CN112877211A

CN112877211A - Organoid culture array and method of use thereof

Info

Publication number: CN112877211A
Application number: CN202110230520.1A
Authority: CN
Inventors: 王丽; 魏文博; 冯可; 王南
Original assignee: Suzhou Jiyan Biomedical Technology Co ltd
Current assignee: Suzhou Jiyan Biomedical Technology Co ltd
Priority date: 2021-03-02
Filing date: 2021-03-02
Publication date: 2021-06-01

Abstract

The invention belongs to the field of biomedical engineering, and particularly discloses an organoid culture array and a using method thereof, wherein the organoid culture array consists of three parts, including a sheet structure (100) containing a micropore array, a bottom structure (200) for placing the sheet structure (100) and a cover structure (300); the organoid culture array designed by the invention can meet the requirement of preparing uniform 3D organoids in high flux, is simple and convenient to operate, can perform fluid stimulation on the organoid culture array in modes of swinging and the like to form dynamic culture, thereby promoting the proliferation and differentiation of organoids, and can be applied to research work in the fields of biomedicine, developmental science, new drug research and development, toxicology and the like.

Description

Organoid culture array and method of use thereof

Technical Field

The invention belongs to the field of biomedical engineering, and particularly discloses an organoid culture array and a using method thereof.

Background

In 2009, the Hans Clevers doctrine, the institute for Hubrecht, the netherlands, demonstrated that intestinal stem cells were able to form organoids, opening the era of organoid research. Nature Methods thus evaluate organoid (Organoids) technology: the stem cell is directly induced to generate a three-dimensional tissue model, a powerful method is provided for the biological research of human beings, and the research on the tool is continuously developing and progressing. In recent years, the technology not only makes a breakthrough in the construction of a wider variety of tissues and organs, but also shows application potential as a tissue model in the fields of disease research, drug screening, drug toxicology testing and the like. This time, the technology is determined by Nature Methods to be an annual technology in 2017, the huge development potential of the technology is highlighted again, and the field is expected to cause a wider research hot tide all over the world in the future.

However, the current organoid culture method still relies on the culture of matrigel-plated cells to form three-dimensional organoid micro-tissues by cell self-assembly. The organoids formed by the method cannot form organoids with uniform size, and the secondary separation and selection process after organoid formation is complicated.

Disclosure of Invention

In order to overcome the defects, the invention discloses an organoid array and a using method thereof, wherein the micropore array structure on the sheet structure can realize uniform inoculation, so that organoids with more uniform size can be formed in high flux. In addition, the array structure is also convenient for observation and subsequent organoid selection.

The technical scheme of the invention is as follows:

an organoid culture array, said organoid culture array consisting of three parts, including a sheet structure containing an array of microwells, a base structure on which the sheet structure is placed and a lid structure; the structure comprises a sheet structure and is characterized in that micro through hole structures of an array structure are arranged on the sheet structure, an annular opening is formed in the inner side of the bottom of each micro through hole structure, concave structures are arranged on two sides of the sheet structure respectively, a convex structure is arranged at each of four corners of the bottom of the sheet structure respectively, a plurality of groove structures are further arranged in the sheet structure and are located between two adjacent rows of micro through hole structures, and the cross section of each groove structure is V-shaped. The sheet structure is a main culture device of the organoid, the annular opening of the circular micro-through hole structure is beneficial to the retention of the matrigel containing the organoid, and the matrigel containing the organoid cannot be separated from the sheet structure downwards through the annular opening under the action of tension due to the small diameter of the annular opening and the high viscosity of the matrigel, and the sample adding process can be controlled; when the organoid is cultured in the micro-through hole structure, the organoid can be contacted with culture medium from top to bottom, and the nutrient exchange is promoted. The groove structure can promote liquid flow in the culture medium replacement process, so that bubbles are prevented from being generated; the bottom structure and the cover structure are used for bearing organoids and culture medium, and dynamic culture, such as shake culture, can be realized; compared with the traditional culture dish and culture mode, the organoids cultured in the matrigel have culture mediums at the bottom and the upper part, so that the nutrition supply can be accelerated.

Further, in the above organoid culture array, the sheet structure is a rectangular sheet having a length of 60 to 80 mm, a width of 15 to 30mm, and a thickness of 0.5 to 3 mm.

Further, in the above organoid culture array, the diameter of the micro via structures on the sheet structure is 1.5 to 10 mm, the diameter of the annular opening at the bottom of the micro via structures is 0.1 to 2.5 mm, the thickness is 0.1 to 0.4 mm, and the distance between the micro via structures is 1 to 2 mm.

Furthermore, in the above organoid culture array, the inner wall of the micro-via structure is frosted or has a structure with a plurality of annular protrusions.

Furthermore, in the above organoid culture array, the concave structures on both sides of the sheet structure have a long side of 10-20 mm and a short side of 1-3 mm, the height of the convex structures at the four corners of the bottom of the sheet structure is 0.2-0.5 mm, and the width of the top of the groove structure is 0.1-0.5 mm.

Furthermore, in the above-mentioned organoid culture array, the four corner regions below the bottom structure and above the cover structure are respectively provided with symmetrical foot pad structures, which can play a role in fixing when a plurality of such arrays are stacked; four sides of the bottom surface of the cover structure are respectively provided with a foot pad structure with the height of 0.2-0.5 mm, and the structure can ensure that the bottom structure and the cover structure flow out of gaps to exchange gas when being combined.

Further, in the above organoid culture array, the bottom structure and the lid structure of the sheet placement structure are both rectangular; the length of the bottom structure is 62-85 mm, the width is 16-35 mm, the thickness of the bottom side wall is 0.5-1.5 mm, and the height is 5-10 mm; the cover structure is 63-90 mm long, 17-40 mm wide, 0.5-1.5 mm thick and 5-10 mm high.

Further, in the above organoid culture array, the sheet structure is a circular sheet; existing commercial cell culture well plates and culture dishes can be matched, including and not limited to: 6-well plate, 12-well plate, 24-well plate, 48-well plate, 30mm culture dish, 60mm culture dish, 100mm culture dish.

Further, the above-mentioned organoid culture array, the sheet structure, the bottom structure, and the cover structure are made of hard materials including but not limited to PS, PC, PMMA, by injection molding or machining.

Further, the method for using the above organoid culture array comprises the following steps:

s1, placing the sheet structure in a bottom structure, covering a cover structure and then performing sterilization treatment, wherein the sterilization mode is as follows: ray sterilization or gas sterilization;

s2, preparing the required cell suspension with the cell concentration of 10⁴-10⁶Each per milliliter; the required matrigel ingredients are added to achieve a matrigel concentration of 0.1-10 milligrams per milliliter, where matrigel ingredients include, but are not limited to: matrigel, collagen;

s3, adding the prepared cell and matrix gel suspension into each micro-through hole structure along the annular opening of the micro-through hole structure by using a liquid transfer device; after all the micro-through hole structures are added with the samples, curing the matrigel by using a temperature or ultraviolet irradiation method;

and S4, adding a required cell culture medium into the bottom structure through gaps of the concave structures on the two sides of the sheet structure, enabling the culture medium to sink through the sheet structure, covering the cover structure, and placing the cover structure in an incubator for culture.

According to the technical scheme, the invention has the following beneficial effects

1. The invention mainly aims at the defects that one side of the mixture of the organoid and the matrigel is contacted with the bottom of the culture dish and the exchange of nutrient substances is poor in the traditional culture method, and the micro through hole structure on the organoid culture array disclosed by the invention can realize that the organoid and the matrigel mixture can be contacted with the nutrient substances from top to bottom;

2. the organoid culture arrays disclosed herein can achieve dynamic, e.g., shaking culture;

3. compared with the traditional method, the application of the organoid culture array disclosed by the invention has the advantages that the sample adding and dispensing processes are simple, and the connection between the glue blocks or the adhesion with the side wall of the culture dish is avoided.

4. Compared with the traditional dispensing, the traditional dispensing has thick middle, thin periphery and uneven nutrition supply; the thickness of the rubber block is uniform in the culture of the micro through hole structure, and the thickness of the rubber block is adjustable, so that the growth of organoids is facilitated.

Drawings

FIG. 1 is a schematic view of an organoid culture array according to the present disclosure prior to assembly;

FIG. 2 is a schematic top view of a organoid culture array sheet configuration according to the present disclosure;

FIG. 3 is a schematic cross-sectional view of a micro-via structure in an organoid culture array according to the present invention;

FIG. 4 is a schematic top view of a circular sheet structure of an organoid culture array according to the present disclosure;

FIG. 5 is a pictorial illustration of an organoid culture array for culturing endometrial cancer cells, in accordance with the present invention;

wherein: a sheet structure 100, a micro-via structure 101, a ring opening 102, a recessed structure 103, a raised structure 104, a recessed structure 105, a bottom structure 200, a lid structure 300.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

In the description of the present invention, it is to be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "clockwise", "counterclockwise", and the like, indicate orientations and positional relationships based on those shown in the drawings, and are used only for convenience of description and simplicity of description, and do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, are not to be considered as limiting the present invention.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, unless otherwise specified, "a plurality" means two or more unless explicitly defined otherwise.

In the present invention, unless otherwise expressly specified or limited, the terms "mounted," "connected," "secured," and the like are to be construed broadly and can, for example, be fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.

In the present invention, unless otherwise expressly stated or limited, "above" or "below" a first feature means that the first and second features are in direct contact, or that the first and second features are not in direct contact but are in contact with each other via another feature therebetween. Also, the first feature being "on," "above" and "over" the second feature includes the first feature being directly on and obliquely above the second feature, or merely indicating that the first feature is at a higher level than the second feature. A first feature being "under," "below," and "beneath" a second feature includes the first feature being directly under and obliquely below the second feature, or simply meaning that the first feature is at a lesser elevation than the second feature.

Example 1

1-3, said organoid culture array being comprised of three parts, including a sheet structure 100 containing an array of microwells, a base structure 200 and a lid structure 300 on which the sheet structure 100 is placed; the sheet structure 100 is provided with micro through hole structures 101 of an array structure, the inner side of the bottom of each micro through hole structure 101 is provided with an annular opening 102, two sides of the sheet structure 100 are respectively provided with a concave structure 103, four corners of the bottom of the sheet structure 100 are respectively provided with a convex structure 104, a plurality of groove structures 105 are further arranged in the sheet structure 100, the groove structures 105 are located between two adjacent rows of micro through hole structures 101, and the cross section of each groove structure 105 is V-shaped.

Further, the sheet structure 100 is rectangular;

further, the sheet structure 100 has a length of 74 mm, a width of 24 mm, and a thickness of 1 mm;

further, the diameter of the micro via structures 101 on the sheet structure 100 is 2 mm, the diameter of the annular opening 102 at the bottom of the micro via structure 101 is 0.2 mm, the thickness is 0.2 mm, and the pitch of the micro via structures 101 is 1.5 mm;

further, the inner wall of the micro through hole structure 101 is a frosted structure;

further, the width of the concave structures 103 on both sides of the sheet structure 100 is 10 mm, the length thereof is 2 mm, the height of the convex structures 104 at the four corners of the bottom of the sheet structure 100 is 0.5 mm, and the width of the top of the groove structure 105 is 0.1 mm;

further, the bottom structure 200 and the cover structure 300 for placing the sheet structure 100 are rectangular;

further, the length of the bottom structure 200 is 75 mm, the width is 25 mm, the thickness of the bottom side wall is 1 mm, and the height is 8 mm;

further, the cover structure 300 has a length of 77 mm, a width of 27 mm, a thickness of 1 mm and a height of 9 mm;

furthermore, four corner areas below the bottom structure 200 and above the cover structure 300 are respectively provided with symmetrical foot pad structures, which can play a role in fixing when a plurality of the arrays are stacked;

furthermore, a foot pad structure with a height of 0.3 mm is respectively arranged on four sides of the bottom surface of the cover structure 300, and the structure can enable the bottom structure 200 and the cover structure 300 to flow out of the gap for gas exchange when being combined;

further, the sheet structure 100, the base structure 200 and the lid structure 300 of the organoid culture array are made of a hard material by machining, wherein the hard material is PMMA.

Example 2

Further, the sheet structure 100 is rectangular;

further, the diameter of the micro via structures 101 on the sheet structure 100 is 1.5 mm, the diameter of the annular opening 102 at the bottom of the micro via structure 101 is 0.2 mm, and the distance between the micro via structures 101 with the thickness of 0.2 mm is 1 mm;

further, the width of the concave structures 103 on both sides of the sheet structure 100 is 8 mm, the length thereof is 2 mm, the height of the convex structures 104 at the four corners of the bottom of the sheet structure 100 is 0.5 mm, and the width of the top of the groove structure 105 is 0.1 mm;

the bottom structure 200 and the cover structure 300 for placing the sheet structure 100 are both rectangular;

furthermore, foot pad structures with the height of 0.3 mm are respectively arranged on four sides of the bottom surface of the cover structure 300, and the foot pad structures can enable the bottom structure 200 and the cover structure 300 to flow out of gaps for gas exchange when being combined;

the sheet structure 100, the base structure 200 and the lid structure 300 of the organoid culture array are made of a hard material, which is PS, by injection molding.

Example 3

An organoid culture array as shown in FIGS. 1, 3 and 4, said organoid culture array being comprised of three parts, including a sheet structure 100 containing an array of microwells, a base structure 200 on which the sheet structure 100 is placed, and a lid structure 300; the sheet structure 100 is provided with micro through hole structures 101 of an array structure, the inner side of the bottom of each micro through hole structure 101 is provided with an annular opening 102, two sides of the sheet structure 100 are respectively provided with a concave structure 103, four corners of the bottom of the sheet structure 100 are respectively provided with a convex structure 104, a plurality of groove structures 105 are further arranged in the sheet structure 100, the groove structures 105 are located between two adjacent rows of micro through hole structures 101, and the cross section of each groove structure 105 is V-shaped.

Further, the sheet structure 100 is circular;

further, the sheet structure 100 has a diameter of 15 mm and a thickness of 1 mm;

further, the diameter of the micro via structures 101 on the sheet structure 100 is 1.5 mm, the diameter of the annular opening 102 at the bottom of the micro via structures 101 is 0.2 mm, the thickness is 0.2 mm, and the pitch of the micro via structures 101 is 1 mm;

further, the sheet structure 100 may be placed in a commercial 24-well plate for use;

further, the sheet structure 100 of the organoid culture array is manufactured by injection molding from a hard material, wherein the hard material is PS.

Example 4

Cell culture was performed using the organoid culture array of example 2, comprising the following steps:

s1, placing the sheet structure 100 in a bottom structure 200, covering a cover structure 300, and then performing sterilization treatment, wherein the sterilization mode comprises the following steps: ray sterilization or gas sterilization;

s2, preparing a primary endometrial cancer cell suspension of the patient, wherein the cell concentration is 10⁶Each per milliliter; then the mixture was added to a concentration of 0.1 mg/mmRaised matrigel;

s3, adding the prepared endometrial cancer cells and matrigel suspension into each micro-through hole structure 101 along the annular opening 102 of each micro-through hole structure 101 by using a liquid transfer device; after all the micro through hole structures 101 are filled with the samples, putting the micro through hole structures into an incubator to solidify matrigel;

s4, adding a high-sugar culture medium containing 10% serum into the bottom structure 200 through gaps of the concave structures 103 on the two sides of the sheet structure 100, enabling the culture medium to sink through the sheet structure 100, covering the cover structure 300, and then placing the sheet structure in a 37-degree and 5% carbon dioxide incubator for culture; as shown in figure 5, for the second generation of endometrial cancer cells, the second generation of endometrial cancer cells is cultured for 6 days, the cells are aggregated into lumps, the number of the cells is increased, and the cell passage cycle is 6-7 days under the culture system.

The above are only preferred embodiments of the present invention, and the scope of the present invention should not be limited thereby, and all the equivalent changes and modifications made by the claims and the summary of the invention should be covered by the protection scope of the present patent application.

Claims

1. An organoid culture array, characterized in that said organoid culture array is composed of three parts, including a sheet structure (100) comprising an array of microwells, a base structure (200) on which the sheet structure (100) is placed and a lid structure (300); the novel micro-through hole structure is characterized in that micro-through hole structures (101) of an array structure are arranged on the sheet structure (100), an annular opening (102) is formed in the inner side of the bottom of each micro-through hole structure (101), concave structures (103) are arranged on two sides of the sheet structure (100) respectively, protruding structures (104) are arranged at four corners of the bottom of the sheet structure (100) respectively, a plurality of groove structures (105) are further arranged in the sheet structure (100), the groove structures (105) are located between two adjacent rows of micro-through hole structures (101), and the cross section of each groove structure (105) is V-shaped.

2. An organoid culture array according to claim 1, wherein the sheet structure (100) is a rectangular sheet having a length of 60-80 mm, a width of 15-30 mm and a thickness of 0.5-3 mm.

3. An organoid culture array according to claim 1, wherein the diameter of the microperforation structures (101) on the sheet structure is between 1.5 and 10 mm, the diameter of the annular opening (102) at the bottom of the microperforation structures (101) is between 0.1 and 2.5 mm, the thickness is between 0.1 and 0.4 mm, and the spacing between the microperforation structures (101) is between 1 and 2 mm.

4. An organoid culture array according to claim 1, wherein the inner walls of the microperforation structures (101) are frosted or provided with a plurality of annular protrusions.

5. The organoid culture array of claim 1, wherein the concave structures (103) on both sides of the sheet structure (100) have a long side of 10-20 mm and a short side of 1-3 mm, the convex structures (104) on the four corners of the bottom of the sheet structure (100) have a height of 0.2-0.5 mm, and the top of the groove structure (105) has a width of 0.1-0.5 mm.

6. An organoid culture array according to claim 1, wherein the four corner regions below the base structure (200) and above the lid structure (300) are provided with symmetrical footpads, respectively; four sides of the bottom surface of the cover structure (300) are respectively provided with a foot pad with the height of 0.2-0.5 mm.

7. The organoid culture array of claim 1, wherein the base structure (200) and the lid structure (300) of the sheet structure (100) are rectangular; the length of the bottom structure (200) is 62-85 mm, the width is 16-35 mm, the thickness of the bottom side wall is 0.5-1.5 mm, and the height is 5-10 mm; the cover structure (300) is 63-90 mm long, 17-40 mm wide, 0.5-1.5 mm thick and 5-10 mm high.

8. An organoid culture array according to claim 1, wherein the sheet structure (100) is a circular sheet; existing commercial cell culture well plates and culture dishes can be matched, including and not limited to: 6-well plate, 12-well plate, 24-well plate, 48-well plate, 30mm culture dish, 60mm culture dish, 100mm culture dish.

9. Organoid culture array according to any of claims 1-8, wherein the sheet structure (100), base structure (200), lid structure (300) are made of hard materials including but not limited to PS, PC, PMMA, by injection molding or machining.

10. The method of using the organoid culture array of any of claims 1-8, wherein the method of using comprises the steps of:

s1, placing a sheet structure (100) in a bottom structure (200), covering a cover structure (300), and then performing sterilization treatment, wherein the sterilization mode is as follows: ray sterilization or gas sterilization;

s3, adding the prepared cell and matrix gel suspension into each micro-through hole structure (101) along the annular opening (102) of the micro-through hole structure (101) by using a liquid transfer device; after all the micro-through hole structures (101) are added with the samples, curing the matrigel by using a temperature or ultraviolet irradiation method;

s4, adding the required cell culture medium into the bottom structure (200) through the gaps of the concave structures (103) on the two sides of the sheet structure (100), enabling the culture medium to submerge the sheet structure (100), covering the cover structure (300), and placing the sheet structure in an incubator for culture.