culture medium as in Example 1 was used, and the entire volume was replaced with fresh medium on days 2, 4, and 6.

[0050] Comparative Example 2 In this comparative example, instead of PLC / PRF / 5 cells, human liver cancer-derived cell line HepG2 cells (RIKEN BioResource Center, cell number RCB1648) were used, and a single-layer human liver cancer cell sheet consisting only of human liver cancer-derived cell line HepG2 cells was prepared on a glass-bottom dish in the same manner as in Comparative Example 1, without using silica fiber nonwoven fabric.

[0051] Comparative Example 3 In this comparative example, the procedure in Example 1 was repeated to prepare a human hepatoma cell sheet, except that human hepatoma-derived cell line HepG2 cells were used instead of PLC / PRF / 5 cells.

[0052] Immunostaining of human liver cancer cell sheets For each human liver cancer cell sheet prepared in Examples 1-6 and Comparative Examples 1-3, immunohistochemical staining using anti-BSEP antibody and nuclear staining using TO-PRO®-3 (Thermo Fisher Scientific) were performed.

[0053] First, to fix the cultured cells in the silica fiber nonwoven fabric, each human hepatoma cell sheet was treated for 20 minutes with phosphate-buffered saline (PBS) containing 4% paraformaldehyde by volume. Subsequently, membrane permeabilization was performed for 30 minutes with PBS containing 1% Triton-X100 by volume to allow the antibody and nuclear stain to penetrate the cell membrane and enter the cells. Furthermore, to suppress nonspecific reactions of the antibody and nuclear stain, blocking treatment was performed for 30 minutes with PBS containing 10% goat serum and 0.1% Tween20 by volume, respectively.

[0054] After these pretreatments, immunohistochemistry was performed according to standard procedures, using anti-BSEP antibody (F-6) (Santa Cruz Biotechnology; catalog #sc-74500) as the primary antibody and anti-mouse IgG(H+L)F(ab')2 fragment (Alexa Fluor® 488 conjugate; Cell Signaling Technology; catalog #4408) as the secondary antibody. Subsequently, TO-PRO-3 staining (nuclear staining) was performed according to the attached instructions. The human liver cancer cell sheets stained in this manner were then subjected to the aforementioned (Determination Method 1). Furthermore, the human liver cancer cell sheet prepared in Example 6, which was confirmed to have the highest BSEP expression level (BSEP expression level evaluated as ○○○) as a result of being subjected to (Determination Method 1), was subjected to (Determination Method 2) as described above. The graphs created by subjecting the human liver cancer cell sheet prepared in Example 6 to (Determination Method 2) as described above are shown in Figures 1 and 2. In these graphs, the left side of the paper represents the main surface (0 μm) of the human liver cancer cell sheet that was in contact with the culture vessel during culture, and the right side of the paper represents the main surface (59.5 μm) of the human liver cancer cell sheet that was not in contact with the culture vessel during culture.

[0055] The results are shown in Tables 1 and 2. Regarding the "BSEP Expression Level" column in Tables 1 and 2, "×" indicates that when subjected to (Determination Method 1), no areas with different fluorescence colors were observed, meaning that the human hepatoma cell sheet does not contain any human hepatoma cells expressing BSEP. On the other hand, the areas marked "△," "〇," "〇〇," and "〇〇〇" show different fluorescent colors, indicating that these are human liver cancer cell sheets containing human liver cancer cells that express BSEP. Furthermore, the amount of BSEP expression increases from "△" to "〇〇〇." In Tables 1 and 2, the "Presence or Absence of Uneven Distribution" column indicates that the presence of human liver cancer cells expressing BSEP could not be confirmed, and therefore the distribution pattern of BSEP-expressing cells was not examined. Furthermore, "×" indicates a human liver cancer cell sheet in which cells expressing BSEP (such as PLC / PRF / 5 cells) could not be confirmed to be predominantly located on one side of the sheet. On the other hand, "〇" indicates a human liver cancer cell sheet in which cells expressing BSEP (such as PLC / PRF / 5 cells) were confirmed to be predominantly located on one side of the sheet.

[0056] [Table 1]

[0057] [Table 2]

[0058] A comparison of the comparative example and the example revealed that culturing PLC / PRF / 5 cells using a porous material can provide a model hepatocyte that highly expresses BSEP. Furthermore, a comparison of Examples 1-2 and 3-6 revealed that by employing the two-stage culture method according to the present invention, model hepatocytes highly expressing BSEP can be efficiently cultured and provided even with a short culture period. It was also found that when the culture period in the second stage (the number of culture days in a culture environment with a large area for installing porous materials on the bottom surface of the well) is longer than 5 days (specifically, 8 days or more), human hepatoma cell sheets in which the PLC / PRF / 5 cells expressing BSEP are predominantly located on one main surface can be efficiently provided. Furthermore, it has been found that by using the culture method according to the present invention, it is possible to provide a human liver cancer cell sheet in which PLC / PRF / 5 cells are present in the voids of a porous material, and the PLC / PRF / 5 cells expressing BSEP are predominantly located on one main surface side. Furthermore, it was considered possible that cell polarity (different proteins expressed on the apical and basal sides, and different physiological functions) is structurally reproduced in each individual human liver cancer cell that constitutes the human liver cancer cell sheet according to the present invention. Based on the above, it is considered that the human liver cancer cell sheet according to the present invention can be used to evaluate bile excretion function. [Industrial applicability]

[0059] The human liver cancer cell sheet of the present invention can be used to evaluate bile excretion function, and for example, it can be used for in vitro evaluation of drug-induced liver injury, which is one of the side effects of drugs, and in particular cholestatic hepatotoxicity, which is an early symptom thereof.

Claims

1. A human liver cancer cell sheet in which PLC / PRF / 5 cells are present in the voids of a silica fiber nonwoven fabric, The PLC / PRF / 5 cells expressing the bile acid transporter BSEP are predominantly located on one of the main surfaces. Human liver cancer cell sheet.

2. A human liver cancer cell sheet according to claim 1, for use in evaluating bile excretion function.

3. A method for culturing human liver cancer cells using silica fiber nonwoven fabric as a cell culture carrier, The human liver cancer cells are characterized by being PLC / PRF / 5 cells. A method for culturing human liver cancer cells.

4. Step 1: A step of preparing two types of culture vessels having wells (culture vessel (A) and culture vessel (B)), silica fiber nonwoven fabric, PLC / PRF / 5 cells, and culture medium for the PLC / PRF / 5 cells. Step 2: Filling the wells with the culture medium until all of the silica fiber nonwoven fabric placed on the bottom surface of the wells of the culture vessel (A) is submerged. Step 3: A step of sowing the PLC / PRF / 5 cells onto the main surface of the silica fiber nonwoven fabric, which is placed on the bottom surface of the well, on the side opposite to the bottom surface of the well. Step 4: A step of culturing the PLC / PRF / 5 cells in the well on the silica fiber nonwoven fabric. Step 5: A step of filling the wells of the culture vessel (B) with the culture medium so that the entire silica fiber nonwoven fabric is immersed in the wells in the subsequent steps. Step 6: Remove the silica fiber nonwoven fabric together with the cultured PLC / PRF / 5 cells from the well of the culture vessel (A). Step 7: The step of placing the silica fiber nonwoven fabric removed in Step 6 onto the bottom surface of the well of the culture container (B) such that the side of the silica fiber nonwoven fabric on which the PLC / PRF / 5 cells are seeded is opposite to the bottom surface of the well. Step 8: A step of culturing the PLC / PRF / 5 cells in the wells of the culture vessel (B), It is equipped with, The area on which the silica fiber nonwoven fabric can be installed on the bottom surface of the well of culture vessel (B) is larger than the area on which the silica fiber nonwoven fabric can be installed on the bottom surface of the well of culture vessel (A). A method for culturing human liver cancer cells according to claim 3.

5. The culture period of the PLC / PRF / 5 cells in step 8 is characterized by being longer than 5 days. A method for culturing human liver cancer cells according to claim 4.

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