JP2019151747A - Film-like soft material, fluorescent film substrate, manufacturing method of film-like soft material, and stress measurement method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a film-like soft material and a fluorescent film base material capable of visualizing and quantifying stress generated in an object such as a cell, and a stress measuring method using them. A film-like soft material 11 having a three-dimensional network structure made of a polymer compound and having a fluorescent dye introduced into a side chain of a main skeleton of the three-dimensional network structure. The boundary between the high fluorescence intensity region and the low fluorescence intensity region by irradiating at least a part of the film-like soft material with energy rays in a pattern and the process of contacting an object such as a cell on the film-like soft material. A stress measurement method comprising a step of creating a gradient or a step in the fluorescence intensity in the above step and a step of evaluating the deviation of the fluorescence intensity distribution of the film-like soft material. [Selection diagram] Fig. 1

Description

  The present invention relates to a film-like soft material, a fluorescent film substrate, a method for producing a film-like soft material, and a stress measurement method using the film-like soft material.

  In vivo, cells form adhesion points called adhesion spots between proteins constituting the surrounding extracellular matrix (hereinafter referred to as ECM) and membrane proteins on the cell membrane (Non-patent Document 1). Adhesion plaques are connected to the cytoskeleton in the cell, and the cell function is controlled by transmitting stress (hereinafter referred to as traction force) generated in the cell adhered to the ECM to the inside of the cell. It is known that the traction force generated in cells changes depending on the composition and mechanical properties of ECM. For example, in mesenchymal stem cells, the traction force of cells changes according to the elastic modulus of ECM. It is known that the properties of cells differ. That is, it can be seen that cell traction has a strong correlation with cell function. The nature of ECM in a living body changes due to external damage, disease, aging, and the like. Since the influence of such ECM property changes on cell functions lasts for a long period of time, it is expected that changes in cell traction force will be measured stably over a long period of time, and changes in cell functions in response to changes in the properties of ECM will be evaluated. . Also, from the viewpoint of regenerative medicine, a base material is needed as a scaffold for cells during tissue regeneration, and elucidating the effects of the characteristics of the base material on the traction force of cells in the tissue regeneration process over a long period of time It becomes important for the material design of the base material.

  Several techniques for quantitatively detecting the traction force generated in the cells have been proposed so far. For example, the method of Wang et al. Is a widely used method from the viewpoint of cost and practicality (Non-patent Document 2). In this method, a fluorescent bead having a diameter of 1 μm or less is encapsulated in a film gel made of polyacrylamide or the like by chemical crosslinking, and the traction force generated in the cells adhered to the gel surface is detected from the displacement of the fluorescent bead. is there.

Disher et al. 2009, Science, Vol.324, 2009, pp.1673-1677. Dembo et al., Biophysical Journal.Vol.76, 1999, pp.2307-2316.

  However, in the method disclosed in Non-Patent Document 2, it is difficult to precisely control the density of acrylate groups presented on the surface, and thus the crosslink density around the fluorescent beads varies depending on the fluorescent beads. There has been a problem that there may be a difference in elastic modulus around the fluorescent beads and other regions. Further, it is necessary to perform a complicated operation of presenting an active ester group such as an amino group or N-hydroxysuccinimide on the surface of the fluorescent bead and chemically modifying the acrylate group via an amide bond or an ester bond. In order to simplify such a complicated operation, it is possible to embed fluorescent beads in a hydrogel without chemical crosslinking, but in this case, fluorescent beads are diffused in a polyacrylamide gel. The fluctuation of the beads caused a decrease in the accuracy and reproducibility of the traction force measurement in the long-term measurement.

  Therefore, the present invention provides a film-like soft material and a fluorescent film base material capable of visualizing and quantifying the stress generated in an object such as a cell, and a stress measurement method using them.

The present invention includes the following aspects.
[1] A film-like soft material having a three-dimensional network structure made of a polymer compound, and having a fluorescent dye introduced into the side chain of the main skeleton of the three-dimensional network structure.
[2] The film-shaped soft material according to [1], wherein the polymer compound includes a polyacrylamide resin.
[3] A high fluorescence intensity region and a low fluorescence intensity region having a fluorescence intensity lower than that of the high fluorescence intensity region, and a gradient or step in fluorescence intensity at the boundary between the high fluorescence intensity region and the low fluorescence intensity region The film-like soft material according to [1] or [2], wherein:
[4] A fluorescent film substrate comprising the film-like soft material according to any one of [1] to [3] on a substrate.

[5] A method for producing the film-like soft material according to any one of [1] to [3],
The manufacturing method of a film-form soft material which has the process of superposing | polymerizing the monomer liquid which contains the fluorescent pigment | dye which has a polymeric group on a board | substrate, and forms a three-dimensional network structure.
[6] A method for producing the film-like soft material according to [3],
A step of polymerizing a monomer liquid containing a fluorescent dye having a polymerizable group and forming a three-dimensional network structure on a substrate to form a film-like soft material polymer;
Irradiating at least a part of the film-shaped soft material polymer with energy rays in a pattern to generate a gradient or a step in the fluorescence intensity at the boundary between the high fluorescence intensity region and the low fluorescence intensity region; and A method for producing a film-like soft material.
[7] A stress measurement method using the film-like soft material according to [1] or [2],
Contacting an object such as a cell on the soft film material;
Irradiating at least a part of the film-like soft material with energy rays in a pattern to generate a gradient or a step in the fluorescence intensity at the boundary between the high fluorescence intensity region and the low fluorescence intensity region;
Evaluating the displacement of the fluorescence intensity distribution of the film-shaped soft material.
[8] A stress measurement method using the film-like soft material according to [3],
Contacting an object such as a cell on the soft film material;
Evaluating the displacement of the fluorescence intensity distribution of the film-shaped soft material.

  ADVANTAGE OF THE INVENTION According to this invention, the film-like soft material and fluorescent film base material which can visualize and quantify the stress which generate | occur | produces in objects, such as a cell, and the stress measurement method using them can be provided.

It is a schematic sectional drawing which shows the film-form soft material 11 of one Embodiment of this invention, and the fluorescent film base material 1 provided with the film-form soft material 11. FIG. It is a schematic sectional drawing which shows the film-form soft material 11 of one Embodiment of this invention, and the fluorescent film base material 2 provided with the film-form soft material 11. FIG. It is a schematic sectional drawing which shows the fluorescent film base material 3 provided with the film-form soft material 14 and film-form soft material 14 of one Embodiment of this invention. It is the schematic which shows the stress measuring method of one Embodiment of this invention. It is a schematic diagram of the board | substrate which can be used for manufacture of the film-form soft material of one Embodiment of this invention. It is a figure which shows typically the manufacturing procedure of the film-form soft material 11 and fluorescent film base material 2 of one Embodiment of this invention. It is an example of the photomask used for fluorescence intensity distribution structure formation, Comprising: (A) is the plane schematic diagram, (B) is the cross-sectional schematic diagram. It is a figure which shows typically the manufacturing procedure of the film-form soft material 14 and fluorescent film base material 3 of one Embodiment of this invention. The evaluation result of the fluorescence intensity distribution of the film-form soft material of one Embodiment of this invention is represented. The result of having observed the fading by the defocus light of the film-form soft material of one Embodiment of this invention with the fluorescence microscope is represented. The evaluation result of the displacement distribution of the fluorescence intensity of the film-form soft material of one Embodiment of this invention is represented.

Hereinafter, embodiments of a film-shaped soft material, a fluorescent film base material, a film-shaped soft material manufacturing method, and a stress measurement method using the film-shaped soft material of the present invention will be described with reference to the drawings.
In addition, in order to make the features of the present invention easier to understand, the drawings used in the following description may show the main portions in an enlarged manner for convenience, and the dimensional ratios of the respective components are the same as the actual ones. Not necessarily.

≪Film-like soft materials and fluorescent film bases≫
FIG. 1 is a schematic cross-sectional view showing a film-shaped soft material 11 of this embodiment and a fluorescent film substrate 1 including the film-shaped soft material 11. The film-like soft material 11 of this embodiment has a three-dimensional network structure made of a polymer compound, and a fluorescent dye is introduced into the side chain of the main skeleton of the three-dimensional network structure. The film-shaped soft material 11 can be used as the fluorescent film substrate 1 including the film-shaped soft material 11 on the substrate 12 in order to maintain the shape.

  Since the fluorescent dye is introduced into the film-like soft material 11 of the present embodiment, the stress generated in an object such as a cell can be visualized and quantified by a stress measurement method described later. Further, the film-shaped soft material 11 of the present embodiment has a three-dimensional network structure made of a polymer compound, and as described above, the fluorescent dye is chemically bonded to the side chain of the main skeleton of the three-dimensional network structure. Because it has been modified and introduced, the effect of changes in crosslink density can be reduced compared to the conventional film-type soft material in which fluorescent beads are encapsulated by chemical crosslinks. The variation in elastic modulus due to can be reduced. Furthermore, in the film-shaped soft material 11 of this embodiment, since the fluorescent dye introduced into the main skeleton has a small molecular size, the unreacted fluorescent dye is film-shaped by swelling the film-shaped soft material into a good solvent. It can be removed from the soft material. Therefore, fluctuations not dependent on stress due to diffusion movement of uncrosslinked fluorescent molecules do not occur, so that the problem that the accuracy and reproducibility of stress measurement are reduced can be solved, and stable stress measurement can be realized for a long time.

  The polymer compound constituting the film-shaped soft material 11 of the present embodiment has a three-dimensional network structure, and any kind can be used as long as a fluorescent dye can be introduced into the side chain of the main skeleton of the three-dimensional network structure. It doesn't matter. Examples of the polymer compound include elastomers such as polysilicon, water-soluble polymer gels such as polyacrylamide and polysaccharides, silicone hydrogels obtained by blending silicone into hydrophilic gels, hydrogels composed of proteins such as collagen, Etc.

  The polymer compound constituting the film-shaped soft material 11 preferably has biocompatibility. Examples of elastomers include polysilicon, hydrogels include skeletons such as polyacrylamide and polyhydroxyethyl acrylate, acrylic polymers, water-soluble polysaccharides such as dextran and alginic acid, collagen and fibronectin, and the like. And hydrogel composed of a derivative protein. A plurality of these constituent molecules may be mixed. Of these, the polymer compound preferably contains a polyacrylamide resin.

  As shown in FIG. 2, a cell adhesion layer 13 made of a protein such as poly-D-lysine (PDL) is provided on the surface of the film-shaped soft material 11 opposite to the base material 12. Is preferred. Arbitrary cells and tissues can be adhered by the cell adhesion layer 13 provided on the surface of the film-shaped soft material 11. In order to provide the cell adhesion layer 13 made of protein, a solution of sulfosuccinimidyl 6- (4′-azido-2′-nitrophenylamino) hexanoate (hereinafter, sulfo-SANPAH) is dropped on the film-shaped soft material 11 in advance and irradiated with ultraviolet rays. Thus, it is preferable to increase the protein affinity of the film-shaped soft material 11. Although the elastic modulus of the film-like soft material is not limited, ECM mechanical characteristics can be imitated by setting the elastic modulus of the living tissue to 100 Pa to 1 MPa.

  As shown in FIG. 3, the film-shaped soft material has a high fluorescence intensity region 32 and a low fluorescence intensity region 31 having a fluorescence intensity lower than that of the region 32, and at the boundary between the region 32 and the region 31. It is preferable that a gradient or a step is generated in the fluorescence intensity. That is, the film-like soft material preferably has a fluorescence intensity distribution.

The gradient of fluorescence intensity means that the fluorescence intensity changes continuously at the boundary between adjacent regions having different fluorescence intensities. The greater the slope of the change in fluorescence intensity, the more desirable and the fluorescence intensity that can be detected with a fluorescence microscope. It is desirable that a distribution change has occurred.
The step of the fluorescence intensity refers to a boundary where a difference in fluorescence intensity occurs between the high fluorescence intensity region 32 and the low fluorescence intensity region 31 having different fluorescence intensities, and it is desirable that the difference in fluorescence intensity is large. Further, the closer the fluorescence intensity of the low fluorescence intensity region 31 is to 0, the better. The interval between the high fluorescence intensity region 32 and the low fluorescence intensity region 31 can be optimized by adjusting as appropriate according to the size of the object whose stress is to be detected and the magnitude of the stress.

≪Method for manufacturing film-like soft material≫
The method for producing a film-shaped soft material according to the present invention includes a step of polymerizing a monomer liquid that contains a fluorescent dye having a polymerizable group and forms a three-dimensional network structure on a substrate. The method for producing a film-like soft material according to the present invention is a conventional method that does not involve the complexity of a chemical modification process when encapsulating fluorescent beads in a hydrogel, and can be easily prepared. Problems caused by fluctuations derived from unreacted fluorescent beads can also be solved. The substrate used in the step of polymerizing can be used as it is as the base material 12 of the fluorescent film base material 1, and the film-like soft material 11 obtained through the polymerizing step is transferred to the separately prepared base material 12 The fluorescent film substrate 1 can also be used.

  The monomer liquid contains a fluorescent dye having a polymerizable group and forms a three-dimensional network structure. In order to form a three-dimensional network structure, the monomer liquid can contain a fluorescent dye having a polymerizable group and a polymerizable compound having two or more polymerizable groups.

  The method for introducing a fluorescent dye into the side chain of the main skeleton of the three-dimensional network structure of the polymer compound constituting the film-shaped soft material is not particularly limited. However, in the case of a silicone elastomer, hydrosilylation using a platinum catalyst is performed. The method to use is mentioned. When a three-dimensional network structure is formed by chemical polymerization of monomers as in polyacrylamide gel, a monomer in which a fluorescent dye is chemically modified is used.

  The fluorescence intensity of the film-like soft material can be easily controlled by the mixing ratio of the fluorescent dye having a polymerizable group in the monomer liquid forming the three-dimensional network structure when polymerizing the polymer compound having the three-dimensional network structure. Is possible. For unreacted fluorescent dye monomers, by making the molecular size smaller than the network structure of the polymer compound, by re-swelling the produced film-like soft material in a good solvent, It is possible to remove. In the case of a film-like soft material consisting of a three-dimensional network structure by self-assembly of proteins such as collagen and cross-linking of polysaccharides such as alginic acid and hyaluronic acid, chemical bonds such as amide bonds and ester bonds before forming the three-dimensional network structure The chemical modification of the fluorescent dye is performed via As described above, the method of introducing a fluorescent dye into the side chain of the main skeleton having a three-dimensional network structure is simple and does not require complicated chemical modification. Unreacted fluorescent dye can be removed by dialysis either before or after film formation.

  The introduction rate of the fluorescent dye that modifies the side chain of the main skeleton of the three-dimensional network structure is not limited, but is preferably 0.01 to 1 mol% with respect to the unit unit of the main skeleton. The type of fluorescent dye introduced into the side chain of the film main skeleton is not limited, but is preferably water-soluble and does not cause molecular association between the dyes.

  The fluorescent dye having a polymerizable group is preferably a fluorescent modifying monomer such as fluorescein-O-acrylate (formula (1-A), hereinafter referred to as FL acrylate) sold by Sigma-Aldrich, or sold by Polysciences. Nile Blue Acrylamide (Formula (1-F)), Methacryloxyethyl thiocarbamoyl rhodamine B (Formula (1-G)) and the like (Formula (1-A) to Formula (1-G)).

  Although the preparation method of a film-form soft material is not specifically limited, In the case of a silicone type elastomer, hydrosilylation using a platinum catalyst is mentioned. In the case of an acrylic polymer, chemical crosslinking by a polymerization reaction of an acrylic group is exemplified. In the case of polysaccharides and proteins, gelation by self-assembly may be used, or chemical crosslinking by modifying the acrylic group to the main skeleton using the above carbodiimide may be used.

Although the kind of polymerization reaction is not specifically limited, As an example, the radical polymerization using a photoinitiator is mentioned. Examples of a substance that generates a chemical bond constituting itself by light irradiation include a raw material of a polymer compound such as a monomer that is polymerized by a photopolymerization initiator.
As the photopolymerization initiator, a water-soluble photopolymerization initiator is preferable, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) represented by the following formula (2-A): B) 2-hydroxy-4 ′-(2-hydroxyethoxy) -2-methylpropiophenone (Irgacure 2959), EosinY represented by the following formula (2-C), and the following formula (2-D) N-vinylpyrrolidone (NMP) represented by the formula, and triethanolamine (TEOA) represented by the following formula (2-E).

  After the step of polymerizing a monomer liquid containing a fluorescent dye having a polymerizable group and forming a three-dimensional network structure on a substrate to form a film-like soft material polymer, at least the film-like soft material polymer Part of the process is a process of irradiating energy rays in a pattern to create a gradient or step in the fluorescence intensity at the boundary between the high fluorescence intensity region and the low fluorescence intensity region. A step can be formed.

  Examples of a method of causing a gradient or a step in the fluorescence intensity of a film-like soft material include, for example, a method of using a photomask or a liquid crystal film as a photomask to irradiate high-intensity energy rays and fade, a digital mirror device Examples thereof include a method of locally irradiating with energy rays and fading, and a method of fading by light interference caused by laser beam defocusing.

≪Stress measurement method≫
In the stress measurement method according to the present invention, the step of bringing an object 14 such as a cell into contact with the film-like soft material 14 (FIG. 4A) and the displacement of the fluorescence intensity distribution of the film-like soft material 14 are evaluated. And a step (FIG. 4B) (FIG. 4).

  The gradient or step in the fluorescence intensity of the film-shaped soft material may be generated either before or after the object such as a cell is brought into contact with the film-shaped soft material. The stress measurement method according to the present invention includes a step of bringing an object such as a cell into contact with the film-shaped soft material, and irradiating at least a part of the film-shaped soft material with energy rays in a pattern, thereby increasing the fluorescence. You may provide the process of producing the gradient or level | step difference in fluorescence intensity in the boundary of an intensity | strength area | region and a low fluorescence intensity area | region, and the process of evaluating the displacement of the fluorescence intensity distribution of the said film-form soft material.

  As a method for stably measuring stress for a long period of time, it is possible to give an arbitrary fluorescence intensity distribution to a fluorescent film and quantify the amount of displacement due to the stress generated by the object. In a fluorescent film having a uniform fluorescence intensity distribution, it is difficult to quantitatively measure the amount of displacement of the fluorescence intensity distribution. A film-like soft material into which a fluorescent dye is introduced can form a gradient or step in the local fluorescence intensity distribution by irradiation with energy rays, thereby quantitatively measuring the amount of displacement of the fluorescence intensity distribution. Is possible.

  The gradient or step of the fluorescence intensity distribution is preferably optimized according to the size of the target object and the traction force generated. As mentioned above, since the fluorescent dye is modified with a chemical bond on the side chain of the main skeleton of the soft material constituting the fluorescent film, there is no variation in elastic modulus due to local differences in crosslink density, and unreacted Since the fluorescent dye can be removed from the film, fluctuations that do not depend on stress due to the diffusion movement of the fluorescent molecules do not occur, so that stable stress measurement can be realized for a long period of time.

  An in vitro model for elucidating the mechanism by which cells / tissues recognize the mechanical properties of ECM and respond as a traction force by using a film-like soft material into which such a fluorescent dye is introduced as a culture substrate. can do. In addition, it is suggested that the mechanical properties of ECM change at the time of tissue damage, and that the mechanical properties of ECM associated with diseases and aging affect cell / tissue functions. It is possible to use an in vitro model of trauma / disease for screening the mechanical properties of scaffold materials that are optimal for tissue regeneration using a culture substrate.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

<Elastic modulus measurement method>
The elastic modulus of each gel obtained in the examples was determined by the following method.
The film-like soft material swollen with an aqueous PBS solution adjusted to be approximately isotonic with human body fluid was measured using an atomic force microscope under measurement conditions of a temperature of 25 ° C.

[Example 1]
<Production of film-like gel and fluorescent film substrate into which fluorescent dye is introduced>
After placing Saran Wrap (registered trademark) as spacers 17 as shown in FIG. 5 on both ends of glass substrate 16 (hereinafter, silanized glass substrate) surface-modified with 3- (trimethylsilyl) propyl methacrylate, 5% by mass An aqueous solution containing 1 mM of acrylamide, 0.5% by mass of bisacrylamide, 0.05% by mass of FL acrylate, and a photopolymerization initiator LAP (hereinafter, monomer solution 18) was dropped onto the center of the silanized glass substrate (FIG. 6A). ). A cover glass (hereinafter referred to as a seal substrate 19) treated with oxygen plasma was placed on the glass substrate 16 from above, the monomer solution 18 was sandwiched between the silanized glass substrate and the seal substrate, and the excess monomer solution 18 was removed with Kimwipe. Using a bandpass filter with a wavelength of 360 nm, the monomer liquid 18 was irradiated with light at 10 mW / cm ² for 10 minutes (FIG. 6B). After completion of light irradiation, the obtained film-like hydrogel was immersed in a large excess amount of PBS solution to remove unreacted monomer liquid components. After removing the cover glass and removing the excess solution on the surface of the film-like gel into which the fluorescent dye was introduced, 100 μl of 2 mM sulfosuccinimidyl 6- (4′-azido-2′-nitrophenylamino) hexanoate (hereinafter, sulfo-SANPAH) was added. The solution was dropped and irradiated with light for 10 minutes through a band-pass filter having a wavelength of 360 nm (FIG. 6C). After removing the sulfo-SANPAH solution on the surface of the film-like gel, 100 μl of a 0.1 mg / ml poly-D-lysine (hereinafter referred to as PDL) solution was dropped and allowed to stand at room temperature for 2 hours (FIG. 6D). ). The PDL solution on the surface of the film-like gel was removed and permeated with a large excess of PBS solution for overnight or longer to obtain a fluorescent film substrate 2 having a film-like gel having an 18 mm × 18 mm-introduced fluorescent dye (FIG. 6). (E)). The elastic modulus of this film-form gel as the film-form soft material 11 was about 3.5 kPa, and was generally uniform as a whole.

[Example 2]
<Production 1 of film-like gel having fluorescent intensity distribution and fluorescent film substrate>
After producing the fluorescent film base material 2 provided with the film-like soft material into which the fluorescent dye is introduced by the method of Example 1, a photomask 24 (FIG. 7) in which a gold or chromium mask 21 is provided on the light transmission substrate 23. Hereinafter, a photomask.) Was placed on the substrate 16 side of the fluorescent film substrate 2. The size of the mask 21 is 62.5 μm × 62.5 μm. Light irradiation with a xenon light source was performed through a 422 nm high-pass filter (FIG. 8G), and a fluorescent film substrate 3 was obtained (FIG. 8H). After light irradiation for 10 minutes, observation with a fluorescence microscope was performed (FIG. 9). According to the mask pattern of the film-like gel as the film-like soft material 14, the light-irradiated region becomes a low fluorescence intensity region by fading the fluorescent dye, and the masked region becomes a high fluorescence intensity region. A fluorescence intensity distribution with a pitch of 125 μm was formed, indicating that there was a step in the fluorescence intensity between adjacent regions.

[Example 3]
<Preparation 2 of film-like gel having fluorescent intensity distribution and fluorescent film substrate>
After preparing the fluorescent film base material 2 provided with the film-like soft material into which the fluorescent dye was introduced by the method of Example 1, 488 nm laser light was irradiated as defocused light through the objective lens of the microscope. The observation result by the fluorescence microscope at that time is shown in FIG. It was shown that the high-fluorescence intensity region 32 and the low-fluorescence intensity region 31 are formed in an arbitrary region by defocusing light irradiation, and a step difference occurs in the fluorescence intensity between adjacent regions.

[Example 4]
<Measurement Method of Film Displacement and Stress Distribution Measurement Method>
In the measurement of the stress distribution, first, the displacement distribution of the gel is analyzed using a particle image flow velocity measurement method (hereinafter referred to as PIV). As a model system of cell traction force, the gel was displaced 1.6 μm with a glass needle. FIG. 11 shows the observation results of the gel before and after displacement by the fluorescence microscope and the PIV analysis results. An analysis result equivalent to the actual displacement direction and displacement amount was obtained from the displacement mapping. Next, the stress distribution can be calculated by numerically analyzing a theoretical expression based on the linear elasticity theory from the displacement distribution of the gel. ImageJ plug-ins were used for PIV analysis and stress distribution analysis of acquired images.

  When cells are used as an object by the stress measurement method using the film-like soft material and fluorescent film substrate of the present invention, the mechanism of tissue response to changes in the mechanical properties of the extracellular environment, and the disease / damage model of the tissue, etc. Therefore, it is possible to stably measure the stress generated by the cells for a long period of time. Moreover, the film-like soft material and fluorescent film substrate of the present invention may be used as a film substrate for cell culture. The elucidation of cell / tissue recognition / response mechanisms for ECM mechanical properties that have not been clarified so far can be expected. From the viewpoint of regenerative medicine, it is possible to screen the mechanical properties of the scaffold material optimal for tissue regeneration by using a damage / disease model.

  1, 2, 3 ... fluorescent film base material, 11, 14 ... film-like soft material, 12 ... base material, 13 ... cell adhesion layer, 13 '... PDL solution, 13 " ..Sulfo-SANPAH solution, 15 ... object, 16 ... glass substrate, 17 ... spacer, 18 ... monomer solution, 19 ... sealing substrate, 21 ... mask, 22 ... Light transmission area, 23 ... Light transmission substrate, 31 ... Low fluorescence intensity area, 32 ... High fluorescence intensity area

Claims (8)

  A film-like soft material having a three-dimensional network structure made of a polymer compound and having a fluorescent dye introduced into the side chain of the main skeleton of the three-dimensional network structure.   The film-form soft material of Claim 1 in which the said high molecular compound contains polyacrylamide type resin.   A high fluorescence intensity region and a low fluorescence intensity region having a fluorescence intensity lower than that of the high fluorescence intensity region, and a gradient or a step occurs in the fluorescence intensity at the boundary between the high fluorescence intensity region and the low fluorescence intensity region. The film-like soft material according to claim 1 or 2.   A fluorescent film base material provided with the film-like soft material according to any one of claims 1 to 3 on a base material. A method for producing the film-like soft material according to claim 1,
The manufacturing method of a film-form soft material which has the process of superposing | polymerizing the monomer liquid which contains the fluorescent pigment | dye which has a polymeric group on a board | substrate, and forms a three-dimensional network structure. A method for producing the film-like soft material according to claim 3,
A step of polymerizing a monomer liquid containing a fluorescent dye having a polymerizable group and forming a three-dimensional network structure on a substrate to form a film-like soft material polymer; and at least a part of the film-like soft material polymer And a step of irradiating energy rays in a pattern to generate a gradient or a step in the fluorescence intensity at the boundary between the high fluorescence intensity region and the low fluorescence intensity region. A stress measurement method using the film-like soft material according to claim 1 or 2,
Contacting an object such as a cell on the soft film material;
Irradiating at least a part of the film-like soft material with energy rays in a pattern to generate a gradient or a step in the fluorescence intensity at the boundary between the high fluorescence intensity region and the low fluorescence intensity region;
Evaluating the displacement of the fluorescence intensity distribution of the film-shaped soft material. A stress measurement method using the film-like soft material according to claim 3,
Contacting an object such as a cell on the soft film material;
Evaluating the displacement of the fluorescence intensity distribution of the film-shaped soft material.