JP2010200679A - Cell culture container, method for performing cell culture, and method for evaluating cell - Google Patents

Abstract

To provide a cell culture container, a cell culture method, and a cell evaluation method for presenting a culture space suitable for cell growth and differentiation using a minimum amount of cells.
A cell culture container 10 has a plurality of micro containers 11a to 11d on the surface, and the micro containers 11a to 11d are 7.85 × 10 ⁻⁵ mm ^{2 to} 7.10 × 10 ^−4. At least two types of microcontainers, that is, a microcontainer 11d having a minimum bottom area of mm ² and microcontainers 11a to 11c having different bottom areas obtained by multiplying the minimum bottom area by a positive integer of 2 to 15 times Have. The cell culture method is a culture method in which one cell is injected into the microcontainers 11a to 11d and cultured, and the cell evaluation method includes a microcontainer having a bottom area of a size optimal for cell growth and differentiation and differentiation ability. It is something to evaluate.
[Selection] Figure 1

Description

  The present invention relates to a cell culture container, a cell culture method, and a cell evaluation method.

  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. Therefore, the cells used in the biotechnology field have been extremely diversified.

  In some cases, the isolated cells are immediately used for the test in a floating state, but in many cases, the cells are cultured in a state of being attached to a culture dish and used for various tests and examinations. Primary cells and cell lines used for cell culture are required to exhibit the same drug sensitivity and toxic reaction as those in in vivo tests, so-called in vivo tests. That is, a cell function similar to that in the living body is required on the cell culture container. Further, since the isolation procedure for obtaining the primary cells is complicated and the cell culture strain used for the cell culture test is expensive, a test method with a small number of cells is desired.

  In the cell culture test, the amount and concentration of the drug to be evaluated are varied under the same conditions, and the effect is measured. Therefore, the material, shape, etc. of the cell culture container must be the same. As the cell culture container, a plastic petri dish, a glass petri dish, a glass plate fixed in the container, a well plate, or the like is generally used. Well plates include 6-well, 12-well, 48-well, and 96-well plates or petri dishes. In general, the size of the whole plate is substantially the same, and the larger the number of wells, the smaller the size of one well. One well corresponds to one culture dish. In addition, with the recent trend toward micro-volumes, 384-well plates and 1536-well plates that have a larger number of culture dishes with smaller diameters have begun to be used. The bottom of these culture dishes is a flat plate shape, and this bottom is used as the culture surface.

  For culturing with a small number of cells, 96-well, 384-well, and 1536-well plates are effective. However, the plate has a flat bottom surface, and the adhesion bottom surface is too large for the cells. There is a problem that the function is reduced or lost.

  Therefore, a method has been studied in which cell adhesion molecules are arranged on the cell culture bottom surface only in an area where about one cell can adhere, and the outer periphery thereof is arranged with non-adhesion molecules so that the cells are arranged one by one within a specified area. (Patent Document 1).

JP 2003-33177 A

  This method allows culturing with a smaller number of cells than the 96-well, 384-well, and 1536-well plates, and makes it possible to easily observe the behavior of a single cell, but forcibly controls the bottom area to which the cells adhere. However, although the function of the cell is determined by the adhesion spots to be formed and the function lowering is suppressed as compared with the conventional flat bottom, there is a problem that it is greatly different from the original function. In addition, it is possible to place one type of cell at a predetermined position, but it is difficult to place two or more types of cells one by one at an arbitrary position, and this method is expensive. Is a problem.

  The present invention has been made to solve such problems, and uses a minimum amount of cells, a cell culture container for presenting a culture space suitable for cell growth and differentiation, a cell culture method, An object is to provide a cell evaluation method.

One aspect of the cell culture container according to the present invention is a cell culture container having a plurality of micro-containers on the surface, the plurality of micro-containers being 7.85 × 10 ⁻⁵ mm ^{2 to} 7.10. At least two types of microcontainers: a microcontainer having a minimum bottom area of × 10 ⁻⁴ mm ² and a microcontainer having different bottom areas obtained by multiplying the minimum bottom area by a positive integer of 2 to 15 times Have The depth of the plurality of microcontainers is preferably 20 μm to 150 μm, and the major axis of the bottoms of the plurality of microcontainers is preferably in the range of 1 to 1.5 times the minor axis.

  In addition, in one embodiment of the cell culture method according to the present invention, at least one cell is injected into each micro container described above and cultured. Alternatively, at least two types of cells are injected into each micro container one by one and cultured.

  Furthermore, in one embodiment of the cell evaluation method according to the present invention, undifferentiated cells are cultured by the above-described cell culture method, and the size and differentiation capacity of the micro container bottom area optimal for differentiation are evaluated.

  According to the present invention, it is possible to provide a cell culture container, a cell culture method, and a cell evaluation method for presenting a culture space suitable for cell growth and differentiation using a minimum amount of cells.

It is a front view which shows the structural example of the cell culture container used for the cell culture method which concerns on embodiment of this invention. It is II sectional drawing of the cell culture device of FIG. 2 is a photograph showing a cell culture container and a micromanipulator according to Example 1. FIG. 2 is an electron microscopic image of the upper surface showing the configuration of a cell culture container used in the cell culture method according to Example 1. FIG. 2 is an electron microscope image obtained by enlarging a container of each size of a cell culture container used in the cell culture method according to Example 1. FIG. 2 is a phase contrast microscopic image of bone marrow mesenchymal stem cells according to Example 1. FIG.

  The cell culture container according to the present invention is formed with an uneven pattern, that is, a plurality of micro containers, that is, a culture space. By optimizing the bottom area and depth of the micro container, cells can be cultured in the micro container, and an environment suitable for cell growth and differentiation can be constructed.

  When the bottom surface area of the micro container is too large, the cells partially grow thin like the culture on the flat plate and do not exhibit the intended function. On the other hand, if the bottom surface area of the micro container is too small, cells cannot be accommodated. Therefore, it is preferable that the size of the space is within a range that can be accommodated and can exhibit a desired function and grow according to the cell type to be cultured.

  Also, the depth of the micro container needs to be in an optimum range for culturing cells. If the depth is too large, it will be difficult to produce, and it will be difficult for the substance to diffuse, and the culture environment will deteriorate. If the depth is too small, the cells will cross the side wall and are not suitable for culture. In addition, according to the cell type to culture | cultivate, it can also apply to various culture systems by setting the bottom area and depth of a micro container conveniently.

  Embodiments of the present invention will be described below. However, the present invention is not limited to the following embodiment. In addition, for clarity of explanation, the following description and drawings are simplified as appropriate. “X to Y” means X or more and Y or less.

Embodiment FIG. 1 is a front view showing a configuration example of a cell culture container according to the present embodiment, and FIG. 2 is a cross-sectional view taken along the line II in FIG. As shown in FIGS. 1 and 2, the cell culture container 10 includes micro containers 11a to 11d. Hereinafter, the micro containers 11a to 11d are referred to as the micro container 11 when it is not necessary to distinguish them. A plurality of microcontainers 11 are formed on the culture surface of the cell culture container 10.

  1 and 2, the width a of the bottom of the micro container 11 and the depth b of the micro container 11 are shown.

The bottom shape of the micro container 11 is not particularly limited, and various shapes other than a square, a circle, and a polygon can be adopted. The minimum value of the bottom area is preferably 7.85 × 10 ⁻⁵ mm ^{2 to} 7.10 × 10 ⁻⁴ mm ² . Since the minimum equivalent diameter of the cell is about 10 μm, the cell cannot be cultured in the micro container at a bottom area smaller than 7.85 × 10 ⁻⁵ mm ² . Further, since the maximum equivalent diameter of the cells of about 30 [mu] m, greater than a bottom area 7.10 × ¹⁰ -4 mm ² is not a minimum range. Therefore, the micro container 11 minimizes the bottom area of 7.85 × 10 ⁻⁵ mm ^{2 to} 7.10 × 10 ⁻⁴ mm ^{2 and has} a bottom area that is a positive integer multiple of 2 to 15 times the bottom area. Is preferred. Further, an isotropic shape is preferable, and when the bottom surface is rectangular, the long side is preferably 1 to 1.5 times the short side.

  The depth b of the micro container 11 is preferably 20 to 150 μm when cells are cultured in the micro container 11, for example, when bone marrow stromal cells are cultured. If the depth is less than 20 μm, it becomes difficult for the cells to stay in the micro container at the time of cell seeding, and if the depth is more than 150 μm, the production is difficult.

FIG. 1 shows four types of microcontainers 11 having different bottom areas, including a minimum bottom area of 7.85 × 10 ⁻⁵ mm ^{2 to} 7.10 × 10 ⁻⁴ mm ² . FIG. 1 shows microcontainers 11c, 11b, and 11a having a bottom area of the microcontainer 11d that has different bottom areas that are two to fifteen times the minimum bottom area. However, the number of types of the bottom area is not limited to four. The cell culture vessel 10 preferably includes at least two types of microcontainers 11 having different bottom areas.

  The method for producing the microcontainer 11 on the cell culture container of the present invention is not particularly limited. For example, transfer molding using a mold, three-dimensional stereolithography, precision mechanical cutting, wet etching, dry etching, laser processing, Examples of the method include electric discharge machining. It is preferable to appropriately select these production methods in consideration of the use of the cell culture container, required processing accuracy, cost, and the like.

  As a specific example of the transfer molding method using a mold, there is a method of forming a concavo-convex pattern 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 concavo-convex pattern on the resin at a high transfer rate, and the cost of the material 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. What is necessary is just to select suitably in consideration of a use, the required process precision, cost, etc.

  Examples of a method for forming a concavo-convex pattern on 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.

  The material constituting the cell culture container of the present invention is not particularly limited as long as it is transparent and has a self-supporting property, and examples thereof include synthetic resin, silicon, and glass. From the viewpoint of cost and cell visibility by microscopic observation, it is preferable to use a transparent synthetic resin as a material. Examples of the transparent synthetic resin include acrylic resins such as polymethyl methacrylate and methyl methacrylate-styrene copolymer, styrene resins such as polystyrene, olefin resins such as cycloolefin, polyethylene terephthalate, and polylactic acid. Examples thereof include ester resins, silicone resins such as polydimethylsiloxane, and polycarbonate resins. Such a resin may contain various additives such as a colorant, a diffusing agent, and a thickener as long as the transparency is not impaired.

  The cell culture container of the present invention is subjected to a surface treatment on the surface of the concavo-convex pattern for the purpose of improving the hydrophilicity, biocompatibility, cell affinity, etc. of the container surface, and the modified layer and / or coating layer is disposed. May be.

  The method for providing the modified layer is not particularly limited unless it is a method for losing self-supporting property or a method for causing extreme surface roughness of 100 μm or more. For example, a graft polymer by chemical treatment, solvent treatment, or surface graft polymerization is used. And methods such as chemical treatment such as introduction of carbon, physical treatment such as corona discharge, ozone treatment, and plasma treatment.

  The method for providing the coating layer is not particularly limited, and examples thereof include dry coating such as sputtering and vapor deposition, wet coating such as inorganic material coating and polymer coating.

  It is desirable to impart hydrophilicity to the concavo-convex pattern in order to inject the culture solution without mixing bubbles, and inorganic vapor deposition is preferred as a method for forming a uniform hydrophilic film.

  In consideration of cell affinity, it is more preferable to coat a cell affinity protein such as collagen or fibronectin. In order to uniformly coat a collagen aqueous solution or the like, it is preferable to coat after forming the above-mentioned hydrophilic film. Usually, in cell culture, it is desirable to mimic the in vivo environment and culture on the surface of the extracellular matrix. Therefore, after the uniform hydrophilic inorganic membrane is disposed as described above, the cell is composed of an extracellular matrix suitable for cultured cells. It is particularly preferable to arrange an organic film.

  The cells cultured by the cell culture method of the present invention are not particularly limited, and cells that dedifferentiate on a flat plate are preferable. The animal species may be selected according to the purpose, such as rat, mouse, chicken, dog, monkey, human. For example, chondrocytes, osteoblasts, odontoblasts, enamel blasts, mammary epithelial cells, ciliated epithelial cells, intestinal epithelial cells, adipocytes, hepatocytes, mesangial cells, glomerular epithelial cells, sinusoidal endothelial cells, kuppa Cells, myoblasts, nerve cells, glial cells, fibroblasts, smooth muscle cells, ES cells, bone marrow stromal cells, bone marrow mesenchymal stem cells), stem cells such as neural stem cells, and the like.

  The method for injecting cells into the microcontainer by the cell culture method of the present invention is not particularly limited as long as it is a method capable of stably injecting one cell into one microcontainer without loading the cells. For example, an apparatus such as an inkjet or a dispenser can be used.

  Next, examples of the cell culture container according to the present invention will be described, but the present invention is not limited to these examples.

<Preparation of bone marrow mesenchymal cells>
Bone marrow mesenchymal stem cells (BMSC) used for culture were prepared as follows. Femurs were removed from 10-week-old C57BL / 6J mice, bone marrow cells removed from the bone lumen were seeded on cell culture plates, and cultured in αMEM medium supplemented with 15% fetal bovine serum and penicillin / kanamycin / amphotericin B. did. Spontaneous immortalization was induced by long-term culturing of cells attached to the plate, and a single cell line was prepared by cell cloning using the cylinder method and limiting dilution method.

<Culture method>
The culture medium used for the culture was αMEM containing 20% fetal bovine serum and 1% penicillin / streptomycin, and the culture medium was changed every two days. Culturing was performed at 37 ° C. in a 5% carbon dioxide atmosphere.

<Cell seeding method in micro container>
Inoculation of one cell into a micro container was performed under a microscope using a micro manipulator installed on a microscope stage shown in FIG. Cells were seeded from a microglass needle attached to the tip of a micromanipulator, and then a water flow was generated by water absorption from the tip of the needle to induce cells into the chamber and seed in a micro container.

[Example 1]
1 is a concavo-convex pattern shape having a bottom width a = 20 μm, 40 μm, 80 μm, 160 μm, and a depth b = 30 μm produced by photolithography, Ni electroplating, and having a corresponding concavo-convex shape I got a mold. Using the mold, pattern transfer was performed on polymethyl methacrylate 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 base material by vacuum deposition, and γ-ray sterilization was performed to obtain an uneven pattern base material. Bone marrow mesenchymal stem cells were cultured on the uneven substrate by the above culture method. FIG. 4 shows an electron microscope image of the upper surface showing the configuration of the cell culture container. Moreover, the electron microscope image which expanded the container of each size of a cell culture container in FIG. 5 is shown. In FIG. 5, (a) is a = 160 μm, (b) is a = 80 μm, (c) is a = 40 μm, and (d) is a = 20 μm. Is different.

[Comparative Example 1]
A commercially available dish (Becton Dickinson, Falcon (registered trademark)) sterilized by γ-ray was used as an example of a dish on a flat plate. Bone marrow mesenchymal stem cells were seeded in a dish using the micromanipulator and cultured by the above culture method.

  FIG. 6 shows a phase-contrast microscope image of bone marrow mesenchymal stem cells seeded in the microcontainer of Example 1. After 4 hours of seeding, the cell size was about 20 μm when the diameter of the micro container was 20 μm and 40 μm, but hardly changed at 80 μm, but about 40 μm at 80 μm and about 50 μm at 160 μm. In this way, culture is possible while maintaining a three-dimensional shape at diameters of 20 μm and 40 μm, but at 80 μm and 160 μm, flattening progresses with the passage of culture time, and changes from bone marrow mesenchymal stem cells to cells with different forms. did. In Comparative Example 1, flattening occurred quickly and showed a spindle shape. In Example 1, since the incubators of different sizes are formed on one chip, it is possible to determine an appropriate culture environment within one chip.

  In addition, this invention is not limited to embodiment shown above. Within the scope of the present invention, it is possible to change, add, or convert each element of the above-described embodiment to a content that can be easily considered by those skilled in the art.

10 Cell culture vessel 11 Micro vessel

Claims (6)

A cell culture vessel having a plurality of micro-vessels on the surface,
The plurality of microcontainers include a microcontainer having a minimum bottom area of 7.85 × 10 ⁻⁵ mm ^{2 to} 7.10 × 10 ⁻⁴ mm ² , and a minimum base area that is 2 to 15 times positive. A cell culture vessel comprising at least two types of microcontainers, with a microcontainer having a bottom area that is an integral multiple of.   The cell culture container according to claim 1, wherein a depth of the plurality of micro containers is 20 μm to 150 μm.   3. The cell culture container according to claim 1, wherein a major axis of the plurality of micro container bottoms is within a range of 1 to 1.5 times a minor axis.   A cell culture method comprising injecting and culturing at least one cell into each micro container according to any one of claims 1 to 3.   The cell culture method according to claim 4, wherein at least two types of cells are injected into each micro container one by one and cultured.   A cell evaluation method for culturing undifferentiated cells by the cell culture method according to claim 4 or 5, and evaluating the size and differentiation capacity of a micro-vessel bottom area optimal for differentiation.