CN113308376A

CN113308376A - Clamping type multi-concave cell culture sheet

Info

Publication number: CN113308376A
Application number: CN202110794150.4A
Authority: CN
Inventors: 郭浩翔; 张乐帅; 祖鹤; 夏亚男; 喻欢
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2021-07-14
Filing date: 2021-07-14
Publication date: 2021-08-27

Abstract

The application relates to a clamping type multi-concave cell culture sheet, which comprises a culture sheet body, a plurality of concave surfaces formed by inwards sinking from the surface of the culture sheet body, bulges formed by outwards protruding from the center of the culture sheet body, and convex rings formed upwards from the periphery of the culture sheet body; the clamping type multi-concave cell culture plate is simple in use method, and is beneficial to collecting the obtained cell spheres into a required container, so that the acceptance degree of three-dimensional cell culture is enlarged, and the culture cost of the three-dimensional cell spheres is reduced.

Description

Clamping type multi-concave cell culture sheet

Technical Field

The invention relates to a clamping type multi-concave cell culture sheet.

Background

The conventional cell culture is an adherent two-dimensional culture mode, but because the communication among cells is very little, the cell group cannot have the structure and physiological and biochemical characteristics similar to those in vivo. For example, liver cells lose the hepatic ductuli and function of secreting albumin/urea after several days in adherent culture, or cancer cells lack biomarkers in vivo in two-dimensional culture. In contrast, the three-dimensional culture of the cells can effectively improve and simulate the in vivo environment, for example, the three-dimensional culture of the hepatocytes has the three-dimensional structure and the liver function of a natural liver, and more accurate hepatotoxicity data is obtained; the cancer cells can reproduce some biomarkers and certain metastasis and infiltration functions under three-dimensional culture. Therefore, the importance of three-dimensional cell culture is self evident.

However, the current three-dimensional culture method is too complex and high in economic cost (such as a scaffold method), so that the three-dimensional culture cannot be widely accepted by common researchers; or lack of high throughput features, such as the use of agarose or ultra-low adsorption plates in 96-well plates, where only one cell three-dimensional sphere can be formed in one well; since each cell ball has about 1000 cells, cell ball staining, Western Blot protein detection, qPCR quantification of mRNA transcript levels and methods based on detection kits all require at least 50K more cells to obtain more reliable data.

Therefore, in the process of three-dimensional cell culture promoted by the actual market, due to the complex pretreatment mode, high economic cost, small amount of balls and lack of high-flux characteristics, the multi-concave cell pellet culture plate of the three-dimensional cell pellet which can be used by simple treatment is promoted to appear, but at present, when the cultured cell pellet is collected into a required container, the culture plate is difficult to transfer, and the operation is inconvenient.

Disclosure of Invention

The invention aims to provide a clamping type multi-concave cell culture sheet which is simple in structure and convenient to extract and move.

In order to achieve the purpose, the invention provides the following technical scheme: the utility model provides a can press from both sides formula of getting many concave surface cell culture piece for place in the cell culture apparatus, can press from both sides formula of getting many concave surface cell culture piece includes the culture plate body, a plurality of from the concave surface of the surface of culture plate body inwards sunken formation, the bulge loop of culture plate periphery outwards protruding formation, and from the surface of culture plate body outwards protruding the arch of formation, grip the arch in order to draw can press from both sides formula of getting many concave surface cell culture piece.

Further, the projection is provided at a central position of the surface of the culture sheet body.

Further, the diameter of the bulge is 0.5-2.0mm, and the height is 1.0-3.0 mm.

Furthermore, the periphery of the culture plate body protrudes outwards to form a convex ring, and the convex ring and the surface of the culture plate are surrounded to form a containing space.

Further, the height of the convex ring is 0.2-2.0 mm.

Further, the concave surface is of an inverted quadrangular frustum pyramid structure or an inverted quadrangular pyramid structure, and the cross section of the concave surface is in an inverted trapezoid shape or an inverted triangle shape.

Furthermore, the depth range of the concave surface is 0.2-2.5mm, in the height direction of the clamping type multi-concave surface cell culture sheet, the side length of the upper bottom surface of the concave surface is 0.4-5.0mm, and the side length of the lower bottom surface of the concave surface is 0-2.0 mm.

Further, the concave surface has a concave end, and the shape of the cross section of the concave end is semicircular, arc-shaped, rectangular or conical.

Furthermore, the concave surface further comprises a cylindrical cavity or a conical cavity for increasing the depth of the concave surface, and in the height direction of the clamping type multi-concave cell culture sheet, the tail end of the concave surface is positioned below the cylindrical cavity or the conical cavity and is connected with the cylindrical cavity or the conical cavity.

Furthermore, the clamping type multi-concave cell culture sheet is formed by integral injection molding, and the smoothness of the clamping type multi-concave cell culture sheet is nano-scale.

The invention has the beneficial effects that: the concave surface of the culture sheet is used for culturing three-dimensional cell spheres, the protrusions facilitate the tweezers to clamp the multi-concave cell culture sheet, and the convex rings and the surface of the culture sheet are surrounded to form an accommodating space for assisting cells to gather on the surface of the culture sheet without falling into a gap between the culture sheet and a cell culture tool. The concave surface of the culture sheet provides two design schemes, including a round bottom and a conical bottom, wherein the conical bottom concave surface greatly increases the quantity and the concentration of the concave surface on the basis of meeting the requirement of cell balling, and the condition that the cells fall at the position outside the concave surface and can not be balling is also avoided. And the clamping type multi-concave cell culture sheet can be directly placed in a cell culture tool, a large number of cell balls can be obtained through simple treatment, and the clamping type multi-concave cell culture sheet is suitable for mechanism research with high cell quantity demand. The clamping type multi-concave cell culture sheet can obtain a large number of cell balls, is simple in using method and convenient and fast to clamp, and is beneficial to collecting the obtained cell balls into a required container, so that the acceptance of three-dimensional cell culture is expanded, and the culture cost of the three-dimensional cell balls is reduced.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.

Drawings

FIG. 1 is a schematic structural view of a grippable multi-concave cell culture plate according to a first embodiment of the present invention;

FIG. 2 is a schematic view of the removable multi-concave cell culture sheet shown in FIG. 1 in another orientation;

FIG. 3 is a cross-sectional view of the clip-on multi-concave cell culture sheet shown in FIG. 1;

FIG. 4 is an enlarged view of the configuration of the clip-on multi-concave cell culture sheet shown in FIG. 3;

FIG. 5 is a schematic structural view of a grippable multi-concave cell culture plate according to a second embodiment of the present invention;

FIG. 6 is a cross-sectional view of the grippable multi-concave cell culture plate shown in FIG. 5;

FIG. 7 is an enlarged view of the layout of the grippable multi-concave cell culture plate shown in FIG. 6.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In addition, the technical features involved in the different embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

Referring to fig. 1 to 4, the clip-on cell culture sheet 100 according to the first embodiment of the present invention can be placed in a cell culture tool (not shown). The cell culture tool is any one of a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96-well plate, and a 35mm cell culture dish and a 60mm cell culture dish, and it is needless to say that the cell culture tool may be other in other embodiments, and is not particularly limited herein depending on the actual situation. The diameter of the removable multi-concave cell culture sheet 100 depends on the specifications of the cell culture tool, and when the cell culture tool is a 24-well plate, for example, the diameter of the removable multi-concave cell culture sheet 100 is in the range of 14.5 to 15.5mm, preferably 15.0 mm. Indeed, in other embodiments, the diameter of the removable multi-concave cell culture sheet 100 may be other, and is not limited herein, depending on the actual situation. The thickness of the grippable multi-concave cell culture sheet 100 is not particularly limited, and can be selected according to actual needs.

The clamping type multi-concave cell culture sheet 100 comprises a culture sheet body 1, a plurality of concave surfaces 2 formed by inwards recessing the surface of the culture sheet body 1, and bulges 3 formed by outwards protruding the surface of the culture sheet body 1, wherein the bulges 3 are held to extract the clamping type multi-concave cell culture sheet 100. Note that the projections 3 and the recessed surfaces 2 are formed on the same surface of the sheet body 1. The clamping type multi-concave cell culture sheet 100 can be directly placed in a cell culture tool, a large number of cell balls can be obtained in a single hole of the cell culture tool through simple treatment, 128 or 472 cell balls can be conveniently and quickly generated, the detection requirements of cell ball staining, Western Blot quantification, mRNA transcription level qPCR quantification and the like are met, the operation method is simple, and the using process is convenient and quick.

In this embodiment, the main body 1 of the culture plate is a circular structure with a diameter of 15.0mm and a height of 1.45 mm. A plurality of concave surfaces 2 are evenly distributed on the surface of the culture plate body 1. Indeed, in other embodiments, the plurality of concave surfaces 2 may be unevenly distributed, and the arrangement of the plurality of concave surfaces 2 on the culture sheet body 1 is not limited herein, depending on the actual situation. In this embodiment, the number of the concave surfaces 2 is 128. The number of the concave surfaces 2 is not specifically limited, and it is needless to say that the number of the concave surfaces 2 may be other in other embodiments, and is not specifically limited, depending on the actual situation.

Each concavity 2 has a concave end 21. The cross section of the concave end 21 is semicircular, arc, rectangular or conical or other shapes, which are not limited herein, according to the actual situation. The concave tip 21 is most effective in forming a cell sphere when the cross-section is curved, and in one embodiment, the curve may be semicircular. The diameter of the concave end 21 ranges from 0.4 to 5.0mm and the depth of the concave end 21 ranges from 0.2 to 2.5 mm. Indeed, in other embodiments, the diameter range and the depth range of the concave end 21 may be other, and are not limited specifically herein, as the case may be. The concave surface 2 further comprises a cylindrical cavity 22 or a conical cavity 22 for increasing the depth of the concave surface 2, and the concave end 21 is located below the cylindrical cavity 22 or the conical cavity 22 and connected with the cylindrical cavity 22 or the conical cavity 22 in the height direction of the clamping-type multi-concave cell culture plate 100. The depth range of the cylindrical cavity 22 or the conical cavity 22 is 0-2.0mm, that is, the cylindrical cavity 22 or the conical cavity 22 may be provided or not, which is determined according to the actual requirement. The cylindrical cavity 22 or the conical cavity 22 may be a cavity of other shapes, and is not particularly limited herein, depending on the actual situation.

In this embodiment, protruding 3 sets up the central point on the surface of culture plate body 1 and puts to when conveniently holding this protruding 3 and drawing the culture plate, the culture plate can keep at the ascending balance of horizontal direction, avoids being located the material outflow on culture plate body 1 surface, does benefit to and collects the required container with the cell ball that obtains. Indeed, in other embodiments, the protrusions 3 may be disposed at other positions on the surface of the culture sheet body 1, and are not limited herein. The protrusions 3 may be cylindrical in configuration, with the protrusions 3 having a diameter of 0.5-2.0mm and a height of 1.0-3.0 mm. The shape and size of the protrusion 3 are not limited in particular, and may be other shapes and sizes, such as a rectangular parallelepiped. In a preferred embodiment, the protrusions 3 have a diameter of 1.2mm and a height of 1.5 mm.

The periphery of the culture sheet body 1 protrudes outwards to form a convex ring 4, the convex ring 4 and the surface of the culture sheet body 1 are surrounded to form a containing space, cells can fall into the central area of the culture sheet in an auxiliary manner, and the cells are not easy to fall off from the culture sheet into a gap between a cell culture tool and the culture sheet to cause that part of the cells do not participate in balling. The height of the convex ring 4 is 0.2-2.0mm, and the specific height of the convex ring 4 is not limited in detail and can be selected according to actual needs. In a preferred embodiment, the height of the collar 4 is 0.5 mm.

Referring to fig. 5 to 7, the removable multi-concave cell culture sheet 200 according to the second embodiment of the present invention has a structure substantially the same as the removable multi-concave cell culture sheet 100 according to the first embodiment, except that: the concave surface 2 is of an inverted quadrangular frustum pyramid structure or an inverted quadrangular pyramid structure, and the cross section of the concave surface 2 is of an inverted trapezoid or an inverted triangle. The depth range of the concave surface 2 is 0.2-2.5mm, and in the height direction of the grippable multi-concave cell culture sheet 200, the side length of the upper bottom surface of the concave surface 2 is 0.4-5.0mm, and the side length of the lower bottom surface of the concave surface 2 is 0-2.0 mm. In this embodiment, the number of the concave surfaces 2 is 472, and the 472 concave surfaces 2 are closely arranged adjacent to each other, so that the space on the surface of the culture sheet body 1 is fully utilized, the depth of the concave surface 2 is 0.5mm, the side length of the upper bottom surface of the concave surface 2 is 0.55mm, and the side length of the lower bottom surface of the concave surface 2 is 0.18 mm.

The specific structure of the concave surface 2 is not particularly limited, and may be other structures, which are not listed here.

The clamping type multi-concave cell culture sheet is integrally formed by injection molding, so that the method is fast, simple and convenient, the manufacturing process is simplified, the production cost is reduced, the production scale can be enlarged, the cell culture sheet can be produced in batch, and the traditional cell culture mode is changed. And the injection molding method can conveniently adjust the size of the culture plate, thereby meeting various practical applications.

The smoothness of the clamping type multi-concave cell culture sheet is in a nanometer level, so that the adsorbability of cells on the surface of the clamping type multi-concave cell culture sheet is greatly reduced, the cells can smoothly slide to the concave surface, and the efficiency and the quality of cell balling are improved.

The invention also provides a culture method of the three-dimensional cell balls, which adopts the clamping type multi-concave cell culture sheet and comprises the following steps:

placing the clamping type multi-concave cell culture sheet in a cell culture tool;

adding an ethanol solution into the cell culture tool to perform infiltration and sterilization treatment on the clamping type multi-concave cell culture sheet;

adding cell culture medium to wash off ethanol solution, and repeating the operation for three times;

adding the cell culture medium again to enable the cell culture medium to infiltrate the clamping type multi-concave cell culture sheet, and reserving 0.2-2ml of the cell culture medium;

adding the cell suspension into the cell culture tool, standing for 48h, and allowing the cells to settle in the concave surface under the action of gravity to form a cell ball.

Wherein, adding ethanol solution into the cell culture tool to infiltrate and sterilize the grippable multi-concave cell culture sheet specifically comprises the following steps:

and adding an ethanol solution into the cell culture tool, so that the ethanol solution is over the clamping type multi-concave cell culture sheet, and keeping for 15 minutes, so that the clamping type multi-concave cell culture sheet is infiltrated and sterilized by the ethanol solution. Indeed, in other embodiments, other solutions may be used for the soaking and sterilization effects, which are determined according to the actual situation and are not specifically limited herein.

The concentration range of the ethanol solution is 75-100%, and the volume range of the ethanol solution is 0.2-2 ml. Indeed, in other embodiments, the concentration and volume of ethanol may be other, and are not specifically limited herein, depending on the actual situation. The purpose of using the ethanol solution is: the concave surface can be both sterilized and infiltrated to remove air bubbles that may be present in the concave surface to prevent affecting cell balling.

In conclusion, the concave surface of the culture plate is used for culturing three-dimensional cell spheres, the protrusions facilitate the tweezers to clamp the multi-concave cell culture plate, and the convex ring and the surface of the culture plate are surrounded to form a containing space for assisting cells to gather on the surface of the culture plate without falling into the gap between the culture plate and the cell culture tool. The concave surface of the culture sheet provides two design schemes, including a round bottom and a conical bottom, wherein the conical bottom concave surface greatly increases the quantity and the concentration of the concave surface on the basis of meeting the requirement of cell balling, and the condition that the cells fall at the position outside the concave surface and can not be balling is also avoided. And the clamping type multi-concave cell culture sheet can be directly placed in a cell culture tool, a large number of cell balls can be obtained through simple treatment, and the clamping type multi-concave cell culture sheet is suitable for mechanism research with high cell quantity demand. The clamping type multi-concave cell culture sheet can obtain a large number of cell balls, is simple in using method and convenient and fast to clamp, and is beneficial to collecting the obtained cell balls into a required container, so that the acceptance of three-dimensional cell culture is expanded, and the culture cost of the three-dimensional cell balls is reduced.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. The clamping type multi-concave cell culture sheet is placed in a cell culture tool and is characterized by comprising a culture sheet body, a plurality of concave surfaces formed by inwards recessing the surface of the culture sheet body and bulges formed by outwards protruding the surface of the culture sheet body, and the bulges are held to extract the clamping type multi-concave cell culture sheet.

2. The clampable multi-concave cell culture plate according to claim 1, wherein the projection is provided at a central position of the surface of the plate body.

3. The clampable multi-concave cell culture plate according to claim 1, wherein the protrusions have a diameter of 0.5 to 2.0mm and a height of 1.0 to 3.0 mm.

4. The clampable multi-concave cell culture plate of claim 1, wherein the outer periphery of the plate body is formed with a convex ring protruding outwards, and the convex ring and the surface of the plate are surrounded to form a containing space.

5. The clampable multi-concave cell culture plate according to claim 4, wherein the height of the convex ring is 0.2-2.0 mm.

6. The clampable multi-concave cell culture plate according to claim 1, wherein the concave surface has an inverted quadrangular pyramid structure or an inverted quadrangular pyramid structure, and the cross section of the concave surface has an inverted trapezoid shape or an inverted triangle shape.

7. The grippable multi-concave cell culture plate according to claim 6, wherein the depth of the concave surface is in the range of 0.2 to 2.5mm, and the side length of the upper bottom surface of the concave surface is 0.4 to 5.0mm and the side length of the lower bottom surface of the concave surface is 0 to 2.0mm in the height direction of the grippable multi-concave cell culture plate.

8. The clampable multi-concave cell culture plate according to claim 1, wherein the concave surface has a concave end, and the cross-section of the concave end has a shape of a semicircle, an arc, a rectangle or a cone.

9. The grippable multi-concave cell culture plate of claim 8, wherein the concave surface further comprises a cylindrical cavity or a conical cavity for increasing the depth of the concave surface, and the end of the concave surface is located below and connected with the cylindrical cavity or the conical cavity in the height direction of the grippable multi-concave cell culture plate.

10. The grippable multi-concave cell culture plate according to claim 1, wherein the grippable multi-concave cell culture plate is formed by injection molding, and the smoothness of the grippable multi-concave cell culture plate is nanometer.