JP2009065945A - Cell attaching/culturing base material capable of imparting cell attaching property by irradiation of light - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell attaching/culturing base material (substrate) capable of imparting cell attaching property by irradiation of light and which is suitable for cell culture, over a long time. <P>SOLUTION: The cell attaching/culturing base material has a photodegradable group and a cell attachment suppressing group bonded, in the order, to the surface of the base material through a base material binding group by covalent bond. The photodegradable group is e.g. a bivalent organic group, represented by structure formula shown by chemical formula (1), and the cell attachment suppressing group is, preferably, a polyethylene glycol (PEG) group represented by the structure formula: -(CH<SB>2</SB>CH<SB>2</SB>O)<SB>m</SB>R (m is an integer of 3-1,200; and R is H or CH<SB>3</SB>). <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a cell adhesion / culture substrate in which a photodegradable group and a cell adhesion-inhibiting group are bonded to the surface of a substrate (or substrate) via a substrate binding group, and the cell adhesion / culture substrate. And a substrate for cell attachment / culture with a predetermined pattern of cell adhesion region obtained by using light irradiation, or a substrate with a predetermined pattern of cell mass, and further, the cell mass of the predetermined pattern The present invention also relates to a method of growing a cell mass along a predetermined pattern on a base material using an attached base material and light irradiation. These substrates or methods are useful as the basis for cell patterning techniques.

Cell patterning technology is a culture substrate in which two types of chemical species are patterned: those that have adhesion to cells (mainly animal-derived adherent cells) and those that do not have adhesion (non-adhesive). (Or substrate) to define cell attachment and non-attachment regions, and is not only useful for basic cell biological research, but also tissue engineering such as tissue regeneration, drug sensors, and environmental pollution This technology is useful for new development of cell-based sensors such as substance sensors. In particular, cell adhesion conversion technology in response to external stimuli, which has attracted attention in recent years, causes conversion reactions such as phase transition, oxidation / reduction, and photoreaction on the surface by external stimuli such as temperature (heat), electricity, and light. Functional materials with chemical species are used.
The following several methods are known as “cell attachment switching technology” using such a functional material.

(1) Method using temperature-responsive polymer membrane by Okano et al. (Yamada, N. et al., 1990; Nandkumar, MA et al., 2002)
This is a method using a temperature-responsive polymer membrane poly (N-isopropylacrylamide) (polyNIPAAm). PolyNIPAAm is hydrophilic below the lower critical solution temperature and hydrophobic above it, but in culture dishes grafted with polyNIPAAm, cells are non-adherent below the lower critical solution temperature, and cells are above it. Okano et al. Found it to adhere. Utilizing this property, the technology has been developed mainly for detaching sheet-like cells to be used for transplantation from the surface of the culture dish, and a number of tissues are being tested for practical use. This technique is also applied to co-culture of multiple cells.

(2) A method using a monolayer having electrochemical activity such as Mrksich et al. And Whitesides et al. (Yousaf, MN et al., 2001; Yeo, WS et al., 2001; Yeo, WS et al., 2003; Jiang , XY et al., 2003)
(I) Mrksich et al .: a method using an alkanethiol self-assembled film having electrochemically active hydroquinone formed on a gold-deposited cover glass. Utilizing the fact that hydroquinone is reversibly oxidized to benzoquinone according to the applied potential, cell attachment / desorption is performed by binding / dissociating cell-adhesive RGD peptides. Recently, a technology for changing cell adhesion in two stages of “adhesion → non-adhesion → adhesion” on one substrate has been developed.
(Ii) Whitesides et al .: A method for converting cell adhesion by utilizing the fact that the bond between gold and thiol itself is dissociated by a reduction potential. A patterned self-assembled membrane in which the cell attachment region was surrounded by a cell non-adhesive ethylene glycol derivative was produced, and the cell attachment property was restricted to a specific region. When an electric potential is applied to the entire substrate, ethylene glycol is dissociated from the surface, so that the cell movement restriction is released. They are using this method to evaluate drugs that inhibit cell motility.

(3) A technique for generating an oxidant locally using a microelectrode by Nishizawa et al. (Kaji, H. et al., 2004)
This is a method in which cell adhesion-inhibiting bovine serum albumin (BSA) previously adsorbed on a substrate is denatured and converted into cell adhesion by generating an oxidizing agent such as HBrO locally using a microelectrode. . In this method, it is possible to overwrite the surface on which the cells are first patterned, and it is possible to form a cell adhesion region even on a substrate with unevenness, which has been difficult in the past.

(4) A method using a photoresponsive polymer having spiropyran in the side chain of Kanamori et al. (Edahiro, J. et al., 2005)
This is a method for controlling adhesion after cell seeding using a photoresponsive polymer (compound that is reversibly isomerized by light) having spiropyran as a photochromic compound in the side chain.

(5) A method of Nakanishi et al. (See Patent Document 1 and Non-Patent Document 1) in which a cell adhesion inhibitor is physically adsorbed on a substrate modified with a photodegradable group.
In this method, a cell adhesion inhibitor is physically adsorbed on a substrate modified with a photodegradable group. As a result, co-culture of the same or different cells by light irradiation and light induction of cell migration can be realized, but the cell adhesion inhibitor physically adsorbed on the substrate can be easily combined with proteins derived from cell culture medium. Due to the exchange, maintenance of the cell pattern is limited to 2-3 days.

JP 2006006214 A Nakanishi, J. et al., J. Am. Chem. Soc. 126: 16314-16315 (2004)

  As described above, the base material that allows cells to be selectively attached to the surface of the base material (or the substrate) and cultured can be used for basic research in cell biology, tissue engineering, or cells. Useful for new development of base sensors. In particular, a substrate capable of converting cell adhesion (adhesiveness) on the surface by external stimuli such as heat, electric potential, and light makes it possible to induce cell proliferation and migration, and repeats external stimuli and cell seeding. This makes it possible to coexist (co-culture) heterogeneous cells on the same substrate, and thus has a wide range of applications. Considering the remote operability and temporal / spatial resolution, light is considered to be the most ideal external stimulus. However, the cell adhesion conversion base materials of the above (4) to (5) are known so far. Although the cell adhesion was converted by light, the maintenance time was about several days, and it was difficult to keep the cells in a specific position on the substrate over a long period of time. Accordingly, an object of the present invention is to develop a cell adhesion / cultivation substrate (substrate) that can impart cell adhesion by light irradiation and is suitable for long-term cell culture.

In order to achieve the above object, the present inventors have made various studies focusing on covalent bonding of a photodegradable group and a cell adhesion-inhibiting group on a base material via a base material binding group. The present invention has been completed.
That is, the present invention is a cell adhesion / culture substrate in which a photodegradable group and a cell adhesion-inhibiting group are sequentially bonded to a substrate surface via a substrate binding group via a covalent bond.

  Further, the present invention is obtained by irradiating the cell adhesion / culture substrate with light in a predetermined pattern and then washing to remove the cell adhesion inhibitory group. In other words, the cell adhesion inhibitory group is provided in the light irradiated part. Therefore, the light-irradiated portion also provides a cell adhesion / culture base material with a cell adhesion region having a predetermined pattern.

  The present invention also provides a base material with a cell pattern having a predetermined pattern obtained by attaching and proliferating cells to the cell adhesion region of the base material for cell attachment / culture.

Further, the present invention is a substrate with an aligned cell mass of a predetermined pattern in which the same or different types of cell clusters are each attached to and present in an island-like manner on the substrate. Also provided is a cell mass-attached substrate obtained by a method comprising the following steps.
(1) A part of the surface of the substrate for cell attachment / culture is irradiated with light in a predetermined pattern, washed, seeded with a first cell type, incubated in a nutrient medium, and irradiated with the light. Obtaining a substrate with a first cell type mass, wherein a first cell type is propagated in a part;
(2) The other part of the surface of the obtained base material with the first cell type mass is irradiated with light in a predetermined pattern, washed and seeded with the second cell type, and this is in a nutrient medium. A step of obtaining a base material with a second cell type mass, which is kept warm and proliferates the second cell type in the light irradiation part;
(3) to (n) Hereinafter, similarly, third to n-th cell type masses are sequentially formed on the surface of the base material with cell type masses.

  In the present invention, light irradiation is performed in a predetermined pattern in the vicinity of the cell mass of the cell mass-attached base material, and after washing to remove the cell adhesion inhibiting group of the light irradiation part, this is kept warm in the nutrient medium. The present invention also provides a method for growing a cell mass along a predetermined pattern on a cell attachment / culture substrate.

  In the cell adhesion / culture substrate of the present invention, the cell adhesion inhibitor that is covalently bonded to the substrate does not easily adhere to proteins derived from the cell culture solution, and the cells (or (The pattern) can be stably maintained. Therefore, the cell adhesion / cultivation substrate of the present invention, the cell adhesion / culture substrate with a cell adhesion region, or a substrate with a cell aggregate of a predetermined pattern, or The method of growing cell clusters along a predetermined pattern on a cell adhesion / culture substrate can provide a useful means for basic research of cell biology or new development of tissue engineering, cell-based sensors, etc. .

In the cell adhesion / culture substrate of the present invention, as described above, a photodegradable group and a cell adhesion inhibitory group are sequentially covalently bonded to the surface of the substrate via a substrate binding group. Cell adhesion / culture substrate.
Here, the “substrate” means a material (substrate) in which cells become an anchor for growth and proliferation, and the shape thereof is a flat plate (substrate), concave or convex. There may be one having R in the shape, a stick-like one, a spherical one, etc., but a flat plate-like one (substrate) is preferred.
The base material is not particularly limited as long as it is suitable for cell attachment. There are glass, gold, silicon, diamond, metal oxide, plastic (styrene, polyethylene, polypropylene, PET, etc.) and the like.

The “substrate binding group” means a chemical group that can be bonded to the substrate surface by a covalent bond. The chemical group varies depending on the substrate used, but when the substrate is glass, a chemical group represented by the following structural formula is preferably used.

When the substrate is gold, a chemical group represented by the following structural formula is preferably used.

The “photodegradable group” means a divalent chemical group that easily decomposes when irradiated with high-energy light such as ultraviolet rays. For example, the following divalent organic groups are preferably used.

The following divalent organic groups can also be used.

“Cell adhesion inhibitory group” means a chemical group that inhibits (or inhibits) cell adhesion (or adhesion), and representative ones are polyethylene glycol (PEG), polyacrylamide, or poly N-isopropylamide. An organic group having (NIPAAm), preferably an organic group having polyethylene glycol (PEG) represented by the following chemical formula.
(Here, m is an integer of 3 to 1200, and R is H or CH _3. )

  The “cells” to be attached and cultured include various animal cells and plant cells, and preferably adherent animal cells.

Example 1 is an example in which the base material is glass.
(1) Synthesis of 1- [3-methoxy-6-nitro-4- (3-trimethylsilyl) propylphenyl] ethyl N-succinimidyl carbonate The title compound (silane coupling agent) represented by the chemical structural formula of Chemical Formula 10 is: The compound was synthesized according to the method described in JP-A-2007-186472.

(2) Production of Cell Immobilization Substrate FIG. 1 shows a surface treatment procedure (scheme) of a glass substrate using a chemical formula.
A glass substrate (cover glass: 18 × 18 mm ² , 0.12 to 0.17 mm) is mixed with a mixed solution of H ₂ SO ₄ : H ₂ O ₂ (7: 3, volume ratio) (Pyrania solution; explosive) Therefore, it is necessary to take care not to handle the organic substances in the vicinity of the organic substances). The organic substances were removed by heating in 20 mL at 90 to 100 ° C. for 1.5 hours. Subsequently, it was washed with pure water and further subjected to ultrasonic cleaning treatment. The cover glass is refluxed for 24 hours in a 1.5 mM benzene solution of 1- [3-methoxy-6-nitro-4- (3-trimethylsilyl) propylphenyl] ethyl N-succinimidyl carbonate (silane coupling agent). As a result, the glass surface was chemically modified. The obtained chemically modified substrate was added to an aqueous solution (0.4 M K ₂ SO ₄ /0.1 M NaH ₂ PO ₄ , pH 7.1) of 0.1 M polyethyleneglycol amine (PEG-amine, molecular weight 5,000). It was soaked for a time to bind PEG.
In addition, the chemical modification of the glass substrate (cover glass) with the compound of the chemical formula 10 and the chemical modification with PEG-amine increase the contact angle of water on the substrate surface from 12 ° (cover glass used) → 67 ° → 46 °. And changed. This change in the contact angle signifies that the surface chemical species of the cover glass has changed as shown in FIG. 1 (upper three stages).

(3) Production of Cell Fixing Substrate with Cell Adhesive Region in Predetermined (Circular) Pattern On one surface of the cell fixing substrate thus obtained, a circular region (diameter of about 500 μm) is formed on one surface under a fluorescence microscope. Irradiated with light (λ = 365 nm, 1 W / cm ² ) for ² seconds, and then washed with purified water to wash out the PEG to obtain a cell fixing substrate with a cell adhesion region having a predetermined (circular) pattern.
The contact angle of water on the substrate surface before and after the light irradiation changed from 46 ° (front) to 64 ° (rear). This change in the contact angle signifies that the surface chemical species of the cover glass has changed as shown in FIG. 1 (bottom two steps).

(4) Production of a substrate with a cell cluster having a predetermined (circular) pattern On a cell fixing substrate with a cell adhesion region, 2 × 10 ⁵ NIH3T3 cells were seeded using a serum-free medium, and a carbon dioxide incubator The temperature was kept at 37 ° C. After 1 hour of seeding, the medium was replaced with a serum medium, and cells on the substrate were observed with a phase contrast microscope 1 day and 14 days after sowing. NIH3T3 cells adhere only to the cell-adhesive circular region provided on the cell-fixing substrate, and a confluent cell group (colony) that has proliferated as shown in FIG. Then, the cells continued to proliferate, and the pattern of the cell group was maintained even after a long period of 7 days after seeding (FIG. 2 (b)).

(5) Induction of mobile growth of cells selectively attached to the area The cells were attached to a circular area obtained by carrying out the same steps as in (2) to (4) above, and after overnight culture, Light irradiation (secondary light irradiation) was performed again on a circular portion of similar size close to the cell group (colony) (FIG. 3 (a)), and the culture was continued. Then, the cells proliferated while moving to the region irradiated with the secondary light, reached a confluent pattern (peanut shape), and the pattern was maintained for 5 days or more after sowing (FIG. 3B). This result indicates that the cell attachment region can be sequentially increased by further irradiating the substrate with light during cell culture. In this example, the allogeneic cell adhesion region is expanded, but it is also possible to place multiple types of cells on the same substrate (co-culture of different types of cells) by seeding different types of cells after secondary light irradiation. .

Example 2 is an example where the base material is gold.
(1) Synthesis of Bis (12- (4-succinimidyloxycarbonylethyl-2-methoxy-5-nitrophenyl) oxydedecyl) disulphide The title disulfide compound represented by the structural formula of Chemical Formula 11 was synthesized by the method described in Japanese Patent Application No. 2006-120002. did.

(2) Production of Cell Immobilization Substrate FIG. 4 (a) shows a surface treatment procedure (Scheme I) for a gold substrate shown using a chemical formula.
The gold sputtered substrate was immersed in 50 mL of a mixed solution of H ₂ SO ₄ : H ₂ O ₂ (2: 1, volume ratio) (Pyraniya solution) for 1 minute to remove organic substances. Subsequently, it was washed with pure water and further subjected to ultrasonic cleaning treatment. This substrate was immersed in a DMSO solution of Bis (12- (4-succinimidyloxycarbonylethyl-2-methyoxy-5-nitrophenyl) oxydedecyl) disulphide (0.5 mM) to form an organic monomolecular film on the surface. Subsequently, this substrate was immersed in a phosphate buffer containing 1 wt% PEG-amine (molecular weight 5,000) and 0.4 M K ₂ SO ₄ overnight to modify PEG on the surface.

(3) Production of cell fixing substrate with cell adhesion region having a predetermined (circular) pattern Light is applied to the circular region (diameter: about 500 μm) on one surface of the obtained cell fixing substrate (gold substrate) under a fluorescence microscope. Irradiation (λ = 365 nm, about 1 W / cm ² , 60 sec), and then washed with purified water to wash out the PEG, thereby obtaining a cell fixing substrate with a cell adhesion region having a predetermined (circular) pattern.
In addition, the chemical modification of the gold substrate with the organic reagent, and then the chemical modification with PEG-amine, the contact angle of water on the substrate surface is 65 ° (gold substrate on which an organic monomolecular film is formed) → 45 ° → 70 °. Changed. This change in contact angle is consistent with the change in the surface chemical species of the gold substrate as shown in FIG.

(3) Preparation of a substrate with a cell cluster having a predetermined (circular) pattern 2 × 10 ⁵ NIH3T3 cells were seeded in a serum-free medium on the cell-fixing substrate with the cell adhesion region, and serum was obtained after 1 hour. The culture medium was replaced with a medium containing, and kept at 37 ° C. in a carbon dioxide incubator. The cells showed adhesion corresponding to the irradiation pattern (FIG. 5 (a)). When the culture was further continued, the cells continued to grow and formed aggregates, but the pattern of the cell group was maintained even after a long period of time on the seventh day after cell seeding (FIG. 5 (b)). ).

Example 3 is also an example in which the base material is gold as in Example 2.
(1) Production of Cell Immobilization Substrate A surface treatment procedure (Scheme II) for a gold substrate is shown in FIG.
0.5 mM Bis (12- (4-succinimidoxycarbonylethyl-2-methyl-5-nitrophenyl) oxydedecyl) disulphide (molecular weight 10,000) was reacted overnight in a DMSO solution containing 1 mM triethylamine. Inside, a piranha-cleaned gold sputtered substrate was immersed overnight as in Example 2 to form an organic monomolecular film on the surface thereof.
(2) Production of cell fixing substrate with a cell adhesion region having a predetermined (circular) pattern Light is applied to a circular region (diameter of about 500 μm) on one surface of the obtained cell fixing substrate (gold substrate) under a fluorescence microscope. Irradiation (λ = 365 nm, about 1 W / cm ² , 60 sec), and then washed with purified water to wash out the PEG, thereby obtaining a cell fixing substrate with a cell adhesion region having a predetermined (circular) pattern.
(3) Preparation of a substrate with a cell cluster having a predetermined (circular) pattern 2 × 10 ⁵ NIH3T3 cells were seeded in a serum-free medium on the cell-fixing substrate with the cell adhesion region, and serum was obtained after 1 hour. The culture medium was replaced with a medium containing, and kept at 37 ° C. in a carbon dioxide incubator. The cells showed adhesion corresponding to the irradiation pattern (FIG. 6 (a)). When the culture was further continued, the cells continued to grow and formed aggregates, but the pattern of the cell group was maintained even after a long period of time on the seventh day after cell seeding (FIG. 6 (b)). ).

4 is a “substrate surface treatment procedure (scheme)” according to Example 1; A in the figure means a substrate (glass). A phase-contrast micrograph showing selective attachment / proliferation of cells on a cell-fixing substrate (glass substrate) with a cell-adhesive region of a predetermined pattern (circular), (a) is one hour after seeding, (b ) Is after 7 days of sowing. A bright-field photomicrograph showing (light) induction of mobile growth on a cell-fixing substrate (glass substrate), (a) is one day after sowing, the dotted line is the secondary light irradiation region, (B) is after 5 days after sowing. In the “substrate surface treatment procedure (scheme)” according to Example 2 and Example 3, (a) relates to Example 2, and (b) relates to Example 3. A in the figure means a base material (gold). It is a phase-contrast micrograph which shows the selective adhesion | attachment and proliferation (I) of the cell on the cell-fixing board | substrate (gold substrate) which concerns on Example 2, (a) is a thing 1 day after sowing, (b) is seeding 7 A day later. FIG. 3 is a phase contrast micrograph showing selective adhesion / proliferation (II) of cells on a cell fixing substrate (gold substrate) according to Example 3, wherein (a) is one hour after seeding, (b) is seeding. Shown after 14 days.

Claims (7)

  A substrate for cell attachment / culture, wherein a photodegradable group and a cell adhesion-inhibiting group are sequentially bonded to a substrate surface via a substrate-binding group via a covalent bond. The photodegradable group has the following chemical structural formula
The substrate for cell attachment / culture according to claim 1, which is a divalent organic group represented by the formula: The cell adhesion inhibiting group has the following chemical structural formula:

(M is an integer of 3 to 1200, R is H or CH ₃ )

The cell adhesion / culture substrate according to claim 1, wherein the substrate is a polyethylene glycol (PEG) group represented by the formula:   A cell adhesion region having a predetermined pattern obtained by irradiating the cell adhesion / culture substrate according to any one of claims 1 to 3 with light in a predetermined pattern and then washing to remove the cell adhesion inhibitory group. Cell adhesion / culture base material.   A substrate with a cell pattern having a predetermined pattern obtained by attaching and growing cells on the cell adhesion region of the cell adhesion / culture substrate according to claim 4. Cell aggregates of a predetermined pattern in which the same or different types of cell clusters, which are the same or different, adhere to and exist on the base material separately from each other, are obtained by a method including the following steps: Substrates with cell clumps:
(1) A portion of the surface of the cell adhesion / culture substrate according to any one of claims 1 to 3 is irradiated with light in a predetermined pattern, washed, seeded with the first cell type, and nourished. Incubating in the medium and proliferating the first cell type in the light irradiation part (step of obtaining a substrate with a first cell type mass);
(2) Other part of the surface of the obtained base material with the first cell type mass is irradiated with light in a predetermined pattern, washed, and seeded with the second cell type. Incubating and proliferating the second cell type in the light irradiation part (a step of obtaining a substrate with a first and second cell type cluster);
(3) to (n) Hereinafter, in the same manner, a step of sequentially forming third, fourth,..., And nth cell type masses on the surface of the cell type mass-attached base material.   Light irradiation is performed in a predetermined pattern in the vicinity of the cell mass of the substrate with cell mass according to claim 5, and after washing to remove the cell adhesion-inhibiting group of the light irradiation part, this is kept warm in a nutrient medium. A method of proliferating a cell mass along a predetermined pattern on a cell adhesion / culture substrate.