Cell culture dish

Abstract

This cell culture dish comprises a container (101), a stress-buffering layer (103), and a film (104). An opening (102) is formed in the bottom of the container (101). The opening (102) passes through the bottom of the container (101). The stress-buffering layer (103) is formed at the edge of the opening (102) around the entirety of the outer circumferential direction of the container (101) and is formed from a resin that has greater flexibility than polystyrene. The film (104) is stuck to the bottom of the container (101) with the stress-buffering layer (103) therebetween so as to cover the opening (102).

Description

Cell culture dishes

[0001] The present invention relates to a cell culture dish.

[0002] Traditionally, biological tissues have been observed primarily using optical microscopes, which widely use staining methods to distinguish between different chemical properties of biological tissues by changing their optical properties using chemicals.

[0003] In recent years, ultrasonic microscopes with resolutions equivalent to those of optical microscopes have been put into practical use, making it possible to observe biological tissues at the cellular level. Using ultrasonic microscopes, it is possible to obtain ultrasonic images (such as sound velocity images and acoustic impedance images) of measured acoustic parameters (such as tissue sound velocity and acoustic impedance) as color images. Thus, ultrasonic microscopes can distinguish differences in physical properties such as sound velocity and acoustic impedance without the use of chemicals or other staining, thereby enabling non-invasive measurements with minimal impact on the subject. Therefore, ultrasonic microscopes have attracted attention.

[0004] Among ultrasonic microscopes, an ultrasonic imaging inspection device has been proposed that displays acoustic impedance images of cells using a pulsed excitation ultrasonic microscope. In the ultrasonic imaging inspection device, a reference portion 403 is provided at a position different from a region 402 in which biological tissue is placed within the range scanned by ultrasonic waves on a bottom portion 401 of a cell culture dish containing cells to be observed. The cell culture dish contains biological tissue cells together with a culture medium. Ultrasound is irradiated via the bottom portion 401 to the region 402 in which the biological tissue cells are placed and the reference portion 403 (FIG. 4).

[0005] The ultrasonic wave (incident wave) S0 irradiated at an angle perpendicular to the surface of the reference portion 403 and the reflected wave Sr have the relationship "Sr = (Zs - Zr) / (Zs + Zr) S0 (1)." In formula (1), Zs is the acoustic impedance of the bottom portion 401, and Zr is the acoustic impedance of the culture medium in the reference portion 403.

[0006] The relationship between the ultrasonic wave S0 irradiated at an angle perpendicular to the surface of a cell and the reflected wave St is expressed as follows: St = (Zs - Zt) / (Zs + Zt) S0 (2). In equation (2), Zt is the acoustic impedance of the cell.

[0007] Therefore, from the above equations (1) and (2), the acoustic impedance Zt of the cell can be calculated by "Zr = {(1 - St / Sr) (Zs - Zr) / (Zs + Zr)} / {(1 + St / Sr) (Zs - Zr) / (Zs + Zr)} ... (3)."

[0008] In the above-described ultrasonic imaging test, a two-dimensional acoustic impedance image is obtained by two-dimensionally scanning the irradiation point of the ultrasonic wave S0 while measuring the acoustic impedance Zt. The acoustic impedance Zt is a parameter related to the stiffness of the cell, and the properties of the cell can be observed from the acoustic impedance image.

[0009] To observe the shape of a cell, a positional resolution smaller than that of a cell, for example, of about 10 μm, is required, and the spot diameter of the ultrasonic beam must be narrowed down to about several μm. The spot diameter and focal length of the ultrasonic beam are correlated, and when the spot diameter is narrowed down to the desired size, the focal length from the transducer that emits the ultrasonic beam becomes about several hundred μm.

[0010] Commercially available cell culture dishes containing cells to be observed have a bottom thickness of approximately 1 mm, which is larger than the focal length of the beam. This makes it impossible to focus the ultrasound beam on the location of the cells, making observation impossible. In response to this, a measurement technique has been proposed that uses a cell culture dish with a different bottom thickness only in the area where ultrasound measurement is performed (Non-Patent Document 1). Specifically, a through-hole is drilled in a portion of the cell culture dish, and a polystyrene film is attached to the hole. In this way, a cell culture dish is produced in which the bottom thickness of a portion is reduced to the thickness of the film, and cells are observed in the area where the film is attached. The film is attached by heating using laser irradiation to melt the polystyrene film and the dish bottom, which are then fused to the dish bottom.

[0011] N. Hozumi et al., "Three-dimensional acoustic impedance mapping of cultured biological cells", Ultrasonics, vol. 99, no. 105966, 2019.

[0012] However, in laser beam fusion, the film is locally heated and melted, making it impossible to uniformly adjust the heating conditions, resulting in a spatially complex stress distribution in the film. This results in minute distortions and acoustic impedance distributions throughout the film, making it difficult to accurately measure the acoustic impedance of the object being measured. Cell observation requires a positional resolution of several micrometers or less, and measurements at a position other than the target observation area are used as reference data. Therefore, the effects of film distortion and acoustic impedance distribution cannot be ignored.

[0013] In particular, if the distortion of the film is not uniform, the acoustic impedance image of the cells being observed will change depending on the selection of the reference points, making it necessary to measure multiple reference points and correct the distortion, making automatic and continuous measurements difficult.As such, a configuration in which the film is fused to the bottom of the container by laser beam irradiation has the problem that accurate cell observation using an ultrasonic microscope cannot be performed.

[0014] The present invention has been made to solve the above problems, and its object is to enable a film to be attached to the bottom of a container so that cells can be accurately observed using an ultrasonic microscope.

[0015] The cell culture dish of the present invention comprises a container, an opening formed in the bottom of the container, a stress buffer layer formed around the entire circumferential outer edge of the container at the edge of the opening and made of a resin having greater flexibility than polystyrene, and a film made of polystyrene that covers the opening and is attached to the bottom of the container via the stress buffer layer.

[0016] As described above, according to the present invention, a film made of polystyrene that covers the opening at the bottom of the container is attached via a stress buffer layer made of a resin that is more flexible than polystyrene, so that the film is attached to the bottom of the container so that cells can be accurately observed using an ultrasonic microscope.

[0017] Fig. 1 is a cross-sectional view showing the configuration of a cell culture dish according to an embodiment of the present invention. Fig. 2 is a cross-sectional view showing the configuration of another cell culture dish according to an embodiment of the present invention. Fig. 3A is a distribution diagram showing a two-dimensional map of the reflection intensity of ultrasound from a film of a cell culture dish according to an embodiment. Fig. 3B is a distribution diagram showing a two-dimensional map of the reflection intensity of ultrasound from a film of a conventional cell culture dish. Fig. 4 is an explanatory diagram explaining measurement using an ultrasound image inspection device.

[0018] A cell culture dish according to an embodiment of the present invention will be described below with reference to FIG. 1. The cell culture dish includes a container 101, a stress buffer layer 103, and a film 104. The container 101 is made of, for example, polystyrene and has a cylindrical shape. An opening 102 is formed in the bottom of the container 101. The opening 102 penetrates the bottom of the container 101. The opening 102 has, for example, a circular shape in a plan view.

[0019] The stress buffer layer 103 is formed on the entire outer circumferential surface of the container 101 around the edge of the opening 102 and is made of a resin having greater flexibility than polystyrene. The stress buffer layer 103 may have a planar shape with an opening corresponding to the opening 102, which is circular in plan view. The stress buffer layer 103 may be made of an adhesive resin. The stress buffer layer 103 may be made of polydimethylsiloxane (PDMS), for example. The stress buffer layer 103 may also be made of an adhesive with a fluororesin base material, for example.

[0020] The film 104 covers the opening 102 and is attached to the bottom of the container 101 via a stress buffer layer 103. The film 104 may be made of polystyrene. The film 104 may have a thickness of, for example, about 70 μm.

[0021] The film 104 may have a rectangular outer shape in a planar view. In this case, the stress buffer layer 103 may have an opening corresponding to the opening 102 which is circular in a planar view, and may have a rectangular outer shape corresponding to the film 104 which is rectangular in a planar view. The film 104 may also have a circular outer shape in a planar view. In this case, the stress buffer layer 103 may have an opening corresponding to the opening 102 which is circular in a planar view, and may have a circular outer shape corresponding to the film 104 which is circular in a planar view.

[0022] 2, the cell culture dish can also include a fixing jig 105 that presses and fixes the film 104 against the stress buffer layer 103. The fixing jig 105 is disposed opposite the stress buffer layer 103 and presses the film 104 against the stress buffer layer 103. The fixing jig 105 can also press the film 104 against the stress buffer layer 103 with a sealing material 106 sandwiched therebetween. The sealing material 106 can have the same shape as the stress buffer layer 103 in a plan view.

[0023] Next, we will explain the results of experiments using cell culture dishes that were actually prepared. First, a commercially available "Corning® 35 mm untreated dish, model number 430588" was used as the container. The container used was cylindrical, 10 mm high, with an inner diameter of 33.9 mm. A circular hole with a diameter of 4 mm was drilled in the bottom of this container, and PDMS was used as a stress buffer layer. A 30 mm square polystyrene film was attached to the bottom to cover the hole. The elastic modulus of the polystyrene film used was approximately 3 GPa. There were no restrictions on the thickness or dimensions of the polystyrene film.

[0024] To briefly explain the fabrication method, a pre-crosslinked PDMS polymer solution was applied to the outer surface of the bottom of a container, and a polystyrene film was placed on top of this and attached to the outer surface of the bottom of the container. The applied PDMS polymer solution contained 10 wt% crosslinking agent. The entire container, applied PDMS layer, and polystyrene film were then heated to crosslink and harden the PDMS, forming a stress buffer layer. The modulus of elasticity of the PDMS stress buffer layer fabricated under these conditions was approximately 1.5 MPa. While the conditions for the stress buffer layer are not limited to those used in this study, they must be such that the modulus of elasticity is smaller than that of the polystyrene film to be bonded.

[0025] Next, the results of measuring the strain of the polystyrene film in the cell culture dish prepared as described above will be described. This measurement was performed on the cell culture dish according to the embodiment and a comparative cell culture dish to which the polystyrene film was attached by laser welding. In the measurement, the opening blocked by the polystyrene film was filled with water. The acoustic impedance of the filled water was known. The strain of the polystyrene film was measured using an ultrasonic microscope while the filled water was in contact with the polystyrene film. The measurement results for the cell culture dish according to the embodiment are shown in Figure 3A. The measurement results for the comparative cell culture dish are shown in Figure 3B.

[0026] Compared to Figure 3A, Figure 3B shows that the polystyrene film attached to the bottom of the container has a large strain of approximately 40 μm. As is clear from these results, attaching the film using a stress buffer layer results in a cell culture dish with minimal distortion of the attached film. Furthermore, fusion by laser beam irradiation limits the welded (adhesive) area to the edge of the laser beam, resulting in a small adhesive area, which may result in insufficient adhesive strength during cell culture for several days or longer. In contrast, cell culture dishes with films attached using a stress buffer layer can be used for ultrasound measurements even after approximately one week of cell culture without interruption due to leakage.

[0027] As described above, according to the present invention, a film made of polystyrene that covers the opening at the bottom of the container is attached via a stress buffer layer made of a resin that is more flexible than polystyrene, so that the film can be attached to the bottom of the container so that cells can be accurately observed using an ultrasonic microscope.

[0028] It should be noted that the present invention is not limited to the embodiments described above, and it is clear that many modifications and combinations can be made by a person having ordinary knowledge in the art within the technical concept of the present invention.

[0029] 101...container, 102...opening, 103...stress buffer layer, 104...film.

Claims

1. A cell culture dish comprising: a container; an opening formed in a bottom of the container; a stress buffer layer formed around the edge of the opening in the entire circumferential direction of the outside of the container and made of a resin having greater flexibility than polystyrene; and a film made of polystyrene covering the opening and affixed to the bottom of the container via the stress buffer layer.

2. A cell culture dish according to claim 1, wherein the stress buffer layer is made of an adhesive resin.

3. A cell culture dish according to claim 1, further comprising a fixing jig arranged opposite said stress buffer layer for pressing and fixing said film against said stress buffer layer.

4. A cell culture dish according to any one of claims 1 to 3, wherein the container is made of polystyrene.

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