CN117402818B - Embryoid body packaging material, packaging device and preparation method of packaging device - Google Patents
Embryoid body packaging material, packaging device and preparation method of packaging device Download PDFInfo
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- CN117402818B CN117402818B CN202311729913.2A CN202311729913A CN117402818B CN 117402818 B CN117402818 B CN 117402818B CN 202311729913 A CN202311729913 A CN 202311729913A CN 117402818 B CN117402818 B CN 117402818B
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- 238000004806 packaging method and process Methods 0.000 title claims abstract description 115
- 210000002242 embryoid body Anatomy 0.000 title claims abstract description 85
- 239000005022 packaging material Substances 0.000 title claims abstract description 29
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 239000003607 modifier Substances 0.000 claims abstract description 49
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 44
- 239000000741 silica gel Substances 0.000 claims abstract description 44
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 44
- 238000002156 mixing Methods 0.000 claims abstract description 23
- 229920002134 Carboxymethyl cellulose Polymers 0.000 claims abstract description 14
- 239000000783 alginic acid Substances 0.000 claims abstract description 14
- 235000010443 alginic acid Nutrition 0.000 claims abstract description 14
- 229920000615 alginic acid Polymers 0.000 claims abstract description 14
- 229960001126 alginic acid Drugs 0.000 claims abstract description 14
- 150000004781 alginic acids Chemical class 0.000 claims abstract description 14
- 239000001768 carboxy methyl cellulose Substances 0.000 claims abstract description 14
- 235000010948 carboxy methyl cellulose Nutrition 0.000 claims abstract description 14
- 239000008112 carboxymethyl-cellulose Substances 0.000 claims abstract description 14
- 229920002307 Dextran Polymers 0.000 claims abstract description 13
- 239000000758 substrate Substances 0.000 claims description 36
- 239000000203 mixture Substances 0.000 claims description 26
- 238000000034 method Methods 0.000 claims description 23
- 238000005538 encapsulation Methods 0.000 claims description 17
- 238000007711 solidification Methods 0.000 claims description 17
- 230000008569 process Effects 0.000 claims description 15
- 230000008023 solidification Effects 0.000 claims description 13
- 238000001816 cooling Methods 0.000 claims description 12
- 239000003795 chemical substances by application Substances 0.000 claims description 11
- 238000004381 surface treatment Methods 0.000 claims description 11
- 239000011248 coating agent Substances 0.000 claims description 10
- 238000000576 coating method Methods 0.000 claims description 10
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 5
- 229920002338 polyhydroxyethylmethacrylate Polymers 0.000 claims description 4
- 239000002202 Polyethylene glycol Substances 0.000 claims description 2
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 2
- 229920001223 polyethylene glycol Polymers 0.000 claims description 2
- 239000004065 semiconductor Substances 0.000 claims description 2
- 238000012546 transfer Methods 0.000 abstract description 4
- 239000000499 gel Substances 0.000 abstract description 3
- 108010082117 matrigel Proteins 0.000 description 23
- 230000000052 comparative effect Effects 0.000 description 15
- 230000000694 effects Effects 0.000 description 15
- 210000004556 brain Anatomy 0.000 description 14
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 10
- 230000007547 defect Effects 0.000 description 10
- 238000007789 sealing Methods 0.000 description 9
- 210000002220 organoid Anatomy 0.000 description 6
- 238000010586 diagram Methods 0.000 description 5
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 5
- 238000003795 desorption Methods 0.000 description 4
- 210000001654 germ layer Anatomy 0.000 description 4
- 239000003292 glue Substances 0.000 description 4
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- 238000009826 distribution Methods 0.000 description 3
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- 210000003014 totipotent stem cell Anatomy 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 229920001503 Glucan Polymers 0.000 description 2
- 229920000289 Polyquaternium Polymers 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 210000001671 embryonic stem cell Anatomy 0.000 description 2
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
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- 238000005259 measurement Methods 0.000 description 2
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- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- 238000012605 2D cell culture Methods 0.000 description 1
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- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
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- 241001465754 Metazoa Species 0.000 description 1
- 241000282579 Pan Species 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0604—Whole embryos; Culture medium therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2513/00—3D culture
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/20—Small organic molecules
Abstract
The invention discloses an embryoid body packaging material, a packaging device and a preparation method of the packaging device, and belongs to the technical field of embryoid body packaging. The packaging material comprises a modifier solution and silica gel, wherein the modifier solution is prepared by mixing 15-25mg/L of carboxymethyl cellulose solution, 70-90mg/L of dextran solution and 5-15mg/L of alginic acid solution according to the volume ratio of 1-2:1-2:1-2; the volume ratio of the modifier solution to the silica gel is 10-35:100. The packaging device can effectively solve the problems of gel breaking, degumming, difficult transfer and the like existing in the existing embryoid body packaging mode, and has the advantages of good stability, high packaging efficiency, high packaging success rate and the like.
Description
Technical Field
The invention belongs to the technical field of embryoid body packaging, and particularly relates to embryoid body packaging material, packaging device and preparation method of the packaging device.
Background
Under certain in vitro induction conditions, totipotent or pluripotent stem cells can form a structure with three germ layers, namely an inner germ layer, an intermediate germ layer and an outer germ layer, which is highly similar to a cell structure in the early stage of embryo development, and the three-dimensional spherical structure is called Embryoid Body (EB). EB formation is an important stage of differentiation of totipotent or multipotent stem cells, and the obtaining of various functional cells can firstly prepare the totipotent or multipotent stem cells into EB, and then make directional induction differentiation under different culture conditions, such as natural killer cells, mesenchymal stem cells, neural stem cells, brain organs and the like.
The brain, particularly the human brain, is a complex tissue whose cellular composition and spatial distribution are greatly different from the cellular composition, spatial distribution, developmental mechanisms and physiological functions of the animal brain. Even the chimpanzees and gorillas closest to human relatives have significant differences in their brain physiological functions (as intended). Studies show that embryonic stem cells (ES) or Induced Pluripotent Stem Cells (iPSCs) are prepared into EB first, and then are packaged by matrigel and subjected to suspension culture to form whole brain or regional brain organoids. This form of brain organoid highly restores the cellular composition and spatial distribution of the human brain and is highly similar to the developmental process of the human brain. The brain organoid technology provides a unique platform for researching the basic mechanism, physiological functions, the establishment of various brain disease models, drug screening, cell replacement therapy, regenerative medicine and the like of brain development, overcomes the defects of 2D cell culture and animal models, and has wide scientific research and clinical application prospects.
Despite the great potential advantages and applications of brain organoids, there are a number of shortcomings in the preparation of brain organoids. For example, most of the current documents report that when using matrigel to package EB in the process of preparing brain organoids, a common packaging mold is a pit formed by manually pressing on a sealing film temporarily, and the EB packaging mold can generate large differences according to different operators, different operation proficiency and different preparation batches, so that consistency and stability of EB packaging effect cannot be ensured, and meanwhile, amplification operation cannot be performed. In addition, the EB packaging by this method has a high failure rate, which is mainly expressed in that:
1) The die for preparing the sealing film is lighter, and is easy to move when the EB packaging is carried out, so that the damage of the EB or the operation difficulty of the packaging are caused, and the time and the labor are wasted.
2) Because the sealing film has certain viscosity, and the matrigel is also more viscous, the integrity of the glue is easily damaged when the packaged EB is transferred, and even the EB is degummed and damaged, so that the preparation success rate is greatly reduced.
3) Even if the encapsulation and transfer of the EB are successfully completed, the EB often falls off from the matrigel in the later culture process, so that the culture fails, and the main reason for the phenomenon is that the EB is not well positioned in the inner central position of the matrigel during encapsulation, but is positioned at the bottom or the edge of the matrigel, and falls off along with the increase of the culture time.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides an embryoid body packaging material, a packaging device and a preparation method of the packaging device, and the packaging device can effectively solve the problems of gel breaking, degumming, difficult transfer and the like existing in the existing embryoid body packaging mode and has the advantages of good stability, high packaging efficiency, high packaging success rate and the like.
In order to achieve the above purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
an embryoid body packaging material comprises a modifier solution and silica gel;
the modifier solution is prepared by mixing 15-25mg/L of carboxymethyl cellulose solution, 70-90mg/L of dextran solution and 5-15mg/L of alginic acid solution according to the volume ratio of 1-2:1-2:1-2; the volume ratio of the modifier solution to the silica gel is 10-35:100.
In the scheme, the silica gel is used as a main material, and the solidified silica gel has certain hardness and elasticity, can be made into a specific shape, and is convenient for packaging the embryoid body; the carboxymethyl cellulose solution, the dextran solution and the alginic acid solution are used as modifiers, the carboxymethyl cellulose is used for adjusting the strength of the silica gel, so that the silica gel has certain strength, the dextran is used for adjusting the viscosity of the silica gel, the alginic acid is used for adjusting the viscosity and the solidification time of the silica gel, so that the silica gel is quickly solidified, and the silica gel is modified by adding three materials, so that the encapsulation effect is further improved.
An embryoid body packaging device comprises the packaging material.
The preparation method of the embryoid body packaging device comprises the following steps:
(1) Uniformly mixing the modifier solution and silica gel, placing the mixture into a vessel, and curing to obtain a semi-solidified packaging substrate;
(2) Forming a concave cavity on the surface of the packaging substrate in a semi-solidification state by adopting a mould;
(3) And (3) carrying out surface treatment on the packaging substrate with the concave cavities by adopting a surface treating agent.
In the scheme, the modifier solution and the silica gel mixture are solidified to form a semi-solidification state so as to manufacture a cavity, the cavity is formed by extruding the surface of the semi-solidification state by using a die, then the cavity with a specific angle is formed after the solidification is continued, the cavity is used for containing the embryoid body and the matrigel, and finally the surface of the cavity is treated by adopting the surface treating agent, so that the adhesion of the surface of the material is reduced, the packaged embryoid body is conveniently demoulded, and the packaging success rate of the embryoid body is improved.
Further, in the step (1), the curing temperature is 60-70 ℃ and the curing time is 1-2min.
Further, the preparation process of the concave cavities in the step (2) comprises the following steps: and vertically placing the mold on the surface of the semi-solidified packaging substrate, then placing the mold in an environment of 60-70 ℃ for solidification for 1-2min, cooling, and taking down the mold to obtain the semiconductor package.
Further, in the step (2), the diameter of the recess is 3-5mm, and the radian of the recess is 0-20 degrees.
In the scheme, the diameter of the concave cavity is too large, so that the subsequent demolding is difficult, the problems of base adhesive breakage and the like are easily caused, and the demolding effect is influenced; radian refers to the included angle between the horizontal plane of the concave cavity and the inclined plane; the radian of the concave cavity is too large, so that the embryoid body packaged by the matrigel is difficult to detach from the packaging device, and even if the embryoid body is detached, the embryoid body is damaged.
Further, the surface treating agent in the step (3) comprises any one of methoxy polyethylene glycol, polyquaternium-51 and poly (2-hydroxyethyl methacrylate).
In the scheme, the surface active agent can increase the lubricity of the surface of the concave cavity, so that the packaged embryoid body can be conveniently demoulded and transferred after packaging is finished.
Further, the surface treatment process in the step (3) is as follows: coating the surface treating agent on the surface of the encapsulation substrate with the concave cavities, and standing for 1-3 h.
Further, the amount of the surface treating agent used in the step (3) is 0.1 to 7g/cm 2 。
Further, the amount of methoxypolyethylene glycol is 6-7X10 -3 g/ cm 2 The amount of polyquaternium-51 is 0.1-0.2X10 -3 g/ cm 2 The amount of poly (2-hydroxyethyl methacrylate) is 0.5-1.5X10 -3 g/ cm 2 。
The beneficial effects of the invention are as follows:
1. the packaging device has the advantages of high consistency and good stability, can be applied to a large number of laboratories, avoids manual hole making operation on the sealing film, and is convenient for standardized operation.
2. The packaging device has high success rate for packaging the EB, effectively solves the problems of easy glue breaking, degumming, difficult transfer and the like existing in the existing packaging method, and can greatly improve the packaging efficiency and the experimental efficiency.
3. The packaging device has simple and easily obtained raw materials; the preparation method of the device is simple, convenient to operate and convenient for mass production.
Drawings
FIG. 1 is a physical diagram of an matrigel encapsulated embryoid body;
FIG. 2 is a statistical graph of the success rate of packaging the embryoid bodies in example 3 and comparative examples 1-5;
FIG. 3 is a physical diagram of the embryoid body of example 3 before and after encapsulation;
FIG. 4 is a schematic illustration of a measurement of radian of a cavity of an embryoid body encapsulation device;
fig. 5 is a physical view of the sealing film package of comparative example 5 and the package device package of example 3.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the particular embodiments described herein are illustrative only and are not intended to limit the invention, i.e., the embodiments described are merely some, but not all, of the embodiments of the invention.
Thus, the following detailed description of the embodiments of the invention, as provided, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by a person skilled in the art without making any inventive effort, are intended to be within the scope of the present invention.
It is noted that relational terms such as "first" and "second", and the like, are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises an element.
The features and capabilities of the present invention are described in further detail below with reference to the examples and figures.
Example 1
An embryoid body packaging material comprises a modifier solution and silica gel;
the modifier solution is prepared by mixing 15mg/L of carboxymethyl cellulose solution, 70mg/L of dextran solution and 5mg/L of alginic acid solution according to the volume ratio of 1:2:2; the volume ratio of the modifier solution to the silica gel is 10:100.
The embryoid body packaging device comprises the packaging material, and the preparation method comprises the following steps:
(1) Uniformly mixing the modifier solution and silica gel, sucking 2ml of the mixture, placing the mixture in a vessel with the diameter of 35mm, and then curing the mixture at 60 ℃ for 2min to prepare a semi-solidified packaging substrate;
(2) Vertically placing the die on the surface of a semi-solidified packaging substrate, then integrally placing the die in an environment of 60 ℃ for solidification for 2min, cooling, and taking down the die to obtain a concave cavity, wherein the diameter of the concave cavity is 3mm, and the radian of the concave cavity is 10 degrees;
(3) Coating methoxypolyethylene glycol on the surface of packaging substrate at an amount of 6.25X10 -3 g/ cm 2 Standing for 1h, finishing the surface treatment process, and capping and packaging.
Example 2
An embryoid body packaging material comprises a modifier solution and silica gel;
the modifier solution is prepared by mixing 25mg/L of carboxymethyl cellulose solution, 90mg/L of dextran solution and 15mg/L of alginic acid solution according to the volume ratio of 2:1:1; the volume ratio of the modifier solution to the silica gel is 35:100.
The embryoid body packaging device comprises the packaging material, and the preparation method comprises the following steps:
(1) Uniformly mixing the modifier solution and silica gel, sucking 2ml of the mixture, placing the mixture in a vessel with the diameter of 35mm, and then placing the vessel in a condition of 70 ℃ for curing for 1min to prepare a semi-solidified packaging substrate;
(2) Vertically placing the die on the surface of a semi-solidified packaging substrate, then integrally placing the die in an environment of 70 ℃ for solidification for 1min, cooling, and taking down the die to obtain a concave cavity, wherein the diameter of the concave cavity is 5mm, and the radian of the concave cavity is 20 degrees;
(3) Coating polyquaternium on the surface of the packaging substrate with the dosage of 0.16X10 -3 g/ cm 2 Standing for 3h, finishing the surface treatment process, and capping and packaging.
Example 3
An embryoid body packaging material comprises a modifier solution and silica gel;
the modifier solution is prepared by mixing 20mg/L of carboxymethyl cellulose solution, 80mg/L of dextran solution and 10mg/L of alginic acid solution according to the volume ratio of 1:1:1; the volume ratio of the modifier solution to the silica gel is 20:100.
The embryoid body packaging device comprises the packaging material, and the preparation method comprises the following steps:
(1) Uniformly mixing the modifier solution and silica gel, sucking 2ml of the mixture, placing the mixture in a vessel with the diameter of 35mm, and then placing the vessel in a condition of 65 ℃ for curing for 1.5min to prepare a semi-solidified packaging substrate;
(2) Vertically placing the die on the surface of a semi-solidified packaging substrate, then integrally placing the die in an environment of 65 ℃ for solidification for 1min, cooling, and taking down the die to obtain a concave cavity, wherein the diameter of the concave cavity is 3mm, and the radian of the concave cavity is 20 degrees;
(3) Coating methoxypolyethylene glycol on the surface of packaging substrate at an amount of 6.25X10 -3 g/ cm 2 Standing for 2h, finishing the surface treatment process, and capping and packaging.
Example 4
An embryoid body packaging material comprises a modifier solution and silica gel;
the modifier solution is prepared by mixing 20mg/L of carboxymethyl cellulose solution, 70mg/L of dextran solution and 15mg/L of alginic acid solution according to the volume ratio of 1:2:1; the volume ratio of the modifier solution to the silica gel was 25:100.
The embryoid body packaging device comprises the packaging material, and the preparation method comprises the following steps:
(1) Uniformly mixing the modifier solution and silica gel, sucking 2ml of the mixture, placing the mixture in a vessel with the diameter of 35mm, and then curing the mixture at 68 ℃ for 2min to prepare a semi-solidified packaging substrate;
(2) Vertically placing the die on the surface of a semi-solidified packaging substrate, then integrally placing the die in an environment of 68 ℃ for solidification for 1.5min, cooling, and taking down the die to obtain a concave cavity, wherein the diameter of the concave cavity is 4mm, and the radian of the concave cavity is 15 degrees;
(3) Coating the surface of the packaging substrate with poly (2-hydroxyethyl methacrylate) in an amount of 0.99X10 -3 g/ cm 2 Standing for 1.5h, finishing the surface treatment process, and capping and packaging.
Example 5
An embryoid body packaging material comprises a modifier solution and silica gel;
the modifier solution is prepared by mixing 20mg/L of carboxymethyl cellulose solution, 80mg/L of dextran solution and 5mg/L of alginic acid solution according to the volume ratio of 2:2:1; the volume ratio of the modifier solution to the silica gel is 15:100.
The embryoid body packaging device comprises the packaging material, and the preparation method comprises the following steps:
(1) Uniformly mixing the modifier solution and silica gel, sucking 2ml of the mixture, placing the mixture in a vessel with the diameter of 35mm, and then placing the vessel in a condition of 62 ℃ for curing for 2min to prepare a semi-solidified packaging substrate;
(2) Vertically placing the die on the surface of a semi-solidified packaging substrate, then integrally placing the die in an environment of 65 ℃ for solidification for 1min, cooling, and taking down the die to obtain a concave cavity, wherein the diameter of the concave cavity is 4mm, and the radian of the concave cavity is 15 degrees;
(3) Coating polyquaternium on the surface of the packaging substrate with the dosage of 0.16X10 -3 g/ cm 2 Standing for 2h, finishing the surface treatment process, and capping and packaging.
Comparative example 1
An embryoid body packaging material comprises a modifier solution and silica gel;
the modifier solution is prepared by mixing 20mg/L of carboxymethyl cellulose solution and 10mg/L of alginic acid solution according to the volume ratio of 1:1; the volume ratio of the modifier solution to the silica gel is 20:100.
The embryoid body packaging device comprises the packaging material, and the preparation method comprises the following steps:
(1) Uniformly mixing the modifier solution and silica gel, sucking 2ml of the mixture, placing the mixture in a vessel with the diameter of 35mm, and then placing the vessel in a condition of 65 ℃ for curing for 1.5min to prepare a semi-solidified packaging substrate;
(2) Vertically placing the die on the surface of a semi-solidified packaging substrate, then integrally placing the die in an environment of 65 ℃ for solidification for 1min, cooling, and taking down the die to obtain a concave cavity, wherein the diameter of the concave cavity is 3mm, and the radian of the concave cavity is 20 degrees;
(3) Coating methoxypolyethylene glycol on the surface of packaging substrate at an amount of 6.25X10 -3 g/ cm 2 Standing for 2h, finishing the surface treatment process, and capping and packaging.
Comparative example 2
An embryoid body packaging material comprises a modifier solution and silica gel;
the modifier solution is prepared by mixing 20mg/L of carboxymethyl cellulose solution, 80mg/L of dextran solution and 10mg/L of alginic acid solution according to the volume ratio of 1:1:1; the volume ratio of the modifier solution to the silica gel is 20:100.
The embryoid body packaging device comprises the packaging material, and the preparation method comprises the following steps:
(1) Uniformly mixing the modifier solution and silica gel, sucking 2ml of the mixture, placing the mixture in a vessel with the diameter of 35mm, and then placing the vessel in a condition of 65 ℃ for curing for 1.5min to prepare a semi-solidified packaging substrate;
(2) Vertically placing the die on the surface of a semi-solidified packaging substrate, then integrally placing the die in an environment of 65 ℃ for solidification for 1min, cooling, and taking down the die to obtain a concave cavity, wherein the diameter of the concave cavity is 6mm, and the radian of the concave cavity is 20 degrees;
(3) Coating methoxypolyethylene glycol on the surface of packaging substrate at an amount of 6.25X10 -3 g/ cm 2 Standing for 2h, finishing the surface treatment process, and capping and packaging.
Comparative example 3
An embryoid body packaging material comprises a modifier solution and silica gel;
the modifier solution is prepared by mixing 20mg/L of carboxymethyl cellulose solution, 80mg/L of dextran solution and 10mg/L of alginic acid solution according to the volume ratio of 1:1:1; the volume ratio of the modifier solution to the silica gel is 20:100.
The embryoid body packaging device comprises the packaging material, and the preparation method comprises the following steps:
(1) Uniformly mixing the modifier solution and silica gel, sucking 2ml of the mixture, placing the mixture in a vessel with the diameter of 35mm, and then placing the vessel in a condition of 65 ℃ for curing for 1.5min to prepare a semi-solidified packaging substrate;
(2) And vertically placing the mold on the surface of the semi-solidified packaging substrate, integrally placing the mold in an environment of 65 ℃ for solidification for 1min, cooling, taking down the mold to obtain a concave cavity, wherein the diameter of the concave cavity is 3mm, the radian of the concave cavity is 60 degrees, and capping and packaging.
Comparative example 4
An embryoid body packaging material comprises a modifier solution and silica gel;
the modifier solution is prepared by mixing 20mg/L of carboxymethyl cellulose solution, 80mg/L of dextran solution and 10mg/L of alginic acid solution according to the volume ratio of 1:1:1; the volume ratio of the modifier solution to the silica gel is 20:100.
The embryoid body packaging device comprises the packaging material, and the preparation method comprises the following steps:
(1) Uniformly mixing the modifier solution and silica gel, sucking 2ml of the mixture, placing the mixture in a vessel with the diameter of 35mm, and then placing the vessel in a condition of 65 ℃ for curing for 1.5min to prepare a semi-solidified packaging substrate;
(2) Vertically placing the die on the surface of a semi-solidified packaging substrate, then integrally placing the die in an environment of 65 ℃ for solidification for 1min, cooling, and taking down the die to obtain a concave cavity, wherein the diameter of the concave cavity is 3mm, and the radian of the concave cavity is 20 degrees;
(3) Coating agarose on the surface of the packaging substrate with the dosage of 6.25X10 -3 g/ cm 2 Standing for 2h, finishing the surface treatment process, and capping and packaging.
Comparative example 5
And packaging by adopting a sealing film.
Test examples
Taking the packaging device in example 3 as an example, the packaging devices prepared in example 3 and comparative examples 1 to 5 were taken respectively, 20 were taken for each case, and the packaging effect was verified, and the specific operation was as follows:
1. pre-cooling the packaging device on ice;
2. selecting EB with proper size, transferring to a recess of a packaging device, and removing excessive liquid to ensure that 2-5 mu L of liquid is remained around the EB to ensure that the EB is in a wet state;
adding 20 μl Matrigel (Corning) to wrap EB, inverting the packaging device on ice for 1min, and transferring to 37 deg.C carbon dioxide incubator for 40min;
3. adding 2mL of minimal medium (DMEM/F12, gibco), blowing off EB encapsulated by matrigel, and transferring to an ultra-low adhesion plate (Corning);
4. the minimal medium was removed, then rinsed 3 times with ultrapure water, and stained with 2mL of swiss-giemsa stain (Baso) for 5min;
5. washing with ultrapure water for 3 times, and then placing under a body microscope for photographing and observing;
6. dividing the EB after the matrigel is packaged into complete, partial defect, serious defect and degumming according to the integrity of the matrigel, wherein the complete refers to that the EB is in the matrigel and the matrigel is not defective; the partial defect means that EB is in the matrigel, and the matrigel edge has defect; the serious defect means that the edge defect of the matrigel is larger, so that part of EB is not in the matrigel; degumming refers to that EB is not coated in matrix glue; see in particular fig. 1, and make statistics. Wherein, the complete mark is successful encapsulation, and the partial defect, the serious defect and the degumming mark are failed encapsulation;
package success = complete glue EB number/total EB number packaged 100%; the specific results are shown in FIG. 2.
Fig. 2 is a statistical graph of the packaging success rate of the packaging device in example 3 and comparative examples 1-5, and it can be seen that the packaging success rate in example 3 is higher, more than 90%, and the packaging success rate in comparative examples 1-5 is lower than 80%, and the packaging effect is obviously worse than that in examples, which proves that the packaging device prepared according to the method of the invention has better use effect.
Compared with the embodiment 3, the comparative example 1 omits the use of glucan, and the effect of fig. 2 shows that the packaging success rate of the embryoid body is reduced, and presumably, the glucan can change the microscopic morphology of the surface of the packaging material, so that the surface is smoother, and the packaged embryoid body is convenient to detach;
compared with the embodiment 3, the diameter of the recess is adjusted to be 6mm in the comparative example 2, and as can be seen from the effect of fig. 2, the packaging success rate of the embryoid body is reduced, presumably, the contact between the matrigel and the packaging device is increased after the diameter is increased, so that the difficulty of desorption is increased, the desorption effect is poor, and the packaging success rate is reduced;
comparative example 3 compared with example 3, the treatment of the surface of the encapsulating material was omitted, and it can be seen from the effect of fig. 2 that the encapsulation success rate of the embryoid body was reduced, and it was presumed that the surface possibly untreated was unfavorable for matrix gel desorption, affecting the desorption effect, resulting in a reduction in the encapsulation success rate.
Compared with the embodiment 3, the comparative example 4 adjusts the surface treating agent, and the effect of fig. 2 shows that the packaging success rate of the embryoid body is reduced, and it is presumed that the adjusted surface treating agent cannot achieve the effect of improving the surface smoothness of the packaging material, and thus the packaging success rate cannot be improved;
compared with the embodiment 3, the comparison example 5 adopts the sealing film as the packaging device to package the embryoid body, and the effect of the sealing film is obviously lower than that of the embodiment 3, so that the packaging effect of the packaging device prepared in the invention is better.
Fig. 3 is a physical diagram of the embryoid body of example 3 before and after encapsulation, and it can be seen that the matrigel is uniformly round in the encapsulation device.
Fig. 4 is a schematic diagram of measurement of radian of a cavity of an embryoid body packaging device, wherein the radian of the cavity refers to an included angle between a horizontal plane and an inclined plane of the cavity.
Fig. 5 is a physical diagram of the sealing film package in comparative example 5 and the packaging device package in example 3, and the embryoid bodies are all arranged in the middle of the matrigel from the appearance, and the matrigel is round and indistinct.
Claims (7)
1. An embryoid body packaging material is characterized by comprising a modifier solution and silica gel;
the modifier solution is prepared by mixing 15-25mg/L of carboxymethyl cellulose solution, 70-90mg/L of dextran solution and 5-15mg/L of alginic acid solution according to the volume ratio of 1-2:1-2:1-2;
the volume ratio of the modifier solution to the silica gel is 10-35:100.
2. An embryoid body encapsulation device comprising the encapsulation material of claim 1.
3. The method of manufacturing an embryoid body encapsulation device of claim 2, comprising the steps of:
(1) Uniformly mixing the modifier solution and silica gel, placing the mixture into a vessel, and curing to obtain a semi-solidified packaging substrate;
(2) Forming a concave cavity on the surface of the packaging substrate in a semi-solidification state by adopting a mould;
(3) Surface treating the packaging substrate with the concave cavity by adopting a surface treating agent;
the diameter of the concave cavity in the step (2) is 3-5mm, and the radian of the concave cavity is 0-20 degrees;
the surface treating agent in the step (3) comprises any one of methoxy polyethylene glycol, polyquaternium-51 and poly (2-hydroxyethyl methacrylate);
the radian of the concave cavity refers to the included angle between the horizontal plane of the upper part of the concave cavity and the inclined plane.
4. The method of claim 3, wherein the curing temperature in step (1) is 60-70 ℃ and the curing time is 1-2min.
5. The method of claim 3, wherein the cavity preparation in step (2) comprises: and vertically placing the mold on the surface of the semi-solidified packaging substrate, then placing the mold in an environment of 60-70 ℃ for solidification for 1-2min, cooling, and taking down the mold to obtain the semiconductor package.
6. The method of manufacturing an embryoid body encapsulation device according to claim 3, wherein the surface treatment process in step (3) is as follows: coating the surface treating agent on the surface of the encapsulation substrate with the concave cavities, and standing for 1-3 h.
7. The method for producing an embryoid body encapsulating device according to claim 3, wherein the amount of the surface treating agent used in the step (3) is 0.1 to 7g/cm 2 。
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