CN117734075A

CN117734075A - Manufacturing method of 3D cell culture plate and 3D cell culture plate

Info

Publication number: CN117734075A
Application number: CN202311787031.1A
Authority: CN
Inventors: 朱海林; 沈昊炜; 陈慧敏
Original assignee: Huaxia Siyin Shanghai Biotechnology Co ltd
Current assignee: Huaxia Siyin Shanghai Biotechnology Co ltd
Priority date: 2023-12-22
Filing date: 2023-12-22
Publication date: 2024-03-22

Abstract

The invention discloses a manufacturing method of a 3D cell culture plate and the 3D cell culture plate, which comprises the following steps: s1, respectively preparing 10% of methacrylic acylated gelatin and 1.6% of polyethylene oxide solution for preparing a bracket, mixing the solutions according to a ratio of 1:1, and swirling for 5-20S; s2, taking a cell culture plate, placing a limiting ring of a casting mold into a culture hole of the cell culture plate, attaching the limiting ring to the bottom of the culture hole, attaching the peripheral side of the limiting ring to the wall of the culture hole, arranging the top of the limiting ring in a flush manner, arranging the inner wall of a containing cavity of the limiting ring for containing a bracket in a flush manner, and casting a solution into the containing cavity according to a preset value of the top height of the limiting ring in a flush manner; s3, irradiating the solution in the accommodating cavity to form a bracket, shaping and solidifying, ultrasonically cleaning the solidified bracket, taking out the limiting ring, then putting the cell culture plate with the bracket into a freeze dryer for freeze drying, and sterilizing the cell culture plate after freeze drying is finished.

Description

Manufacturing method of 3D cell culture plate and 3D cell culture plate

Technical Field

The invention relates to the technical field of cell, tissue and organoid culture, in particular to a manufacturing method of a 3D cell culture plate and the 3D cell culture plate.

Background

In the existing in vitro cell, tissue and organoid culture techniques, the culture modes are divided into suspension culture and adherent culture, the latter being divided into 2D (planar) and 3D (stereoscopic) culture. 2D adherent culture is a common technique for cell expansion, but in 2D culture, it is difficult for cells to simulate in vivo 3D environment, the interaction between cells and matrix is limited, and at the same time, cells receive surface tension action, which can lead to differentiation of cells. To overcome these drawbacks, researchers have begun to employ 3D culture methods that more closely approximate in vivo environments. Unlike conventional 2D cell culture, 3D cell culture reproduces the in vivo environment of cells, and the multi-layered three-dimensional structure can form gradients of oxygen, nutrients, metabolites, and soluble signals, thereby forming diverse cell populations, tissues, or organs.

Currently, in 3D cell culture techniques, two methods, i.e., stentless and stented culture, are generally employed. The bracket-free 3D culture method mainly enables cells to self-aggregate to form spheres, but the self-aggregated spheres have different sizes, uneven spatial distribution and larger cell sphere difference among all holes, so that the stability of high-throughput screening is poor. Whereas 3D culture with scaffolds mimics cell-to-cell interactions and cell-to-extracellular matrix interactions in the in vivo environment well, while allowing cell aggregation, proliferation, differentiation and migration on the scaffolds.

However, whether or not the 3D culture plate includes a bracket, after the liquid culture medium is added, the liquid near the wall surface can rise due to capillary phenomenon, i.e. adhesion of the surface of the wall to the liquid, so as to form a concave liquid surface (concave meniscus), and an included angle formed between the liquid surface far from the wall and the wall is smaller than 90 degrees. Thus allowing more cells and culture medium to adhere to the walls of the wells, resulting in a significant reduction in the number of cells in the center of the wells and aggregation at the edges of the wells. In addition, due to the uneven liquid surface, imaging is blurred under microscopic observation, particularly for 96-well plates and 384-well plates for high-throughput screening, the smaller the well, the more serious the meniscus, and the more blurred the imaging.

Aiming at the problem of concave liquid level, in the prior art, the formation of concave liquid level is reduced by coating the surface of a hole wall to change the contact angle, or the design of a hole plate is increased on the basis of surface coating, a hydrophobic layer is coated on the upper half part of the hole, and the wall of the upper half part of the hole is designed into a saw-tooth shape, so that the concave liquid level formed by capillary phenomenon is further reduced, but under the condition that no bracket exists in the hole, the cell aggregation mode is self aggregation, the cell balls are different in size, uneven in spatial distribution and larger in difference among the holes, and meanwhile, the 3D support of the cells is difficult to complete because the bracket does not exist in the hole. When the bracket is arranged in the hole to support the cells in a 3D mode, the shape of the bracket is difficult to control, so that the positions of the cells in the bracket are different, on one hand, the observation of the cells by a microscope is not facilitated, and on the other hand, when the fixing device is arranged in a narrow space of the culture hole to fix the bracket, the fixing device can form a concave liquid surface at the joint of the fixing device and the bracket, so that the cells are aggregated to the fixing device to be difficult to image, and the use is not facilitated.

Disclosure of Invention

The invention aims to overcome the defects that a concave liquid surface exists in a culture hole in a cell culture plate and cell culture is not facilitated in the prior art, and provides a manufacturing method of a 3D cell culture plate and the 3D cell culture plate.

The invention solves the technical problems by the following technical scheme:

a method for manufacturing a 3D cell culture plate, the method for manufacturing a 3D cell culture plate being used for manufacturing a cell culture plate, the method for manufacturing a 3D cell culture plate specifically comprising the steps of:

s1, respectively preparing 10% of methacrylic acylated gelatin and 1.6% of polyethylene oxide solution for preparing a bracket, mixing the solutions according to a ratio of 1:1, and swirling for 5-20S;

s2, taking a cell culture plate, placing a limiting ring of a casting mold into a culture hole of the cell culture plate, attaching the limiting ring to the bottom of the culture hole, attaching the peripheral side of the limiting ring to the hole wall of the culture hole, arranging the top of the limiting ring in a flush manner, arranging the limiting ring in a flush manner on the inner wall of a containing cavity for containing the bracket at the same time, casting the vortexed solution into the containing cavity according to a preset value of the top height of the limiting ring in a flush manner, and oscillating to enable the solution to be tiled;

S3, carrying out illumination on the solution in the accommodating cavity to form a bracket, shaping and solidifying, carrying out ultrasonic cleaning on the solidified bracket, taking out the limiting ring, then, putting the cell culture plate with the bracket into a freeze dryer for freeze drying, and carrying out sterilization treatment on the cell culture plate after the freeze drying is finished.

In this scheme, can form the hole that supplies cell culture after the pouring at self inside through configuration solution messenger support, carry out intensive mixing and vortex to the solution and then make the hole distribution in the support more even after the pouring, before pouring to the support, need put into the cell culture board's cultivation hole with the spacing ring, cut off the intercommunication relation of holding the chamber and cultivating the pore wall of hole through the spacing ring, top parallel and level setting with the spacing ring simultaneously, the inner wall of holding the chamber also sets up the parallel and level, later pour the solution into holding the chamber and carry out the support pouring, it is to take care that the lateral part of support is through holding the inner wall in chamber in order to guarantee the parallel and level, guarantee with the top of spacing ring through the solution volume of pouring into holding the chamber looks parallel and level, for the cell culture that makes the support can be applicable to high flux screening, and pour the solution volume according to the default, just can avoid the top of support to appear the recess or protruding inaccurately. Can assist the mode of concussion when the pouring support with solution tiling, make it solidify and form the support after carrying out the illumination to the solution at last, through taking out the spacing ring and make cell culture plate finally form the cell culture plate that has the support, realize 3D cultivates when the support can guarantee cell culture, simultaneously, the top of support is set up to the parallel and level and can make the cell mainly gather in the central region of support, and the support passes through spacing ring direct shaping in the central region of culture hole bottom again, and then can concentrate the central region of observing the support when observing the cell after the expansion, make the microscope can see through the support of parallel and level and observe the cell culture condition, the image is clear and the position is fixed, compare in the condition of "concave liquid level", imaging quality improves and provides abundant sample when screening to high flux, avoid the unable by observing of cell in a plurality of culture holes and influence the stability of high flux screening.

In addition, the bracket is directly solidified and formed in the central area of the bottom of the culture plate, is attached to the bottom and has good adhesion. The position of the bracket in the culture hole is prevented from changing while the transportation is convenient, a gap is formed between the side part of the bracket and the wall of the culture hole after the limiting ring is taken out, and the gap can promote the exchange of nutrient substances and metabolic products and promote the growth of cells.

Preferably, in step S3, the method specifically further comprises the following steps:

s31, carrying out jack and surface cutting on the top and the side of the support after the support is shaped and solidified, and carrying out ultrasonic cleaning on the support after carrying out jack and surface cutting.

In this scheme, through carrying out the jack to the support to dredge the hole in support top or lateral part parallel and level part, get into the inside speed of support when in order to improve cell culture, cut and then remain the top and the lateral part parallel and level of support all the time to the surface behind the jack, get rid of remaining piece through adopting ultrasonic cleaning's mode, avoid damaging the support simultaneously.

Preferably, in step S1, the method specifically further comprises the following steps:

s11, when the solution is prepared, the freezing speed of the hydrogel precursor solution obtained by mixing the methacryloylated gelatin and the polyethylene oxide is regulated to keep the diameter range of the holes of the stent within 50-400 mu m.

In the scheme, the diameter of the hole is changed by adjusting the freezing speed of the precursor solution, so that the bracket can still be suitable for culturing different cells or tissues or organoids, and the application range of the cell culture plate is improved.

The 3D cell culture plate is manufactured by the manufacturing method of the 3D cell culture plate, a plurality of culture holes are arrayed on the 3D cell culture plate, the 3D cell culture plate can be used for high-throughput screening of cells, a support is arranged in the culture holes, the support is cast in the central area of the bottom of the culture holes and integrated with the culture holes, holes for cell culture are formed in the support after casting, the holes are arranged in the support along the horizontal direction and the vertical direction, the top of the support and the side parts of the support are flush, gaps are formed between the side parts of the support and the walls of the culture holes, and the top of the support is provided with the following structures: when the cells and the culture solution are injected into the bracket, the cells and the culture solution are mutually leveled and uniformly distributed at each position on the top of the bracket.

In the scheme, the bracket is arranged in the culture hole of the cell culture plate for 3D culture of cells, the top and the side parts of the bracket are arranged in a flush way, so that when a cell suspension contacts with the top of the bracket, the cell suspension can be uniformly spread on the top of the bracket, the situation that the cells are concentrated at the edge of the bracket and the cells in the central area of the bracket are few, namely a concave liquid surface is avoided, in addition, a gap is formed between the bracket and the hole wall of the culture hole, and the gap can promote the exchange of nutrient substances and metabolites and promote the growth of the cells; meanwhile, the preparation is simple, the operation is convenient, the cost is low, the method is particularly suitable for observing and analyzing cell samples in a plurality of culture holes during high-flux screening, cells in each culture hole can effectively grow, compared with other cell culture plates with or without brackets, clearer cells can be observed under a microscope, and the stability under high-flux screening is ensured.

Preferably, the 3D cell culture plate further comprises a casting mold, wherein the casting mold is arranged in the gap and is removed from the culture hole after the bracket is formed, and the bottom of the casting mold is set as: and when the bracket is poured, the bottom of the pouring mould keeps a communicated relation between the bracket and the hole wall of the culture hole.

In this scheme, pour into a mould the support through casting die for support and cell culture board fixed connection avoid the support to suspend in the culture hole, make the lateral part parallel and level setting of support and avoid being connected with the pore wall of culture hole through casting die, further avoid "concave liquid level" phenomenon. And the casting mould is removed in time after casting is finished, so that the influence on the exchange of nutrient substances during cell culture is avoided.

Preferably, the casting mold comprises a limiting ring, the limiting ring is provided with a containing cavity, the top of the limiting ring is flush, the side wall of the containing cavity is flush, and the support is cast in the containing cavity according to a preset value flush with the top of the limiting ring.

In this scheme, the spacing ring sets up in the clearance between support and pore wall, and the spacing ring has and holds the chamber to make the support can pour into a mould in holding the chamber and with the pore wall between the interval setting, through the top with the spacing ring with hold the lateral wall parallel and level setting in chamber for the top and the lateral part parallel and level of support after the casting molding need be noted, need pour into a mould according to the default with the top parallel and level of spacing ring for the solution volume that is used for the pouring support when guaranteeing the top parallel and level of support when pouring, in order to avoid the uneven phenomenon to appear on the support surface.

Preferably, the casting mold further comprises a supporting bar and a connecting arm, the supporting bar is arranged on the top of the bottom plate of the 3D cell culture plate in a covering mode and covers the culture hole, the supporting bar is used for being clamped with the top of the bottom plate of the 3D cell culture plate, one end of the connecting arm is connected to the top of the limiting ring and far away from the accommodating cavity, and the other end of the connecting arm is connected to the supporting bar.

In this scheme, casting mold still can be provided with support bar and linking arm, and support bar, linking arm and spacing ring connect gradually, drive the linking arm through the support bar and take out the spacing ring from cultivateing the hole to form the cell culture board that only has the support after the pouring, through setting up support bar and linking arm, avoid setting up the condition that the spacing ring probably catches in cultivateing the hole when taking out the spacing ring alone.

Preferably, the limiting ring is a permanent magnet.

In this scheme, through setting up the spacing ring into the permanent magnet to in time take out it from cultivateing the hole through electromagnetic adsorption or reverse repulsive characteristic, avoid setting up the condition that the spacing ring probably catches in cultivateing the hole when taking out the spacing ring alone.

Preferably, the diameter of the holes is in the range of 50-400 μm.

In the scheme, the diameter of the holes is not smaller than 50 mu m by limiting the diameters of the holes to meet the requirements of cell attachment and migration, so that the condition that the holes are too small to block the cells to pass through is avoided, and the cell culture efficiency is ensured.

Preferably, the material of the bracket is hydrogel, the hydrogel comprises a 1:1 mixed methacrylic acid gelatin and polyethylene oxide solution, wherein the concentration range of the methacrylic acid gelatin is 10% -20%, and the concentration range of the polyethylene oxide is 1% -2%.

In the scheme, the hydrogel solution containing the methacryloylated gelatin and the polyethylene oxide is prepared, wherein the polyethylene oxide is a space occupying solution for forming the bracket, so that holes are formed in the bracket after casting, the time required for hole forming in the bracket by mechanical processing is reduced, and the manufacturing efficiency of the cell culture plate is improved.

The invention has the positive progress effects that: the invention can form holes for cell culture in the bracket after casting by configuring the solution, fully mixing and swirling the solution so as to ensure that the holes in the bracket are distributed more uniformly after casting, before casting the bracket, placing the limiting ring into a culture hole of a cell culture plate, isolating the communication relation between a containing cavity and the wall of the culture hole by the limiting ring, simultaneously arranging the top of the limiting ring in a flush way, arranging the inner wall of the containing cavity in a flush way, pouring the solution into the containing cavity for casting the bracket, taking care that the side part of the bracket is flush with the top of the limiting ring through the inner wall of the containing cavity, ensuring that the top of the bracket is flush with the top of the limiting ring through the solution injected into the containing cavity, casting the solution according to a preset value for ensuring that the bracket can be suitable for cell culture of high-throughput screening, avoid the middle concave or convex condition of the top of the bracket, the solution can be spread in an auxiliary oscillation mode when the bracket is poured, finally the solution is solidified to form the bracket after being irradiated, the limiting ring is taken out to finally form the cell culture plate with the bracket, the bracket can ensure the 3D culture when the cell culture is realized, meanwhile, the top of the bracket is set to be flush to ensure that the cell is mainly accumulated in the central area of the bracket, the bracket is fixed in the central area of the bottom of the culture hole through the limiting ring, the central area of the bracket can be intensively observed when the amplified cell is observed, the microscope can observe the cell culture condition through the flush bracket, the imaging is clear and the position is fixed, compared with the 'concave liquid surface' condition caused by the bracket-free culture, the imaging quality is improved, and abundant samples are provided for high-throughput screening, so that the influence on the stability of high-throughput screening caused by the fact that cells in a plurality of culture holes cannot be observed is avoided.

Drawings

FIG. 1 is a top view of a 3D cell culture plate according to a preferred embodiment of the invention.

FIG. 2 is a side view of a 3D cell culture plate according to a preferred embodiment of the invention.

FIG. 3 is a diagram showing the relationship between the top and bottom plates of a 3D cell culture plate according to a preferred embodiment of the present invention.

Fig. 4 is a schematic structural view of a casting mold according to a preferred embodiment of the present invention.

Fig. 5 is a schematic structural view of a housing chamber according to a preferred embodiment of the present invention.

FIG. 6 is a side view of a casting mold according to a preferred embodiment of the present invention.

FIG. 7 is a schematic diagram of the hole structure of a bracket according to a preferred embodiment of the present invention.

FIG. 8 is a flow chart of a method for manufacturing a 3D cell culture plate according to a preferred embodiment of the invention.

Reference numerals illustrate:

cell culture plate 1

Culture well 11

Bottom plate 12

Bracket 2

Hole 21

Casting mold 3

Stop collar 31

Support bar 32

Connecting arm 33

Accommodating chamber 311

Upper cover 4

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention.

Example 1

The present embodiment provides a method for manufacturing a 3D cell culture plate, as shown in fig. 8, where the method for manufacturing a 3D cell culture plate is used for manufacturing a cell culture plate 1, and the method for manufacturing a 3D cell culture plate specifically includes the following steps:

s1, respectively preparing 10% of methacrylic acid gelatin and 1.6% of polyethylene oxide solution for preparing a bracket 2, mixing the solutions according to a ratio of 1:1, and swirling for 5-20S;

s2, taking the cell culture plate 1, placing a limiting ring 31 of a casting mold 3 into a culture hole 11 of the cell culture plate 1, attaching the limiting ring 31 to the bottom of the culture hole 11, attaching the peripheral side of the limiting ring 31 to the wall of the culture hole 11, arranging the top of the limiting ring 31 in a flush manner, arranging the inner wall of a containing cavity 311 for containing the bracket 2 in a flush manner by the limiting ring 31, casting the vortexed solution into the containing cavity 311 according to a preset value of the top height of the limiting ring 31 in a flush manner, and oscillating to enable the solution to be tiled;

s3, irradiating the solution in the accommodating cavity 311 to form a bracket, shaping and solidifying, ultrasonically cleaning the solidified bracket 2, taking out the limiting ring 31, then putting the cell culture plate 1 with the bracket 2 into a freeze dryer for freeze drying, and sterilizing the cell culture plate 1 after freeze drying.

Specifically, when the solution for casting the stent 2 is prepared, the solution is prepared from non-cytotoxic methacryloylated gelatin and polyethylene oxide, so that cell culture can be performed after the stent 2 is molded, and the growth of cells is not affected, the concentration of the solution of the methacryloylated gelatin and the polyethylene oxide is limited, so that the hole 21 is formed inside the cast stent 2, wherein the polyethylene oxide is used as a space occupying solution of the stent 2, also called a pore-forming agent, so as to be dissolved after the stent 2 is molded, and further the hole 21 is formed inside the cast stent 2, as shown in fig. 7, the hole 21 is a round irregular hole 21 and is distributed along the horizontal and vertical directions, the hole 21 is fully mixed with the solution and further the hole 21 in the stent 2 is distributed more uniformly after the casting, the cell liquid can reproduce the in vivo environment of the cells when the stent 2 is injected, the three-dimensional stent 2 structure can form gradients of oxygen, nutrients, metabolites and soluble signals, so that diversified cell groups, tissues or organs are formed, and the solution of the stent 2 can be used in the prior art to manufacture the stent 2, such as high-molecular materials (e.g. collagen, such as high-polymer, polylactic acid, collagen, and the like), and the like, and the fibrous materials such as the collagen, the fibrous material, the fibrous adhesive layer, and the adhesive layer, etc.

Before casting the bracket 2, the limiting ring 31 needs to be placed in the culture holes 11 of the cell culture plate 1, the culture holes 11 are arranged on the bottom plate 12 of the cell culture plate 1, and the cell culture plate 1 such as 24, 48, 96 holes and the like and the form of inserting the cell culture plate 1 in the 8 or 12 holes in parallel rows can be used for manufacturing the 3D cell culture plate, and the number and the form of the culture holes 11 are the prior art and are not repeated herein. The material of the limiting ring 31 for limiting can be stainless steel, the material which can be used for manufacturing 3D printing materials, such as PLA or ceramic materials, permanent magnets, silica gel and the like, and the materials are required to be processed in a cell-free manner, the size of the limiting ring 31 is required to be processed according to the size of the culture hole 11, 3D printing or die pouring and the like can be adopted in the processing, the top of the limiting ring 31 is arranged in a flush manner, the inner wall of the accommodating cavity 311 is also arranged in a flush manner, the flush is arranged in the horizontal direction, the bottom of the culture hole 11 is contacted with the bottom of the limiting ring 31, the top of the limiting ring 31 and the inner wall of the accommodating cavity 311 are not influenced, the limiting ring 31 can be placed and taken out after the processing through automatic mechanical operation or manual operation, the connection relationship between the accommodating cavity 311 and the wall of the culture hole 11 is cut off by the limiting ring 31, so that the poured bracket 2 is not connected with the wall of the culture hole 11, and then the solution is poured into the accommodating cavity 311 to pour the bracket 2, it can be understood that the side part of the bracket 2 is flush with the top of the limiting ring 31 by the inner wall of the accommodating cavity 311, the top of the bracket 2 is flush with the top of the limiting ring 31 by the amount of the solution poured into the accommodating cavity 311, for the cell culture plate 1 suitable for high throughput screening of 96 holes, 384 holes and the like, the bracket 2 can be suitable for high throughput screening of cells, the amount of the solution is poured according to a preset value, the condition that the top of the bracket 2 is concave or convex in the middle is avoided, the embodiment is illustrated by taking the volume of the bracket 2 as 30 mu l as an example, but the limiting ring 31 is not limited, the accommodating cavity 311 is arranged in the central area, the accommodating cavity 311 is also of a cylindrical structure, so that the support 2 is of a cylindrical structure after casting molding, the accommodating cavity 311 penetrates through the upper end and the lower end of the limiting ring 31, the top of the limiting ring 31 can be prefabricated according to cells to be cultured, the volume of the accommodating cavity 311 is ensured to be consistent with that of the support 2, the liquid level of the solution is flush with the top of the limiting ring 31 after the solution is injected into the accommodating cavity 311, and a pipette with higher precision can be adopted during the injection of the solution, so that excessive description is omitted. The method comprises the steps that a solution is paved in an auxiliary vibration mode when the bracket 2 is poured, the solution is finally solidified to form the bracket 2 after being irradiated by 405nm, the limiting ring 31 is taken out to enable the cell culture plate 1 to finally form the cell culture plate 1 with the bracket 2, 3D culture can be achieved when the cells are cultured, meanwhile, the top of the bracket 2 is arranged to be flush, the cells can be mainly accumulated in the central area of the bracket 2, the bracket 2 is fixed in the central area of the bottom of the culture hole 11 in a pouring molding mode, the central area of the bracket 2 can be intensively observed when the amplified cells are observed, the microscope can observe the cell culture condition through the flush bracket 2, imaging is clear and the position is fixed, compared with the situation of 'concave liquid level', imaging quality is improved, abundant samples are provided when high-throughput screening is achieved, the cells in a plurality of culture holes cannot be observed to influence the stability of high-throughput screening, and the 3D cell culture plate manufactured by the manufacturing method is easy to achieve automatic and short period and high-throughput screening and high-clinical medicine-related models when the cells are cultured.

In addition, the bracket 2 is fixedly connected with the cell culture plate 1 after casting, so that the position of cells in the culture hole 11 is prevented from being changed while transportation is facilitated, a gap is formed between the side part of the bracket 2 and the wall of the culture hole 11 after the limiting ring 31 is taken out, and the gap can enable nutrient solution to be injected into the culture hole 11 to promote the exchange of the cells in the bracket 2 with nutrient substances and metabolites and promote the growth of the cells. In addition, the coating of the scaffold 2 with various extracellular matrix components, such as collagen, laminin, fibronectin, matrigel, etc., in this example can better simulate the in vivo environment, improve the adhesion and differentiation of normal and transformed anchorage-dependent epithelial cells and other cell types.

The interior of the bracket 2 is subjected to impurity removal in an ultrasonic cleaning mode after the bracket 2 is poured, then the cell culture plate 1 with the bracket 2 is placed into a freeze dryer for freeze drying for 12 hours, so that the stability of the bracket 2 is further improved, and the cell culture plate 1 is subjected to sterilization treatment after freeze drying is finished.

Further, the step S3 specifically further includes the following steps:

s31, after the support 2 is shaped and solidified, inserting holes and surface cutting are carried out on the top and the side of the support 2, and after inserting holes and surface cutting are carried out, ultrasonic cleaning is carried out on the support 2.

Specifically, jack is carried out through plum blossom to support 2 to dredge the hole 21 in support 2 top or lateral part parallel and level part, get into the inside speed of support 2 when improving cell culture, cut and then remain the top and the lateral part parallel and level of support 2 all the time to the surface behind the jack, avoid the condition that "concave liquid level" appears when cell sap pours into, can carry out preliminary degerming edulcoration to support 2 inside through adopting ultrasonic cleaning 30min, avoid damaging support 2 simultaneously. Preferably, a parallel laser may be used to detect the presence or absence of irregularities on the surface of the support 2 to keep its top and side portions flush after casting of the support 2.

In other embodiments, the spacing ring 31 is overfed to raise the surface, and the upper surface is flattened by planing after light curing, so that the bracket 2 with a completely transparent and flat surface can be obtained, so that the imaging of the microscope is clearer.

In this embodiment, the step S1 specifically further includes the following steps:

s11, when the solution is prepared, the diameter range of the holes 21 of the bracket 2 is kept within 50-400 mu m by adjusting the freezing speed of the hydrogel precursor solution after the methacryloylated gelatin is mixed with polyethylene oxide.

Specifically, the adjustment of the freezing speed of the precursor solution can be performed by using the adjustment mode and structure in the prior art, which are not described in detail herein, and the diameter of the hole 21 is changed by adjusting the freezing speed of the precursor solution, so that the bracket 2 can still be applied when culturing different cells, and the application range of the cell culture plate 1 is improved.

In other embodiments, the hole size of the hole 21 of the support 2 can be adjusted by adding ingredients in the solution of the casting support 2 to the ingredients in the prior art, or adjusting the mixing ratio of the methacryloylated gelatin and polyethylene oxide to adjust the hole size, which is the prior art, and it should be noted that the hole size 21 always needs to be kept larger than 50 μm to avoid affecting the cell passage, and the ingredients belong to the prior art and are not described in any detail herein. It will be appreciated that for different cell cultures, photosensitive materials such as CSMA, HAMA, etc. may also be added to the above solutions to accommodate photosensitive cell growth.

As shown in fig. 1-7, the present embodiment further provides a 3D cell culture plate, the 3D cell culture plate is manufactured by adopting the manufacturing method of the 3D cell culture plate, a plurality of culture holes 11 are arrayed on the 3D cell culture plate, the 3D cell culture plate is used for high-throughput screening of cells, as shown in fig. 2, a support 2 is provided in the culture hole 11, the support 2 is cast in a central area of a bottom of the culture hole 11 and is integrated with the culture hole 11, holes 21 for cell culture are formed after the casting of the support 2, the holes 21 are arranged in the support 2 along the horizontal and vertical directions, the top of the support 2 and the side of the support 2 are flush, the side of the support 2 and the wall of the culture hole 11 form a gap, and the top of the support 2 is set as: when the cells and the culture solution are injected into the bracket 2, the cells and the culture solution are mutually leveled and evenly distributed at each position on the top of the bracket 2.

Specifically, as shown in fig. 3, the cell culture plate 1 includes a bottom plate 12 and an upper cover 4, a plurality of culture holes 11 are arrayed on the bottom plate 12, and a clamping portion is disposed at the edge of the bottom plate 12 near the opening of the culture holes 11, the clamping portion is used for being matched with the upper cover 4 and preventing external dust from entering the culture holes 11 when the cell culture plate 1 is stored or transported, meanwhile, a support 2 is poured into the culture holes 11, the support 2 is fixedly connected with the culture holes 11, namely, fixedly connected with the bottom plate 12, holes 21 formed by the support 2 can be used for cell culture along the horizontal direction and the vertical direction, compared with the 2D of cells, gradients of oxygen, nutrient substances, metabolites and soluble signals can be formed, so that diversified cell populations, tissues or organs can be formed, and many defects of monolayer 2D culture can be made up. In addition, the top and side portions of the bracket 2 are flush, in this embodiment, the bracket 2 is in a cylindrical structure, however, in other embodiments, the accommodating cavity 311 may be in a rectangular structure or other shaped surface flush structures, which will not be described herein. The top and the side parts which are arranged in parallel can ensure that cells and nutrient solution are evenly spread on the top of the bracket 2 when contacting with the top of the bracket 2, so that the situation that the cells are concentrated on the edge of the bracket 2 and the cells in the central area of the bracket 2 are few, namely a concave liquid surface is avoided, in addition, a gap is formed between the bracket 2 and the wall of the culture hole 11, and the gap can promote the exchange of nutrient substances and metabolites and promote the growth of the cells; meanwhile, the manufacturing cost of the cell culture plate 1 is reduced, the structure is simple, the operation and the observation are convenient during the high-throughput screening, especially, the observation and the analysis are performed on the cell samples in the plurality of culture holes 11 during the high-throughput screening, the cells in each culture hole 11 can effectively grow, compared with other cell culture plates 1 with or without brackets, the cell growth is more consistent with the original environment and the formed cell colony is more uniform, clearer cells can be observed under a microscope, and the stability under the high-throughput screening is ensured.

In other embodiments, the scaffold 2 of hydrophobic material is subjected to a surface modification technique to increase cell attachment. Preferably, the surface modification technique is a physicochemical treatment, including plasma treatment, glow discharge treatment, surface modification technique treatment, to further improve cell attachment.

In this embodiment, the 3D cell culture plate further comprises a casting mold 3, wherein the casting mold 3 is disposed in the gap and is removed from the culture hole 11 after the support 2 is formed, and the bottom of the casting mold 3 is set as: the bottom of the casting mold 3 maintains a communicating relationship between the closed support 2 and the wall of the culture well 11 when the support 2 is cast.

Specifically, the casting mold 3 is used when the support 2 is cast in the culture hole 11, and the casting mold 3 is removed in time after casting is completed to avoid the influence on nutrient exchange during cell culture. The cell culture plate 1 manufactured is made to comprise a bracket 2, a bottom 12 and an upper cover 4 from the appearance, and as cells and culture solution can be kept flush rather than gathered at the edge of the bracket 2 when being injected into the bracket 2, the periphery side of the bracket 2 is isolated from the hole wall when being poured, so that the problem that liquid is gathered on the hole wall due to tension in the connection condition is avoided, namely, the problem of concave liquid level is avoided, the concave liquid level not only enables the cell growth to be influenced, but also the microscope imaging when observing cells is influenced, the bracket 2 is poured through a pouring die 3, the bracket 2 is fixedly connected with the cell culture plate 1, the bracket 2 is prevented from being suspended in the culture hole 11, and the microscope imaging difficulty is reduced.

Further, in this embodiment, the casting mold 3 includes a spacing ring 31, the spacing ring 31 has a receiving cavity 311, the top of the spacing ring 31 is flush, the side wall of the receiving cavity 311 is flush, and the solution of the casting support 2 is cast into the receiving cavity 311 according to a preset value flush with the top of the spacing ring 31 when the support 2 is cast into the receiving cavity 311.

Specifically, the spacing ring 31 is arranged in the clearance between the support 2 and the hole wall, the spacing ring 31 is provided with the accommodating cavity 311, so that the support 2 can be poured in the accommodating cavity 311 and is arranged at intervals between the spacing ring and the hole wall, the height of the support 2 is adjusted through the spacing ring 31, the side wall of the accommodating cavity 311 is arranged at the same level, the top of the support 2 after pouring molding is enabled to be flush with the side part, and attention is paid to the fact that the solution amount for pouring the support 2 needs to be poured according to the preset amount of the top of the spacing ring 31 when the top of the support 2 is flush with the top of the spacing ring 31 for guaranteeing that the top of the support 2 cannot be uneven when cells and culture solution are poured.

In other embodiments, as shown in fig. 4, 5 and 6, the casting mold 3 further includes a supporting bar 32 and a connecting arm 33, the supporting bar 32 is covered on the top of the bottom plate 12 of the 3D cell culture plate and covers the culture hole 11, the supporting bar 32 is used for being clamped with the top of the bottom plate 12 of the 3D cell culture plate, one end of the connecting arm 33 is connected to the top of the limiting ring 31 and is far away from the accommodating cavity 311, and the other end of the connecting arm 33 is connected to the supporting bar 32.

Specifically, support bar 32, linking arm 33 and spacing ring 31 fixed connection in proper order, wherein support bar 32 is located the top of linking arm 33 and spacing ring 31, support bar 32 is the planar sheet material and support bar 32 is provided with corresponding turn-ups to the block portion of bottom plate 12 for be used for with support bar 32 block on bottom plate 12, linking arm 33 is cylindrical structure and be located between support bar 32 and the spacing ring 31, the junction of linking arm 33 and spacing ring 31 is kept away from and is held the chamber 311 setting, in order to avoid pouring support 2's solution part adhesion and influence the roughness at its top when support 2 pours. When in use, the support bar 32 is clamped at the top of the bottom plate 12 and covers the culture hole 11, the connecting arm 33 and the limiting ring 31 correspondingly extend into the culture hole 11, the solution is injected into the limiting ring 31 to pour the support frame 2, and after pouring is completed, the connecting arm 33 is driven by the support bar 32 to take the limiting ring 31 out of the culture hole 11, so that the cell culture plate 1 only provided with the support frame 2 is formed after pouring, and the situation that the limiting ring 31 is possibly clamped in the culture hole 11 when the limiting ring 31 is taken out is avoided by arranging the support bar 32 and the connecting arm 33.

In other embodiments, the stop collar 31 is a permanent magnet.

Specifically, through setting up spacing ring 31 as the permanent magnet to in time take out it from cultivateing hole 11 through electromagnetic adsorption or reverse repulsive characteristic, for example, put into cultivate hole 11 in the back with spacing ring 31 and make it be located cultivate hole 11 bottom through electromagnetic adsorption, outage after the pouring is accomplished and invert cell culture board 1 and can take out the spacing ring, or use the bar magnet that corresponds cultivate hole 11, stretch into the inside suction spacing ring 31 of cultivateing hole 11, probably the condition of blocking in cultivateing hole 11 when taking out spacing ring 31 when avoiding setting up alone spacing ring 31, compared with the mode that sets up support bar 32 and linking arm 33, its cost is lower, the structure is simpler.

In another embodiment, the material of the limiting ring 31 may be a metal material that can be attracted by an electromagnet, at this time, the limiting ring 31 is placed into the culture hole 11 and the electromagnet is used to attract the limiting ring 31 to perform the casting of the bracket 2, and after casting, the cell culture plate 1 is inverted to take out the limiting ring 31, so that the limiting ring 31 is taken out in time after the casting of the bracket 2, and redundant description is omitted herein.

In this embodiment, as shown in FIG. 7, the diameter of the hole 21 is in the range of 50 to 400. Mu.m.

Specifically, the holes 21 in the bracket 2 are directly formed after casting, so that the problem that the manufacturing period of the cell culture plate 1 is too long due to the fact that the bracket 2 needs to additionally process the holes 21 is avoided, the requirements of cell attachment and migration are met by limiting the diameters of the holes 21, the diameters of the holes 21 are not smaller than 50 mu m, the situation that the holes 21 are too small to block the passage of cells is avoided, and the cell culture efficiency is ensured. Preferably, the diameter of the holes 21 is in the range of 100-300 μm.

In this embodiment, the material of the stent 2 is hydrogel, and the hydrogel includes a 1:1 mixture of methacryloylated gelatin and polyethylene oxide solution, wherein the concentration range of the methacryloylated gelatin is 10% -20%, and the concentration range of the polyethylene oxide is 1% -2%.

Specifically, by preparing a hydrogel solution containing methacryloylated gelatin and polyethylene oxide so that the pores 21 are formed inside the scaffold itself after casting, the time required for the mechanical processing to make the pores inside the scaffold 2 is reduced, and the manufacturing efficiency of the cell culture plate 1 is improved. Preferably, the concentration of methacryloylated gelatin is 10% and the concentration of polyethylene oxide is 1.6%.

Example 2

The embodiment provides a detection method for removing the influence of a 'concave liquid surface' of a 3D cell culture plate, so as to detect the 3D cell culture plate in embodiment 1, which specifically comprises the following steps:

first, cells are cultured in the scaffold 2 and stained.

And secondly, respectively inoculating the stained cells into the 3D cell culture plate and a common 3D culture plate containing the influence of a concave liquid surface.

After that, the cells were allowed to stand for 24 hours and 48 hours, and then the colonies were observed.

Finally, the plates containing "meniscus" and the present 3D cell culture plates were observed by microscopy.

According to the detection method, cells of the culture plate with the concave liquid level can be obtained to be more at the edge of the culture plate, the cells in the central area of the culture plate are fewer, and the cells cultured by the 3D cell culture plate are uniformly distributed on the bracket 2, and the imaging is clearer.

Example 3

The embodiment provides a method for culturing lung cancer cells by using a 3D cell culture plate, so as to detect the 3D cell culture plate in embodiment 1, specifically comprising the following steps:

first, a549 cells were cultured on a 2D culture plate, and after the cells were grown, the cells were digested and counted.

Next, the cells were counted to 2X 10 ⁷ And each ml. Centrifuging to precipitate cells, removing culture medium, and re-suspending cells to give cell density of 5×10 ⁶ And each ml.

After that, the upper lid 4 was opened, and after the cell suspension was homogenized, 150. Mu.l of the cell suspension was sucked up by a pipette, and was added dropwise from directly above the holder 2.

Again, after completion, the lid 4 was closed at 37 ℃ with 5% CO ₂ Culturing in incubator for 1 hr.

Then, the medium was preheated, and after the incubation was completed, the medium was added along the culture well 11, and the culture was continued.

Finally, all the steps are carried out under aseptic conditions.

It should be noted that the cell culture plate 1 of this example was a 48-well plate having dimensions of 1cm long by 1cm wide by 2mm thick. By the culture method and observing the cells, uniform cell growth distribution, clear observation under a mirror and stable structure can be realized, the exchange of nutrient substances and metabolites can be better promoted, the efficiency and the stability of cell growth are improved, and the high-throughput screening is facilitated.

Example 4

The embodiment provides a method for culturing a mouse intestinal organoid by using a 3D cell culture plate, so as to detect the 3D cell culture plate in embodiment 1, specifically comprising the following steps:

first, after the mice were sacrificed by cervical scission, sections of the intestine approximately 15cm from the terminal ileum were collected and placed in PBS wash at 4 ℃.

Next, the membranes, vessels and fat outside the intestine were removed using pointed forceps, and PBS was injected from one end of the small intestine with a syringe for rinsing.

Then, the cells are extracted by the way of extracting the digested tissue cells in the prior art, the cells are counted, and then the cells are centrifugally resuspended to have a cell density of 5X 10 ⁶ And each ml.

And finally, selecting a 3D cell culture plate with a proper size, directly planting the 3D cell culture plate into the bracket 2 without using matrigel mixed cells, and carrying out subsequent culture and observation. Thereby reducing the cost required for cultivation.

Example 5

The embodiment provides a method for culturing tumor cells by using a 3D cell culture plate, so as to detect the 3D cell culture plate in embodiment 1, specifically comprising the following steps:

firstly, removing fat, connective tissue and necrotic part from the obtained tumor tissue, washing with Hanks solution 3 times in a plate, shearing the tissue, and cutting into 1-2mm pieces ³ Small blocks.

Next, 0.25% trypsin or 2000U/ml collagenase was added.

Then, digestion was performed in a water bath at 37℃for 30min or prolonged, the digestion solution was removed, the solution was washed 3 times with the washing solution, the medium was washed 1 time, resuspended in complete medium, dispersed into a cell suspension by pipetting, and counted.

Finally, the cells are directly planted in the bracket 2 without using matrigel mixed cells, subsequent culture and observation are carried out, high-flux drug screening can be carried out, and the cost required by culture is reduced. It should be noted that the cell culture plates used in this example were 96-well plates to ensure the high throughput screening requirements.

While specific embodiments of the invention have been described above, it will be appreciated by those skilled in the art that this is by way of example only, and the scope of the invention is defined by the appended claims. Various changes and modifications to these embodiments may be made by those skilled in the art without departing from the principles and spirit of the invention, but such changes and modifications fall within the scope of the invention.

Claims

1. A method for manufacturing a 3D cell culture plate, the method for manufacturing a 3D cell culture plate being used for manufacturing a cell culture plate, characterized in that the method for manufacturing a 3D cell culture plate specifically comprises the following steps:

2. The method of manufacturing a 3D cell culture plate according to claim 1, further comprising the steps of, in step S3:

3. The method of manufacturing a 3D cell culture plate according to claim 1, wherein in step S1, the method further comprises the steps of:

4. A 3D cell culture plate, characterized in that, 3D cell culture plate adopts the manufacturing approach of 3D cell culture plate in claim 1-3 to make, a plurality of culture holes have been arranged on the 3D cell culture plate array, 3D cell culture plate is used for high flux screening cell, be provided with the support in the culture hole, the support pour into a mould in the central region of the bottom of culture hole and with culture hole forms wholly, the support forms the hole that is used for cell culture after passing through the pouring, the hole arrange along horizontal and vertical direction and set up in the support, the top of support and the lateral part parallel and level of support set up, just the lateral part of support with the pore wall of culture hole forms the clearance, the top of support is set up to: when the cells and the culture solution are injected into the bracket, the cells and the culture solution are mutually leveled and uniformly distributed at each position on the top of the bracket.

5. The 3D cell culture plate of claim 4, further comprising a casting mold disposed in the gap and removed from the culture well after the scaffold is formed, a bottom of the casting mold being configured to: and when the bracket is poured, the bottom of the pouring mould keeps a communicated relation between the bracket and the hole wall of the culture hole.

6. The 3D cell culture plate of claim 5, wherein the casting mold comprises a limiting ring, the limiting ring comprises a receiving cavity, the top of the limiting ring is flush, the side walls of the receiving cavity are flush, and the amount of solution for casting the bracket when the bracket is cast in the receiving cavity according to a preset value flush with the top of the limiting ring.

7. The 3D cell culture plate of claim 6, wherein the casting mold further comprises a supporting bar and a connecting arm, the supporting bar is covered on the top of the 3D cell culture plate and covers the culture hole, the supporting bar is used for being clamped with the top of the bottom plate of the 3D cell culture plate, one end of the connecting arm is connected to the top of the limiting ring and is far away from the accommodating cavity, and the other end of the connecting arm is connected to the supporting bar.

8. The 3D cell culture plate of claim 6, wherein the stop collar is a permanent magnet.

9. The 3D cell culture plate of claim 4, wherein the holes have a diameter ranging from 50 to 400 μm.

10. The 3D cell culture plate of claim 4, wherein the scaffold is made of a hydrogel, the hydrogel comprises a 1:1 mixture of a methacryloylated gelatin and a polyethylene oxide solution, wherein the concentration of the methacryloylated gelatin is in the range of 10% to 20%, and the concentration of the polyethylene oxide is in the range of 1% to 2%.