JP2013039071A - Cell culture substrate and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell culture substrate excellent in formation efficiency of a cell aggregate and excellent in durability and stability, and to provide a method for producing the same.SOLUTION: The cell culture substrate arranged with a plurality of concave parts having an opening in a funnel shape and a pore at a bottom thereof on one part of a glass substrate via a process flow including at least: a glass substrate cleaning process; a Cr film mask formation process; a resist mask formation process; a Cr film mask opening formation process by etching; and an etching process for performing wet etching on the glass substrate. The cell aggregate is formed by using the cell culture substrate.

Description

  The present invention relates to a cell culture substrate used for cell culture and a method for producing the same.

In drug discovery research, when screening for drugs, cell aggregates formed by spreading a medium on a cell culture substrate (abbreviation: substrate) and culturing cells or cell lines are used. It is used for evaluation of pharmacological activity. The structure of cell aggregates formed by cell culture techniques is affected not only by the chemical properties of the substrate surface but also by the surface structure.
In the culture of cells by conventional cell culture techniques, it is difficult to form cell aggregates that are made closer to living organisms, and improvements in accuracy and efficiency are required in drug screening. For this reason, research for making a three-dimensional structure close to a living tissue has been conducted, and various methods have been proposed regarding the shape of the substrate and the surface microstructure. For example, a three-dimensional tissue culture method using a honeycomb structure (Patent Document 1), a cell culture substrate using a honeycomb structure (Patent Document 2), a method for forming a cell aggregate (Patent Document 3), a cell culture structure The body (refer patent document 4) etc. are proposed.

  In the case of Patent Documents 1 and 2, both a biodegradable polymer and an amphiphilic polymer are cast on a substrate, and minute water droplets generated by condensation are evaporated to form a honeycomb structure and a cell culture substrate. Is. That is, in Patent Document 1, a honeycomb structure that can be economically prepared by combining a biodegradable polymer and an amphiphilic polymer in an appropriate ratio, is self-supporting, and is structurally stable. Can be given. Further, in Patent Document 2, formation of ordered three-dimensional aggregates of cells similar to living tissue is formed in vitro by using a honeycomb structure in which a mixing ratio of biodegradable polymer and amphiphilic polymer is defined. It is said that it is effective as a culture method for performing. However, in Patent Documents 1 and 2, it is difficult to control the size of the honeycomb structure and to form a shape other than the honeycomb structure, and there are problems such as restrictions on usable materials. In addition, precise conditions for forming a stable honeycomb structure are required, and this is not suitable for mass production.

  On the other hand, in Patent Document 3, a plurality of single cells are seeded in a funnel, hemisphere, or a culture vessel having a hydrophilic shape with a bottom surface having a water contact angle of 30 degrees or less in a shape in which the center of the bottom surface is a flat surface, By culturing cell aggregates (spheroids) by aggregating and dividing the single cells on the bottom surface, a large number of uniform-sized cell aggregates can be formed simultaneously and easily. However, when a commercially available multi-plate is used as the culture vessel in Patent Document 3, there is a difficulty in that it is difficult to fix spheroids and medium replacement work is difficult, and it is difficult to maintain a culture environment suitably.

  Further, in Patent Document 4, a cell adhesion surface in which a plurality of cells having a width between cells of 3 μm or less, a depth of 10 nm or more, a planar shape of a polygon, and a minimum inner diameter of 3 μm or less are continuously formed. A cell culture structure having a functioning concavo-convex structure can be used to manufacture a cell culture structure and a cell culture container in which the shape, size, and material of the microstructure are controlled, and this is suitable for screening evaluation of drugs. It is said that spheroids can be cultured easily and in a short time at a lower cost. As a specific example of a cell culture structure, a material formed by nanoimprint technology using resin as a material is used. However, when a high melting point material (quartz glass, etc.) is used as a base material, an uneven structure by nanoimprint technology is used. The formation of the process involves technical difficulties and remains a problem in productivity.

As described above, in Patent Document 4, a resin such as cycloolefin polymer and polystyrene is used as a specific example of the cell culture structure. In Patent Document 3, as a specific example of the culture vessel, a commercially available U-shaped multiplate whose surface is hydrophilized by surface treatment with polyhydroxyethyl methacrylate, ethylene vinyl alcohol copolymer or the like is used. In Patent Documents 1 and 2, as a specific example of the cell culture substrate, a glass substrate formed by casting a biodegradable polymer and an amphiphilic polymer to form a honeycomb structure is used. However, these cell culture substrates have problems in durability and stability.
As for a method for forming a minute structure, for example, a technique for forming a minute structure containing a sulfur compound and silicon oxide using thermal lithography (Patent Document 5) has been proposed. This method includes a step of forming a layer containing a sulfur compound, silicon oxide, and a material that improves light absorption on a substrate, a step of irradiating the layer formed on the substrate with laser light, It consists of a process of etching the irradiated layer, and is said to be useful for forming information recording media, masters, optical elements, optical communication devices, DNA chips, light emitting elements, photoelectric conversion elements, optical lenses, and the like.

  As mentioned above, proposals have been made to improve the accuracy and efficiency of screening in drug discovery by devising the shape and surface microstructure of the base material, but it is still not sufficient, and screening efficiency can be further improved. Cell culture technique, particularly cell culture with excellent durability and stability capable of forming cell aggregates (three-dimensional tissue bodies: sometimes referred to as “spheroids”) that are made to be tissues closer to living bodies A substrate is desired.

  As described above, it is known that by providing a concavo-convex structure on a base material, it is possible to promote growth in a three-dimensional direction (three-dimensional direction) when cells are cultured on the base material. In other words, when cells are cultured on a flat substrate with no concavo-convex structure, the cells spread only in the two-dimensional direction (planar direction) and grow into a single-layered cell organization (monolayer cells). On the other hand, it has been reported that when a substrate having a concavo-convex structure is used, it grows in a three-dimensional structure (spheroid) in order to spread in a three-dimensional direction (stereoscopic direction). In other words, by using a base material having a concavo-convex structure, a three-dimensional tissue of cells is formed from a single cell by adhering to the base material, so compared with single layer cells or spheroids cultured from multiple cells , Has the potential to make the tissue closer to the living body. However, in order to increase the efficiency of screening, further improvement in aggregate formation efficiency is desired, and the development of a more suitable cell culture substrate is required.

  The present invention has been made in view of the above prior art, and an object thereof is to provide a cell culture substrate having good cell aggregate formation efficiency, excellent durability and stability, and a method for producing the same. .

As a result of intensive studies on the cell culture substrate, focusing on the formation origin of the cell aggregate and the extension of the cell aggregate, and a structure suitable for improving the aggregate formation efficiency, one surface of the glass substrate was obtained. In addition, the inventors have invented to arrange a plurality of recesses having a bowl shape, a so-called funnel shape, and having pores (cavities) at the bottom thereof, and have found that the above-mentioned problems can be solved, thereby achieving the present invention.
That is, the cell culture substrate of the present invention is a cell culture substrate in which a plurality of recesses having an opening in a funnel shape and pores at the bottom are arranged on one surface of a glass substrate,
The concave portion is formed through a process flow including at least a glass substrate cleaning process, a Cr film mask forming process, a resist mask forming process, a Cr film mask opening forming process by etching, and a wet etching process of the glass substrate. It is characterized by.
Further, the method for producing a cell culture substrate of the present invention is a method for producing a cell culture substrate in which a plurality of recesses having an opening in a funnel shape and pores in the bottom are arranged on one surface of a glass substrate. The recess is formed by a process flow including at least the following steps.
・ Glass substrate cleaning process,
A step of forming a Cr film mask on the cleaned glass substrate;
A step of forming a resist mask on the Cr film mask;
A step of opening the Cr film mask on which the resist mask is formed by etching;
-Wet etching process in which etching of the glass substrate and etching of the Cr film mask from the glass substrate are performed in parallel using the etchant to form a recess.

  The cell culture substrate of the present invention is excellent in stability and durability because it has a structure in which a plurality of concave portions having openings formed in a funnel shape and pores at the bottom thereof are arranged on a glass substrate by fine processing. . By using the cell culture substrate of the present invention, cell aggregate formation efficiency (aggregate formation efficiency: plate efficiency and spheroid efficiency) can be improved, and cell aggregates close to a living body can be formed. The accuracy and efficiency of screening can be improved.

It is the perspective view (FIG. 1a) which shows the base-material surface which observed the cell culture substratum concerning this invention by SEM, and sectional drawing (FIG. 1b) which shows the base-material side surface observed by SEM. It is a schematic diagram which shows the characteristic cross-sectional shape of the recessed part in the cell culture substratum which concerns on this invention as an example. It is a figure which shows the shape reproduced by simulation and the shape of the recessed part of the cell culture substratum which concerns on this invention formed by experiment.

  As described above, the cell culture substrate in the present invention is a cell culture substrate in which a plurality of recesses having an opening in a funnel shape and pores in the bottom are arranged on one surface of a glass substrate, The concave portion is formed through a process flow including at least a glass substrate cleaning step, a Cr film mask forming step, a resist mask forming step, a Cr film mask opening forming step by etching, and a wet etching step of the glass substrate. It is a feature.

  The pores (cavities) provided in the recesses of the cell culture substrate serve as a starting point for aggregation of cells (for example, single cells or cell lines) seeded on the substrate, and have a funnel-shaped slope structure. Can provide a medium suitable for extending cells in the three-dimensional direction (three-dimensional direction). Thereby, the efficiency of cell culture can be improved and good cell aggregate formation efficiency (plate efficiency, spheroid efficiency) can be obtained. In general, a plurality of recesses are regularly arranged.

Since the cell culture substrate of the present invention is a glass substrate provided with recesses by microfabrication, stability (dimensional stability, heat resistance, damage resistance, etc.) and durability (environmental resistance and deterioration resistance) Etc.) are also excellent. By using the cell culture substrate of the present invention, cell aggregate formation efficiency (cell aggregate formation efficiency) can be improved, and a cell aggregate close to a living body can be formed. Can be improved.
In other words, the use of a cell culture substrate (substantially, substrate) with excellent durability and stability that optimizes the surface structure improves the plate efficiency and spheroid efficiency, so that the three-dimensional tissue body is transformed from the cell to the substrate. A tissue body that is formed by adhering to a living body and close to a living body. In addition, the medium can be easily replaced and the culture operation is facilitated, and the accuracy and efficiency of screening in drug discovery is improved.
The recesses are preferably connected (adjacent openings are connected). Thereby, it is possible to suppress the formation of non-uniform cell aggregates between the recesses (the region where no opening is formed).

In the present invention, a cell aggregate having a tissue closer to a living body may be referred to as a “three-dimensional tissue” or “spheroid”.
Here, the cell aggregate formation efficiency (abbreviation: formation efficiency) can be evaluated by the plate efficiency and spheroid efficiency defined below.
Plate efficiency: Value (%) calculated from the number of colonies (cell aggregates) formed after the lapse of a predetermined number of days with respect to the number of cells seeded (per unit area)
Spheroid efficiency: Value (%) calculated from the number of spheroids (cell aggregates) formed after the lapse of a predetermined number of days with respect to the number of seeded cells (per unit area)

Here, in the wet etching step, it is preferable to control the Cr film mask so that the Cr film mask is peeled off from the glass substrate in parallel with the etching of the glass substrate to form a recess.
As will be described later, the degree of peeling of the Cr film mask from the glass substrate is a peeling rate higher than the mask undercut seen in normal wet etching, that is, the opening is funnel-shaped due to the progress of etching of the glass substrate from the peeling portion. Therefore, it is necessary to control so that pores (cavities) are formed at the bottom. In addition, it is preferable that the bottom surface of the pore has a spherical surface or a curved surface structure. Assuming that the funnel shape of the opening in the recess and the pore (cavity) are connected in a gentle continuous shape, so that the extension from the cell aggregation origin to the three-dimensional direction (three-dimensional direction) of the cell proceeds smoothly. Is done. Note that the slope angle of the funnel is selected within a range where a single cell aggregate can be formed.
The glass substrate is preferably made of quartz glass. If a substrate made of quartz glass is used, a fine pattern (for example, a grid-like arrangement, a staggered pattern, etc.) is formed by applying an established microfabrication technique used for semiconductors and the like to form a funnel-shaped recess having pores at the bottom. A plurality of arrays can be formed in the arrangement.
The cavity is preferably a fine hole having a bottom surface made of a spherical surface or a curved surface. The bottom surface of the spherical surface or curved surface is set in a range where a plurality of cell aggregates are not formed in cell culture. Usually, it is 100 μm or less and preferably 20 μm or less, but is not limited thereto.
The kind of cell cultured using the cell culture substratum (base material) of this invention is not specifically limited. In addition, the type of medium used for cell culture is not particularly limited, and an appropriate medium can be selected according to the type of cell. Therefore, when cells are cultured using the substrate of the present invention, cell aggregates (three-dimensional tissue bodies: spheroids) that are made to be tissues closer to living bodies can be formed.
The culture conditions are appropriately selected according to the cell type and are not limited, but generally the pH, temperature, atmosphere and the like are adjusted. For example, the culture can be performed under conditions such as pH 6 to 8, temperature 30 to 40 ° C., and presence of CO ₂ (about 5% concentration).

Hereinafter, the shape of the cell culture substrate (substrate) of the present invention and the formation method thereof will be described with reference to the drawings. First, the characteristics of the shape are shown, and then the manufacturing method is shown.
FIG. 1 shows a perspective view (FIG. 1a) of the surface of the substrate when the cell culture substrate of the present invention is observed by SEM, and a sectional view (FIG. 1b) of the side surface of the substrate observed by SEM.
1 is a glass substrate cleaning process, a Cr film mask forming process, a resist mask forming process, a Cr film mask opening forming process by etching, a wet etching process of a glass substrate (in parallel, the Cr film mask is peeled off from the glass substrate) It is formed through a process flow consisting of control. The opening diameter near the surface of the funnel shape of FIG. 1 is about 20 μm, the pore diameter at the bottom is about 10 μm, and the depth of the recess is about 10 μm.
In the schematic diagram of FIG. 2, the characteristic cross-sectional shape of the recessed part in the cell culture substratum which concerns on this invention is shown as an example. In FIG. 2, reference numeral 1 denotes a recess, reference numeral 2 denotes a funnel-shaped opening, and reference numeral 3 denotes a pore. The pores have a spherical bottom surface or a curved surface structure.
The cell culture substrate of the present invention has a plurality of recesses as described above arranged on a glass substrate, preferably a recess is regularly arranged. As an arrangement pattern, the plurality of recesses are arranged in a lattice or staggered pattern. Can be selected.
The diameter of the opening of each of the recesses is not particularly limited, but it is more preferable that the diameter is 10 μm or more as a cell aggregate (spheroid) used to increase the accuracy and efficiency of screening in drug discovery. It can be about 200 μm.

  Screening for cell aggregates (spheroids) formed using the cell culture substrate of the present invention by adding a drug or the like to cells that form spheroids and exhibiting action, effect, etc. it can. Spheroids can also be used in fields such as disease malignancy discrimination, food or drug safety assessment, or regenerative medicine.

As described above, the method for producing a cell culture substrate of the present invention comprises a cell culture substrate comprising a plurality of recesses having an opening in a funnel shape and pores at the bottom on one surface of a glass substrate. In the manufacturing method, the concave portion is formed by a process flow including at least the following steps.
・ Glass substrate cleaning process,
A step of forming a Cr film mask on the cleaned glass substrate;
A step of forming a resist mask on the Cr film mask;
A step of opening the Cr film mask on which the resist mask is formed by etching;
-Wet etching process in which etching of the glass substrate and etching of the Cr film mask from the glass substrate are performed in parallel using the etchant to form a recess.

As a process for forming the recess, a normal fine process based on mask formation and etching can be used. An example will be described below.
(1) Glass substrate washing step: For example, the glass substrate is washed with sulfuric acid / hydrogen peroxide and dried.
(2) Cr is vacuum-deposited (about 100 nm thick) on the cleaned glass substrate by sputtering to form a Cr film mask.
(3) A resist is formed on the Cr film mask using a photoresist material (thickness of about 3 μm), and the resist mask is formed by exposure and development using the photomask.
(4) The Cr film mask on which the resist mask has been formed is opened by etching using a Cr etchant (for example, a mixed solution of diammonium cerium nitrate, acetic acid and water).
(5) Using an etchant (for example, HF etchant), the glass substrate is etched from the opening and the Cr film mask is peeled from the glass substrate in parallel to perform wet etching to form a recess.

In normal wet etching, isotropic etching is performed, and a hemispherical recess is formed. In the present invention, as described in (5), in wet etching, the Cr film mask is peeled off from the glass substrate along with the etching of the glass substrate. By utilizing this, a concave portion having a funnel-shaped opening and a cavity (a fine hole having a bottom surface formed of a spherical surface or a curved surface) is formed at the bottom thereof.
Here, the degree to which the Cr film mask is peeled off from the glass substrate refers to a peeling speed equal to or higher than the mask undercut observed in normal wet etching. Specifically, the peeling rate of the Cr film mask from the glass substrate is preferably 2 to 6 times the etching rate of the glass substrate, and more preferably 3 to 5 times.

  The shape of the recess in the cell culture substrate of the present invention formed by experiment and the shape reproduced by simulation are shown in FIG. In FIG. 3, the part indicated by dots (•) is the experimental result, and the part indicated by the solid line (−) is the calculation result. In addition, the part shown by the rectangle shows a Cr film mask. In the simulation, it is calculated that the mask peels off from the glass substrate at a rate four times as fast as the glass substrate etching rate, and the etching of the glass substrate proceeds from the peeled portion, so that the opening is funnel-shaped and at the bottom. A recess having a cavity is reproduced.

The resist mask material used in the step of forming a resist mask on the Cr film mask is preferably a photoresist material. By using a photoresist material, there is a merit that the productivity becomes high with a simple process.
In the wet etching step, it is preferable to perform wet etching without removing the resist mask. Thereby, since the Cr mask can be reinforced, desired etching can be performed even when there is a defect in the Cr mask such as a pinhole.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further more concretely, this invention is not limited to an Example at all.

[Example 1]
In accordance with the process flow shown in Table 1 below, a cell culture substrate in which a plurality of concave portions having openings in a funnel shape on one side of a glass substrate and cavities in the bottom thereof are arranged regularly (lattice) Produced. The SEM observation drawing of the formed cell culture substrate was the same as FIG.

Each process is as follows.
(1) Glass substrate washing step: A quartz glass substrate was used as a glass substrate, washed with sulfuric acid / hydrogen peroxide and dried.
(2) Cr was sputtered on the cleaned quartz glass substrate surface under the sputtering conditions shown in Table 2 below to form a Cr film mask having a thickness of 100 nm.

  (3) After forming a 3 μm thick resist using a photoresist material [OFPR800 (manufactured by Tokyo Ohka)] on a Cr film mask, exposure is performed using a photomask in which openings having a diameter of 8 μm are formed at a pitch of 50 μm. Then, a resist mask was formed by development. Resist application and resist shape preparation were performed according to the preparation conditions shown in Table 3 below.

  (4) Using a Cr etchant having a formulation described in Table 4 below, a Cr film mask on which a resist mask was formed was opened by etching. The etching processing time was 5 to 6 minutes.

  (5) As described in Table 1, the etching of the glass substrate and the peeling of the Cr film mask from the glass substrate are proceeded in parallel using the HF etchant, and the Cr film mask from the glass substrate at this time. A recess was formed with a peeling rate of about 4 times the etching rate of the glass substrate.

Next, 1000 cells of human glioma-derived cell line U251 were seeded together with the α-MEM medium using the cell culture substrate prepared above, and were cultured for 7 days in the atmosphere of 37 ° C. and 2% CO ₂ (on the way 2 The culture medium was exchanged to form spheroids as cell aggregates, and the plate efficiency and spheroid efficiency were calculated to examine the culture efficiency. The results are shown in Table 5 below.
The plate efficiency is a value (%) calculated from the number of colonies (cell aggregates) formed after a predetermined number of days (3 days) after culturing with respect to the number of cells seeded (per unit area).
The spheroid efficiency is a value (%) calculated from the number of formed spheroids (cell aggregates) when a predetermined number of days have elapsed (7 days) after culturing with respect to the number of cells seeded (per unit area).

[Comparative Example 1]
In Example 1, a process other than the glass substrate cleaning process is not applied, and a so-called flat quartz glass substrate with no recesses formed on the surface is used as a cell culture substrate, and cell culture is performed in the same manner as in Example 1. The culture efficiency (plate efficiency and spheroid efficiency) was examined. The results are shown in Table 5 below.

From the results shown in Table 5, when using a cell culture substrate in which a plurality of recesses (openings having a funnel shape and pores at the bottom thereof) of the present invention are arranged, recesses are formed on the surface. Compared to the case where a flat quartz glass substrate with no flat plate was used as a cell culture substrate, both plate efficiency and spheroid efficiency showed large values, indicating that the culture efficiency was improved.
That is, the cell culture substrate having pores (cavities) that serve as the origin of cell aggregation according to the present invention and a concave structure having a funnel shape (spot shape) suitable for cell expansion in the three-dimensional direction is used. As a result, cell aggregate formation efficiency (cell aggregate formation efficiency) can be improved, cell aggregates close to living bodies can be formed, and screening precision and efficiency in drug discovery can be improved.

1 Concave part 2 Funnel-shaped opening 3 Pore

JP 2002-335949 A Japanese Patent No. 443233 Japanese Patent No. 2716646 Japanese Patent No. 4159103 JP 2008-290227 A

Claims (6)

On one surface of the glass substrate, a cell culture substrate in which a plurality of recesses having an opening in a funnel shape and pores at the bottom thereof are arranged,
The concave portion is formed through a process flow including at least a glass substrate cleaning process, a Cr film mask forming process, a resist mask forming process, a Cr film mask opening forming process by etching, and a wet etching process of the glass substrate. A cell culture substrate characterized by the above.   2. The cell culture substrate according to claim 1, wherein in the wet etching step, a recess is formed by controlling the Cr film mask to be peeled from the glass substrate in parallel with the etching of the glass substrate.   The cell culture substrate according to claim 1 or 2, wherein the bottom surface of the pore has a spherical surface or a curved surface structure.   The cell culture substrate according to any one of claims 1 to 3, wherein the glass substrate is made of quartz glass. A method for producing a cell culture substrate, wherein a plurality of recesses having an opening in a funnel shape and pores at the bottom thereof are arranged on one surface of a glass substrate, wherein the recess includes at least the following steps. A process for producing a cell culture substrate characterized by comprising the steps of:
・ Glass substrate cleaning process,
A step of forming a Cr film mask on the cleaned glass substrate;
A step of forming a resist mask on the Cr film mask;
A step of opening the Cr film mask on which the resist mask is formed by etching;
-Wet etching process in which etching of the glass substrate and etching of the Cr film mask from the glass substrate are performed in parallel using the etchant to form a recess.   The method for producing a cell culture substrate according to claim 5, wherein the resist mask material used in the step of forming a resist mask on the Cr film mask is a photoresist material.