JP2019054781A - Particle-containing three-dimensional tissues, and shrinkage measurement method for particle-containing three-dimensional tissues - Google Patents

Abstract

To provide particle-containing three-dimensional tissues that can measure the shrinkage of cells on not only the surface of, but the inside of three-dimensional tissues with high accuracy and readily.SOLUTION: Provided is a particle-containing three-dimensional tissue which is attached to the basal plate having cell adhesion and contains at least particles in at least one of the vicinity of one surface and the vicinity of the opposite surface in the thickness direction, and in the vicinity of the center in the thickness direction. The aspect is preferable that the average thickness is 30 μm or more, the particle density is sufficiently sparse at a degree that the projections of the particles do not overlap each other in planar view, and the cells have shrinkage ability.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a particle-containing three-dimensional structure and a method for measuring contraction of the particle-containing three-dimensional structure.

  Conventionally, when handling cells in vitro, it has been the mainstream to handle the cells planarly.

In recent years, three-dimensional tissue construction technology has been developed, and it has become possible to artificially produce a three-dimensional tissue body in vitro. With the development of research on these three-dimensional tissues, it is expected that artificially produced three-dimensional tissues will be applied to the fields of regenerative medicine and drug discovery.
In order to use the three-dimensional tissue in the field of regenerative medicine and drug discovery, a technique for evaluating the quality is important in order to ensure the quality of the three-dimensional tissue. In particular, as an evaluation of the produced three-dimensional tissue cardiomyocytes or cardiomyocyte sheets, the pulsation state of the cardiomyocytes is evaluated by an image processing technique, and the pulse for an evaluation electrical stimulation combining an image processing technique and a staining technique. Evaluation is based on dynamic response.

  In addition, various technical developments aiming to evaluate the influence of the drug on the living body by observing changes in the pulsatile state of the myocardium with respect to the drug administration have been performed. Among these techniques, a technique for evaluating the amount of contraction of a three-dimensional myocardial tissue has attracted particular attention. Typical methods for determining the contractile force of cells include (1) a method of calculating the contractile force from the movement of particles in the substrate, and (2) particles on the cell membrane surface. And a method for calculating the contractile force from the movement of the particles on the surface is already known.

  However, (1) in the method in which particles are contained in the cell adhesion substrate and the contraction force is calculated from the movement of the particles in the substrate, the measurement sample is assumed to be a single layer (two-dimensional). For this reason, when multi-layer (three-dimensional) measurement is attempted with this method, only the movement of the substrate portion is captured, and the original motion of the three-dimensional tissue body cannot be captured. In addition, (2) the method of adding particles to the cell membrane surface and calculating the contractile force from the movement of the particles on the surface assumes a single layer (two-dimensional) as in (1) above. There was a problem that only the movement of the surface of the three-dimensional organization could be captured.

  Therefore, a cardiotoxicity evaluation apparatus has been proposed in which a cardiomyocyte population is arranged on a transparent substrate and the quality of the cardiomyocytes is evaluated from the response of the cardiomyocytes to the forced pulsatile stimulation given to the myocardial pulsation cells (for example, a patent Reference 1).

  It is an object of the present invention to provide a particle-containing three-dimensional tissue that can easily measure not only the surface of the three-dimensional tissue but also the contraction of cells inside with high accuracy.

  The particle-containing three-dimensional tissue of the present invention as a means for solving the problems is adhered to a substrate having cell adhesiveness, and at least one of the vicinity of one surface in the thickness direction and the vicinity of the opposite surface thereof. In the vicinity of the center in the thickness direction, at least particles are contained.

  ADVANTAGE OF THE INVENTION According to this invention, not only the surface of a three-dimensional structure | tissue but the particle | grain containing three-dimensional structure | tissue which can measure the shrinkage | contraction of the cell inside is highly accurate and can be provided.

FIG. 1 is a schematic diagram showing an example of a particle-containing three-dimensional structure of the present invention. FIG. 2 is a schematic diagram showing an example of a conventional method for measuring a tissue body. FIG. 3 is a schematic diagram showing another example of a conventional method for measuring a tissue body. FIG. 4 is a schematic diagram showing an example in which the particles of the particle-containing three-dimensional structure are projected when the particle-containing three-dimensional structure is viewed in plan. FIG. 5 is a block diagram illustrating an example of a method for measuring contraction of a particle-containing three-dimensional tissue. FIG. 6A is a schematic diagram illustrating an example of a shrinkage measurement method for a particle-containing three-dimensional tissue according to the present invention. FIG. 6B is a schematic diagram showing the positions of particles in the particle-containing three-dimensional tissue at time t ₀ . FIG. 6C is a schematic diagram showing the positions of the particles in the particle-containing three-dimensional structure at time t ₁ . FIG. 6D is a schematic diagram showing the amount of movement of particles from time t ₀ to t ₁ . FIG. 7 is a schematic diagram illustrating an example of a Fourier domain type particle-containing three-dimensional tissue shrinkage measurement apparatus. FIG. 8 is a graph showing an example of a cross-correlation function obtained by the method for measuring contraction of a particle-containing three-dimensional structure according to the present invention. FIG. 9 is a schematic diagram illustrating an example of a tomographic image relating to another cross section in a direction intersecting with one cross section. FIG. 10 is a schematic diagram illustrating an example of a state of the particle-containing three-dimensional mouse myocardial tissue in Example 1. FIG. 11 is a schematic diagram showing an example of the state of the three-dimensional mouse myocardial tissue in Comparative Example 1. FIG. 12 is a schematic diagram showing an example of a state of iPS-derived cardiomyocytes in Example 2. FIG. 13 is a schematic diagram showing another example of the state of iPS-derived cardiomyocytes in Example 2. FIG. 14 is a schematic diagram illustrating an example of a state of a particle-containing three-dimensional structure in Example 3.

(Particle-containing three-dimensional structure)
The particle-containing three-dimensional tissue of the present invention is adhered to a substrate having cell adhesiveness, and at least one of the vicinity of one surface in the thickness direction and the vicinity of the opposite surface thereof, and the vicinity of the center in the thickness direction, at least It contains particles and further contains other components as necessary.
The particle-containing three-dimensional tissue of the present invention has a problem that the conventional cardiotoxicity evaluation apparatus only captures two-dimensional cell movement and cannot capture the original movement of the three-dimensional tissue. It is based on the knowledge that. In addition, in the conventional technique, the cell population constituting the three-dimensional tissue has a uniform refractive index. Therefore, when observing using light, it is difficult to observe the movement inside the tissue. It is based on the knowledge that there is.
The particle-containing three-dimensional structure of the present invention includes a particle contained in the three-dimensional structure, and particles having different optical characteristics from the three-dimensional structure are contained in the three-dimensional structure, thereby refracting incident light. The rate can be changed, and the contraction (displacement, movement) in the depth direction of the three-dimensional tissue can be easily observed. In addition, since the three-dimensional movement of the three-dimensional tissue can be captured, the original contraction (displacement, movement) of the tissue can be captured without being affected by the adhesion of the substrate or the surface. It is possible to observe drug penetration and cell response to stimulation in the depth direction.
The thickness direction means a direction perpendicular to the bottom surface of the substrate.

  In the cell aggregate, the cells in each site do not contract in the same way (displacement, movement), and contract (displacement, displacement) in different directions and different contraction amounts on the surface, inside, and lower layers of the cell aggregate. Moving). Further, even in the same layer of the cell aggregate, the cells are contracted in different directions and with different contraction amounts. It is important to grasp the contraction of cells in each part because the function and state of the cell aggregate can be grasped more accurately by accurately evaluating the contraction of the cell aggregate for each part.

The particle-containing three-dimensional structure means a structure having a three-dimensional shape containing particles.
An organization is a unit of a structure in which cells are connected via proteins such as connexon, cadherin, and extracellular matrix (ECM), and means an aggregate having a single role as a whole.

FIG. 1 is a schematic diagram showing an example of a particle-containing three-dimensional structure of the present invention. As shown in FIG. 1, in the particle-containing three-dimensional tissue body 20, a tissue body 22 formed of cells is adhered to a substrate 21 having cell adhesiveness. Moreover, the particle-containing three-dimensional tissue body 20 may contain a medium as necessary.
The tissue contains particles 23. In the present embodiment, the particle-containing three-dimensional structure 20 has a thickness direction near one side surface (for example, T1), an opposite side surface vicinity (for example, T3), and a thickness direction center vicinity (for example, T2). 1 shows an example containing the particles 23. Further, in this embodiment, the solvent (medium) 24 is placed on T3.
In the present embodiment, since each of T1 to T3 has the particle 23, the movement of the particle 23 contained in the one-side surface vicinity T1 and the particle 23 contained in the center vicinity T2 in the thickness direction are measured by a measurement method described later. It is possible to confirm that the displacement of the former (T1) is smaller than that of the latter (T2). This is presumed that T1 of the tissue body is affected by the fixed wall (substrate) more than T2, and the original tissue body displacement is not shown.
Further, by comparing the movement of the particle 23 contained in the vicinity T3 on the opposite side surface with the movement of the particle 23 contained in the center vicinity T2 in the thickness direction, the former (T3) has a smaller displacement than the latter (T2). It is possible to confirm. This is presumed that T3 of the tissue body is affected by the viscosity of the adjacent solvent (medium) 24, and as a result, the original tissue body displacement is not shown.
In this example, since the particles 23 are contained not only in the vicinity of the surface such as T1 and T3 but also in the vicinity of the center T2 in the thickness direction, the displacement of the tissue body not affected by the surface can be confirmed. That is, it is possible to know the accurate pulsating power by the measurement method described later.
The vicinity of one surface in the thickness direction means a range close to the surface in the thickness direction.
The vicinity of the reverse side surface means a range close to the surface on the opposite side to the vicinity of the one side surface.
The vicinity of the center in the thickness direction means a range close to the center in the thickness direction.

FIG. 2 is a schematic diagram showing an example of a conventional method for measuring a tissue body. FIG. 3 is a schematic diagram showing another example of a conventional method for measuring a tissue body.
As shown in FIG. 2, in the conventional method of measuring a tissue body, a coating layer 34 including particles 33 is provided on a substrate 31, and a three-dimensional cell body 30 having a tissue body 32 is provided on the coating layer 34. use. The pulsation (movement) of the tissue body 32 is measured by observing the movement of the particles 33 contained in the coating layer 34. However, in this method, only the pulsation (movement) of the tissue adhered to the coating layer 34 can be measured, and the three-dimensional motion of the surface and the interior of the tissue cannot be measured.
As shown in FIG. 3, another conventional method for measuring a tissue uses a particle-containing three-dimensional tissue 40 having particles 43 on the surface of the tissue 42 on the substrate 41. By observing the movement of particles on the surface of the tissue body 42, the pulsation (movement) of the tissue body 42 is measured. However, in this method, only the pulsation (movement) of the surface of the tissue body 42 can be measured, and the pulsation (movement) inside and at the bottom of the tissue body cannot be measured.
On the other hand, by using the configuration of the particle-containing three-dimensional tissue body 20 described above and measuring with a measurement method described later, it is possible to measure the movement inside the tissue body.

  There is no restriction | limiting in particular as average thickness of a particle | grain containing three-dimensional structure | tissue, Although it can select suitably according to the objective, 30 micrometers or more are preferable, 50 micrometers or more are more preferable, and 100 micrometers or more are especially preferable. When the average thickness is 30 μm or more, it is possible to detect a response to stimulation in the depth direction, such as drug penetration. Thickness can be measured with an optical microscope by using a method using optical coherence tomography as a non-invasive observation technique, or by preparing a section of a prepared tissue as an invasive observation technique. . The average thickness can be determined from the average value of the thicknesses at any five locations of the particle-containing three-dimensional structure.

  As the particle-containing three-dimensional structure, it is preferable that the particle density is sufficiently sparse so that the projections of the particles do not overlap each other when viewed in plan. When viewed from above, the density of the particles is sufficiently sparse so that the projections of the particles do not overlap each other, so that the particles do not overlap the incident light. Movement can be easily detected.

  FIG. 4 is a schematic diagram showing an example in which the particles of the particle-containing three-dimensional structure are projected when the particle-containing three-dimensional structure is viewed in plan. As shown in FIG. 4, the particles 53 of the particle-containing three-dimensional structure when viewed in plan can be visually recognized as single particles because the particle density is sufficiently sparse. Since the distance is sufficiently large, even when the particles 53 move due to the behavior of the tissue body, they do not overlap each other, and are continuously recognized as single particles, and the behavior of the particles 53 is captured by the measurement method described later. You can know the exact beat power.

<Particle>
The particles are not particularly limited and can be appropriately selected according to the purpose. However, it is preferable that the particles do not adversely affect the cells and do not inhibit the contraction (movement) of the cells.

  The volume average particle diameter of the particles is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1 μm or more and 50 μm or less, more preferably 2 μm or more and 20 μm or less, further preferably 5 μm or more and 15 μm or less, and more preferably 7 μm. The thickness is particularly preferably 10 μm or less.

The particles are not particularly limited and may be appropriately selected depending on the purpose. However, particles having optical properties different from those of the tissue body are preferable.
Examples of particles having optical properties different from those of the tissue body include polystyrene particles, glass particles, gold particles, silver particles, and alumina particles. These may be used individually by 1 type and may use 2 or more types together. Among these, polystyrene particles are preferable.
Further, particles different in particle size and optical characteristics may be arranged in each layer (for example, near one surface in the thickness direction (upper layer) and near the center in the thickness direction (middle layer)) when viewed in the thickness direction.

The particles preferably have a cell adhesive component on the surface.
There is no restriction | limiting in particular as a component which has cell adhesiveness, According to the objective, it can select suitably, For example, fibronectin, collagen, fibrinogen, laminin, vitronectin etc. are mentioned.

There is no restriction | limiting in particular as particle | grains, According to the objective, it can select suitably, For example, it is preferable that it can detect using an optical measuring means.
The optical measuring means is not particularly limited and can be appropriately selected according to the purpose. For example, means using moire interferometry, means using speckle interferometry, means using holography, digital Examples include means using an image correlation method. Among these, means using speckle interferometry is preferable.
The means using the speckle interferometry is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a phase contrast microscope and an optical coherence tomography (OCT) apparatus. Among these, an optical coherence tomography apparatus is preferable.

The content of the particles is preferably 1% by mass or more and 50% by mass or less, more preferably 1% by mass or more and 30% by mass or less, and more preferably 5% by mass or more and 20% by mass or less with respect to the total amount of the particle-containing three-dimensional structure. Particularly preferred. When the content is 1% by mass or more, the pulsation (movement) of the tissue can be accurately measured, and when it is 50% by mass or less, the particles do not inhibit the adhesion and extension of the tissue.
In addition, the content of particles in each unit in the vicinity of one surface in the thickness direction, in the vicinity of the surface on the opposite side, and in the vicinity of the center in the thickness direction of the particle-containing three-dimensional structure is preferably 1% by mass or more and 50% by mass or less. 1 mass% or more and 30 mass% or less are more preferable, and 5 mass% or more and 20 mass% or less are especially preferable. When the content is 1% by mass or more, the pulsation (movement) of the tissue can be accurately measured, and when it is 50% by mass or less, the particles do not inhibit the adhesion and extension of the tissue.

<Cells that make up the tissue>
The cells constituting the tissue body are not particularly limited as long as they are cells of a multicellular organism forming the tissue, and can be appropriately selected according to the purpose. Examples thereof include animal cells and plant cells.

  As the cells constituting the tissue body, cells having contractile ability are preferable, cardiomyocytes having pulsatile ability are more preferable, and when measuring contractile force against an exogenous stimulus, it is not necessary to be a cardiomyocyte, and three-dimensional Cells capable of forming a tissue can also be used.

There is no restriction | limiting in particular as a cardiac muscle cell which has pulsation ability, According to the objective, it can select suitably, For example, iPS origin cardiac muscle cell, a mouse cardiac muscle cell, a rat cardiac muscle cell etc. are mentioned.
The cells capable of forming a three-dimensional tissue body are not particularly limited and can be appropriately selected according to the purpose. Examples thereof include iPS cells, various differentiated cells derived from iPS cells, and various fibroblasts.

There is no restriction | limiting in particular as the number of the cells which comprise a tissue body, Although it can select suitably according to the objective, 0.01 * 10 < ⁴ > / cm < ² > -100 * 10 < ⁴ > / cm < ² > is preferable.

<Substrate with cell adhesion (culture vessel)>
The substrate having cell adhesiveness (culture container) is not particularly limited as long as it has cell adhesiveness, and can be appropriately selected according to the purpose. For example, it adheres as a container having cell adhesiveness or a coating layer of the substrate. Examples include containers capable of imparting cell adhesiveness with sex proteins.
There is no restriction | limiting in particular as a container which has cell adhesiveness, According to the objective, it can select suitably, For example, a surface-treated plastic dish etc. are mentioned.
There is no restriction | limiting in particular as a container which can provide cell adhesiveness with adhesive protein, According to the objective, it can select suitably, For example, a glass bottom dish etc. are mentioned.
There is no restriction | limiting in particular as adhesive protein, According to the objective, it can select suitably, For example, fibronectin, collagen, fibrinogen, laminin, vitronectin etc. are mentioned.
The shape of the culture vessel is not particularly limited and can be appropriately selected according to the purpose. Examples thereof include not only a planar shape but also a shape with a hole, a mesh shape, an uneven shape, and a honeycomb shape. It is done. Among these, a planar shape is preferable.
There is no restriction | limiting in particular as a material of a culture container, According to the objective, it can select suitably, For example, glass, the plastics which consist of various polymers, filter paper, a metal, hydrogel etc. are mentioned.
Examples of culture containers include non-porous substrates, porous substrates, fibers, fabrics such as woven fabrics and non-woven fabrics, hydrogel surfaces, medical material surfaces, artificial organ surfaces, phospholipid membranes such as cell membranes, etc. It can also be formed on top.

  The medium is not particularly limited and can be appropriately selected depending on the purpose. For example, Eagle's MEM medium, Dulbecco's Modified Eagle medium (DMEM), Modified Eagle medium (MEM), Minimum Essential medium, RDMI , GlutaMax medium, serum-free medium and the like.

[Method for producing particle-containing three-dimensional structure]
Cells suspended in phosphate buffered saline (PBS) are added to an Eppendorf tube containing particles to obtain a particle-containing cell suspension. Thereafter, a particle-containing cell suspension can be applied to the coated substrate to obtain a particle-containing three-dimensional tissue.
In addition, cells are discharged onto a substrate having a coating layer using a droplet forming device (for example, an in-house developed device). Thereafter, a particle-containing liquid containing particles in phosphate buffered saline (PBS) is discharged onto the cells using a droplet forming device (for example, an in-house developed device). Next, by repeating this step after a predetermined time has elapsed, a particle-containing three-dimensional structure in which polystyrene particles are arranged at arbitrary locations inside the particle-containing three-dimensional structure can be obtained.
The predetermined time is preferably 2 hours to 48 hours, and more preferably 3 hours to 4 hours. When the predetermined time is 2 hours to 48 hours, the cells are clogged to some extent without any gaps, so that sedimentation of particles can be prevented.

  As the particle-containing three-dimensional structure, it is preferable to produce a three-dimensional structure by discharging cells and particles using an inkjet method. By using the ink jet method, the particles are precisely arranged in the three-dimensional tissue with an angle with respect to the measurement surface of the optical measuring means for calculating the contraction amount (movement amount) of the three-dimensional tissue. (Position so that they do not overlap).

(Method for measuring contraction of particle-containing three-dimensional structure and device for measuring contraction of particle-containing three-dimensional structure)
The method for measuring contraction of a particle-containing three-dimensional structure of the present invention is a particle displacement amount calculating step of measuring the particle-containing three-dimensional structure of the present invention using an optical coherence tomography apparatus and calculating the displacement amount of the particle. And a cell contraction amount calculating step of calculating the contraction amount of the cells from the particle displacement amount obtained by the particle displacement amount calculating step, and further including other steps as necessary.
The particle-containing three-dimensional tissue contraction measuring device measures the particle-containing three-dimensional tissue of the present invention using an optical coherence tomography apparatus, and calculates a particle displacement amount calculating means, Cell shrinkage amount calculating means for calculating the amount of contraction of the cells from the amount of particle displacement obtained by the particle displacement amount calculating means, and further having other means as necessary.
The method for measuring the contraction of the particle-containing three-dimensional tissue can be preferably carried out by a device for measuring the contraction of the particle-containing three-dimensional tissue.

FIG. 5 is a block diagram illustrating an example of a method for measuring contraction of a particle-containing three-dimensional tissue. As shown in FIG. 5, in the method for measuring contraction of a particle-containing three-dimensional tissue, first, particles are added to isolated cardiomyocytes (isolated cardiomyocytes) to obtain a cardiomyocyte suspension. The obtained cardiomyocyte suspension is seeded on a substrate having cell adhesiveness to obtain a particle-containing three-dimensional tissue. The culture is then continued until pulsation begins. Next, using an optical coherence tomography apparatus, pulsation (particles) is observed and the amount of particle movement is analyzed. Using the image acquired by the optical coherence tomography apparatus, the movement amount per unit time of each particle contained in the three-dimensional myocardial tissue is derived by image analysis (for example, software name: ImageJ). Next, using the physical property value of the cell (spring constant: 100 mN / m), the contractile force of the particle-containing three-dimensional structure calculated from the following calculation formula based on Hooke's law can be obtained. ImageJ can be used for image analysis.
F = G × L Expression In the expression, F represents contractile force, G represents a spring constant, and L represents a particle movement amount.

There is no restriction | limiting in particular as culture | cultivation of a tissue body, According to the objective, culture | cultivation temperature, culture | cultivation time, etc. can be selected suitably.
As culture | cultivation temperature, 4 to 60 degreeC is preferable, 20 to 40 degreeC is more preferable, and 30 to 37 degreeC is especially preferable.
The culture time is preferably 1 hour to 240 hours, more preferably 72 hours to 120 hours, and particularly preferably 96 hours to 120 hours. When the culture time is 1 to 240 hours, the binding state between the cells is stabilized.

<Particle displacement calculation step and particle displacement calculation means>
The particle displacement amount calculation step includes backscattered light information acquisition processing, and further includes other processing as necessary.
The particle displacement amount calculation means includes a backscattered light information acquisition unit, and further includes other members as necessary.
The particle displacement amount calculating step can be preferably performed by a particle displacement amount calculating means.

<< Backscattered light information acquisition process and backscattered light information acquisition unit >>
The backscattered light information acquisition process is a process of irradiating light on the particle-containing three-dimensional tissue at at least two different times to acquire information on the backscattered light from the particle-containing three-dimensional tissue.
The backscattered light information acquisition unit is a member that acquires information on backscattered light from the particle-containing three-dimensional tissue by irradiating the particle-containing three-dimensional tissue with light at at least two different times.
A backscattered light information acquisition part means the member for acquiring the information of backscattered light.
The light information means, for example, the light intensity. In the spectroscopic detector, the light information is light intensity information for each frequency.

  The at least two different times include, for example, the time at the time of n o'clock of time Δt to nΔt in time series. The at least two different times are not particularly limited and may be appropriately selected according to the purpose. However, a larger number is preferable because the displacement amount of the particle-containing three-dimensional structure can be evaluated in more detail. By acquiring information on a plurality of backscattered light at times Δt to nΔt, the amount of contraction of the particle-containing three-dimensional tissue body can be calculated in more detail.

  In addition, the region for irradiating light to the particle-containing three-dimensional structure is not particularly limited and can be appropriately selected according to the purpose. For example, when it is desired to evaluate the state of the particle-containing three-dimensional structure only for a specific part. In order to evaluate the state of the entire particle-containing three-dimensional structure, it is sufficient to irradiate the entire particle-containing three-dimensional structure.

FIG. 6A is a schematic diagram illustrating an example of a shrinkage measurement method for a particle-containing three-dimensional tissue according to the present invention. FIG. 6B is a schematic diagram showing the positions of particles in the particle-containing three-dimensional tissue at time t ₀ . FIG. 6C is a schematic diagram showing the positions of the particles in the particle-containing three-dimensional structure at time t ₁ . FIG. 6D is a schematic diagram showing the amount of movement of particles from time t ₀ to t ₁ .
As shown in FIG. 6A, a particle-containing three-dimensional tissue body 90 has a collagen gel 94 as a coating layer on a base material (culture vessel) 91 as necessary, and a tissue containing particles 93 on the collagen gel 94. It has a body 92. The tissue body 92 may be appropriately immersed in the medium 95 as necessary. By measuring the particle-containing three-dimensional structure 90 using the particle-containing three-dimensional structure shrinkage measuring apparatus 100, the position of the particle at time Δt to nΔt can be calculated. As shown in FIG. 6B, with respect to the position of the particle 93 in the particle-containing three-dimensional structure at time t ₀ , as shown in FIG. 6C, the particle 93 ′ in the particle-containing three-dimensional structure at time t ₁ The position is displaced (moved) as the tissue body 92 is displaced.
As shown in FIG. 6D, the position (X ₀ ) of the particle 93 in the particle-containing three-dimensional structure at time t ₀ shown in FIG. 6B and the particle in the particle-containing three-dimensional structure at time t ₁ shown in FIG. 6C. From the position of 93 ′ (X ₁ ), the particle movement amount can be calculated as shown in the following formula (1). Further, the moving speed can be calculated as shown in the following formula (2).
Particle movement amount = X ₁ −X ₀ Formula (1)
Movement speed = (X ₁ −X ₀ ) × (t ₁ −t ₀ ) (2)

In the backscattered light information acquisition process and backscattered light information acquisition unit, when irradiating light to the particle-containing three-dimensional tissue, the light to be irradiated is divided into measurement light and reference light, and the measurement light contains particles It is preferable to obtain information on the backscattered light from the particle-containing three-dimensional structure by irradiating the three-dimensional structure and obtain information on the interference light between the backscattered light and the reference light.
There is no restriction | limiting in particular as light, According to the objective, it can select suitably, For example, when performing optical coherence tomography, incoherent light etc. which have various wavelengths are mentioned.
The division means to divide the irradiated light into measurement light and reference light.
The light is not particularly limited and can be appropriately selected depending on the purpose. For example, a superluminescent diode operating at a center wavelength of 900 nm, 930 nm, 1,300 nm, or 1,325 nm, a center wavelength of 1,060 nm, Examples include a wavelength sweep type laser that operates at 1,310 nm or 1,550 nm.
Irradiation means a state in which light remains emitted at a predetermined position for a certain period of time.
Backscattered light refers to light that reflects back and returns to light traveling forward in the optical fiber.
The measurement light means light for measuring the particle-containing three-dimensional tissue.
The reference light means a wavefront that is a reference for examining how much the other wavefront (optical path) is different from one wavefront (optical path) by dividing the light beam into two.
Interference light means superimposed light of backscattered light and reference light.

There is no restriction | limiting in particular as acquisition of backscattered light, According to the objective, it can select suitably, For example, an optical coherence tomography (Optical Coherence Tomography: OCT) apparatus can be used.
An optical coherence tomography (OCT) apparatus is widely used as a method for acquiring a high-resolution tomographic image of a living tissue in ophthalmology because it can be measured non-invasively and non-contactly.
In optical coherence tomography, one-dimensional depth measurement data is called A scan data, a two-dimensional image is called a B scan image, and a three-dimensional image is called a C scan image.

In an optical coherence tomography (OCT) apparatus, for example, a time domain method, a Fourier domain method, or the like can be given. Among these, the Fourier domain method is preferable.
Examples of the time domain method include time domain OCT.
Examples of the Fourier domain method include spectral domain OCT, optical frequency sweep OCT, and the like.
As the time domain OCT, the reference light is obtained while moving the mirror to mechanically change the optical path length of the reference light, and the tomographic image is obtained by superimposing the reference light and the backscattered light to obtain information on the interference light.
As the spectral domain OCT, the spectral information is detected using a spectroscope / detector to obtain a tomographic image.
As an optical frequency sweep OCT, a tomographic image is acquired by detecting a spectrum interference signal using a wavelength scanning light source.
The interference signal means three-dimensional information having frequency and time elements.
A tomographic image means an image obtained by clearly photographing only an arbitrary cross-sectional layer.

  In the Fourier domain type OCT, the acquired spectral interference signal is Fourier transformed to obtain depth information (transmitted light or reflected light intensity signal), so that the reference light is moved by moving the mirror during OCT imaging as in the time domain type. No operation is required to change the optical path length (this is usually referred to as A scan). Thereby, a tomographic image can be acquired at a remarkably high speed compared with time domain OCT. Therefore, in recent years, mainly Fourier domain OCT has been adopted.

FIG. 7 is a schematic diagram illustrating an example of a Fourier domain type particle-containing three-dimensional tissue shrinkage measurement apparatus. As shown in FIG. 7, a contraction measuring apparatus 100 for a particle-containing three-dimensional tissue conforming to the principle of Fourier-domain OCT includes a light source 101 that generates a light bundle 102, a Michelson interferometer apparatus, and a lens. The measurement light 106 is irradiated to a beam deflecting device 150 for spatially scanning the inside of the particle-containing three-dimensional tissue, and the measurement light 106 is directed toward the particle-containing three-dimensional structure 140 having a three-dimensional shape to be investigated. And a lens 160 that condenses the measurement light 106.
When the measurement light 106 is irradiated onto the particle-containing three-dimensional tissue, it is divided into multiple scattered light 111, multiple forward scattered light 113, and back scattered light 112.
The Michelson interferometer apparatus can separate the beam of the light beam 102 into reference light 104 and measurement light 106. Further, the Michelson interferometer apparatus superimposes the reference light 104 deflected in the reverse direction by the mirror 130 and the backscattered light 112 reflected from the particle-containing three-dimensional tissue forming the reflected wavefront 128 through the lens 160. And a device (beam splitter) 120 that forms interference light (superimposed light) 108.
The particle-containing three-dimensional tissue contraction measuring apparatus 100 further includes a spectroscope 170 including a diffraction grating and an array detector, and backscattered light 112 reflected by the reference light 104 and the particle-containing three-dimensional tissue 140 and returned. The interference light 108 forming the interference wavefront 118 formed by combining the two is detected as an interference signal, and the obtained spectral spectrum is Fourier transformed to obtain depth information of the particle-containing three-dimensional structure 140. it can.

The depth resolution in optical coherence tomography (OCT) is determined by the measurement resolution of the time delay of light.
Depth resolution in low coherence interference is given by the width of the autocorrelation function of the electric field of light. The autocorrelation function is proportional to the reciprocal of the wavelength spectrum width of the light source. In the case of a Gaussian spectrum, the depth resolution can be obtained by the following equation (3) (in the air).

In Expression (3), Δz represents the full width at half maximum of the autocorrelation function, Δλ represents the full width at half maximum of the wavelength power spectrum, and λc represents the center wavelength of the light source.

The lateral resolution is determined by the numerical aperture (NA) of the lens to be used and the center wavelength (λ) of the light source, and the spot size W ₀ when condensed by the lens can be obtained by the following equation (4). Yes (in the air)

The measurement light 106 irradiated to the particle-containing three-dimensional structure 140 is subjected to the multiple scattered light 111, the multiple forward scattered light 113, the multiple backscattered light, the single forward scattered light, Reflected by the backscattered light 112 or the like in a state where the wavefront is disturbed. The interference signal obtained by combining the reflected backscattered light 112 and the reference light 104 is detected as a speckle image. The speckle image is contracted (displaced, moved) in accordance with the contraction (displacement) of the particle-containing three-dimensional tissue when the particle-containing three-dimensional tissue is displaced.

  There is no restriction | limiting in particular as a light source, According to the objective, it can select suitably, For example, the super luminescence diode etc. of wavelength 930nm are mentioned.

The at least two different times are preferably times before and after the reagent is administered to the particle-containing three-dimensional tissue. By acquiring tomographic images at the time before and after the reagent is administered to the particle-containing three-dimensional tissue, the drug is three-dimensional based on the evaluation result of the movement of the particle-containing three-dimensional tissue having a three-dimensional shape. The influence on the particle-containing three-dimensional structure having a shape can be evaluated.
The time before and after the reagent is administered means the time before the reagent is administered and the time after the reagent is administered.
The time after administration of the reagent may be immediately after administration, or may be the time when the reaction due to stimulation has settled.

  There is no restriction | limiting in particular as a reagent, According to the objective, it can select suitably, For example, the reagent which evaluates medicinal effect, the reagent which evaluates toxicity, etc. are mentioned.

The reagent for evaluating the medicinal effect is not particularly limited and can be appropriately selected according to the purpose. For example, oxazolone, benzoquinone, 2,4-dinitrocolobendin, 4-phenylenediamine, glutaraldehyde, benzoyl peroxide, 4-methylaminophenol sulfate, formaldehyde, cinnamaldehyde, ethylenediamine, 2-hydroxyethyl acrylate, isoeugenol, nickel (II) sulfate, benzylideneacetone, methyl 2-noninate, benzyl salicylate, diethylenetriamine, thioglycerol, 2- Mercaptobenzothiazole, phenylacetaldehyde, hexylcinnamaldehyde, dihydroeugenol, benzisothiazolyone, citral, resorcinol, phenyl benzoate, euge Lumpur, abietic acid, ethyl aminobenzoate, benzyl cinnamate, cinnamyl alcohol, hydroxy citronellal, imidazolidinyl urea, butyl glycidyl ether, ethylene glycol dimethacrylate, glyoxal, and 4-nitrobenzyl bromide and the like.
The reagent for evaluating toxicity is not particularly limited and may be appropriately selected depending on the intended purpose. For example, zinc chloride, 1-butanol, benzoic acid, ethyl vanillin, 4-hydroxybenzoic acid, sulfanilic acid, tartaric acid , Methyl salicylate, salicylic acid, sodium lauryl sulfate, lactic acid, benzyl alcohol, dextran, diethyl phthalate, glycerol, propylparaben, Tween 80, dimethyl isophthalate, phenol, chlorobenzene, sulfanilamide, octanoic acid and the like.

<Cell contraction amount calculating step and cell contraction amount calculating means>
The cell contraction amount calculating step is a step of calculating the cell contraction amount from the particle displacement amount obtained by the particle displacement amount calculating step.
The cell contraction amount calculating means is a means for calculating the contraction amount of the cell from the particle displacement amount obtained by the particle displacement amount calculating means.
The cell contraction amount calculating step can be suitably performed by a cell contraction amount calculating means.

The cell contraction amount calculating step is a step of calculating the contraction amount of the particle-containing three-dimensional tissue based on the information of the backscattered light at at least two different times.
The cell contraction amount calculation means is a means for calculating the contraction amount of the particle-containing three-dimensional tissue based on the information of the backscattered light at at least two different times.
The cell contraction amount calculating step can be suitably performed using a cell contraction amount calculating means.

The cell contraction amount calculating step and the cell contraction amount calculating means obtain a tomographic image from the interference signal based on the interference light, and compare the plurality of tomographic images at at least two different times to contract the particle-containing three-dimensional tissue body. It is preferred to calculate the amount.
Comparing a plurality of tomographic images means comparing tomographic images using a plurality of tomographic images.
Calculation of the amount of contraction means determining the contraction of the particle-containing three-dimensional tissue.
The cell contraction amount calculation means means for calculating the contraction amount of cells.

In the cell contraction amount calculation step and the cell contraction amount calculation means, the tomographic image is obtained by capturing a speckle pattern of a particle-containing three-dimensional tissue having a three-dimensional shape represented by a plurality of pixels (hereinafter, “speckle image”). The comparison between the plurality of tomographic images is preferably performed by calculating a cross-correlation function (hereinafter also referred to as “correlation pattern”) between the plurality of speckle images. .
A plurality of pixels means a portion of pixels obtained by cutting out a region for calculating correlation from the original image.
In the cell contraction amount calculation step and the cell contraction amount calculation means, it is preferable that the calculation of the cross-correlation function between the plurality of speckle images is performed for each specific pixel in the speckle image.
The calculation of the cross-correlation function means calculating the degree of the relationship with the original signal when the signal is shifted for a certain time, and obtaining a result value.
To be performed for each specific pixel means to be performed for a specific pixel.

The displacement (movement) of the particle-containing three-dimensional tissue body can be obtained from the temporal change in the pattern of the speckle image by performing a correlation operation between the speckle images. Note that the total displacement can be obtained by integrating the obtained displacements. Moreover, the contraction (movement) speed and acceleration of contraction (displacement, movement) of the particle-containing three-dimensional tissue can be obtained by dividing the obtained displacement by the frame rate. The speckle pattern imaging result (hereinafter also referred to as “image pattern”) measured at time t = nΔt is expressed as f (n) (= f (n) ij). However, the subscripts i and j each represent the position of the pixel of the detected speckle image. For example, f (3) 23 means the pixel value of the second pixel in the i direction and the third pixel in the j direction that is the orthogonal direction at the time t = 3Δt. Unless otherwise specified, the image pattern is expressed as f (n). When the pixel value of a specific pixel i, j is meant, the image pattern is expressed as f (n) ij.
When the image pattern f (n) is received, a cross-correlation function is obtained from the following equation (5) using the image pattern f (n) and the previously received image pattern f (n−1).
f (n) * f (n-1) ≡F-1 [LCF [f (n)] F [f (n-1)] *] (5)
However, the symbol “★” is a cross-correlation operator, the function LC is a low frequency removal pattern, the function F [f] is a discrete Fourier transform of the function f, the function F-1 [f] is an inverse discrete Fourier transform of the function f, a symbol F * is the complex conjugate of F. According to Equation (5), high-speed calculation of the cross-correlation function is possible by using the discrete Fourier transform.

  The low frequency removal pattern is a pattern in which the pixel value of the central pixel and the surrounding 3 × 3 pixels out of 128 × 32 pixels are set to zero, and the other pixel values are set to 1. In equation (5), the background removal processing of the image pattern is performed by taking the product with the low frequency removal pattern LC. Note that the low frequency removal pattern LC is determined in advance so that the background distribution (background) can be satisfactorily removed by experiments or the like. Thereby, contraction (displacement) of the particle-containing three-dimensional tissue can be obtained with high accuracy.

  FIG. 8 is a graph showing an example of a cross-correlation function obtained by the method for measuring contraction of a particle-containing three-dimensional structure according to the present invention. In the correlation pattern that does not perform low-frequency removal, only a sharp peak tip derived from the speckle pattern appears in a gentle mountain-shaped distribution, whereas, as shown in FIG. In the correlation pattern, a gentle mountain distribution does not appear, and almost the entire sharp peak derived from the speckle pattern appears in noise with a small amplitude. In this way, by performing the background removal processing of the image pattern, the background distribution (background) is removed from the correlation pattern, and the peak derived from the speckle pattern appears clearly. As a result, the peak position can be accurately determined. Can be sought.

  The displacement of the particle-containing three-dimensional structure between the different times is obtained by analyzing the cross-correlation function and obtaining the first displacement in the pitch unit of the plurality of pixels and the second displacement below the pitch unit. be able to. The displacement can be obtained in pixel pitch units from the position (peak position) of the pixel having the maximum pixel value derived from the speckle pattern. Further, the displacement below the pixel pitch can be obtained by the following subpixel processing. By adding the pixel pitch of these OCT images, the displacement of the particle-containing three-dimensional tissue having a three-dimensional shape can be obtained.

In sub-pixel processing, polygonal approximation (primary and zeroth order binomial approximation), parabolic approximation (secondary and zeroth order binomial approximation), approximation by sum of polygonal line and parabola (secondary to zeroth order ternary) A displacement equal to or smaller than the pixel pitch is obtained by interpolating the cross-correlation function f (n) * f (n−1) by applying (approximation) or the like. Here, the pixel i0 having the maximum pixel value P (0) among the cross-correlation functions f (n) * f (n−1) is set, and the pixel value of the pixel i = i0 + m adjacent to the pixel i0 is set to P ( m (= i−i0)). In the polygonal line approximation, the pixel value is approximated as P (i−i0) = a | i−i0 | + P0 using the first-order and zero-order binomial expressions in the vicinity of P (0). Thereby, the displacement x below the pixel pitch can be obtained by the following formula (6a) or the following formula (6b).
x = {P (-1) -P (1)} / {2P (-1) -2P (0)} Expression (6a)
However, in Expression (6a), P (−1) ≧ P (1).
x =-{P (1) -P (-1)} / {2P (1) -2P (0)} Expression (6b)
However, in the formula (6b), P (-1) <P (1).

In the parabolic approximation, the pixel value is approximated to P (i−i0) = b (i−i0) ² + P0 in the vicinity of P (0) by using a binomial expression of second order and zero order. Thereby, the displacement x can be calculated | required by following formula (7).
x = − {P (1) −P (−1)} / {2P (−1) −4P (0) + 2P (1)} (7)

In the approximation by the sum of the polygonal line and the parabola, the pixel value is approximated as P (i−i0) = b (i−i0) ² + a | i−i0 | + P0 using a quadratic polynomial in the vicinity of P (0). Thereby, the displacement x can be calculated | required by following formula (8a) or following formula (8b).
x = {P (-1) -P (1)} / {P (-1) -P (0) -P (1) + P (2)} Expression (8a)
However, in the formula (8a), P (−1) ≧ P (1).
x = {P (1) -P (-1)} / {P (-2) -P (-1) -P (0) + P (1)} (8b)
However, in the formula (8b), P (-1) <P (1).

  By integrating the obtained displacements, the total displacement of the particle-containing three-dimensional tissue having a three-dimensional shape can be obtained. In the present embodiment, the subcorrelation function is interpolated by adopting the approximation based on the sum of the polygonal line and the parabola in the subpixel processing. However, the interpolation is performed by adopting higher order approximation such as a cubic polynomial. It is good.

  In the method for measuring contraction of a particle-containing three-dimensional tissue of the present invention, in the cell contraction amount calculation step, the speckle image at each time nΔt is partitioned into a plurality of partition images composed of a plurality of pixels, Thus, it is preferable to calculate the displacement value x. In this case, the cross-correlation function, displacement amount, and speed are calculated for each section image to obtain a two-dimensional mapping image of these parameters, and the movement of the particle-containing three-dimensional tissue having a three-dimensional shape. Will be evaluated.

As the amount of cell contraction, using the image acquired by the optical coherence tomography apparatus, the amount of movement per unit time of each particle contained in the 3D myocardial tissue is derived by image analysis (software name: ImageJ). Then, using the physical property value of the cell (spring constant: 100 mN / m), the contractile force of the particle-containing three-dimensional tissue calculated from the following calculation formula based on Hooke's law can be obtained. ImageJ can be used for image analysis.
F = G × L Expression In the expression, F represents contractile force, G represents a spring constant, and L represents a particle movement amount.

In the cell contraction amount calculating step and the cell contraction amount calculating means, the comparison of a plurality of tomographic images is not only performed on the tomographic image relating to one cross section in the particle-containing three-dimensional tissue, but also intersects with one cross section. It is also preferable to perform tomographic images relating to other cross sections in the direction.
FIG. 9 is a schematic diagram illustrating an example of a tomographic image relating to another cross section in a direction intersecting with one cross section. As shown in FIG. 9, in the particle-containing three-dimensional structure 300, the contraction evaluation is also performed on the tomographic image 301 of another cross section in the direction intersecting with one cross section, thereby further reducing the contraction of the particle-containing three-dimensional structure. It can be measured in detail.
The other cross section in the direction intersecting with one cross section means that the cross section partially intersects with one cross section and is different from the one cross section in the plane direction.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

(Average thickness of particle-containing three-dimensional structure)
The thickness of the particle-containing three-dimensional tissue was measured at 5 points using an optical coherence tomography (OCT) apparatus. The average value of 5 points was calculated and used as the average thickness.

Example 1
Cardiomyocyte culture kit using cardiomyocytes obtained by cryopreserving a cell group obtained by removing most non-cardiomyocytes from mouse fetal heart (trade name: Cardiomyocyte Culture kit (Mouse, Cryopreserved Cell), manufactured by Cosmo Bio) Was used. First, in order to capture the pulsation of the three-dimensional myocardial tissue more accurately, a collagen gel (trade name: Type IA / Cell matrix, manufactured by Kurashiki Boseki Co., Ltd.) is used according to the procedure manual so that the average thickness becomes 500 μm. The bottom of a 24-well culture plate (trade name: MICRO PLATE 24 Well, manufactured by AGC Techno Glass Co., Ltd.) was coated. Thereafter, phosphoric acid was added to a 1.5 mL Eppendorf tube (trade name: HYPER MICRO TUBE, manufactured by Watson) containing polystyrene particles having a diameter of 7.7 μm (trade name: Duke Standard 2008A, manufactured by Thermo Fisher Scientific KK). 1 mL of isolated mouse cardiomyocytes suspended in buffered saline (PBS) was added to obtain a particle-containing mouse cardiomyocyte suspension. Cardiomyocyte density in the obtained particle-containing mouse cardiomyocyte suspension was 1 × 10 ⁷ cells / mL, and the particle concentration was 2 × 10 ⁶ particles / mL. Subsequently, the cells are seeded in a 24-well culture plate that has been coated with the particle-containing mouse cardiomyocytes (particle-containing three-dimensional tissue) so as to be 2 × 10 ⁶ cells / mL, and particles having an average thickness of 100 μm are contained. Mouse myocardial tissue was obtained. Thereafter, the cells were cultured for 72 hours in a CO ₂ incubator at 37 ° C. until pulsation was started.

The state of pulsation of the particle-containing three-dimensional mouse myocardial tissue was measured using a glass heater (device name: cell culture compact chamber, manufactured by BLAST) while controlling the temperature of the culture plate part to 37 ° C. Observation was performed using an coherence tomography device (device name: Ganymede, manufactured by Thorlabs). Observation was performed at an imaging interval of 6 msec (= 167 FPS, Frame Per Second) to capture the maximum pulsation value of the myocardial tissue. The results are shown in FIG.
As shown in FIG. 10, the tissue body 62 in the particle-containing three-dimensional mouse myocardial tissue body (particle-containing three-dimensional tissue body) 60 adhered to the substrate 61 having the coating layer 64 has a one-side surface vicinity T1 in the thickness direction, The particles 63 were contained in the vicinity of the reverse surface T3 and the central vicinity T2 in the thickness direction.

Then, using the image acquired by the optical coherence tomography device of the particle-containing mouse myocardial tissue obtained in Example 1, the amount of contraction per unit time of each particle contained in the three-dimensional myocardial tissue image analysis ( As a result of deriving with software name: ImageJ), the maximum was 5 μm. Thereafter, using the physical property value of the cell (spring constant: 100 mN / m), the maximum contractile force value of the particle-containing mouse myocardial tissue calculated from the following formula based on Hooke's law was 500 pN.
F = G × L Expression In the expression, F represents contractile force, G represents a spring constant, and L represents a particle movement amount.

(Comparative Example 1)
A three-dimensional mouse myocardial tissue having an average thickness of 100 μm was obtained in the same manner as in Example 1 except that polystyrene particles were not used.
In the same manner as in Example 1, the three-dimensional mouse myocardial tissue was observed. The results are shown in FIG.
As shown in FIG. 11, the tissue body 72 in the particle-containing three-dimensional mouse myocardial tissue body 70 adhered to the substrate 71 having the coating layer 74 does not contain particles.

  From the results of FIG. 10 and FIG. 11, the particle-containing three-dimensional tissue produced using the same type of mouse cardiomyocytes, but as shown in FIG. It was confirmed that the situation was observed more clearly. On the other hand, as shown in FIG. 11, the particle-containing three-dimensional tissue of Comparative Example 1 did not contain particles, and thus the pulsation of the tissue could not be observed.

(Example 2)
iPS-derived cardiomyocytes (trade name: iCell Cardiomyocytes, manufactured by CDI) were used. First, in order to adhere the three-dimensional myocardial tissue body to the substrate, fibronectin (trade name: Fibronectin from human plasma, manufactured by SIGMA) is used on the surface of a glass bottom dish (trade name: glass base dish, manufactured by AGC Techno Glass Co., Ltd.). The coating treatment used was performed. Thereafter, iPS-derived cardiomyocytes were seeded at 1.5 × 10 ⁶ cells / cm ^{2 on} the coated dish. After culturing at 37 ° C. for 24 hours, adhesion of iPS-derived cardiomyocytes was confirmed using a phase contrast microscope (device name: CKX41, manufactured by Olympus Corporation). Next, a PBS solution (trade name: Duke Standard 2008A, manufactured by Thermo Fisher Scientific KK) containing polystyrene particles (PS particles) having a diameter of 10 μm was placed on the iPS-derived cardiomyocytes at 1 × 10 ⁴ particles / cm ^2. It was dripped so that it might become. Thereafter, the cells were cultured in a CO ₂ incubator at 37 ° C. for 3 hours. Thereafter, iPS-derived cardiomyocytes were again seeded onto the aggregate in which particles were present on the myocardial tissue so as to be 1.5 × 10 ⁶ cells / cm ² . Thereafter, the particles were added dropwise so as to be 3 × 10 ⁴ particles / cm ² and cultured in a CO ₂ incubator at 37 ° C. for 3 hours. By repeating this step a plurality of times, a particle-containing iPS-derived myocardial tissue (particle-containing three-dimensional tissue) having an average thickness of 200 μm in which polystyrene particles (PS particles) are regularly arranged was produced. Thereafter, the cells were cultured for 72 hours in a CO ₂ incubator at 37 ° C. until pulsation was started.

In the same manner as in Example 1, the three-dimensional mouse myocardial tissue was observed. As a result, as in Example 1, it was confirmed that the pulsation of the tissue was observed more clearly by containing particles. The results are shown in FIG. 12 and FIG.
As shown in FIG. 12, a tissue body 82 in a particle-containing three-dimensional mouse myocardial tissue body 80 adhered to a substrate 81 having a coating layer 84 has a thickness near one surface T1, a surface T3 on the opposite side, and a thickness. The particles 83 were contained in the vicinity of the center T2 in the direction.

  Further, when the maximum contractile force value of iPS-derived cardiomyocytes was measured in the same manner as in Example 1, it was 0.5 mN.

(Example 3)
iPS-derived cardiomyocytes (trade name: iCell Cardiomyocytes, manufactured by CDI) were used. In order to enhance cell adhesion to the substrate, a fibronectin solution (0.2 mg / mL, Fibronectin from human plasma, SIGMA) is applied to the entire surface of the substrate (glass bottom dish, trade name: glass base dish, manufactured by AGC Techno Glass Co., Ltd.). Coated) was applied to produce a coating layer. Then, after incubating for 12 hours in a 37 ° C. incubator, about 10 layers (1 × 10 ⁶ cells / cm ² ) are used on the substrate having the coating layer by using a droplet forming device (in-house developed device). Cardiomyocytes were discharged. Thereafter, polystyrene (PS) particles having a diameter of 7 μm (trade name: Duke Standard 2008A, manufactured by Thermo Fisher Scientific KK) were adjusted to a concentration of 1 × 10 ⁵ particles / mL in phosphate buffered saline (PBS). A particle-containing liquid was prepared, and discharged to cardiomyocytes using a droplet forming device (in-house developed device). Thereafter, by repeating this step, a particle-containing three-dimensional structure having an average thickness of 100 μm in which polystyrene particles are arranged at arbitrary positions inside the particle-containing three-dimensional structure was obtained. Thereafter, the cells were cultured for 72 hours in a CO ₂ incubator at 37 ° C. until pulsation was started.

In the same manner as in Example 1, the myocardial tissue was observed. As a result, as in Example 1, it was confirmed that the pulsation of the tissue was observed more clearly by containing particles. Moreover, the particle | grain containing three-dimensional organization | tissue which has arrange | positioned particle | grains in arbitrary positions has been produced. The results are shown in FIG.
As shown in FIG. 14, the structure 88 in the particle-containing three-dimensional structure 85 adhered to the substrate 86 having the coating layer 89 has a one-side surface vicinity T1 in the thickness direction, a reverse-side surface vicinity T3, and a thickness direction The particles 87 were contained in the vicinity of the center T2.

  Further, when the maximum contractile force value of iPS-derived cardiomyocytes was measured in the same manner as in Example 1, it was 0.1 mN.

As an aspect of this invention, it is as follows, for example.
<1> It is adhered to a substrate having cell adhesiveness, and contains at least particles in at least one of the vicinity of one surface in the thickness direction and the vicinity of the opposite surface thereof, and the vicinity of the center in the thickness direction. It is a particle-containing three-dimensional structure.
<2> The particle-containing three-dimensional structure according to <1>, wherein the average thickness is 30 μm or more.
<3> The particle-containing three-dimensional structure according to any one of <1> to <2>, wherein the particle density is sufficiently sparse so that the projections of the particles do not overlap each other when seen in a plan view.
<4> The particle-containing three-dimensional structure according to any one of <1> to <3>, which has contractility.
<5> The particle-containing three-dimensional tissue body according to <4>, wherein the particle-containing three-dimensional tissue body having contractility is formed by cardiomyocytes having pulsatile capacity.
<6> The particle-containing three-dimensional tissue body according to <5>, wherein the cardiomyocytes having pulsating ability are at least one selected from iPS-derived cardiomyocytes, mouse cardiomyocytes, and rat cardiomyocytes.
<7> The particle-containing three-dimensional tissue according to any one of <1> to <6>, wherein the particle has a component having cell adhesiveness on a surface.
<8> The particle-containing three-dimensional structure according to any one of <1> to <7>, wherein the particle is detectable using an optical measurement unit.
<9> The particle-containing three-dimensional structure according to any one of <1> to <8>, wherein the particle is at least one selected from polystyrene particles, glass particles, gold particles, silver particles, and alumina particles. Is the body.
<10> The particle-containing three-dimensional structure according to <9>, wherein the particle is a polystyrene particle.
<11> The particle-containing three-dimensional structure according to any one of <1> to <10>, wherein the particle has a volume average particle diameter of 1 μm or more and 50 μm or less.
<12> A particle displacement amount calculating step of measuring the particle-containing three-dimensional structure according to any one of <1> to <11> using an optical coherence tomography apparatus, and calculating a displacement amount of the particles; ,
A method for measuring contraction of a particle-containing three-dimensional tissue, comprising: a cell contraction amount calculating step of calculating a contraction amount of the cell from a particle displacement amount obtained by the particle displacement amount calculating step.

  <1> to <11> the particle-containing three-dimensional structure according to any one of <11> and the particle-containing three-dimensional structure measurement method according to the above <12>, the conventional problems are solved, The object of the present invention can be achieved.

International Publication No. 2014/098182 Pamphlet

20, 30, 40, 60, 80, 85 Particle-containing three-dimensional structure 21, 31, 41, 51, 61, 71, 81, 86 Substrate 23, 33, 43, 53, 63, 83, 87 Particles 100 Contains particles Three-dimensional tissue shrinkage measurement device

Claims (7)

Containing a particle characterized in that it is adhered to a substrate having cell adhesiveness and contains at least particles in the vicinity of one surface in the thickness direction and in the vicinity of the opposite surface and in the vicinity of the center in the thickness direction. A three-dimensional organization.   The particle-containing three-dimensional structure according to claim 1, wherein the average thickness is 30 μm or more.   3. The particle-containing three-dimensional structure according to claim 1, wherein the density of the particles is sufficiently sparse so that the projections of the particles do not overlap each other when seen in a plan view.   The particle-containing three-dimensional structure according to any one of claims 1 to 3, which has contractility.   The particle-containing three-dimensional tissue according to any one of claims 1 to 4, wherein the particle has a component having cell adhesion on the surface.   The particle-containing three-dimensional tissue according to any one of claims 1 to 5, wherein the particle can be detected using an optical measurement means. A particle displacement amount calculating step of measuring the particle-containing three-dimensional tissue body according to any one of claims 1 to 6 using an optical coherence tomography apparatus, and calculating a displacement amount of the particles;
A method for measuring contraction of a particle-containing three-dimensional tissue, comprising: a cell contraction amount calculating step of calculating a contraction amount of the cell from a particle displacement amount obtained by the particle displacement amount calculating step.