Tissue culture dish and method for producing same

Abstract

This tissue culture dish comprises: a resin film (2) on which a biological tissue (100) is placed; a joining member (3) which is disposed on the resin film (2) and in which a through-hole (30) for forming a biological tissue (100) storage space is formed; and a dish body (1) which is disposed on the joining member (3). The materials for the resin film (2), the joining member (3) and the dish body (1) are selected in such a manner that the joining member (3) adheres to the resin film (2) by electrostatic attraction and the dish body (1) adheres to the joining member (3) by electrostatic attraction.

Description

Tissue culture dish and method for manufacturing same

[0001] The present invention relates to a tissue culture dish used in an ultrasound image acquisition device that acquires an acoustic impedance image of a measurement target such as biological tissue using ultrasound, and a method for manufacturing the tissue culture dish.

[0002] Observation of biological tissues has traditionally been performed using optical microscopes and electron microscopes. In recent years, ultrasonic microscopes with resolution equivalent to that of optical microscopes have been put to practical use, making it possible to observe biological tissues at the cellular level. When using ultrasonic microscopes, ultrasonic images such as sound velocity images and acoustic impedance images can be obtained from measured acoustic parameters such as tissue sound velocity and acoustic impedance.

[0003] Development of ultrasonic microscopes as diagnostic devices for biological tissues is progressing, and it is now possible to observe biological tissues with a resolution approaching that of optical microscopes. Optical microscopes use a staining method to distinguish between different chemical properties in biological tissues by changing their optical properties using chemicals. On the other hand, ultrasonic microscopes can distinguish between different physical properties, such as sound speed and acoustic impedance, without the use of chemicals or staining, which minimizes the impact on the subject and allows for non-invasive observation of biological tissues.

[0004] An ultrasonic imaging inspection device that displays acoustic impedance images of cells using a pulsed excitation ultrasonic microscope has been proposed (Non-Patent Document 1). The configuration of the ultrasonic imaging inspection device is shown in Figure 9. In this ultrasonic imaging inspection device, a reference member 101 is provided at the peripheral position of biological tissue 100 within the range scanned by ultrasonic waves, and ultrasonic waves are irradiated onto the biological tissue 100 and the reference member 101 via a resin film 2. In Figure 9, 102 denotes an ultrasonic beam irradiated from a transducer 4. In Figure 9, 5 denotes a scanning stage that moves the transducer 4 to change the position to which the ultrasonic beam is irradiated, 6 denotes an acoustic lens that focuses the ultrasonic beam and irradiates it onto the biological tissue 100 to be measured, and 103 denotes water, which is the ultrasonic propagation medium.

[0005] The relationship between the ultrasonic wave S0 irradiated to the reference member 101 at an angle perpendicular to its surface and the reflected wave Sr from the reference member 101 is expressed by the following equation (1): Sr=(Zs-Zr) / (Zs+Zr)S0 (1)

[0006] Zs is the acoustic impedance of the resin film 2, and Zr is the acoustic impedance of the reference member 101. The relationship between the ultrasonic wave S0 irradiated to the biological tissue 100 at an angle perpendicular to its surface and the reflected wave St from the biological tissue 100 satisfies the following equation (2): St = (Zs - Zt) / (Zs + Zt) S0 (2)

[0007] Zt is the acoustic impedance of the biological tissue 100. Therefore, from equations (1) and (2), the acoustic impedance Zt of the biological tissue 100 can be calculated by equation (3).

[0008]

[0009] 9, a two-dimensional acoustic impedance image can be obtained by measuring the acoustic impedance Zt while moving the transducer 4 using the scanning stage 5. The acoustic impedance Zt is a parameter related to the stiffness of the biological tissue 100. The properties of the biological tissue 100 can be observed using the acoustic impedance image.

[0010] In an ultrasound imaging inspection device, in order to calculate the absolute value of the acoustic impedance Zt, it is necessary to place the biological tissue 100 and the reference member 101 on a thin resin film 2 having a thickness of several tens of μm. A polystyrene film is often used as the resin film 2. When observing the biological tissue 100, it is necessary to attach the resin film 2 to a tissue culture dish.

[0011] In the ultrasonic imaging inspection device, due to the principle of converging and irradiating ultrasonic beams, it is desirable that the resin film 2 be flat and without distortion. Furthermore, in the ultrasonic imaging inspection device, due to the principle of calculating the acoustic impedance Zt using the reflected wave from the interface between the resin film 2 and the biological tissue 100, it is desirable that the biological tissue 100 and the resin film 2 are in close contact with each other.

[0012] Tissue culture dishes are often made of polystyrene. Methods for attaching the resin film 2 to the dish include thermal attachment using a laser or the like, chemical attachment using an adhesive or the like, and mechanical attachment using a flange or the like.

[0013] When the resin film 2 is attached to the dish by laser welding, there is a problem that liquid leakage is likely to occur due to damage to the resin film 2. In addition, since it is difficult to keep the resin film 2 evenly attached to the dish during processing, there are problems such as uneven welding causing gaps between the dish and the resin film 2, which leads to liquid leakage, or stress concentration causing damage to the resin film 2 during use, which leads to liquid leakage.

[0014] When the resin film 2 is attached to the dish with an adhesive, uneven application pressure of the adhesive can distort the resin film 2, making it impossible to measure the acoustic impedance correctly.When the resin film 2 is mechanically fixed to the dish, stress generated at the interface between the dish and the resin film 2 can distort the resin film 2, making it impossible to measure the acoustic impedance correctly.

[0015] Regardless of the method of welding, adhesion, or mechanical fixation, polystyrene, due to its characteristics, is only negatively charged, so in order to control the charge, it is necessary to modify the surface of the resin film 2 with an anionic polymer or a cationic polymer.

[0016] Yoshifumi Saijo, et al., “Ultrasound Speed ​​and Impedance Microscopy for in vivo Imaging”, Proceedings of the 29th Annual International Conference of the IEEE EMBS, 2007

[0017] The present invention has been made to solve the above problems, and an object of the present invention is to provide a tissue culture dish with little distortion of the resin film, and a method for producing the same.

[0018] The tissue culture dish of the present invention is composed of a resin film for placing biological tissue thereon, a joining member placed on the resin film and having a first through hole formed therein for forming a space for storing the biological tissue, and a dish body placed on the joining member, and is characterized in that the resin film, the joining member, and the dish body are made of materials selected so that the joining member adheres to the resin film by electrostatic adsorption, and the dish body adheres to the joining member by electrostatic adsorption.

[0019] Furthermore, the method for manufacturing a tissue culture dish of the present invention includes a first step of placing a joining member, which has a through hole formed therein for forming a space for storing the biological tissue, on a resin film for placing the biological tissue, and a second step of placing a dish body on the joining member, wherein the resin film, the joining member, and the dish body are made of materials selected so that the joining member adheres to the resin film by electrostatic adsorption, and the dish body adheres to the joining member by electrostatic adsorption.

[0020] According to the present invention, it is possible to prepare a tissue culture dish in which the resin film is less distorted and the electrostatic charge state of the resin film is appropriately adjusted.

[0021] FIG. 1 is a cross-sectional view of a tissue culture dish according to a first embodiment of the present invention. FIGS. 2A and 2B are perspective views of the tissue culture dish according to the first embodiment of the present invention. FIG. 3 is a diagram showing an example of a triboelectric series. FIGS. 4A to 4C are cross-sectional views illustrating a method for manufacturing the tissue culture dish according to the first embodiment of the present invention. FIG. 5 is an exploded perspective view of a tissue culture dish according to a second embodiment of the present invention. FIG. 6 is an exploded perspective view of a tissue culture dish according to a third embodiment of the present invention. FIG. 7 is an exploded perspective view of a tissue culture dish according to a fourth embodiment of the present invention. FIG. 8 is an exploded perspective view of a tissue culture dish according to a fifth embodiment of the present invention. FIG. 9 is a diagram showing the configuration of a conventional ultrasound image acquisition device.

[0022] [First Example] An example of the present invention will be described below with reference to the drawings. Fig. 1 is a cross-sectional view of a tissue culture dish according to an example of the present invention. The tissue culture dish is composed of a dish body 1, a resin film 2 (substrate) made of, for example, polystyrene, that supports a biological tissue 100 and a reference member 101, and a joining member 3 that bonds the dish body 1 and the resin film 2 together.

[0023] The dish body 1 has, for example, a cylindrical outer shape, which allows for easy handling of the biological tissue 100. Of the two parallel bottom surfaces of the dish body 1, the surface opposite the resin film 2 (top surface) is formed with a recess 10 for receiving the biological tissue 100 and the reference member 101. The material of the dish body 1 is polystyrene, which is commonly used in cell culture.

[0024] The resin film 2 is, for example, a polystyrene film having a disk-like outer shape. The joining member 3 is, for example, a disk-like outer shape, and has through-holes 30 formed between the dish body 1 and the resin film 2 to form a space for storing the biological tissue 100 and the reference member 101. Silicon rubber is used as the material for the joining member 3. Fig. 2A is a perspective view of the tissue culture dish as seen from above, and Fig. 2B is a perspective view of the tissue culture dish as seen from below.

[0025] The joining member 3 is attached to the bottom surface of the dish body 1 by electrostatic adsorption. Similarly, the resin film 2 is attached to the bottom surface of the joining member 3 by electrostatic adsorption. The electrostatic adsorption used in this embodiment will be explained below. Some substances are easily charged positively, while others are easily charged negatively. A diagram showing the order in which substances are easily charged positively and negatively is called a triboelectric series. An example of the triboelectric series is shown in Figure 3.

[0026] When two substances shown in the triboelectric series are brought into contact, the one that tends to be positively charged will become positively charged, and the one that tends to be negatively charged will become negatively charged. The static electricity generated increases the further apart the two substances are on the triboelectric series. The position of a substance on the triboelectric series is determined by its work function. A substance with a large work function tends to be negatively charged, and a substance with a small work function tends to be positively charged.

[0027] When two materials with different work functions are brought into contact, electrons are transferred between the two substances, causing the substance that releases the electrons to become positively charged and the substance that receives the electrons to become negatively charged. This phenomenon is called contact electrification. For example, suppose the resin film 2 is a polystyrene film and the bonding member 3 is silicone rubber. When the resin film 2 and bonding member 3 are brought into contact with each other, the bonding member 3 becomes negatively charged and the resin film 2 becomes positively charged.

[0028] In this way, by utilizing the difference in work function (difference in position on the triboelectric series), two substances can be bonded together by electrostatic adsorption. Furthermore, by adjusting the difference in work function between the resin film 2 and the bonding member 3, the charge on the surface of the resin film 2 can be adjusted. In other words, the charge on the resin film 2 can be adjusted by the magnitude of the work function of the bonding member 3. In this embodiment, silicone rubber, which is more likely to be negatively charged than the resin film 2, is used as the material for the bonding member 3, but a material that is more likely to be positively charged than the resin film 2 may also be used.

[0029] 4A to 4C are cross-sectional views illustrating a method for manufacturing a tissue culture dish. When manufacturing a tissue culture dish, a resin film 2 is placed on an assembly base 7 as shown in FIG. 4A. Next, as shown in FIG. 4B, a joining member 3 is placed on the resin film 2, and a biological tissue 100 and a reference member 101 are placed in the space formed by the through-holes 30 of the joining member 3. The reference member 101 (culture medium) can be water or a culture solution. The joining member 3 adheres to the resin film 2 by electrostatic adsorption.

[0030] Finally, as shown in Fig. 4C, the dish body 1 is placed on the joining member 3. The dish body 1 is attached to the joining member 3 by electrostatic adsorption. By placing the dish body 1 on the joining member 3, the space containing the biological tissue 100 and the reference member 101 can be sealed, thereby preventing the biological tissue 100 and the reference member 101 from drying out.

[0031] Thus, in this embodiment, by simply stacking the bonding member 3 and the dish body 1 on top of the resin film 2 in this order, it is possible to produce a tissue culture dish with little distortion of the resin film 2 and with an appropriately adjusted charged state of the resin film 2. The configuration of the ultrasound imaging inspection device that uses the tissue culture dish is as described in Figure 9.

[0032] When the magnitude of distortion on the surface of the resin film 2 of a tissue culture dish made by the conventional technology and the tissue culture dish of this example was examined, it was found that the tissue culture dish made by the conventional technology had distortion of about 3 μm on the surface of the resin film 2. On the other hand, in the case of the tissue culture dish of this example, the distortion on the surface of the resin film 2 was suppressed to within 1 μm.

[0033] 5 is an exploded perspective view of a tissue culture dish according to a second embodiment of the present invention. Of the two parallel bottom surfaces of the dish body 1 of this embodiment, the surface opposite the resin film 2 (the upper surface) is formed with a recess 10 for receiving the biological tissue 100 and the reference member 101, and a through-hole 11 communicating with the through-hole 30 of the joining member 3 is formed at the bottom of the recess 10. By forming the through-hole 11 in the dish body 1 at a position communicating with the through-hole 30 of the joining member 3, the biological tissue 100 and the reference member 101 can be replaced with other ones via the through-hole 11.

[0034] 6 is an exploded perspective view of a tissue culture dish according to a third embodiment of the present invention. In this embodiment, a plurality of through-holes 11a and 11b communicating with the through-holes 30 of the joining member 3 are formed in the bottom of the recess 10 of the dish body 1. By forming a plurality of through-holes 11a and 11b in this manner, one of the through-holes 11a and 11b can be used as an inlet for feeding the biological tissue 100 and the reference member 101, and the other can be used as an outlet.

[0035] 7 is an exploded perspective view of a tissue culture dish according to a fourth embodiment of the present invention. In this embodiment, a through-hole 31 for forming a space for storing pure water or the like is formed in the joining member 3, in addition to the through-hole 30. The through-hole 31 is formed at a position where the upper opening is covered by the dish body 1 and the lower opening is covered by the resin film 2.

[0036] In this way, by forming a through hole 31 in the bonding member 3 independent of the through hole 30, pure water or the like that serves as a reference for acoustic impedance can be sealed in the space formed by the through hole 31. This allows stable quantification of acoustic impedance even when the biological tissue 100 secretes secretions. The step of pouring pure water is performed after placing the bonding member 3 on the resin film 2 as shown in FIG. 4B , and after pouring the pure water, the dish body 1 can be placed on the bonding member 3.

[0037] In this embodiment, the through hole 31 is applied to the configuration of the third embodiment, but it goes without saying that it may also be applied to the configurations of the first and second embodiments.

[0038] 8 is an exploded perspective view of a tissue culture dish according to a fifth embodiment of the present invention. The tissue culture dish of this embodiment has a pressure adjustment tube 12, one end of which is connected to the through-hole 11 of the dish body 1 and the other end of which is connected to a pump 13. In this embodiment, the pressure adjustment tube 12 and the pump 13 pressure-feed the reference member 101 into the space formed by the through-hole 30 of the joining member 3, thereby adjusting the pressure within the space and controlling the distortion of the resin film 2. A silicone tube or the like can be used as the pressure adjustment tube 12. A syringe pump can be used as the pump 13.

[0039] Some or all of the above embodiments can be described as, but are not limited to, the following supplementary notes.

[0040] (Note 1) The tissue culture dish of the present invention is composed of a resin film for placing biological tissue thereon, a joining member placed on the resin film and having a first through-hole formed therein for forming a space for storing the biological tissue, and a dish body placed on the joining member, and the resin film, the joining member, and the dish body are made of materials selected so that the joining member adheres to the resin film by electrostatic adsorption, and the dish body adheres to the joining member by electrostatic adsorption.

[0041] (Appendix 2) In the tissue culture dish described in Appendix 1, the dish body has a cylindrical outer shape, and of the two parallel bottom surfaces of the dish body, a recess for receiving the biological tissue is formed on the surface opposite the resin film, and a second through-hole communicating with the first through-hole is formed at the bottom of the recess.

[0042] (Supplementary Note 3) In the tissue culture dish according to Supplementary Note 2, a plurality of the second through-holes are formed.

[0043] (Appendix 4) In the tissue culture dish described in Appendix 1, the joining member has a second through-hole formed at a position where the upper opening is covered by the dish body and the lower opening is covered by the resin film, and pure water is sealed in the space formed by the second through-hole.

[0044] (Appendix 5) In the tissue culture dish described in Appendix 1, the dish body has a columnar outer shape, and a recess for receiving the biological tissue is formed on one of the two parallel bottom surfaces of the dish body opposite the resin film, and a second through-hole communicating with the first through-hole is formed at the bottom of the recess, and the dish further includes a pressure adjustment tube having one end connected to the second through-hole.

[0045] (Appendix 6) The method for manufacturing a tissue culture dish of the present invention includes a first step of placing a joining member, which has a through hole formed therein for forming a space for storing the biological tissue, on a resin film for placing the biological tissue, and a second step of placing a dish body on the joining member, wherein the resin film, the joining member, and the dish body are made of materials selected so that the joining member adheres to the resin film by electrostatic adsorption, and the dish body adheres to the joining member by electrostatic adsorption.

[0046] (Appendix 7) In the method for manufacturing a tissue culture dish described in Appendix 6, the first step includes placing the joining member on the resin film and then placing the biological tissue in the space formed by the first through hole.

[0047] (Appendix 8) In the method for manufacturing a tissue culture dish described in Appendix 6, the joining member has a second through-hole formed at a position where the upper opening is covered by the dish body and the lower opening is covered by the resin film, and the first step includes a step of placing the joining member on the resin film and then pouring pure water into the space formed by the second through-hole.

[0048] The present invention can be applied to techniques for generating ultrasound images.

[0049] REFERENCE SIGNS LIST 1...dish body, 2...resin film, 3...joining member, 10...recess, 11, 11a, 11b, 30, 31...through holes, 12...pressure adjusting tube, 13...pump

Claims

1. A tissue culture dish comprising a resin film for placing a biological tissue, a joining member placed on the resin film and having a first through-hole formed therein for forming a space for storing the biological tissue, and a dish body placed on the joining member, wherein the resin film, the joining member, and the dish body are selected such that the joining member is attached to the resin film by electrostatic adsorption and the dish body is attached to the joining member by electrostatic adsorption.

2. The tissue culture dish according to claim 1, wherein the dish body has a columnar outer shape, and a recess for receiving the biological tissue is formed in a surface of two parallel bottom surfaces of the dish body opposite to the resin film, and a second through-hole communicating with the first through-hole is formed at the bottom of the recess.

3. The tissue culture dish according to claim 2, wherein a plurality of the second through-holes are formed.

4. The tissue culture dish according to claim 1, wherein the joining member has a second through-hole formed at a position where the upper opening is covered by the dish body and the lower opening is covered by the resin film, and pure water is enclosed in a space formed by the second through-hole.

5. The tissue culture dish according to claim 1, wherein the dish body has a columnar outer shape, and a recess for receiving the biological tissue is formed in a surface of two parallel bottom surfaces of the dish body opposite to the resin film, and a second through-hole communicating with the first through-hole is formed at the bottom of the recess, and further comprises a pressure adjustment tube having one end connected to the second through-hole.

6. A first step of placing a joining member having a first through-hole formed therein for forming a space for storing the biological tissue on a resin film for placing the biological tissue, and a second step of placing a dish body on the joining member, wherein the resin film, the joining member, and the dish body are selected such that the joining member is attached to the resin film by electrostatic adsorption and the dish body is attached to the joining member by electrostatic adsorption. A method for manufacturing a tissue culture dish, characterized by the above.

7. In the method for manufacturing a tissue culture dish according to claim 6, the first step includes a step of placing the biological tissue in the space formed by the first through-hole after placing the joining member on the resin film. A method for manufacturing a tissue culture dish, characterized by the above.

8. In the method for manufacturing a tissue culture dish according to claim 6, the joining member has a second through-hole formed at a position where the upper opening is covered by the dish body and the lower opening is covered by the resin film. The first step includes a step of placing pure water in the space formed by the second through-hole after placing the joining member on the resin film. A method for manufacturing a tissue culture dish, characterized by the above.

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