JP2023067487A - Plate-like substrate for observing or culturing cells and method for producing plate-like substrate for observing or culturing cells - Google Patents

Abstract

To provide a plate-like substrate capable of forming a cell adhesion region of arbitrary size and shape in a designated region on a substrate and of allowing cells to adhere only to the limited region, without using a mask.SOLUTION: Produced is a plate-like substrate for observing or culturing cells that comprises a cell non-adhesive region and a cell adhesive region on one surface of a laser-permeable substrate, the cell non-adhesion region comprising: a first layer formed of a substance that generates heat or plasmon resonance by laser irradiation on one surface of the substrate; and a laminate composed of a second layer that is disposed on the first layer and has at least a surface formed of a cell non-adhesive substance, the cell adhesion region being a region provided with a substance having cell adhesion on the surface.SELECTED DRAWING: Figure 5

Description

TECHNICAL FIELD The present invention relates to a plate-like substrate for observing or culturing cells, and a method for producing a plate-like substrate for observing or culturing cells.

Techniques for adhering or arranging cultured cells to predetermined minute regions on the surface of culture substrates such as cover glasses and petri dishes are currently being researched all over the world. Using this technology, it becomes possible to observe cell movement, proliferation, and cell death. For example, with regard to cell movement, if an adhesion region is created so as to have a linear shape with a width narrower than that of a cell, it is possible to control cells that move in random directions to move linearly. Furthermore, the direction of cell division can also be restricted on the line. In addition, by preparing a cell adhesion area that fits within the microscopic field of view, it is possible to limit the movement area when observing cells with high motility. It becomes possible. Moreover, if a cell sheet with an arbitrary shape is produced, it can be applied to regenerative medicine and the like.

So far, several techniques have been developed for adhering or arranging cultured cells in predetermined microregions. For example, by coating polyethylene glycol (PEG) on a solid-phase substrate to prepare a hydrophilic solid-phase substrate that does not adhere to cells, and then printing extracellular matrix (ECM) on the PEG upper surface, cell adhesion can be achieved. A technique for producing a micropattern of regions has been disclosed (see Patent Document 1). However, there is a problem that PEG cannot completely inhibit cell adhesion. In addition, the formation of the micropattern requires an ink-jet printing technique, making the production of the substrate very complicated and expensive.

In addition, a method has been proposed in which a patterned adhesion region is produced by coating a coverslip with a cell adhesion-changing material that changes the adhesion of cells by the action of a photocatalyst, then placing a mask on it and irradiating it with light ( See Patent Document 2). According to this method, it is possible to temporarily fix cells on the substrate, but there is a problem that it is difficult to maintain the cells in the adhesion region for a long time. Therefore, when observing motile cells, for example, there is a problem that the cells come out of the microscope's field of view, making it impossible to continue observing the same cells for a long period of time.

Furthermore, a substrate for cell culture using a photomodifiable polymer that changes to cell adhesion upon UV irradiation has been disclosed (see Patent Document 3). Although cell patterning was possible using photomodifiable polymers, cells did not completely lose their adhesiveness even in the non-cell-adhesive areas.

The present inventors coated a polymer of 2-methacryloyloxyethylphosphorylcholine (MPC) on a substrate, covered it with a mask with holes of various shapes, and plasma-treated it from above. A method for selectively producing a cell adhesion region on a substrate has been disclosed (see Patent Document 4). In Patent Document 1, a method of removing the MPC coat by plasma treatment is adopted, but in this method, it is necessary to prepare a mask, and for shapes that are difficult to form with a mask, it is necessary to prepare a cell adhesion region. There was a problem that it was not possible to

Furthermore, the present inventors placed cells on a cover glass thin film coated with a thin film of a light-harvesting agent on the top surface, and irradiated the thin film with a laser to open holes in the cell membrane and introduce foreign substances. (Patent Documents 5 and 6, Non-Patent Document 1). This method is a technique for introducing a foreign substance such as a plasmid into a single cell, and no consideration is given to the adherence of cells to a predetermined position.

As an application of the technique of adhering or arranging cultured cells to predetermined microregions, it is also possible to produce a "cell microarray" by arranging a large number of cell adhesion regions on a base material at predetermined intervals. Conventionally, a 96-well incubator was used to examine the effects of drugs, cells were placed in each well, drugs were added, and the effects of drugs were observed. It takes time and effort.

Therefore, a plurality of cell adhesive regions formed on the upper surface of the bottom surface of the culture vessel formed of a cell non-adhesive substance and adhered to the cell group are provided, and the plurality of cell adhesive regions are attached to one of the cell groups. There is disclosed a culture vessel characterized by having a size sufficient for cell adhesion and arranging the cell adhesive regions at predetermined intervals so that they do not contact each other (see Patent Document 7). . According to this method, cells can be adhered to the substrate in a state of being separated one by one in a dot pattern, but it is difficult to control the size and shape of the cell adhesion region.

In addition, a method for noninvasively recovering cell spheroids formed from a cell culture substrate having cell adhesive domains on the surface of the cell non-adhesive substrate on the order of micrometers is disclosed (Patent Document 8). This method is a technique whose task is to collect spheroids. It is equipped with a cell non-adhesive site, and since it is a technology that separates and collects spheroids by degrading or dissolving the cell non-adhesive site, it is not suitable for observation of specific cell movement, proliferation, cell death, etc. I didn't. Also, a cell culture substrate having a substrate and a 2-methacryloyloxyethylphosphorylcholine layer, the substrate having a plurality of rough surface portions not covered with the 2-methacryloyloxyethylphosphorylcholine layer on the surface of the substrate Here, a cell culture substrate is disclosed in which the shape of the rough surface portion is a spot with a diameter of 20 μm to 100 μm or a groove with a width of 3 μm to 30 μm (see Patent Document 9). With this substrate, it is possible in vitro to generate cell populations with the innate biological properties of cancer tissue, such as morphological polarity and tissue motility polarity, and to perform live imaging of microtumors, but cell-to-cell communication was difficult to assess.

JP 2012-187072 A JP 2004-344025 A Japanese Patent Application Laid-Open No. 2020-029468 JP 2019-110819 A International Publication No. 2018/105748 pamphlet JP 2015-167551 A JP-A-2000-41659 JP 2006-67987 A International Publication No. 2018/182044 pamphlet

Yumura, S., A novel low-power laser-mediated transfer of foreign molecules into cells. Scientific Reports, 6:22055(2016)

An object of the present invention is to provide a plate-shaped substrate that has a cell adhesion region of any size and shape in a designated region on the substrate without using a mask, and that allows cells to adhere only to the limited region. to do.

As a result of intensive studies aimed at solving the above problems, the present inventors have found that a cell adhesion region can be formed on a substrate by utilizing heat generated by laser irradiation or plasmon resonance, and completed the present invention.

That is, the present invention is as follows.
[1] A plate-shaped substrate for observing or culturing cells, comprising a cell non-adhesive region and a cell adhesive region on one side of a laser-permeable substrate,
The cell non-adhesion region is arranged on one surface of the base material, and is arranged on a first layer formed of a substance that generates heat or plasmon resonance by laser irradiation, and is arranged on the first layer, at least A laminate comprising a second layer whose surface is formed of a cell non-adhesive substance,
The plate-shaped substrate, wherein the cell adhesion region is a region having a cell adhesion property on the surface.
[2] The above, wherein the substrate is a laser-permeable cell adhesive substrate, and the cell adhesion region is an exposed region of the laser-permeable cell adhesive substrate. The plate-like substrate according to [1].
[3] The plate-like substrate according to [1] or [2] above, wherein the cell-adhesive substrate capable of transmitting laser light is a substrate containing glass as a main component.
[4] The plate-shaped substrate according to any one of [1] to [3] above, wherein the cell non-adhesive substance is a polymer of 2-methacryloyloxyethylphosphorylcholine (MPC).
[5] The plate-shaped substrate according to any one of [1] to [4] above, wherein the cell adhesion region is a region formed by irradiating the first layer with a laser. .
[6] A part of the cell adhesion region is composed of a substance in which the cell non-adhesive substance is denatured or dispersed by laser irradiation and loses cell non-adhesive property, [1] to [5] The plate-shaped substrate according to any one of [5].
[7] The plate-like substrate according to any one of [1] to [6] above, wherein the cell adhesion region has a pattern of arbitrary shape.
[8] The plate-shaped substrate according to any one of [1] to [7] above, wherein the cell adhesion region and the cell non-adhesion region are distinguishable under visible light [9] Laser irradiation The plate-shaped substrate [10] according to any one of [1] to [8] above, wherein the substance that generates heat or plasmon resonance by the cell is a substance that is impermeable to visible light. A method for producing a plate-like substrate for observation or culture, comprising:
(a) forming a first layer by coating or spraying a substance that generates heat or plasmon resonance upon laser irradiation onto one surface of a substrate that can transmit a laser;
(b) applying or spraying a cell non-adhesive substance onto the first layer to laminate a second layer;
(c) irradiating the first layer with a laser, and denaturing or dispersing the cell non-adhesive substance of the second layer by heat generation or plasmon resonance due to laser irradiation, and cell adhesion regions that have lost cell non-adhesiveness forming a;
A method for producing a plate-like substrate, characterized in that it includes steps (a) to (c) in order.
[11] The method for producing a plate-like substrate according to [10] above, wherein the cell adhesion region is a region in which a laser-permeable cell-adhesive substrate is exposed.
[12] The method for producing a plate-shaped substrate according to [10] or [11] above, wherein the cell-adhesive region and the cell-non-adhesive region are distinguishable under visible light.

The plate-shaped substrate of the present invention enables observation of the cell adhesion region under visible light, and is useful for observation of cells. Moreover, the plate-like substrate of the present invention can have a width of 1.5 μm as the cell adhesion area, and it is possible to control the movement of one or several extremely small cells. In addition, by using the plate-like substrate of the present invention, the plate-like substrate can be further irradiated with a laser during or after cell culture to add or expand the cell adhesion region in real time, thereby inducing or controlling cell movement. It becomes possible to Furthermore, according to the method for producing a plate-shaped substrate of the present invention, it is possible to form a desired cell adhesion region only on a designated region on a substrate in a simpler and shorter time than conventional methods without using a mask. It becomes possible.

1 is a diagram showing a plate-like substrate produced in Example 1. FIG. 1 is a diagram showing a state in which a plate-like substrate is irradiated with a laser in Example 1. FIG. 1 is a diagram showing a state in which a cell adhesion region is formed on a plate-like substrate after laser irradiation in Example 1. FIG. In Example 2, the last 2 hours of culturing Dictyostelium Dictyostelium Dictyostelium for 24 hours using a plate-shaped substrate on which a linear cell adhesion region with a width of 1.5 μm was formed as a pattern of arbitrary shape. It is a figure which shows the image which averaged the moving image. The straight line in the figure corresponds to the cell adhesion area observed by the difference in appearance with visible light between the cell adhesion area and the cell non-adhesion area. In Example 2, cellular slime mold Dictyostelium Dictyostelium was grown using a plate-like substrate on which a swirling cell adhesion area was formed in which a straight line with a width of 1.5 μm was rotated clockwise from the center as a pattern of arbitrary shape. FIG. 10 is a diagram showing an image obtained by averaging the last 2 hours of moving images when cultured for 24 hours. The spiral line in the figure corresponds to the cell-adhesion region observed by the difference in appearance with visible light between the cell-adhesion region and the cell-non-adhesion region. In Example 2, the plate-shaped substrate on which a single linear cell adhesion region with a width of 1.5 μm was formed was used to culture Dictyostelium Dictyostelium Dictyostelium for 2 hours (upper) and for 24 hours. FIG. 10 is a diagram showing an image obtained by averaging the moving images for the last two hours (lower stage) of the case. The straight line in the figure corresponds to the cell adhesion area observed by the difference in appearance with visible light between the cell adhesion area and the cell non-adhesion area. FIG. 10 is a diagram showing an image after Cos1 cells were cultured for 24 hours on a plate-like substrate on which a circular cell adhesion region was formed as a pattern of arbitrary shape in Example 3; FIG. 10 is a diagram showing an image after Cos1 cells were cultured for 24 hours on a plate-shaped substrate on which cell adhesion regions of square regions were formed as a pattern of arbitrary shape in Example 3. FIG. In Example 4, an image of Dictyostelium Dictyostelium Dictyostelium was cultured for 24 hours on a plate-shaped substrate in which 10 square cell adhesion regions each having a size of 15 μm×15 μm were formed in a lattice pattern as a pattern of arbitrary shape. FIG. 4 is a diagram showing; In Example 5, it is a figure which shows the image after culture|cultivating the cellular slime mold Dictyostelium discoideum for 24 hours on the plate-shaped base|substrate of the shape which consists of a square area|region and a circular area|region as an arbitrary-shaped pattern. In addition, since this figure is a still image and the number of cells is small, in order to facilitate recognition of the range of the area, square and circular auxiliary lines are added at the boundary between the cell adhesion area and the cell non-adhesion area. there is FIG. 10 is a diagram showing the results of measurement of cell movement speed on a plate-like substrate in Example 9. FIG.

The plate-shaped substrate for observing or culturing cells of the present invention is
A plate-like substrate for observing or culturing cells, comprising a cell non-adhesive region and a cell adhesive region on one side of a laser-permeable substrate,
The cell non-adhesion region is arranged on one surface of the base material, and is arranged on a first layer formed of a substance that generates heat or plasmon resonance by laser irradiation, and is arranged on the first layer, at least A laminate comprising a second layer whose surface is formed of a cell non-adhesive substance,
The cell adhesion region is not particularly limited as long as it is the plate-shaped substrate, the surface of which is a region having cell adhesiveness. say. In addition, the method for producing a plate-shaped substrate for observing or culturing cells of the present invention includes:
(a) forming a first layer by coating or spraying a substance that generates heat or plasmon resonance upon laser irradiation onto one surface of a substrate that can transmit a laser;
(b) applying or spraying a cell non-adhesive substance onto the first layer to laminate a second layer;
(c) irradiating the first layer with a laser, and denaturing or dispersing the cell non-adhesive substance of the second layer by heat generation or plasmon resonance due to laser irradiation, and cell adhesion regions that have lost cell non-adhesiveness forming a;
is not particularly limited as long as it is a method for producing a plate-shaped substrate for observing or culturing cells, which is characterized by sequentially comprising the steps (a) to (c) of (a) to (c), and hereinafter referred to as "plate for observing or culturing the subject cells It is also referred to as a “method for producing a similar substrate”. Hereafter, the details of the "present plate-shaped substrate" or the "method for producing the present plate-shaped substrate" will be described. In the present specification, a state in which the cell non-adhesive region loses cell non-adhesive properties due to heat generated by laser irradiation or plasmon resonance is not included in the cell non-adhesive region but is a cell adhesive region.

(Base material)
In the present specification, the laser-transmissible substrate is not particularly limited as long as it is made of a laser-transmissive material, but a non-cytotoxic material is preferred. Furthermore, from the viewpoint of exposing the substrate and facilitating cell adhesion when all or part of the laminate is removed by laser irradiation, the substrate has cell adhesiveness, that is, a cell adhesive group It is preferable to use the material.

In addition, a material having a laser transmittance of 70% or more, preferably 80% or more, more preferably 90% or more, and further preferably 95% or more can be used. Specifically, from the viewpoint of permeability, cell adhesiveness, and cell growth in microscopic observation of cells on the substrate, glass such as soda glass, quartz, ceramic, and sapphire are used as main components. and transparent inorganic materials such as polystyrene, polypropylene, and polymethacrylacrylamide. Further, the shape of the base material through which the laser can pass is not particularly limited, and examples thereof include a cover slip shape, a petri dish shape, and the like. When a cover slip is used, the cover slip can be adhered to the bottom dish and used as a petri dish. In addition, the shape of the base material through which the laser can be transmitted can be planar, but it may have wells that can hold the cells when they proliferate. You may have it in part or all, and you may combine these.

The thickness of the base material through which the laser can pass is not particularly limited, but may be 0.05 mm to 0.3 mm. Furthermore, the size of the base material through which the laser can pass is not particularly limited, but a width or diameter of 10 to 1000 mm can be mentioned.

When the base material capable of transmitting the laser is adhered to the first layer containing a substance that generates heat or plasmon resonance by laser irradiation, the surface dirt such as oil should be removed from the viewpoint of enhancing the adhesion. may Methods for removing surface contaminants such as oil include plasma treatment, acid or alkali treatment, or organic solvent treatment. In addition, on the above base material, if necessary, cell adhesive proteins such as collagen, gelatin, fibronectin, elastin, hydronectin, and laminin, polysaccharides having an electric charge such as hyaluronic acid, chondroitin sulfate, and chitin, polylysine, Cell adhesion factors such as polyethylenimine, spermidine, spermine and other polycations may be coated.

(first layer)
The cell-non-adhesive region is a cell-non-adhesive region in which a layered product is arranged on one surface of the laser-permeable substrate, and the surface of the layered product is cell-non-adhesive. This laminate is constructed by laminating a first layer and a second layer. The first layer is placed on a laser-transmissible base material, and is made of a substance that generates heat or plasmon resonance upon laser irradiation. In the case where the cell adhesion region and the cell non-adhesion region can be distinguished under visible light, the substance that generates heat or plasmon resonance by the laser irradiation is a substance that constitutes the cell adhesion region or a substrate that can transmit the laser. A substance having low visible light transmittance can be used, and a substance having no visible light transmittance is preferably used. On the other hand, the second layer is arranged on the first layer, and at least the surface thereof is formed of a cell non-adhesive substance, and the surface is cell non-adhesive. In addition, the cell non-adhesiveness refers to the property that cells cannot adhere to the surface in order to fix themselves or move on the substrate, or to create a state where they can grow or proliferate on the substrate. means to have

As the substance that generates heat or plasmon resonance upon laser irradiation, which forms the first layer, the surface of the second layer has cell adhesion by generating heat or plasmon resonance upon laser irradiation. The material is not particularly limited as long as it can be denatured into the material, or can be lost by gasification accompanied by low-molecularization or dispersion including evaporation to expose the base material or the first layer, but it does not cause cytotoxicity. It is preferably a material that does not have Further, as the substance that generates heat or plasmon resonance by laser irradiation, specifically, any metal selected from gold, platinum, and aluminum, and any collection selected from carbon, organic dyes, and inorganic dyes. A light agent can be mentioned, but a substance capable of reflecting or absorbing visible light is preferable from the viewpoint of enhancing the recognizability of a pattern of arbitrary shape. In particular, when the cell adhesion region and the cell non-adhesion region are distinguishable under visible light, the difference in visible light impermeability between the laser-irradiated region and the non-laser-irradiated region is Any metal selected from gold, platinum and aluminum is preferred, with gold being more preferred. When such a metal is used, all or part of the substance itself that generates heat or plasmon resonance upon laser irradiation, which constitutes the first layer, is dispersed or oxidized by laser irradiation, resulting in the above-described comparison before laser irradiation. By changing the density, distribution, shape, or contrast of the metal, it becomes easier to distinguish under visible light the cell-adhered region, which is the laser-irradiated region, and the non-cell-bonded region, which is not irradiated with the laser. The first layer may be a thin film formed from particles of the metal or concentrator. The particle size of the metal or light collector particles is not particularly limited, but may be 2 nm to 200 nm, preferably 5 nm to 100 nm.

In addition, as a method for forming the cell adhesion region, the second layer is directly placed on the base material that can transmit the laser without providing the first layer, and the second layer is irradiated with the laser. Alternatively, a method of exposing the substrate by being lost by dispersion, or modifying the surface of the second layer into a material having cell adhesiveness may be considered. However, in that case, it is necessary to irradiate a high-output laser, which requires an expensive laser device. In other words, by providing the first layer and utilizing the heat generation or plasmon resonance of the material forming the first layer, even if an inexpensive and low-power continuous wave (CW) laser is used, the base material is lost due to dispersion. The material is exposed, or the surface of the second layer can be denatured into a material having cell adhesiveness. In addition, the above-mentioned cell adhesiveness means the property of cells to adhere to a surface in order to fix themselves or move on a substrate, or to create a state in which they can grow or proliferate on a substrate. The contact of floating cells due to their own weight is excluded. Also, the cell adhesion region can be said to be a region having relatively high cell adhesion as compared with the cell non-adhesion region.

The method of disposing the first layer formed of a material that generates heat or plasmon resonance upon laser irradiation on one side of a base material that can transmit laser light is not particularly limited, but may be sputtering, vapor deposition, spin coating, or sol-gel. However, the sputtering method is preferable from the viewpoint of the easiness of the production process.

Examples of the thickness of the first layer include 1 nm to 200 nm, preferably 5 nm to 100 nm, and more preferably 15 nm to 60 nm. Depending on the material of the first layer, if the thickness of the first layer exceeds 200 nm, it is not preferable from the viewpoint of adhesion between the substrate and the first layer, and if it is less than 1 nm, the laser output is reduced. A large facility is required to increase it, resulting in a high cost. The thickness of the first layer and the output of the laser that denatures or disperses the second layer are in an inversely proportional relationship. The layer thickness can be adjusted according to the power of the laser light used. In addition, from the viewpoint of microscopic observation of cells, the first layer has an OD value of 0.005 to 0.1, preferably 0.01 to 0.08 when measured with a spectrophotometer (532 nm). can be mentioned.

(second layer)
A second layer is arranged on the first layer, and at least the surface of the second layer is formed of a cell non-adhesive substance. The cell non-adhesive substance is not particularly limited as long as it has cell non-adhesive properties and can be denatured or dispersed into a cell adhesive substance by heat generation or plasmon resonance of the first layer. Specific examples include amphiphilic polymers such as 2-methacryloyloxyethylphosphorylcholine (MPC), ethylene glycol, hydroxyethylene methacrylate, or dimethylsiloxane and derivatives thereof, or polymers thereof. The MPC polymer may be a homopolymer consisting of the MPC monomer represented by the following formula (I), or a copolymer consisting of the MPC monomer and a monomer other than MPC. . In the area where the cell non-adhesive substance of the second layer is exposed on the surface, when the cells are seeded on a plate-like substrate for observing or culturing the subject cells, the cells are not adhered to the non-adhesive substance. do not adhere to the cells, but preferentially adhere to cell adhesion regions, ie, arbitrary shaped patterns. That is, seeded cells adhere only on patterns of arbitrary arbitrary shape.

Monomers other than MPC include alkyl ( meth) acrylate, benzyl (meth) acrylate, phenoxyethyl (meth) acrylate, cyclohexyl (meth) acrylate, polypropylene glycol mono (meth) acrylate, polytetramethylene glycol mono (meth) acrylate, polypropylene glycol di (meth) acrylate, Examples include polytetramethylene glycol mono(meth)acrylate, polypropylene glycol polyethylene glycol mono(meth)acrylate, sodium methacrylate, 2-hydroxy-3-methacryloyloxypropyltrimethylammonium, and butyl (meth)acrylate is preferred. can be mentioned. In addition, the said "(meth)acrylate" means an acrylate or a methacrylate. An example of a copolymer of 2-methacryloyloxyethylphosphorylcholine (MPC) and butyl (meth)acrylate is shown below in formula (II).

In formula (II), m and n are numbers indicating the molar ratio of each structural unit, and the molar ratio is m/n=70/30 to 90/10.

The second layer can be formed by coating or spraying a cell non-adhesive substance on the first layer. The method of applying or spraying is not particularly limited, but for example, the cell non-adhesive substance is dissolved in an organic solvent such as ethanol, and the solution is applied to the first layer using a rotary coating device such as a spin coater. A method of coating on top can be mentioned. When the MPC polymer is applied, the MPC polymer itself may be applied. The liquid may be applied over the first layer and polymerized by heat or light so that the MPC polymer is formed on the surface of the first layer.

The thickness of the second layer is 5 nm to 200 nm, preferably 8 nm to 50 nm, more preferably 10 nm to 30 nm.

For the second layer, a dye that emits fluorescence may be used from the viewpoint of further enhancing the distinguishability between the cell-non-adhesive region and the cell-adhesive region. For example, when MPC is used as the second layer, since MPC has a phospholipid-like structure, the second layer may be stained with a fluorescent dye that emits red fluorescence when bound to phospholipids and observed under a fluorescence microscope. good.

The substrate capable of transmitting the laser and the first layer, and the first layer and the second layer may be in direct contact, but the laser transmission, heat generation or plasmon resonance due to laser irradiation, heat generation or Other substances or layers may be provided between them as long as they do not affect the denaturation or dispersion of the non-cell-adhesive substance into the cell-adhesive substance by plasmon resonance.

(laser)
The laser light source is not particularly limited, and examples thereof include a continuous wave (CW) laser and a pulse laser.

The wavelength of the laser is not particularly limited, but may be 380 nm to 1100 nm, preferably 450 nm to 780 nm, more preferably 500 nm to 600 nm. By irradiating the laser, the substances constituting the first layer and the second layer adjacent to the first layer are denatured or dispersed due to heat generation and plasmon effect. Further, the frequency of the laser light can be exemplified from 0.1 kHz to 100 kHz, preferably from 1 kHz to 10 kHz. Furthermore, multiple combinations of lasers having the same or different wavelengths or frequencies may be used.

The output of the laser may be in the range that the first layer and the second layer can be modified or dispersed, preferably 0.1 mW to 500 mW, more preferably 1 mW to 300 mW, for example when a CW laser is used. , and more preferably 50 mW to 200 mW. As described above, the thickness of the first layer and the output of the laser for modifying or dispersing the second layer are inversely proportional, and the output of the laser can be adjusted according to the thickness of the first layer. can be done.

It is preferable to irradiate the first layer with the laser through an objective lens. The pupil diameter of the objective lens when passing through the objective lens can be 2 mm to 18 mm, preferably 4 mm to 12 mm, and more preferably 5 mm to 10 mm. In addition, the spot (focus) diameter of the laser when irradiating the laser can be appropriately adjusted according to any pattern shape to be formed, but when forming a predetermined surface, the diameter is 0.1 μm to 5 mm. preferably 0.5 μm to 1 mm, more preferably 1 μm to 100 μm. In addition, in the case of forming a pattern such as predetermined depressions, projections, dots, polygons or lines to the extent that only one or several cells adhere, the laser spot (focus) when irradiating the laser The diameter can be 0.1 μm to 50 μm, preferably 0.5 μm to 30 μm, more preferably 1 μm to 20 μm. The laser irradiation is not particularly limited as long as the substance that generates heat or plasmon resonance by laser irradiation generates heat or plasmon resonance, but it is preferable to focus on the first layer in advance. In addition, even if the laser irradiation is performed from the upper surface side of the base material that can transmit the laser, that is, from the surface on which the laminate is arranged, it is possible to irradiate from the lower surface side, that is, from the surface on which the laminate is not arranged. However, it is preferable to irradiate from the lower surface side.

The irradiation position of the laser is not particularly limited, and the irradiation position can be moved according to a pattern of arbitrary shape to be formed. Further, for example, when forming a linear shape, the laser irradiation may be moved in a straight line, and when forming a square region shape, the laser irradiation may be performed so as to form a square surface. should be moved to . In order to move the irradiation position, the laser irradiation position may be fixed and the laser-transmissive base material may be moved, or the laser-transmissible base material may be fixed and the laser irradiation position may be moved. may

As described above, a pattern of any shape can be formed as a region processed by laser irradiation. However, by the method described in Patent Document 4, a mask is placed on the second layer and plasma treatment is performed. You may combine with the method of forming an arbitrary-shaped pattern.

(pattern shape)
Patterns of any shape include straight lines, curved lines, dots, circles, ellipses, polygons such as triangles and squares, lattices, etc. Represented by arbitrary lines including straight lines and/or curved lines such as indeterminate shape, mesh shape, arc shape, etc., a closed figure composed of these lines, or a state in which multiple arbitrary lines do not intersect each other A shape (for example, a donut shape), a shape in which an arrow or a plurality of arrows are in series, a region formed by these, or a combination thereof can be used. Moreover, the arbitrary-shaped pattern may be concave, convex, or a combination thereof. Since the arbitrary-shaped pattern is formed in the region processed by laser irradiation, the arbitrary-shaped pattern becomes the cell adhesion region.

Alternatively, any predetermined line, figure, shape, or area may be defined as one unit, and may be formed so as to be arranged regularly or randomly at a plurality of locations at predetermined intervals. In particular, when a plate-like substrate for observing or culturing the present cells is used as a microarray, circular regions having a diameter of 10 μm to 30 μm are arranged so that the distance between the outer edges of each circular region is 10 μm to 100 μm, preferably 15 μm to 50 μm. By arranging it in a circular area, it is possible to prevent contact between cells adhered to the circular area, and intercellular communication through cell adhesion and intercellular communication such as diffusible cytokines through the external fluid can be prevented. can be distinguished. Furthermore, since a pattern can be formed by a laser without using a mask, it is possible to form a narrow cell adhesion region. Therefore, by making a linear pattern narrower than the cells to be cultured, it is possible to control the cells moving in random directions so that they move linearly, and to limit the direction of cell division to a line. .

In addition, during or after culturing cells on the present plate-like substrate, the plate-like substrate is further irradiated with a laser to add and/or expand the cell adhesion region in real time to induce or control the movement of the cells. becomes possible. Specifically, predetermined cells are cultured on the present plate-like substrate and in a state where the cells are adhered, a laser is irradiated to the vicinity of the cells to form a cell adhesion region, whereby the cells are newly regenerated. It becomes possible to induce migration to the formed cell adhesion region.

By controlling the laser irradiation position according to arbitrary lines, figures, shapes, or areas designed in advance using design software such as CAD, it is possible to easily form patterns of any shape without using a mask. becomes.

When observing motile cells, the arbitrarily-shaped pattern is preferably a closed shape or region from the viewpoint of preventing cells grown on the arbitrarily-shaped pattern from moving outside the pattern. In this case, the closed shape may further have a cell non-adhesive region. Cells adhere to a pattern of arbitrary shape, and in order to further improve adhesion, a cell-adhesive agent such as collagen, gelatin, fibronectin, elastin, hydronectin, laminin, etc., is placed on the pattern of arbitrary shape. Proteins, charged polysaccharides such as hyaluronic acid, chondroitin sulfate, and chitin, and cell adhesion factors such as polylysine, polyethyleneimine, spermidine, spermine, and other polycations may be coated.

For example, when a visible-light impermeable metal such as gold is used as a substance that causes plasmon resonance, a pattern of arbitrary shape is obtained by laser irradiation, and therefore can be distinguished under visible light. As described above, this is due to the dispersion or scattering of the substance, such as gold, which constitutes the first layer and which generates heat or plasmon resonance when irradiated with laser light, causing the substance to disperse between regions not irradiated with laser light. This is thought to be due to differences in density, distribution, shape, or contrast, as well as differences in reflection and transmittance of visible light.

(cell adhesion region)
The cell-adhesive region is a region having relatively high adhesion to cells compared to the non-cell-adhesive region. Although the method for forming this region is not particularly limited, it can be formed, for example, by irradiating the first layer with a laser. When a cell-adhesive substrate capable of transmitting laser light is used as the substrate, all or part of the substance itself that generates heat or plasmon resonance due to laser irradiation is dispersed by laser irradiation, and the cell non-adhesive substance is dispersed. By dispersing all or part of the above, the cell-adhesive substrate is exposed, and as a result, the surface has cell-adhesiveness, and the cell-adhesiveness is relatively high compared to the cell-non-adhesive region. get higher It should be noted that, as the base material, even if the surface does not have cell adhesiveness and a base material that is permeable to laser is used, the cell adhesion protein that specifically binds to the exposed base material, the charge The surface may be provided with cell adhesiveness by coating with a cell adhesive factor such as polysaccharides and polycations.

In addition, the cell adhesion region generates heat or plasmon resonance by laser irradiation that forms the first layer on all or part of the substrate, regardless of whether the substrate has cell adhesiveness. A substance or its oxidized compound, or a cell non-adhesive substance may be denatured by laser irradiation and may have a substance that has lost cell non-adhesiveness, and these states and a state in which the cell adhesive substrate is exposed may be a combination of

(cell)
The cell herein may be a single cell, a tissue cell, or a plurality of cultured cells, and examples of cell types include protists, yeast, bacteria, and animal cells. be able to. Adhesive cells can be suitably used as the above-mentioned cells.

Examples of protists include cellular slime molds and amoeba, and suitable examples of cellular slime molds include cells of Dictyostelium discoideum.

Animal cells include cells derived from mammals such as humans, mice, rats, dogs, cats, monkeys, rabbits, hamsters, bovines, pigs, horses, and goats, and pluripotent cells such as ES cells and iPS cells. They may be sex stem cells, stem cells, somatic cells, or germ cells.

(Method for preparing plate-shaped substrate for observing or culturing cells)
In the method for producing the plate-shaped substrate, the step of forming the first layer may be any method of applying a substance that generates heat or plasmon resonance upon laser irradiation onto one surface of a substrate that can transmit laser light. Examples include, but are not particularly limited to, sputtering, vapor deposition, spin coating, sol-gel method, plating, coating, printing, and the like. Sputtering is preferred from the viewpoint of ease of production process. In addition, the step of laminating the second layer is not particularly limited as long as it is a method of applying or spraying a cell non-adhesive substance on the first layer. A method of dissolving in a solvent and coating the solution on the first layer using a spin coater such as a spin coater can be used. When the MPC polymer is applied, the MPC polymer itself may be applied. The liquid may be applied over the first layer and polymerized by heat or light so that the MPC polymer is formed on the surface of the first layer.

In the method for producing a plate-like substrate, the first layer is irradiated with a laser, and the cell non-adhesive substance in the second layer is denatured or dispersed by heat generated by laser irradiation or plasmon resonance, resulting in cell non-adhesive properties. As a step of forming a cell adhesion region that has lost It is not particularly limited as long as it forms a cell adhesion region. It should be noted that the method for producing the present plate-like substrate can also be performed while observing or culturing cells on the present plate-like substrate. Therefore, during or after culturing cells on the plate-shaped substrate, the plate-shaped substrate can be further irradiated with a laser to add and/or expand the cell adhesion region in real time, thereby inducing or controlling the movement of the cells. It becomes possible.

EXAMPLES The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples.

[Example 1] Preparation of plate-shaped substrate for observation or culture of cells A coverslip was used as a plate-shaped substrate on which cultured cells can be adhered and arranged only in a predetermined microregion on a substrate such as a cover glass or petri dish. The material was produced as follows.

Borosilicate glass coverslip no. 1 (manufactured by Matsunami Glass Industry Co., Ltd.) is vacuumed for 1 minute with a vacuum device (PB-1: manufactured by Vacuum Device Co., Ltd.), and then plasma-treated (first plasma treatment) for 30 seconds to remove dirt on the surface. Removed. Next, using an ion sputtering device (manufactured by Sanyu Electronics Co., Ltd.), gold (Au) is used as a target under the conditions of 14 mA and 30 to 60 seconds to prepare a coverslip with a gold thin film layer having a thickness of 20 nm to 50 nm. bottom.

Next, using a spin coater, 0.5 mg / ml of a cell non-adhesive substance Lipidure-CM5206 (registered trademark) (2-methacryloyloxyethylphosphorylcholine and butyl (meth) acrylate copolymer: NOF CORPORATION company) was applied onto the cover slip with the gold thin film layer prepared above and dried to form a cell non-adhesive substance layer with a thickness of 20 nm to 50 nm on the gold thin film layer. A plate-shaped substrate 1 was prepared by laminating a cover slip on which a laminate composed of a gold thin film layer and a cell non-adhesive substance layer was arranged. FIG. 1 shows the prepared plate-like substrate 1 . As shown in FIG. 1, a gold thin film layer 12 is in contact with a cover slip 11, and a cell non-adhesive material layer 13 is in contact thereon.

Next, a pattern to be a cell adhesion region was formed on the plate-like substrate 1 produced above. First, patterns were designed in a computer with image editing software (Inkscape). Next, a self-made electric xy stage 21 (rotated by a stepping motor to move the xy stage) was connected to the computer and placed on the stage of a microscope (IX71: Olympus). After that, the plate-like substrate 1 prepared above was placed on the self-made electric xy stage 21 . A laser 23 was introduced from the pupil side of the objective lens 22 (40x magnification), and the laser 23 irradiation was started as shown in FIG. By controlling the electric xy stage 21, it is possible to change the irradiation position of the laser, and by moving the irradiation position of the laser 23 according to a pattern designed in advance, a pattern of arbitrary shape was formed. Further, by fixing the electric xy stage 21 and irradiating the laser 23, the cell adhesion region 24 was formed in a point shape. FIG. 3 shows a plate-like substrate 1 having dot-like cell adhesion regions. A laser with a wavelength of 532 nm and 100 mW was used. The diameter of the laser on the irradiation surface was 0.5 μm to 2 μm. At the site irradiated with the laser, the cell non-adhesive material layer made of Lipidure together with the gold thin film layer, i.e., the laminate, is dispersed or dispersed by plasmons and/or heat generated by the energy absorbed by the gold particles in the gold thin film layer. Denaturation or the like occurs, and the laser irradiation site becomes a cell adhesion region.

[Example 2] Adhesion of cellular slime mold Dictyostelium Dictyostelium A hole with a diameter of 12 mm was made in the bottom of a plastic dish, and a plate-shaped substrate 1 having a pattern of arbitrary shape prepared in the same manner as in Example 1 was adhered. A glass bottom dish (culture substrate) was prepared by sticking with an agent. 2 mL of HL5 culture solution containing 1 to 2×10 ⁶ cells of Dictyostelium discoideum was added to a glass bottom dish and allowed to stand at 22° C. for 10 minutes or longer. As a pattern of arbitrary shape, a plate-like substrate was used in which a straight line with a width of 1.5 μm or a line with a width of 1.5 μm was formed to form a spiral linear figure. Next, the culture medium on the plate-shaped substrate is aspirated, and the cellular slime mold Dictyostelium discoideum that did not adhere to the culture substrate, that is, the cellular slime mold Dictyostelium discoideum floating in a non-adhesive state is included. The medium was replaced by removing the medium and adding fresh medium. After culturing for 1 hour, the culture substrate was observed with a phase-contrast microscope. Figures 4 and 5 show images obtained by averaging the moving images for the last 2 hours of culturing for 24 hours. In FIG. 4, a plate-like substrate having a straight line with a width of 1.5 μm is used, and in FIG. This is the case when

As is clear from FIGS. 4 and 5, the cellular slime mold Dictyostelium discoideum adheres to a pattern of an arbitrary shape on the plate-like substrate, and outside the pattern, that is, at a position where the non-cell adhesion region is exposed to the surface. It was confirmed that the cells were not adhered. In addition, the formed linear or spiral cell adhesion regions were observable with an optical microscope under visible light.

Furthermore, in the case of using a plate-shaped substrate on which a straight line with a width of 1.5 μm was formed, the culture solution was cultured for an additional 2 hours (upper) and the last 2 hours after culturing for 24 hours (upper). FIG. 6 shows the result of averaging the moving images observed with a phase-contrast microscope.

As is clear from FIG. 6, even after culturing for 24 hours, the cellular slime mold Dictyostelium discoideum remains on the pattern, that is, in the cell adhesion region, and the cell non-adhesion region does not contain cellular slime mold Dictyostelium Dictyostelium. was confirmed. In addition, the formed linear cell adhesion region was observable with an optical microscope under visible light.

[Comparative example] Adhesion of cells cultured on a commercially available cell adhesion substrate 1 cell slime mold cultured in HL5 culture medium on a commercially available cell adhesion substrate having a linear cell adhesion control pattern utilizing the action of a photocatalyst 10 ⁶ pieces were placed and left at 22° C. for 10 minutes or longer. The control pattern was 10 μm wide. After that, the culture medium on the culture substrate was removed by aspiration, and the cells were further cultured at 22° C. for 1 hour. Immediately after the culture medium was removed by aspiration, or after culturing for 2 hours, the culture substrate was observed under a phase-contrast microscope. As a result, the cellular slime mold adhered to the cell adhesion control pattern area immediately after the cellular adhesion control pattern was placed, but after 2 hours, it moved to the area other than the cell adhesion control pattern. was confirmed.

[Example 3] Adhesion of Cos1 cells In Example 2, Dictyostelium was used, but Cos1 cells, which are cultured cells of African green monkeys, were cultured in the same manner, and adhesion to the plate-like substrate was confirmed. bottom. A circular area or a square area was used as a pattern of arbitrary shape. 7 and 8 show the results of observation under a phase-contrast microscope after culturing for 24 hours after replacing the medium. Scale bars in FIGS. 7 and 8 are 500 μm and 200 μm, respectively. Although not shown in the figure, a circular region with a diameter of 500 μm and a square region with a side length of 200 μm were visually observed under visible light at the stage before cells adhered to the plate-shaped substrate prepared in this example. I was able to confirm.

As shown in FIGS. 7 and 8, it was confirmed that not only cellular slime molds but also animal cells were cultured by adhering only to the cell adhesion regions. In addition, it was confirmed that the shape of the cell adhesion region can be freely designed by using an arbitrary shape as the pattern of arbitrary shape, and the cells can be cultured in the arbitrary region. In addition, it was confirmed that it is possible to examine cell movement and the like for a long period of time only within the cell adhesion region of this circular or square region.

[Example 4] Fabrication of cell microarray In the method of Example 2, a plate-shaped substrate was prepared by forming 10 square regions of 15 µm × 15 µm in a grid shape (the distance between grids was 21 µm) as a pattern of arbitrary shape. Dictyostelium Dictyostelium was cultured for 24 hours in the same manner as in Example 2 so that only one cell could adhere. The results are shown in FIG. In FIG. 9, the length of the scale bar is 50 μm.

As is clear from FIG. 9, it was possible to adhere the cellular slime mold Dictyostelium discoideum one by one to the patterned positions of the plate-shaped substrate. Therefore, it was confirmed that the plate-like substrate of the present invention can be used for cell microarrays.

[Example 5] Fabrication of Cell Adhesion Region Consisting of Square Region and Circle Region According to the present invention, unlike Patent Document 4, a cell adhesion region can be formed without using a mask. Therefore, a plate-like substrate having a non-cell-adhesive area, which is a figure-shaped area represented by two non-intersecting closed lines having a circle at the center of a square, was prepared by the method of Example 1. Dictyostelium Dictyostelium was cultured for 24 hours. The results are shown in FIG.

As shown in FIG. 10, according to the present invention, a pattern of arbitrary shape can be formed by moving the laser irradiation position according to a predesigned figure without using a mask. It was confirmed that cells could be cultured inside.

[Example 9] Cell motility The cell motility on the plate substrate in Example 8 was measured. For comparison, coverslip No. 1 on which no laminate is arranged. 1 (manufactured by Matsunami Glass Industry Co., Ltd.), the cellular slime mold Dictyostelium Dictyostelium was similarly cultured, and the movement speed was measured. Motion velocity was determined from the length of the trajectory per unit time by taking a moving image. The results are shown in FIG.

As is clear from FIG. 11, there is no significant difference in the movement speed between the control and the control (Student t-test ns:>0.05), and the plate-shaped substrate of the present invention has an effect of suppressing the normal movement of cells. It was confirmed that there was no

INDUSTRIAL APPLICABILITY By using the present invention, it is possible to provide a plate-shaped substrate that can efficiently proceed with research on cell movement, proliferation, cell death, differentiation control, etc., and it is possible to better understand the characteristics of normal cells and the behavior of cancer cells. It is possible to provide a plate-like substrate that can contribute to research efficiency through accurate observation. Furthermore, by arranging a large number of cell adhesion regions on a plate-like substrate, it becomes possible to prepare a "cell microarray" and to understand in detail the reactions to various drugs. It can also be applied to the use of cells as biosensors, bioreactors, and artificial organs. In addition, since cell sheets of any shape can be produced, it can be applied to regenerative medicine and the like.

1 plate-shaped substrate, 11 cover slip, 12 gold thin film layer, 13 cell non-adhesive substance layer, 21 electric xy stage, 22 objective lens, 23 laser, 24 cell adhesion region

Claims (12)

A plate-like substrate for observing or culturing cells, comprising a cell non-adhesive region and a cell adhesive region on one side of a laser-permeable substrate,
The cell non-adhesion region is arranged on one surface of the base material, and is arranged on a first layer formed of a substance that generates heat or plasmon resonance by laser irradiation, and is arranged on the first layer, at least A laminate comprising a second layer whose surface is formed of a cell non-adhesive substance,
The plate-shaped substrate, wherein the cell adhesion region is a region having a cell adhesion property on the surface. 2. The method according to claim 1, wherein the substrate is a laser-permeable cell-adhesive substrate, and the cell-adhesion region is an exposed region of the laser-permeable cell-adhesive substrate. A plate-like substrate as described. 3. The plate-shaped substrate according to claim 1, wherein the cell-adhesive substrate capable of transmitting laser light is a substrate containing glass as a main component. 4. The plate-like substrate according to any one of claims 1 to 3, wherein the cell non-adhesive substance is a polymer of 2-methacryloyloxyethylphosphorylcholine (MPC). The plate-shaped substrate according to any one of claims 1 to 4, wherein the cell adhesion region is a region formed by irradiating the first layer with a laser. 6. The cell adhesion region according to any one of claims 1 to 5, wherein a part of the cell adhesion region is composed of a substance in which the cell non-adhesion substance is denatured or dispersed by laser irradiation and loses cell non-adhesion properties. The plate-shaped substrate according to . The plate-like substrate according to any one of claims 1 to 6, characterized in that said cell-adhering regions are in a pattern of arbitrary shape. The plate-like substrate according to any one of claims 1 to 7, characterized in that the cell-adhesive area and the cell-non-adhesive area can be distinguished under visible light. 9. The plate-like substrate according to any one of claims 1 to 8, wherein the substance that generates heat or plasmon resonance when irradiated with a laser is a substance impermeable to visible light. A method for producing a plate-like substrate for observing or culturing cells, comprising:
(a) forming a first layer by coating or spraying a substance that generates heat or plasmon resonance upon laser irradiation onto one surface of a substrate that can transmit a laser;
(b) applying or spraying a cell non-adhesive substance onto the first layer to laminate a second layer;
(c) irradiating the first layer with a laser, and denaturing or dispersing the cell non-adhesive substance of the second layer by heat generation or plasmon resonance due to laser irradiation, and cell adhesion regions that have lost cell non-adhesiveness forming a;
A method for producing a plate-like substrate, characterized by comprising steps (a) to (c) of . 11. The method for producing a plate-like substrate according to claim 10, wherein the cell adhesion area is an area where a laser-permeable cell adhesive base material is exposed. 12. The method for producing a plate-like substrate according to claim 10, wherein the cell-adhesive area and the cell-non-adhesive area are distinguishable under visible light.

Images