JP5307489B2 - Method for producing three-dimensional cell structure - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple method for producing a high-density three-dimensional cell structure having a uniform diameter. <P>SOLUTION: The method for producing a three-dimensional cell structure includes a process (A) for concentrating and separating a cell population having a survival rate of &ge;70% by a density gradient centrifugation method, a process (B) for seeding the cell population obtained by the process (A) on a substrate having a small region with an effective bottom area of 0.01-0.1 mm<SP>2</SP>and a process (C) for culturing the cell seeded by the process (B). <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

  The present invention relates to animal cell cultures.

  Techniques for testing and examining cells isolated from tissues are indispensable in biotechnology-related fields. It is widely used for diagnosing diseases and pathological conditions, searching for new drugs and determining their efficacy, animal testing, plant testing, and testing for environmental pollutants. The isolated cells may be used immediately for testing, but many are cultured in culture dishes or test tubes by the cell culture method. Various tests are performed in this culture system.

  In these assays, a uniform culture system is usually set, and the effect is observed by changing the amount and concentration of the drug to be evaluated. The cells used at that time are obtained from primary cells separated from the living body, cell banks and cell sales companies. The cells are seeded in various media and incubators and then examined.

  Examples of the culture method used for drug inspection include the method of culturing using a cell culture vessel coated with collagen, as shown in Non-Patent Document 1, and the monolayer culture method of culturing in a sandwich form using collagen and Matrigel. It has been known.

  As described above, although it is a widely used cell culture technique, the conventional cell culture method (monolayer culture method) can maintain a specific function of cells in a living body for a long period of time. There was a problem that it was not possible.

  As a culture method for solving these problems, a three-dimensional culture method has attracted much attention in recent years. Compared with the monolayer culture method, the three-dimensional cell structure obtained by three-dimensional culture can maintain a specific function of cells for a long period of time and can produce a structure similar to that in vivo. Taking advantage of these characteristics, research and development on three-dimensional cell structures in the shape of three-dimensional cell structures has been actively conducted for the purpose of use for examination of drugs and the like. For example, Patent Document 1 proposes a method for producing a three-dimensional cell structure having a uniform diameter. Patent Document 2 proposes a method for culturing a three-dimensional cell structure having a uniform diameter.

JP 2001-226293 A JP 05-227006 A Non-clinical research using extracted human tissues / cells SRC Publishing Co., Ltd. pp261-279

However, the above method has the following problems, and a solution has been desired.
The method described in Patent Document 1 is a method of obtaining a three-dimensional cell structure by co-culturing with a parenchymal cell and one or more types of cells, but it is necessary to prepare two or more types of cells for the culture Therefore, not only a lot of labor is required, but there is a concern about the influence of cells other than hepatocytes on the examination of drugs and the like.

  The method described in Patent Document 2 is a method of culturing by a rotary culture method, a reflux culture method, and a suspension culture method using a high-viscosity culture solution. Therefore, because it uses a high-viscosity culture solution, it is inferior in operability at the time of medium exchange, and further has a disadvantage that a special device is required, which is difficult for everyone to use.

  Therefore, an object of the present invention is to provide a three-dimensional cell structure having a uniform size by a simple method for eliminating these drawbacks.

In the cell culture method according to the present invention, a cell population obtained from the step of (A) concentrating and separating a cell population having a survival rate of 70% or more by density gradient centrifugation, (B) having an effective bottom area of 0.01 It is a step of seeding on a support having a small region of ˜0.1 mm ² and culturing (C). In the step (B), the density gradient centrifugation method preferably has a centrifugal force of 100 × g or less.

Step (C) of the present invention comprises:
(I); culturing to attach the cells;
(Ii); removing non-adherent cells;
(Iii); a step of culturing in order to form a three-dimensional cell structure.
Here, the time of “the step of culturing for attaching cells (i)” is not limited as long as the cells can be attached and can be selected according to the cells used. Absent.

In the step (B) of the present invention, the seeding density of the cells is preferably 1 × 10 ⁴ to 1 × 10 ⁶ cells / cm ² . Moreover, it is preferable that the whole or part of the surface of the small region is provided with an organic film or an inorganic film.

  Furthermore, the cell population in the present invention is preferably human hepatocytes.

  According to the present invention, a high-density three-dimensional cell structure having a uniform size can be obtained by a simple method.

  Hereinafter, an example of an embodiment to which the present invention is applied will be described. It goes without saying that other embodiments may also belong to the category of the present invention as long as they match the gist of the present invention. Moreover, the material, size, and ratio of each member in the following drawings are merely examples, and the present invention is not limited thereto.

  The cell population cultured in the present invention is not particularly limited as long as it forms a three-dimensional cell structure, but from the viewpoint of usefulness, it is preferably a parenchymal cell, particularly preferably a hepatocyte, Furthermore, human hepatocytes are preferable.

  The origin of a cell population having a survival rate of 70% or more may be a primary cell isolated from a living body, a primary cell obtained by thawing a frozen product after being separated from a living body, or a cultured cell passaged from these cells. Well, it may be a cultured cell that has been subcultured from a cell line that is commercially available, but it is known that the function of the cell is reduced or changed immediately after it is isolated from outside the body. In order to obtain a three-dimensional cell structure having a primary cell, a primary cell immediately after separation from a living body or a primary cell obtained by thawing a frozen product after separation from a living body is preferable. Cells are more preferred.

  In the case of a cell population of 70% or less, the viability of the cells used in the present invention is at least because even the surviving cells are damaged by surface membrane proteins and the like and inferior in cell adhesion after cell seeding. 70% or more is necessary, and 80% or more is more preferable from the viewpoint of cell recovery efficiency by pretreatment by density gradient centrifugation. When the survival rate of the initial cell population is 70% or less, it may be used for the present invention after purification to a survival rate of 70% or more. Examples of the method for purifying cells include a method of separating using the specific gravity of cells, or a method of labeling cells and separating them using microbeads or cell sorters, but is not limited thereto. Absent.

The solution used for the density gradient centrifugation in the step ( A ) is, for example, Percoll (G
E Healthcare Bioscience (Percoll is a registered trademark of GE Healthcare Bioscience), Iodixanol solution (Axis-Shield) and the like are commercially available, but are not limited thereto.
In the step ( A ), the density gradient centrifugation is preferably performed at a centrifugal force of 100 × g or less, more preferably 50 × g or less, from the viewpoint of damage to the cells. The lower limit of the centrifugal force is not particularly limited, but is preferably 20 × g or more from the viewpoint of processing time.

  Here, (i) the step of culturing to adhere the cells in step (C) will be described. In this step, the seeded cells only have to adhere, and in the case of 30 minutes or less, unattached cells are removed in the step (ii), and the number of cells decreases. In the case of 24 hours or more, the internal fluid of the cell leaks into the medium, which may lead to cell death of normal cells. Therefore, it is preferably 30 minutes or more and 24 hours or less, and more preferably 1 to 8 hours.

  Examples of the medium used include, but are not limited to, a medium containing serum containing serum and a serum-free medium supplemented with nutrients.

  The medium used in each step (A), (B), or (C) of the present invention may be the same type of medium. For example, (A) is a serum-free medium, and (B) is a serum-containing medium. , (C) serum-free medium may be combined with different media in each step, or (A) serum-free medium, (B), (C) may be combined as serum-free medium. The combination of media is not limited to these.

  Next, the shape of the small area will be described with reference to FIGS. FIG. 1 is a plan view showing a configuration of a support having a small region, and FIG. 2 is a cross-sectional view taken along the line X-X ′ of FIG. 1. As shown in FIG. 1, the support having a small region includes a micro container 11, a side wall 12, and an opening 13. A plurality of side walls 12 are formed in a mesh shape on the culture surface of the support 10 having a small region, and a space surrounded on all sides by the side walls 12 is a micro container 11. Moreover, the opening part 13 is formed in the center part of each edge | side of the side wall 12 formed in the four sides of each micro container 11.

  In FIG. 1, the width a of the bottom part of the micro container 11, the width b and the height c of the side wall 12 for partitioning the micro container 11, and the width d of the opening 13 for the adjacent micro containers 11 to communicate with each other are shown. It was. The bottom area in the present invention refers to a projected area when parallel light is irradiated from above to the bottom of the container in the direction perpendicular to the horizontal surface of the micro container opening (the same surface as the upper surface of the side wall 12). For example, when the bottom of the micro container is U-shaped, the shape projected by parallel light incident on the bottom from above in the direction perpendicular to the opening surface is the bottom area. The major axis and minor axis of the projected bottom are, in the case of circles and ellipses, the distance on each axis at the intersection of the major axis and minor axis and the circumference passing through the center of gravity, called the major axis and minor axis. The difference between the area of the polygon and the extrapolated circle or ellipse that passes through each vertex is the longest and shortest axis. The major and minor axes of an approximate circle or ellipse passing through the vertex

The bottom area and height of the small region of the support can be selected in various sizes depending on the desired diameter of the three-dimensional cell structure, but at least one cell is small from the viewpoint of cell-cell interaction. A minimum diameter of 2 to a maximum of 10 ⁶ per region, with a three-dimensional cell structure size range of 50 μm to 150 μm in diameter is preferred. Therefore, the diameter or one side of the small region is preferably in the range of 70 μm to 300 μm, and the height of the small region is preferably 20 μm to 50 μm.

  Since the angle formed between the horizontal surface of the micro container 11 and the side wall 12 must be an angle at which cells do not ride, the portion of 50% or more from the upper part of the side surface is preferably 80 to 90 °, particularly 85 to 90 °. Preferably there is.

  In FIG. 3, the width d of the opening 13 for communicating adjacent microcontainers 11 with each other only needs to be such that the cultured cells cannot move from the microcontainer 11 seeded first to the adjacent microcontainer 11. Since the diameter of one cell is 10 to 20 μm, it is preferably 5 to 15 μm smaller than this diameter. The opening 13 is not essential, and the four sides of the micro container 11 may be completely surrounded by the side walls 12 as shown in FIGS. 3 and 4. Here, FIG. 3 is a plan view showing the configuration of the support having the small region according to the present embodiment, and FIG. 4 is a cross-sectional view taken along the line Y-Y ′ of FIG. 1.

  Examples of the method for producing a support having a small region include transfer molding using a mold, three-dimensional stereolithography, precision machine cutting, wet etching, dry etching, laser processing, electric discharge machining, and the like, from the viewpoint of accuracy and cost. Transfer molding using a mold is preferred.

  A specific example of the transfer molding method using a mold is a method of forming a small region by resin molding using a metal structure as a mold. This method is preferable because the shape of the metal structure can be reproduced in a small area on the resin with a high transfer rate, and the material cost can be reduced by using a general-purpose resin material. The method using such a metal structure mold is excellent in that it is low in cost and can satisfy high dimensional accuracy.

  Examples of the method for manufacturing the metal structure include plating treatment on a resist pattern produced by photolithography and a resin pattern produced by three-dimensional stereolithography, precision mechanical cutting, wet etching, dry etching, laser processing, electric discharge. Processing etc. are mentioned. From the application, required processing accuracy, cost, etc., the plating process to the resist pattern produced by photolithography is preferable.

  Examples of methods for molding a small region into a resin using the metal structure obtained above as a mold include, for example, injection molding, press molding, monomer cast molding, solvent cast molding, hot emboss molding, roll transfer by extrusion molding, etc. Can be mentioned. It is preferable to employ injection molding from the viewpoint of productivity and mold transferability.

  As a material constituting the support having a small region, a synthetic resin is preferable from the viewpoint of cost, and a synthetic resin having high permeability is preferable from the viewpoint of observing cell morphology during culture. Examples of such synthetic resins include acrylic resins such as polymethyl methacrylate and methyl methacrylate-styrene copolymers, styrene resins such as polystyrene, olefin resins such as cycloolefin, and esters such as polyethylene terephthalate and polylactic acid. Examples of the resin include silicone resins such as polydimethylsiloxane, polycarbonate resins, and the like. From the viewpoints of cost and transparency, acrylic resins or styrene resins are more preferable.

  The surface of the support having a small region is preferably modified or coated from the viewpoint of cell affinity. Substances used for these include inorganic substances such as glass, synthetic substances such as poly-L-lysine and polyethylene glycol (PEG), and extracellular matrices such as collagen and fibronectin. From the viewpoint of cost and operability. Synthetic substances such as poly-L-lysine and PEG are more preferred. More preferably, in the case of a synthetic resin, it is better to hydrophilize the surface with glass or the like in order to uniformly coat the cytophilic substance. As the coating substance, poly-L-lysine or PEG is preferable from the viewpoint of cost. A hydrophilic synthetic substance such as is preferable.

As a result of diligent research, the present inventors have found that (A) a cell population obtained by concentrating and separating a cell population having a survival rate of 70% or more by density gradient centrifugation (B) has an effective bottom area of 0. and a step of seeding the support having a small area 01~0.1mm ^2, the step of culturing (C), led to provide a method for manufacturing a three-dimensional cell structure. Examples of the present invention are shown below.

<Preparation of a support having a small region>
FIG. 3 shows a concavo-convex pattern shape in which a = 200 μm, b = 20 μm, and c = 50 μm.
The pattern was prepared by photolithography and Ni electrolytic plating was performed to obtain a mold having a corresponding uneven shape. The mold was used to transfer the concavo-convex pattern shape onto polystyrene by hot embossing to produce a resin substrate having the above dimensions. A silicon dioxide film having a thickness of 100 nm was formed on the surface of the resin substrate by vacuum deposition, and γ-ray sterilization was performed to obtain a support having a small region. This support was set in each well of a 24-well plate (manufactured by FALCON) with the culture surface facing up. After adding 300 μL of 0.01% poly-L-lysine (manufactured by SIGMA), incubation is performed at 4 ° C. for 24 hours. After 24 hours, the plate was washed 3 times with a phosphate buffer (GIBCO).

<Measurement of cell number and viability>
Stain dead cells using trypan blue (GIBCO), count the total number of cells (number of dead cells and number of living cells) and the number of dead cells, and survive the ratio of the number of living cells to the total number of cells. Rate. The number of living cells was taken as the number of cells.

<Collection of cells>
Six weeks, hepatocytes were isolated from male Wistar rats by a two-stage collagenase perfusion method. Specifically, it was performed as follows. Wistar rats were laparotomized under anesthesia, cannulated from the portal vein, pre-refluxed solution (0.01 M HEPES (manufactured by DOJINDO), 0.5 mM EGTA (manufactured by DOJINDO), penicillin-streptomycin ( Gibco) and potassium and calcium-free phosphate buffer containing 1 g / L glucose (manufactured by Wako Pure Chemical Industries) were started to flow at a flow rate of 25 mL / min, and at the same time, the hepatic artery was cut. Flow 150 mL of pre-reflux liquid. Next, a collagenase solution (0.5 g / L collagenase (manufactured by Wako Pure Chemical Industries), 0.05 g / L trypsin inhibitor (manufactured by GIBCO), 0.01 M HEPES (manufactured by DOJINDO), penicillin, heated to 37 ° C. -After flowing 50 mL of streptomycin (a calcium-containing phosphate buffer solution containing GIBCO) at 30 mL / min, flow 100 mL at 30 mL / min. The liver was excised from the rat, placed in a petri dish with a diameter of 10 cm containing 10 mL of DMEM (GIBCO), and then the liver was finely cut with a scalpel and filtered with gauze, and then the filtrate and 90 mL of DMEM were added to 45 μm. Filter through a metal mesh. The filtrate was dispensed into two 50 mL centrifuge tubes (manufactured by FALCON) and centrifuged at 30 × g for 90 seconds. The supernatant was discarded, and 40 mL of each DMEM solution was added again and stirred, followed by centrifugation at 30 × g for 90 seconds. The supernatant was discarded and a DMEM / F12 solution was added to prepare a cell suspension having a concentration of 0.1 × 10 ⁷ cells / mL. The survival rate at this time was 90 ± 5%.

<Cell culture>
The medium is a seeding medium (10% FBS, 2 mM L-glutamine, 10 mM nicotinamide, 50 μM 2-mercaptoethanol (manufactured by SIGMA), 0.1 μM dexamethasone (manufactured by SIGMA), 26 mM ascorbic acid (manufactured by SIGMA), 5 mM. HEPES (manufactured by DOJINDO), DMEM / F12 (manufactured by SIGMA) containing 0.1 μM insulin (manufactured by Wako Pure Chemical Industries), and maintenance medium (seeding medium containing 20 ng / mL EGF (manufactured by SIGMA)). used. Place 300 μL of the seeding medium containing the cells in each well, and incubate for 4 hours in an incubator at 37 ° C. with a carbon dioxide concentration controlled to 5%. Then, suck the non-adherent cells with the seeding medium, Each 300 μL of the maintenance medium was placed in an incubator controlled at 37 ° C. and 5% carbon dioxide concentration, and cultured for 5 days. The medium was performed once every two days and cultured for 5 days.

<Observation>
The entire culture surface of the support was observed with a transmission inverted microscope (Olympus), and (a) a three-dimensional cell structure having a diameter of 50 to 100 μm and (b) a diameter of less than 50 μm and a diameter of 150 μm or more. The number of three-dimensional cell structures was measured. The number of 1-mm ² three-dimensional cell structures was defined as the three-dimensional cell structure density.

[Example] Treatment Group by Density Gradient Centrifugation Method A DMEM / F12 solution is added, and the cell suspension is 25 mL, and an HBSS solution containing 90% Percoll (90% Percoll (manufactured by GE Healthcare Biosciences)). 33 mM KCl (manufactured by Wako Pure Chemical Industries, Ltd.), 0.441 mM KH ₂ PO ₄ (manufactured by Wako Pure Chemical Industries, Ltd.), 4.17 mM NaHCO ₃ (manufactured by Wako Pure Chemical Industries, Ltd.), 137.9 mM NaCl (manufactured by Wako Pure Chemical Industries, Ltd.), 0.338 mM NaHPO ₄ (manufactured by Wako Pure Chemical Industries, Ltd.), 5.56 mM D-Glucose (manufactured by Wako Pure Chemical Industries, Ltd.), 0.0266 mM phenol red (manufactured by Wako Pure Chemical Industries, Ltd.)) 24 mL were mixed for density gradient centrifugation. A solution was made. The resulting solution for density gradient centrifugation was mixed by inversion 5 times, and then centrifuged at 50 × g for 10 minutes, and the supernatant was discarded (density gradient centrifugation operation). Next, 30 mL of the DMEM / F12 solution was added and stirred, and then centrifuged at 50 × g for 1 minute, and the supernatant was discarded (washing operation). This washing operation was performed twice in total, and the number of cells was adjusted to 4 × 10 ⁵ using a seeding medium, followed by culturing.

[Comparative Example] Density gradient centrifugation untreated group After 10 mL of cell suspension was put into a 50 mL centrifuge tube, centrifuged at 50 × g for 1 minute, and then the supernatant was discarded (washing operation). The cells were cultured after being adjusted to 4 × 10 ⁵ cells / mL. The survival rate at this time was 90 ± 5%.

  Table 1 shows the densities of the three-dimensional cell structures after 5 days of the examples and comparative examples. The example showed about twice the density of the comparative example. In the examples, most of the three-dimensional cell structures were three-dimensional cell structures having a diameter of 50 to 100 μm. On the other hand, in the comparative example, there are many three-dimensional cell structures having a diameter of less than 50 μm and 100 μm or more, and the density was about twice that of the example.

  FIG. 5 shows changes with time in the density of the three-dimensional cell structures of the example and the comparative example. In the examples, formation of a three-dimensional cell structure was confirmed after 2 days of culture, but was hardly observed in the comparative examples. On the other hand, in the comparative example, almost no three-dimensional cell structure was formed after 2 days. From 3 days to 5 days later, the example had a three-dimensional cell structure density more than twice that of the comparative example.

  FIG. 6 shows morphological photographs of the example and the comparative example after 3 days and after 5 days. Three-dimensional cell structures are indicated by arrows. In Examples, clean spherical three-dimensional cell structures were confirmed after 3 days of culture, and clean spherical three-dimensional cell structures were confirmed in almost all small areas on the fifth day. In the comparative example, the three-dimensional cell structure was confirmed after 3 days, but the number of the three-dimensional cell structure was much less than that of the example.

Since the density of the three-dimensional cell structure is more than twice, when the test is performed in 24 wells having a bottom area of about 2 cm ^{2 in} the comparative example, in the example, about 1 cm ² , which is about half of the area, Tests in 48 wells with a bottom area are possible. That is, the number of testable samples is doubled, and the present invention can greatly improve the efficiency of drug tests.

It is a top view which shows the structure of the support body which has a small area | region which concerns on an Example. It is sectional drawing which shows the support body which has a small area | region which concerns on an Example. It is a top view which shows the support body which has a small area | region which concerns on an Example. It is sectional drawing which shows the support body which has a small area | region which concerns on an Example. It is a figure which shows the three-dimensional cell structure density which concerns on an Example and a comparative example. It is a figure which shows the form of the three-dimensional cell structure which concerns on an Example and a comparative example.

Explanation of symbols

10 Hepatocyte culture vessel 11 Micro vessel 12 Side wall 13 Opening

Claims (8)

(A); a step of concentrating and separating a cell population having a survival rate of 70% or more by density gradient centrifugation;
(B); seeding the cell population obtained in step (A) on a support having a small region having an effective bottom area of 0.01 to 0.1 mm ² ;
(C); a method for producing a three-dimensional cell structure, comprising a step of culturing the cells seeded in the step (B). The method for producing a three-dimensional cell structure according to claim 1, wherein the centrifugal force of the density gradient centrifugation in the step (A) is 100 xg or less. The culture step of (C)
(I); culturing to attach the cells;
(Ii); removing non-adherent cells;
(Iii): The method for producing a three-dimensional cell structure according to claim 1, comprising a step of culturing to form the three-dimensional cell structure. ^{2. The} method for producing a three-dimensional cell structure according to claim 1, wherein in the step (B), a seeding density of cells is 1 × 10 ⁴ to 1 × 10 ⁶ cells / cm ² . The method for producing a three-dimensional cell structure according to claim 1, wherein the whole or part of the surface of the small region in the step (B) is provided with an organic film or an inorganic film. The method for producing a three-dimensional cell structure according to any one of claims 1 to 5, wherein the cell population is a parenchymal cell. The method for producing a three-dimensional cell structure according to claim 6, wherein the parenchymal cells are hepatocytes. The method for producing a three-dimensional cell structure according to claim 7, wherein the hepatocytes are human, monkey, chimpanzee, rat, mouse, hamster, cow, pig, sheep or a chimeric animal thereof.