Organ-like inverted culture device

Abstract

The utility model relates to the technical field of organoid culture, and discloses an organoid inverted culture device which comprises a cell culture plate and an inverted culture chamber, the cell culture plate comprises a plate body, and a plurality of rows of counter bores are uniformly formed in the plate body; the inverted small culture chamber comprises supporting frames, a cylinder part, a supporting ring and an adhesion film, the supporting ring is located above the cylinder part, a through hole is formed in the center of the cylinder part, the central aperture of the supporting ring is larger than the diameter of the through hole in the center of the cylinder part, the supporting frames are obliquely arranged, and the bottom end of the supporting ring is connected with the top end of the cylinder part through the multiple supporting frames; an adhesion film is arranged at the bottom end of the cylinder part. The outer diameter of the supporting ring is larger than the inner diameter of the counter bore, and the outer diameter of the cylinder part is smaller than the inner diameter of the counter bore, so that the cylinder part and the supporting frame in the inverted small culture chamber can be placed in the counter bore and supported on the counter bore through the supporting ring. According to the utility model, the number of cells growing in a two-dimensional adherent manner in the organoid forming process can be effectively reduced, and balling and growth of the organoid are facilitated.

Description

Technical Field

[0001] The utility model relates to the technical field of organoid culture, in particular to an organoid inverted culture device. Background Art

[0002] Organoids are three-dimensional miniature organs formed in vitro from cells with the ability to self-renew and self-organize, resembling, to a certain extent, real human tissues and organs. Compared to two-dimensional cell cultures, organoids possess similar cellular and spatial organization to their corresponding organs, sharing all the genetic characteristics of the human body. Therefore, they can be cultured in vitro for long periods of time and simulate the functions of real organs, offering broad application prospects in basic research and clinical diagnosis and treatment. Currently, brain organoids, intestinal organoids, retinal organoids, lung organoids, heart organoids, and various tumor organoids have been successfully established.

[0003] In the past decade of organoid research, a variety of culture methods have been developed to establish practical organoid experimental models. The simplest method is to use a non-stick surface, which prevents cell adhesion by hydrophobic treatment of the culture dish; in addition, there are rotating bioreactor method, hanging drop method, low adhesion cell culture plate method and magnetic levitation method.

[0004] So far, the widely used traditional organoid culture methods are as follows Figure 3 As shown, an appropriate amount of Matrigel / cell suspension is mixed and plated in the center of the bottom well of a cell culture plate. After solidification, sufficient organoid complete medium is added to the well for incubation. Esophageal squamous cell carcinoma organoids grown using this culture method often exhibit two-dimensional cell adhesion during culture, leading to problems with organoid construction. Utility Model Content

[0005] In order to overcome the shortcomings of the existing technology, the purpose of the present invention is to provide an inverted organoid culture device that can significantly reduce the number of adherent cells in organoids and significantly improve the organoid spheroid formation rate.

[0006] The utility model is implemented by the following technical solutions: an organoid inverted culture device, comprising: a cell culture plate and an inverted culture chamber, the cell culture plate comprising a plate body, and a plurality of rows of countersunk holes evenly arranged on the plate body; the inverted culture chamber comprising a support frame, a cylindrical portion, a support ring and an attachment membrane, the support ring being located above the cylindrical portion, a through hole being provided in the center of the cylindrical portion, the central aperture of the support ring being larger than the diameter of the through hole in the center of the cylindrical portion, the support frame being arranged at an angle, the bottom end of the support ring and the top end of the cylindrical portion being connected by a plurality of support frames, and an attachment membrane being provided at the bottom end of the cylindrical portion.

[0007] Furthermore, the outer diameter of the support ring is larger than the inner diameter of the sink hole, and the outer diameter of the cylindrical part is smaller than the inner diameter of the sink hole, so that the cylindrical part and the support frame in the inverted culture chamber can be placed in the sink hole and supported on the sink hole by the support ring.

[0008] Furthermore, the sum of the heights of the cylindrical portion and the support frame is smaller than the height of the countersunk hole, so that there is a certain space between the bottom and outer side of the cylindrical portion and the inner wall of the countersunk hole for storing culture medium.

[0009] Furthermore, the attachment film is a PET film.

[0010] Furthermore, the inverted culture chamber is made of transparent plastic material.

[0011] Furthermore, the cell culture plate is made of medical-grade polystyrene.

[0012] Furthermore, there are three support frames.

[0013] Compared with the prior art, the beneficial effects of the present invention are:

[0014] 1. The present invention relates to an inverted organoid culture device, wherein the inverted culture chamber is made of transparent plastic and can be matched and fitted with a cell culture plate; the attachment film has good transparency and optical clarity, allowing the growth status of the organoid to be clearly observed and recorded through a microscope; and the PET film has a low adsorption rate, which can effectively reduce the loss of small molecule proteins and other compounds.

[0015] 2. The present invention provides an inverted organoid culture device, which cultures organoids through an inverted culture method. The matrix gel is pre-laid on the PET membrane of the chamber, which can isolate the contact between the matrix gel / cell suspension and the PET membrane of the chamber, eliminating the two-dimensional cell adhesion growth caused by gravity during the solidification process of the matrix gel / cell suspension, and can significantly reduce the number of adherent cells in the organoid and significantly improve the organoid spheroidization rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a structural schematic diagram of the cell culture plate in the utility model;

[0017] Figure 2 It is a structural schematic diagram of the inverted culture chamber in the utility model;

[0018] Figure 3 It is a widely used traditional tumor organoid culture method;

[0019] Figure 4 This is a schematic diagram of the utility model when used for tumor organoid culture;

[0020] Figure 5It is a schematic diagram of the inverted culture processing flow of the inverted culture chamber in the utility model.

[0021] In the figure: plate body 11; countersunk hole 12; support frame 21; cylindrical portion 22; support ring 23; attachment film 24. DETAILED DESCRIPTION

[0022] The following describes the implementation of the present invention through specific examples. Those skilled in the art can easily understand the other advantages and functions of the present invention through the content presented in this specification. The present invention can also be implemented or applied in other different specific ways, and the details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the features of the following embodiments can be combined with each other without conflict.

[0023] The purpose of the utility model is to provide an organoid inverted culture device to address the defects of the existing technology.

[0024] Example 1

[0025] This embodiment provides an organoid inverted culture device, comprising: a cell culture plate and an inverted culture chamber,

[0026] Reference Figure 1 As shown, the cell culture plate includes a plate body 11 with several rows of countersunk wells 12 evenly distributed therein. Specifically, in this embodiment, the cell culture plate is made of medical-grade polystyrene and sterilized before use. The distribution and size of each countersunk well 12 facilitate microscopic observation of organoid growth and allows for recording of its growth process.

[0027] Reference Figure 2As shown, the inverted culture chamber includes a support frame 21, a cylindrical portion 22, a support ring 23, and an attachment membrane 24. The support ring 23 is located above the cylindrical portion 22. A through hole is provided in the center of the cylindrical portion 22. The central aperture of the support ring 23 is larger than the diameter of the central through hole of the cylindrical portion 22. The bottom end of the support ring 23 is connected to the top end of the cylindrical portion 22 by multiple support frames 21. The support frames 21 are arranged at an angle, and the attachment membrane 24 is provided at the bottom end of the cylindrical portion 22. The outer diameter of the support ring 23 is larger than the inner diameter of the countersunk hole 12, while the outer diameter of the cylindrical portion 22 is smaller than the inner diameter of the countersunk hole 12. The combined height of the cylindrical portion 22 and the support frame 21 is less than the height of the countersunk hole 12. This allows the inverted culture chamber to be supported on the countersunk hole 12 by the support ring 23. The cylindrical portion 22 and support frame 21 in the inverted culture chamber can be placed within the countersunk hole 12, leaving a certain space between the bottom and outer side of the cylindrical portion 22 and the inner wall of the countersunk hole 12 for storing culture medium. Specifically in this embodiment, the inverted culture chamber is made of transparent plastic material, which is manufactured using 3D printing and can better match and fit with the cell culture plate. The attachment film 24 is a PET film, which has good transparency and optical clarity, and the growth status of the organoid can be clearly observed and recorded through a microscope; in addition, the PET film has the characteristics of low adsorption rate, which can effectively reduce the loss of small molecule proteins and other compounds, and is more suitable for this culture method.

[0028] As an optimized technical solution in this embodiment, the cell culture plate is provided with 24 countersunk wells 12, arranged in four rows of six cells each. The outer diameter of the countersunk wells 12 is 16.28 mm, the inner diameter is 15.67 mm, and the inner height is 18.84 mm. The overall height of the inverted culture chamber is 15.50 mm. The thickness of the support ring 23 is 0.55 mm, and the outer diameter is 18.60 mm. The distance between the three support frames 21 is 15.45 mm. The outer diameter of the cylindrical portion 22 is 9.10 mm, and the diameter of the central through hole is 7.50 mm. The overall diameter of the PET film is 7.50 mm, and the diameter of the pores in the PET film is 0.45 μm. During use, the cylindrical portion 22 and support frame 21 in the inverted culture chamber can be placed within the countersunk wells 12, with a certain space between the bottom and outer side of the cylindrical portion 22 and the inner wall of the countersunk well 12 for storing culture medium.

[0029] Figure 3 The figure shows a traditional culture method for esophageal squamous cell carcinoma organoids. During culture, a certain amount of Matrigel / cell suspension is mixed and evenly plated at the center of the bottom well 12 of a cell culture plate. A sufficient amount of complete organoid culture medium is then added to well 12 for culturing. In traditional organoid culture methods, during the solidification of the Matrigel / cell suspension, some cells settle due to gravity, ultimately growing in a two-dimensional adherent manner. This reduces the organoid spheroid formation rate; on the other hand, adherent cells compete for growth resources during growth, hindering the formation of organoid spheroids.

[0030] Reference Figure 4 、 Figure 5 As shown, the organoid inverted culture device of the present invention is used to culture esophageal squamous cell carcinoma organoids. First, the attachment membrane 24 in the inverted culture chamber is facing upward and placed upside down in a sterile culture dish; then, a pipette is used to suck up 5μl of matrix gel with a pre-cooled pipette tip and vertically dripped on the center of the attachment membrane 24, and the isolation matrix gel is allowed to stand for a certain time to solidify; the matrix gel is pre-spread on the attachment membrane 24 to isolate the contact between the matrix gel / cell suspension and the PET film, eliminating the two-dimensional cell adhesion growth caused by gravity during the solidification process of the matrix gel / cell suspension; then, 70μl of matrix gel is taken with a pre-cooled sterile pipette tip, and it is thoroughly blown and mixed to form a matrix gel / cell suspension. 10μl of matrix gel / cell suspension is taken and dripped on the solidified 5μl isolation matrix gel, and the matrix gel / cell suspension is allowed to stand for a certain time to solidify. Then, add a sufficient amount of preheated organoid complete culture medium into the sink 12, and place the inverted culture chamber as a whole in the sink 12, so that the inverted culture chamber is supported on the upper end of the sink 12 by the support ring 23, and the cylindrical part 22 and the support frame 21 in the inverted culture chamber are placed in the sink 12, and add complete culture medium for culture.

[0031] The inverted organoid culture device of the present invention is used, that is, a certain amount of matrix gel is pre-laid on the PET film of the small chamber for isolation, and then a certain amount of matrix gel / cell suspension is inoculated on the solidified isolation matrix gel, and finally inverted culture is performed by inverting the small chamber. This can significantly reduce the number of adherent cells of the organoid and significantly improve the organoid spheroid formation rate.

[0032] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. An inverted organoid culture device, characterized in that: include: A cell culture plate and an inverted culture chamber, wherein the cell culture plate comprises a plate body (11), and a plurality of rows of countersunk holes (12) are evenly arranged on the plate body (11); the inverted culture chamber comprises a support frame (21), a cylindrical portion (22), a support ring (23) and an attachment membrane (24), wherein the support ring (23) is located above the cylindrical portion (22), a through hole is provided at the center of the cylindrical portion (22), the central aperture of the support ring (23) is larger than the diameter of the through hole at the center of the cylindrical portion (22), the support frame (21) is tilted, the bottom end of the support ring (23) and the top end of the cylindrical portion (22) are connected by a plurality of support frames (21), and the bottom end of the cylindrical portion (22) is provided with an attachment membrane (24).

2. The inverted organoid culture device according to claim 1, characterized in that: The outer diameter of the support ring (23) is larger than the inner diameter of the sink hole (12), and the outer diameter of the cylindrical portion (22) is smaller than the inner diameter of the sink hole (12), so that the cylindrical portion (22) and the support frame (21) in the inverted culture chamber can be placed in the sink hole (12) and supported on the sink hole (12) by the support ring (23).

3. The inverted organoid culture device according to claim 2, characterized in that: The sum of the heights of the cylindrical portion (22) and the support frame (21) is less than the height of the countersunk hole (12), so that a certain space exists between the bottom and outer side of the cylindrical portion (22) and the inner wall of the countersunk hole (12) for storing culture medium.

4. The inverted organoid culture device according to any one of claims 1 to 3, characterized in that: The attachment film (24) is a PET film.

5. The inverted organoid culture device according to claim 1, wherein: The inverted culture chamber is made of transparent plastic material.

6. The inverted organoid culture device according to claim 2, wherein: The cell culture plate is made of medical-grade polystyrene.

7. The inverted organoid culture device according to claim 1, wherein: There are three support frames (21).

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