Organ-like culture pore plate convenient for statistics and positioning

Abstract

The utility model discloses an organoid culture pore plate convenient to count and position, which belongs to the technical field of cell culture and comprises a culture pore plate, a plurality of pore grooves are arranged on the culture pore plate, scale marks are arranged at the bottoms of the pore grooves, and the scale marks comprise a plurality of transverse lines and a plurality of longitudinal lines which are uniformly distributed outwards by taking the circle centers of the pore grooves as centers. According to the utility model, orientation coordinates in the pore plate are arranged, so that the dynamic growth process of the organoid can be conveniently positioned and observed, regular counting of each organoid in each pore is facilitated, the budding number is calculated, and the accuracy of statistical analysis is improved; by setting the grids, the organoid can be located in a grid with a fixed area, so that the area can be conveniently calculated without being limited by different shooting machines or software, the efficiency of experimental data analysis is improved, and calculation or statistical errors are reduced; the same type of organs are continuously observed through scale line positioning, the efficiency of organoid culture experiment operation and data analysis and the data reliability are improved, and the device has very high practicability and wide application prospects.

Description

TECHNICAL FIELD

[0001] The utility model relates to cell culture technical field especially, it relates to a kind of organoid culture well plate of being convenient for statistics and positioning. BACKGROUND

[0002] In vitro cell culture is an important research tool for simulating human development and disease. The widely used traditional monolayer cell culture model lacks tissue structure and cannot accurately reproduce the in vivo biological process. The discovery of organoid technology completely expands the possibility of in vitro culture. Organoids are complex three-dimensional structures that are similar in structure and function to in vivo organs, developed from stem cells or organ-specific progenitor cells through self-organization, and reproduce the cell heterogeneity, structure and function of the original tissue.

[0003] Compared with traditional cell experiments, organoids can better simulate in vivo environment and are suitable for molecular and cellular biology analysis, providing a better solution for tumor research, drug screening, regenerative medicine and other fields between animal and cell levels, and have been widely used in stem cell repair and regeneration, disease model establishment, drug screening, anti-inflammatory test, clinical transformation research and other aspects of research, and have great application prospect in basic research and transformation application. This organoid technology enables researchers to reproduce human organ physiological and disease states in culture dishes, bringing a new round of revolution to research in various fields.

[0004] Currently, the main organoid culture methods are as follows:

[0005] 1. Matrigel culture method: cells are suspended in Matrigel, which provides an extracellular matrix-like environment, forming a three-dimensional structure suitable for the culture of various organoids such as intestinal, liver and pancreatic organoids.

[0006] 2. Air-liquid interface culture method: cells are cultured on a semi-permeable membrane, one side is in contact with air and the other side is in contact with culture medium, suitable for epithelial tissue organoids.

[0007] 3. Micro-well culture method: special micro-well plates are used, cells aggregate in micro-wells to form organoids, suitable for high-throughput screening.

[0008] Among them, Matrigel culture method is the most widely used. Matrigel culture method usually uses standard multi-well plates (such as 24-well plates, 96-well plates, etc.) as culture containers, and records the growth state of organoids by microscope observation and photography. Taking intestinal organoid culture as an example, the following indicators can be detected by culturing in well plates:

[0009] 1. Morphological evaluation of organoids: the growth state, overall structure and distribution of different types of cells of organoids can be observed by microscope;

[0010] 2. Organoid formation and growth: Organoid number (count the number of organoids formed in each well or per unit area to evaluate the efficiency of culture or the overall impact of drug intervention on organoids): Organoid sprouting number and secondary organoid number (evaluate organoid sprouting potential, stem cell self-renewal potential), and organoid size (measure the area of organoids to evaluate the growth status of organoids, sprouting potential);

[0011] 3. Changes in organoid activity: Use live cell staining (such as Calcein AM / PI) to evaluate the survival of organoids after various interventions; or use JC-1 probe to detect changes in mitochondrial function, DCFH-DA probe to detect reactive oxygen species production, Mitotracker to detect mitochondrial number, etc.

[0012] 4. Fluorescent staining and immunohistochemistry: Fluorescent staining or immunohistochemical staining of organoids to detect the expression of specific proteins or markers to evaluate the specific functions and differentiation status of organoids.

[0013] 5. Molecular biology detection of organoids: Evaluate the number of organoids, collect organoids and add TRIzol to extract RNA or RIPA to extract protein according to the proportion of organoid number in each experimental group for molecular level detection.

[0014] However, due to the large number of organoids and their dynamic growth and significant morphological changes, a series of problems are often encountered during experiments:

[0015] (1) We often have difficulty observing the growth and morphological changes of a specific organoid or multiple organoids during the entire culture period, and can only observe the overall sprouting changes in a well. The evaluation of dynamic changes of organoids is limited, and it is difficult to fix the field of view when taking pictures, which has significant limitations on evaluating the impact of drug intervention;

[0016] (2) When observing and counting the number of organoid sprouts under a microscope, it is easy to have repeated fields of view when taking multiple high-power field pictures, which has a negative impact on experimental analysis and data processing, and even may cause serious academic problems of repeated use of pictures;

[0017] (3) When observing and analyzing the number of organoids and sprouts under a microscope, repeated counting, missing or incorrect recording may occur, affecting the authenticity and accuracy of experimental data;

[0018] (4) When the area and other indicators of the light microscope picture of the organoids are measured, due to the difference in the instruments, software and measurement methods used by different laboratories, the difficulty of measurement is different, and the conversion process of the relative area and the actual area is complex, so a simple and unified measurement method is urgently needed.

[0019] The existing organoid culture system is unstable, the culture conditions and methods of the organoids have not been completely standardized and optimized, and the culture results may differ between different laboratories; the composition of the Matrigel is complex, and there may be differences between batches, etc., which result in low repeatability and comparability of the results. Therefore, the same organoid needs to be continuously observed.

[0020] The existing 24-well plate can grow 50-100 organoids in each well at the same time, and sometimes the shapes are similar and the positions are close, so it is difficult to continuously observe and take pictures of the same organoid. At the same time, due to the scattered and irregular distribution of the organoids and the different shapes, counting is time-consuming and prone to errors. When using a 96-well plate for culture, due to the small well area, it is often difficult to form a complete dome with Matrigel, and the number of organoids that can grow smoothly in the same well is too small, and the difference between the wells is too large. Practical new type content

[0021] The purpose of the present application is to provide a kind of organoid culture well plate for easy statistics and positioning, to solve the problem of difficult positioning and inconvenient continuous observation in organoid culture.

[0022] To solve the above technical problems, the present application adopts the following technical solutions:

[0023] The present application provides a kind of organoid culture well plate for easy statistics and positioning, which comprises a culture well plate, a plurality of hole grooves are uniformly arranged on the culture well plate, a scale line is arranged on the bottom of the hole groove, the scale line comprises a plurality of horizontal lines and vertical lines, and the plurality of horizontal lines and the plurality of vertical lines are uniformly distributed outward with the center of the hole groove as the center.

[0024] Further, the horizontal line and the vertical line are arranged as a plurality of lines, the plurality of horizontal lines are sequentially marked, and the plurality of vertical lines are sequentially marked.

[0025] Further, a groove is arranged at the center position of the bottom of the hole groove.

[0026] Further, a groove is arranged at the center position of the bottom of the hole groove.

[0027] Further, a groove is arranged at the center position of the bottom of the hole groove.

[0028] Further, the culture well plate and the well plate cover are connected together through a locking assembly.

[0029] Further, the number of the locking assemblies is two, and the two locking assemblies are arranged on two sides of the culture well plate.

[0030] Further, the locking assembly comprises a locking seat arranged on the side of the culture well plate, the top of the locking seat is provided with a locking groove, the edge of the locking groove is provided with a boss, the side of the well cover is provided with a connecting rod, the lower portion of the connecting rod is provided with a positioning rod, the bottom surface of the positioning rod is provided with a positioning block, the outer side of the positioning rod is provided with a limiting sleeve, the positioning block moves to the lower portion of the boss, the limiting sleeve is in contact with the top surface of the boss, and the limiting pin extends into the insertion hole of the positioning rod after penetrating through the limiting sleeve.

[0031] Compared with the prior art, the beneficial technical effects of the utility model are:

[0032] The utility model discloses the bottom or side of hole groove increases the scale line, utilizes the scale line and divides the grid of hole groove bottom, sets up the inside azimuth coordinate of hole plate and is convenient for positioning observation organoid dynamic growth process, solves the positioning difficult problem of regular observation in the organoid culture process, sets up the inside grid positioning of hole plate, is convenient for regular counting of each organoid in each hole, calculates the number of nodes, improves the precision of statistical analysis, solves the tedious problem of organoid growth state evaluation process, through setting up the grid, organoid can be in a fixed area of the square, and it is convenient for calculating area and is not subject to different shooting machines or software, improves the efficiency of experimental data analysis, reduces the calculation or statistical error, solves the difficult problem of organoid area measurement. The utility model makes up for the deficiency of prior art through the design of the scale line, and the same organoid is continuously observed through the scale line positioning, which can significantly improve the efficiency and data reliability of organoid culture experiment operation, data analysis, has very strong practicality and wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0033] The utility model will be further described in connection with the drawings.

[0034] Figure 1 It is the front view of the organoid culture well plate convenient for statistics and positioning of the utility model;

[0035] Figure 2 It is the top view of the organoid culture well plate convenient for statistics and positioning of the utility model;

[0036] Figure 3 It is the top view of the culture well plate of the utility model;

[0037] Figure 4 It is the enlarged view of the scale line of the utility model;

[0038] Figure 5It is the sectional view of the hole and groove of the utility model;

[0039] Figure 6 It is the three-dimensional structure schematic diagram of the locking assembly of the utility model;

[0040] Figure 7 It is the three-dimensional structure schematic diagram of the positioning rod, positioning block, limiting sleeve and limiting nail of the utility model.

[0041] Mark explanation: 1, culture hole plate;1-1, hole groove;1-1-1, groove;2, scale;2-1, horizontal line;2-2, vertical line;3, hole plate cover;3-1, protrusion;4, locking assembly;4-1, locking seat;4-1-1, locking groove;4-1-2, boss;4-2, connecting rod;4-3, positioning rod;4-3-1, positioning block;4-4, limiting sleeve;4-5, limiting nail;5, matrix glue. Specific implementation

[0042] As Figures 1-7 The utility model discloses a kind of organoid culture hole plate convenient for statistics and positioning, including culture hole plate 1, the culture hole plate 1 is uniformly provided with 24 hole grooves 1-1, the bottom of the hole groove 1-1 is provided with scale 2, the scale 2 includes several horizontal lines 2-1 and vertical lines 2-2, with every hole groove 1-1 bottom center as center, make equidistant 9 horizontal lines 2-1 and 9 vertical lines 2-2 as scale line, 9 horizontal lines 2-1 and 9 vertical lines 2-2 divide 8x8 lattice, transversely mark 1, 2, 3 … 7, 8 in turn, vertically mark A, B, C, D … G, H in turn, horizontal line 2-1 and vertical line 2-2 form square grid, two sides of square are parallel to upper and lower edges of hole plate respectively.Standard 24-hole plate hole diameter is usually 1.56cm, so design each square small grid length is 1mm.

[0043] The processing method of scale 2 is as follows:

[0044] First, before drawing line, glass surface is polished first, to ensure that surface is flat and smooth, to avoid affecting microscope observation.

[0045] Then, using photoetching technology processes, specifically including: gluing: a layer of photoresist (photosensitive material) is coated on the glass surface;Exposure: the designed grid pattern is projected onto the photoresist through a mask using a photoetching machine, and ultraviolet radiation causes the photoresist to undergo a chemical reaction;Developing: after exposure, the glass is immersed in a developing solution, and the unexposed photoresist is dissolved, leaving the grid pattern;Etching: after developing, the glass is placed in an etching solution, which corrodes the glass surface not protected by the photoresist, forming grooves;Stripping: remove the remaining photoresist to expose the glass surface with grid lines engraved.

[0046] Finally, a coating process is performed: in order to enhance the visibility of the grid lines, a layer of metal film (such as chromium or silver) is usually plated on the engraved lines to make them clearer under a microscope.

[0047] As shown in Figure 5 , the bottom center of the hole groove 1-1 is provided with a groove 1-1-1, the edge of the groove 1-1-1 is aligned with the scale line, so that the matrix glue is fixed in a certain range, that is, to ensure that the matrix glue dome is located in the middle of the hole groove 1-1, to ensure that the matrix glue falls within the complete small square grid, and to facilitate counting and area statistics by using the scale line 2.

[0048] The upper side of the culture hole plate 1 is provided with a hole plate cover 3; the top surface of the hole plate cover 3 is provided with a protrusion 3-1, which is arranged to avoid wear of the surface of the hole plate cover 3 during use.

[0049] The culture hole plate 1 and the hole plate cover 3 are connected together through the locking assembly 4 to prevent the hole plate cover 3 from sliding off the culture hole plate 1 during movement, and the hole plate cover 3 plays a role in sealing the culture hole plate 1 to ensure a closed environment for the organ sphere culture process and prevent external interference. The number of the locking assembly 4 is two, and the two locking assemblies 4 are arranged on the two sides of the culture hole plate 1. Figures 6-7 As shown in Figures 6-7 , the locking assembly 4 includes a locking seat 4-1, which is connected to the side of the culture hole plate 1, and the top of the locking seat 4-1 is provided with a locking groove 4-1-1, and the edge of the locking groove 4-1-1 is provided with a boss 4-1-2. The longitudinal section of the locking groove 4-1-1 is T-shaped, and the top surface of the locking groove 4-1-1 is an arc-shaped opening and a rectangular opening connected alternately. The left and right corresponding two bosses 4-1-2 form a rectangular opening, and the adjacent two rectangular openings are arc-shaped openings. The side of the hole plate cover 3 is provided with a connecting rod 4-2, the lower side of the connecting rod 4-2 is connected with a positioning rod 4-3, the bottom surface of the positioning rod 4-3 is connected with a positioning block 4-3-1, and the outer side of the positioning rod 4-3 is sleeved with a limiting sleeve 4-4. The size of the positioning block 4-3-1 is smaller than the arc-shaped opening and larger than the rectangular opening, so that the positioning block 4-3-1 can be inserted into the locking groove 4-1-1 from the arc-shaped opening, and then the positioning block 4-3-1 moves to the rectangular opening, so that the positioning block 4-3-1 is located below the boss 4-1-2. The boss 4-1-2 limits the positioning block 4-3-1 to prevent the positioning block 4-3-1 from moving upward. Then, the limiting sleeve 4-4 moves downward to contact the top surface of the boss 4-1-2, and the limiting pin 4-5 extends into the insertion hole of the positioning rod 4-3 after penetrating the limiting sleeve 4-4, so as to lock the position of the positioning rod 4-3.

[0050] The utility model discloses an improved design of traditional organoid culture well plate (such as 24 well plate), scale line 2 is added to the bottom or side of hole groove 1-1, the bottom of hole groove 1-1 is divided into grid by scale line 2, grid is used as reference coordinate, which is convenient for positioning and observing organoid dynamic growth process, convenient for regularly counting each organoid in each well, calculating the number of buds, improving the accuracy of statistical analysis, through the setting grid, organoid can be in a fixed area of square, which is convenient for calculating area and is not subject to different shooting machines or software, improving the efficiency of experimental data analysis, reducing calculation or statistical error.

[0051] The above-described embodiments are merely preferred modes of the utility model, and do not limit the scope of the utility model, and various modifications and improvements to the technical scheme of the utility model made by those skilled in the art without departing from the design spirit of the utility model shall fall within the protection scope defined by the claims of the utility model.

Claims

1. An organoid culture plate that facilitates statistical analysis and localization, characterized in that: The system includes a culture plate (1), on which a plurality of wells (1-1) are uniformly arranged. The bottom of the wells (1-1) is provided with scale lines (2). The scale lines (2) include a plurality of horizontal lines (2-1) and vertical lines (2-2). The plurality of horizontal lines (2-1) and the plurality of vertical lines (2-2) are uniformly distributed outward from the center of the wells (1-1).

2. The organoid culture plate for easy statistical analysis and localization according to claim 1, characterized in that: Both the horizontal line (2-1) and the vertical line (2-2) are set to a number of lines, and the number of horizontal lines (2-1) are marked in sequence, and the number of vertical lines (2-2) are marked in sequence.

3. The organoid culture plate for easy statistical analysis and localization according to claim 1, characterized in that: A groove (1-1-1) is provided at the bottom center of the hole (1-1).

4. The organoid culture plate for easy statistical analysis and localization according to claim 1, characterized in that: A well cover (3) is provided above the culture plate (1).

5. The organoid culture plate for easy statistical analysis and localization according to claim 4, characterized in that: The top surface of the orifice cover (3) is provided with a protrusion (3-1).

6. The organoid culture plate for easy statistical analysis and localization according to claim 4, characterized in that: The culture plate (1) and the plate cover (3) are connected together by a locking assembly (4).

7. The organoid culture plate for easy statistical analysis and localization according to claim 6, characterized in that: The number of locking components (4) is set to two, and the two locking components (4) are arranged on both sides of the culture well plate (1).

8. The organoid culture plate for easy statistical analysis and localization according to claim 7, characterized in that: The locking assembly (4) includes a locking seat (4-1) disposed on the side of the culture well plate (1). The top of the locking seat (4-1) is provided with a locking groove (4-1-1), and the edge of the locking groove (4-1-1) is provided with a boss (4-1-2). The side of the well plate cover (3) is provided with a connecting rod (4-2), and a positioning rod (4-3) is provided below the connecting rod (4-2). The bottom surface of the positioning rod (4-3) is provided with a positioning block (4-3-1), and the outer side of the positioning rod (4-3) is provided with a limiting sleeve (4-4). The positioning block (4-3-1) moves to the bottom of the boss (4-1-2), and the limiting sleeve (4-4) contacts the top surface of the boss (4-1-2). The limiting pin (4-5) passes through the limiting sleeve (4-4) and extends into the insertion hole of the positioning rod (4-3).

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