WO2007123035A1 - Incubator - Google Patents

Abstract

An incubator in which stress can be uniformly applied to a cell piece. The incubator having a rectangular box-like shape formed of a deformable material comprises a bottom membrane and side walls so installed as to vertically extending from the entire peripheral edge of the bottom membrane. The inner surfaces of the side walls are made porous.

Description

 Specification

 Mouth

 Technical field

 [0001] The present invention relates to an incubator for culturing a sample cell piece while generating stress.

 Background art

 [0002] It is known that when stress is generated in a sample cell piece and this is cultured, a specific change occurs in the cell piece that is cultivated by the stress.

 Conventionally, a method for uniformly applying stress to a sample cell piece has been proposed. For example, according to Patent Document 1, an incubator formed of a deformable material has a rectangular box shape, and includes a bottom membrane and side walls erected from the entire periphery of the bottom membrane, and a pair of opposing side walls. Is formed with an engaging portion on an extension line of the peripheral edge of the bottom membrane. The bottom membrane is provided with a locking portion, and the cell piece is locked to the locking portion to prevent slippage between the bottom membrane and the cell piece when the incubator is extended.

 Patent Document 1: International Publication WO2005 / 087913 A1 Publication

 Disclosure of the invention

 Problems to be solved by the invention

[0003] Even using the above-described conventional incubator, it was possible to uniformly apply stress to the cell pieces.

 However, in the field of cell culture these days, research and development under more precisely controlled conditions is required. Accordingly, the present inventors have been studying an incubator in order to apply a more uniform stress to cell pieces.

 Accordingly, a first object of the present invention is to propose an incubator capable of applying extremely uniform stress to cell pieces.

 Means for solving the problem

[0004] The inventors of the present invention have intensively studied to achieve the above object, and even if a locking portion is provided on the bottom membrane, it is not possible to completely ensure the integrity of the cell piece and the incubator. I realized I couldn't. In particular, the bottom membrane, i.e., the peripheral edge of the cell debris, It was difficult to completely eliminate the bevel.

 Therefore, the present inventors have focused on the side wall of the incubator and realized that the present invention should be locked to the periphery of the cell piece. That is, the first aspect of the present invention is defined as follows.

 An incubator formed of a deformable material,

 A bottom membrane and a side wall standing from the periphery of the bottom membrane,

 An incubator characterized in that the inner surface of the side wall is formed to be porous.

 [0005] According to the incubator configured as described above, since the inner surface of the side wall is formed to be porous, the periphery of the cell piece placed on the bottom membrane enters the pores on the inner surface of the side wall, and enters there Locked. Thereby, when the incubator is extended, the peripheral edge of the cell piece is also extended along with the movement of the side wall thereof, and the peripheral edge of the cell piece does not slide against the bottom membrane of the incubator.

 [0006] In the above, the inner surface of the side wall is preferably formed of a foam material. It is an inexpensive and easy-to-control porous material.

 In the examples, PDMS (Polydimethylsiloxane), which is the same material as the substrate of the incubator (made of silicon elastomer), is used as a powerful foam material in the examples. Any material that has no effect can be selected.

 [0007] It is preferable that the entire circumference of the inner surface of the side wall is porous. However, at least only the inner surfaces of the pair of side walls that are separated from each other when the incubator is extended should be porous. In addition, it is only necessary to make only the portion of the inner surface of the side wall that is in contact with the cell debris porous.

[0008] For example, a rectangular box type incubator is employed. Making the incubator a rectangular box makes it easier to transport and store incubators that are consumables.

 The incubator is made of a deformable material. This is because the sample cell piece is indirectly stressed by extending the incubator.

 A material that does not chemically interfere with the sample cell piece, such as silicone elastomer, is used as the incubator forming material.

[0009] The bottom film has a rectangular shape in plan view, and the entire bottom film is formed to have the same thickness so as to extend uniformly. Is preferred! The bottom membrane is preferably formed of a light-transmitting material so that sample cell fragments can be observed with an optical microscope.

 It is preferable to stand up the side wall from the entire circumference of the bottom film. The side wall is a thick film to give it mechanical rigidity. This can prevent the bottom film from being randomly deformed.

 The bottom film and the side wall are preferably integrated from the standpoint of reducing the number of parts and thus the manufacturing cost, but it does not limit the separation of the bottom film and the side wall.

[0010] Engaging portions are provided on side walls provided at a pair of opposing edges of the rectangular bottom film. This engaging portion may be engaged with the fixed plate and the moving plate of the extension device. Thus, the change in position of the two members whose relative positions can be controlled causes the incubator to be deformed.

 Therefore, it is also possible to provide a side wall projection and engage it with the plate. It is also possible to vary the two plates together.

 [0011] When the bottom membrane is stretched, the peripheral edge in the stretching direction is deformed so that the central portion is depressed.

 When such deformation occurs, the direction of the stress exerted on the sample cell piece on the deformed portion is different from those in other portions. Therefore, it is preferable to form the engaging portion on the extended line of the peripheral edge in the extending direction of the rectangular bottom film. As a result, the force applied to the engaging portion is applied to the peripheral edge in the extending direction of the bottom membrane, and this is reliably pulled. Therefore, the deformation of the peripheral edge is prevented, and uniform stress can be applied to all the sample cell pieces on the bottom membrane.

 There are no particular limitations on the type and method of obtaining the sample cell pieces used in the incubator of this invention. For example, vascular endothelial cells (such as human 'monkey pig' rush 'rat' mouse 'rabbit), smooth muscle cells, cardiomyocytes, skeletal muscle cells, fibroblasts, osteoblasts, chondrocytes, osteoclasts, A nerve cell etc. can be used.

 Brief Description of Drawings

FIG. 1 is a perspective view of an incubator according to an embodiment of the present invention.

 FIG. 2 shows an incubator according to an embodiment of the present invention, (A) is a plan view, (B) is a front view, (C) is a bottom view, and (D) is a right side view.

 FIG. 3 is a cross-sectional view taken along line AA in FIG.

FIG. 4 is a cross-sectional view showing how the incubator of the example is used. FIG. 5 is a photograph showing a PDMS foaming barter.

 [Fig. 6] Fig. 6 is an enlarged photograph of the surface of PDMS foaming barta.

 [FIG. 7] FIG. 7 is a surface electron micrograph of PDMS foaming barta.

 Explanation of symbols

[0013] 21 incubator

 23 Bottom membrane

 25, 26 side wall

 27 Engagement hole

 29 Sample cell debris

 31 Protrusions

 41, 43 Foam

 Example 1

 Hereinafter, the present invention will be described in more detail based on examples.

 FIG. 1 shows an incubator 21 of this example, FIG. 1 is a perspective view thereof, FIG. 2 (A) is a plan view, FIG. 2 (B) is a front view, FIG. 2 (C) is a bottom view, and FIG. D) is a right side view. Note that the left side view is the same as FIG. Fig. 3 is a cross-sectional view taken along line AA in Fig. 2 (A). Figure 4 is a usage diagram.

 The incubator 21 of the embodiment is a box shape molded with a transparent silicone elastomer, and includes a thin bottom film 23 and side walls 25 and 26 erected integrally from the periphery of the bottom film 23. The bottom film 23 has a thickness of about 100 μm to about 200 μm, the side wall 25 has a thickness of about 1 cm, and the side wall 26 has a thickness of about 2 mm. An engaging hole 27 is formed in the side wall 25.

 Fibronectin, collagen, etc. are applied to the surface of the bottom membrane 23 in order to implant cell debris.

 A protrusion 31 is formed as a locking portion from the bottom film 23. The protrusion 31 interferes with the sample cell piece 29 and prevents slippage between the protrusion 31 and the bottom film 23. Accordingly, uniform stress can be applied to the sample cell piece 29. In place of the protrusions 31, irregularities can be formed on the surface of the bottom film 23. The unevenness and protrusion 31 can be omitted.

Porous bodies 41, 41 are laminated on the inner surfaces of the side walls 25, 25, and many The porous bodies 43 and 43 are laminated.

 These porous bodies 43 are formed as follows.

 10% by weight of ultrapure water was added to unpolymerized PDMS (Polydimethylsiloxane) as a foaming agent, and the mixture was stirred for 30 seconds with a stirrer (ARIN, manufactured by THINKY).

 Next, PDMS containing the above water was poured into a hot plate (ASONE, ND-1) heated to 100 ° C. The PDMS was heated to 100 ° C for 15-30 minutes with a thickness of about 0.5-2 mm. As a result, PDMS polymerized while foaming.

 Fig. 5 shows a picture of the PDMS foaming barta removed from the hot plate. Fig. 6 shows an enlarged photograph of the surface of this foamed barta. From Fig. 6, a depression of several millimeters can be confirmed on the surface. Cell debris is locked to the peripheral wall of this depression. Fig. 7 shows a micrograph of the surface. From Fig. 7, it can be seen that the holes in the foamed butter are in communication, and that each hole has a smaller diameter as it goes into the inside where the diameter is larger on the surface side.

[0017] The PDMS foaming barter thus obtained is cut into a predetermined shape to form porous bodies 41, 43.

 The porous bodies 41 and 43 are bonded to the inner surfaces of the side walls 25 and 26 using a general-purpose adhesive.

FIG. 4 shows an extension device 50 according to the embodiment. In the extension device 50, the fixed plate 51 is fixed to the rail plate 56, and the movable plate 53 is slidably mounted on the rail plate 56. Pins 52 and 54 project from the fixed plate 51 and the movable plate 53, respectively, and are inserted into the engagement holes 27 of the incubator 21.

 The movable plate 53 moves through the rod in the direction of the arrow as the step motor 55 rotates. Reference numeral 57 denotes a control device that controls the rotation of the step motor 55. In the embodiment, a computer device is used.

 When the step motor 55 is rotated and the movable plate 53 is moved away from the fixed plate 51, the force is transmitted to the side wall 25 of the incubator 21 via the pin 54. As a result, the bottom membrane 23 is stretched.

At this time, as shown in FIG. 3A, the engagement hole 27 is located on the extended line of the peripheral edges 24, 24 of the bottom film 23. More preferably, as shown in the drawing, the extension line coincides with the outer edge portion of the engagement hole 27. As a result, the force from pin 32 to pin 34 is more directly directed to the peripheral edge 24 of the bottom membrane 23. It will take. Therefore, the depression of the peripheral edge 24 is prevented, and the bottom film 23 is uniformly extended. Therefore, the stress applied to the sample cell piece 29 on the bottom film 23 becomes uniform.

Furthermore, since the porous bodies 41 and 43 are attached to the inner surfaces of the peripheral walls 25 and 26, the periphery of the cell piece 29 is locked to the porous bodies 41 and 43. Thus, when the incubator 21 is extended by the extension device 50, the cell pieces 29 are also extended along the porous bodies 41 and 43 and do not slide against the bottom membrane 23 of the incubator 21. In this embodiment, since the protrusion 31 is formed on the bottom film 23, the slip of the cell piece 29 with respect to the bottom film 23 is more reliably prevented.

 In this embodiment, the rectangular side wall incubator has a porous side wall, but the shape of the incubator is not particularly limited. For example, a side wall may be provided on a culture membrane that is deformed by suction, and the inner surface thereof may be made porous.

The present invention is not limited to the description of the embodiment and examples of the invention described above. Various modifications are also included in the present invention as long as those skilled in the art can easily conceive without departing from the scope of the claims.

Claims

The scope of the claims  [1] An incubator formed of a deformable material,  A bottom membrane and a side wall standing from the periphery of the bottom membrane,  An incubator characterized in that the inner surface of the side wall is formed to be porous.  [2] The incubator according to claim 1, wherein the inner surface of the side wall is made of a foam material. [3] The incubator according to claim 2, wherein the bubbles of the foamed material are open cells whose diameter force decreases as the diameter increases toward the inside on the surface side. [4] The foam material according to claim 2, wherein the foam material comprises PDMS using water as a foaming agent. The incubator according to 3.  [5] The incubator according to any one of [1] to [4], wherein the bottom membrane is formed with a locking portion for locking to a sample cell piece.