CN109016275B - Micropore mould and preparation method and application thereof - Google Patents
Micropore mould and preparation method and application thereof Download PDFInfo
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- CN109016275B CN109016275B CN201811096910.9A CN201811096910A CN109016275B CN 109016275 B CN109016275 B CN 109016275B CN 201811096910 A CN201811096910 A CN 201811096910A CN 109016275 B CN109016275 B CN 109016275B
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- bottom plate
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- handheld rod
- microporous
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- 238000002360 preparation method Methods 0.000 title claims description 17
- 238000004113 cell culture Methods 0.000 claims description 22
- 229920000936 Agarose Polymers 0.000 claims description 15
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 9
- 239000000741 silica gel Substances 0.000 claims description 9
- 229910002027 silica gel Inorganic materials 0.000 claims description 9
- 229920000642 polymer Polymers 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 6
- 239000004800 polyvinyl chloride Substances 0.000 claims description 3
- 229920000915 polyvinyl chloride Polymers 0.000 claims description 3
- 229920002725 thermoplastic elastomer Polymers 0.000 claims description 3
- 239000011248 coating agent Substances 0.000 claims description 2
- 238000000576 coating method Methods 0.000 claims description 2
- 238000002474 experimental method Methods 0.000 abstract description 9
- 231100000956 nontoxicity Toxicity 0.000 abstract description 2
- 238000005336 cracking Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 44
- 210000002242 embryoid body Anatomy 0.000 description 10
- 238000000034 method Methods 0.000 description 10
- 239000000463 material Substances 0.000 description 7
- 210000005036 nerve Anatomy 0.000 description 6
- 230000008569 process Effects 0.000 description 5
- 230000009286 beneficial effect Effects 0.000 description 4
- 230000024245 cell differentiation Effects 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 230000011712 cell development Effects 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 210000001671 embryonic stem cell Anatomy 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000010146 3D printing Methods 0.000 description 1
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 238000010329 laser etching Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000004381 surface treatment Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
- B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
- B29C33/00—Moulds or cores; Details thereof or accessories therefor
- B29C33/38—Moulds or cores; Details thereof or accessories therefor characterised by the material or the manufacturing process
- B29C33/3842—Manufacturing moulds, e.g. shaping the mould surface by machining
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
- C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/12—Well or multiwell plates
Landscapes
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Sustainable Development (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Mechanical Engineering (AREA)
- Manufacturing & Machinery (AREA)
- Clinical Laboratory Science (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The microporous die comprises a bottom plate, a handheld rod and a pull ring, wherein a plurality of protruding structures are arranged on the lower surface of the bottom plate in an orderly manner, the vertical handheld rod and the vertical pull ring are arranged on the upper surface of the bottom plate, and the handheld rod and the pull ring are tightly connected with the bottom plate. The micropore mould has the advantages of wear resistance, no cracking and long service life; no toxicity and smell, and no influence on cells; can be sterilized at high temperature and high pressure, and is convenient to use repeatedly; the cost is relatively low; the biological experiment has the advantage of good repeatability.
Description
Technical Field
The invention belongs to the technical field of preparation of experimental equipment in cell biology, and particularly relates to a microporous die, a preparation method and application thereof.
Background
The cell biology experiment mould has application in various processes, and mainly assists experiment development and obtains stable data. In particular, for the preparation of three-dimensional structures of cells, the preparation is increasingly used in basic biological research. Unlike conventional two-dimensional cell structures, three-dimensional cell culture has become a hotspot in cell biology research because it constructs in vitro a cell growth and development structural system similar to that in vivo. And the preparation of the three-dimensional cell structure is completed by means of a micropore mould. Currently, such microwell molds used in cell biology experiments are produced monopolically by foreign biological companies. The microporous mould produced by foreign company is made of plastic, and needs to use complex technology to carry out special surface treatment so as to be usable and not reusable. A series of matched reagents are also involved in the use process, so that the experimental cost is greatly increased. In conclusion, the micropore mould manufactured abroad is high in price, the use procedure is complicated, and the operation technical requirements on experimental staff are very high.
Disclosure of Invention
The invention aims at solving the problems and provides a microporous die, a preparation method and application thereof, which can obviously reduce the experimental cost.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in one aspect, the invention provides a microporous die, which comprises a bottom plate, a handheld rod and a pull ring, wherein the lower surface of the bottom plate is provided with a plurality of protruding structures which are orderly arranged, the upper surface of the bottom plate is provided with a vertical handheld rod and a vertical pull ring, and the handheld rod and the pull ring are tightly connected with the bottom plate.
Preferably, the bottom plate is circular, rectangular or square.
Preferably, the bottom plate is circular, preferably 20-50mm in diameter and 3-5mm in thickness.
Preferably, the protruding structure is in a pyramid frustum shape or a truncated cone shape. Preferably, the height of the convex structure is 0.1-0.5mm, and the bottom area is 0.5-1.0mm 2 。
Preferably, the hand-held wand is centrally located on the base plate, preferably 35mm to 45mm in length.
Preferably, the diameter of the hand-held wand is tapered from bottom to top, and the diameter of the foot print of the hand-held wand is preferably from 7mm to 12mm.
Preferably, the pull ring is positioned at the edge of the bottom plate, and is in a circular ring or an elliptical ring.
Preferably, the pull ring is in the shape of an elliptical ring, preferably, the major axis of the outer diameter is 17mm to 20mm, the minor axis is 10mm to 13mm, the major axis of the inner diameter is 13mm to 16mm, and the minor axis is 6mm to 9mm.
Preferably, the materials of the base plate, the handheld rod and the pull ring are high molecular polymers, and preferably, the materials of the base plate, the handheld rod and the pull ring are independently selected from silica gel, soft polyvinyl chloride or thermoplastic elastomer.
In another aspect, the present invention provides a method for preparing the micro-porous mold, comprising:
providing a flat plate, wherein the surface of the flat plate is provided with a concave structure;
coating a layer of liquid high molecular polymer on the flat plate, and solidifying the high molecular polymer;
separating the high molecular polymer from the flat plate to obtain a bottom plate of the microporous die;
and the handheld rod and the pull ring are fixedly bonded on the bottom plate.
Preferably, the method further comprises sterilizing the prepared microcellular mold.
In yet another aspect, the invention provides an application of the microporous mould in preparing a three-dimensional cell culture chamber.
Compared with the prior art, the invention has the following beneficial effects:
the microporous silica gel mold disclosed by the invention is wear-resistant, does not crack and has long service life; no toxicity and smell, and no influence on cells; can be sterilized at high temperature and high pressure, and is convenient to use repeatedly; the cost is lower than foreign brands; the repeatability of the biological experiment is good;
the microporous silica gel mold can improve the non-uniformity of the preparation of the three-dimensional cell structure in the cell culture process; the efficiency and the yield of the preparation of the three-dimensional cell structure are improved; the size of the three-dimensional cell structure can be controlled by changing the initial cell quantity; the activity and the survival rate of the cells in the three-dimensional culture process are improved; is particularly beneficial to the preparation of embryoid body three-dimensional structures in embryonic stem cell development and differentiation experiments, and improves the cell differentiation efficiency.
Drawings
FIG. 1 is a schematic view of a microcellular mold according to an embodiment of the present invention;
FIG. 2 is a physical diagram of a microcellular mold according to an embodiment of the present invention;
FIG. 3 is a physical view of a lower surface bump structure in an embodiment of the present invention;
FIG. 4 is a photograph of a microstructure of a lower surface bump structure in an embodiment of the present invention;
FIG. 5 is a flow chart of a method of making a microcellular mold in an embodiment of the present invention;
FIG. 6 is a flow chart of the preparation of agarose cell culture chambers using a microwell die in an embodiment of the invention;
FIG. 7 is a photomicrograph of the three-dimensional structure of embryoid bodies and statistics of their sizes (1000 cells/unit) prepared in the examples of the present invention, wherein (a) is a cell culture chamber with three-dimensional cell mass formed; (b) A three-dimensional cell mass collected in a cell culture chamber, (c) a three-dimensional cell mass size distribution condition of the same batch;
FIG. 8 is a photomicrograph of the three-dimensional structure of embryoid bodies and statistics of their sizes (2000 cells/min) prepared in the examples of the present invention, wherein (a) is a cell culture chamber with three-dimensional cell mass formed; (b) A three-dimensional cell mass collected in a cell culture chamber, (c) a three-dimensional cell mass size distribution condition of the same batch;
FIG. 9 is a photomicrograph of the three-dimensional structure of embryoid bodies and statistics of their size (5000 cells/min) prepared in the examples of the present invention, wherein (a) is a cell culture chamber with three-dimensional cell mass formed; (b) A three-dimensional cell mass collected in a cell culture chamber, (c) a three-dimensional cell mass size distribution condition of the same batch;
FIG. 10 is a photomicrograph of the three-dimensional cell cavity (human embryonic stem cell embryoid body) structure prepared in the examples of the present invention;
FIG. 11 is a photomicrograph of nerve filaments grown on the wall of a neurosphere prepared in the examples of the present invention;
in the drawings, the reference numerals have the following meanings: 1-handheld stick, 2-pull ring, 3-bottom plate.
Detailed Description
The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent.
The microporous mould can be used as an experimental mould for preparing a cell three-dimensional structure culture system. As shown in fig. 1 and 2, in one embodiment, the microcellular mold includes a hand-held bar 1, a tab 2, and a base plate 3. The lower surface of bottom plate 3 has the protruding structure of neat range, and the upper surface of bottom plate 3 has vertical handheld stick 1 and pull ring 2, handheld stick 1 and pull ring 2 and bottom plate 3 fixed connection.
In this embodiment, the bottom plate 3 is circular, with a bottom surface diameter of 35mm and a height of 4mm, as shown in figures 3 and 4. The convex structure is cone frustum-shaped, the height is 0.24mm, and the bottom area is 0.66mm 2 . The convex structure can be fully developed on a soft medium and can be repeatedly cleaned and sterilized for use.
The handheld rod 1 is positioned at the center of the bottom plate 3 and has a length of 40mm. The material is the same as that of the bottom plate 3 and is tightly connected, so that the microporous die is convenient to take, the microporous structure of the bottom surface and the medium below are prevented from being closely contacted, the sterile operation in the manufacturing process is ensured to meet the requirement, and the smooth proceeding of the subsequent cell culture experiment is facilitated.
The diameter of the handheld rod 1 gradually decreases from bottom to top, and the handheld rod is of a cone-like structure, is firmly adhered to the bottom plate 3, is not easy to fall off, is beneficial to experimenters to place the die in a culture dish, and is beneficial to repeated use for many times. The diameter of the bottom area is 10mm.
The pull ring 2 is positioned at the edge of the bottom plate 3 and is in the shape of an elliptical ring. The major axis of the outer diameter is 20mm, the minor axis is 13mm, the major axis of the inner diameter is 16mm, and the minor axis is 9mm. The material is the same as that of the bottom plate 3, and the connection is tight, so that the bottom plate is convenient to separate from a medium below the bottom plate during use.
The materials of the hand-held rod 1, the pull ring 2 and the bottom plate 3 can be independently selected from high molecular polymers such as silica gel, soft polyvinyl chloride, thermoplastic elastomer and the like.
The hand-held rod 1 and the pull ring 2 can be integrally formed with the bottom plate 3, or the hand-held rod 1 and the pull ring 2 are fixedly bonded with the bottom plate 3.
As shown in fig. 5, in one embodiment, the fabrication process of the micro-porous mold includes: the hard flat plate with the concave structure is obtained by utilizing three-dimensional printing or laser etching and other technologies, the concave structure is in a shape of a truncated pyramid, and the hard flat plate is not limited in material. The premixed liquid silica gel is poured into a hard flat plate with a uniform concave structure which is orderly arranged from one side to the other side, and air is prevented from being mixed into the hard flat plate to generate bubbles in the pouring process. Incubate in an oven at 37 ℃ until complete cure. The cured silica gel was separated from the hard plate. Cutting into a size basically consistent with that of a cell culture dish to obtain a die bottom plate, bonding and fixing a hand-held rod and a pull ring which are manufactured in advance on the bottom plate, cleaning with deionized water, wrapping tinfoil paper for high-temperature high-pressure sterilization, and drying for later use.
The liquid silica gel in the embodiment has good fluidity, can be fully contacted and filled with a hard flat plate, and forms a convex structure with clear outline and regular arrangement and high precision. The silica gel material can be sterilized at high temperature and high pressure, and the sterility requirement of the subsequent cell culture experiment is ensured.
In one embodiment, as shown in FIG. 6, agarose cell culture chamber preparation is performed using a sterilized microwell mold. A60 mm dish was charged with 5mL of a 2.5% liquid agarose solution. Holding the handheld rod 1, vertically and rapidly putting the microporous mould into the non-solidified agarose solution, gently shaking the discharged gas, and avoiding air bubbles from remaining. After the agarose solution is cooled and solidified, the pull ring 2 is pulled, and the microporous mould and the agarose are slowly separated.
The prepared agarose cell culture chamber can be used for forming and preparing cell clusters required by various three-dimensional cell cultures, such as embryoid body preparation, neurosphere preparation and the like.
In one embodiment, the agarose cell culture chamber is submerged with DMEM/F-12 medium. After soaking in DMEM/F-12 culture medium for 12 hr, the single cell suspension is directly added into the agarose cell culture chamber to form three-dimensional cell mass.
As shown in FIGS. 7-9, the three-dimensional cell mass prepared by using the agarose cell culture chamber has the advantages of less dead cells, uniform size and high yield, which indicates that the uniform three-dimensional cell mass with high quality and high quantity can be obtained by the invention. The method can achieve the purpose of controlling and preparing the three-dimensional cell mass size by controlling the total cell quantity (1000 cells/cell, 2000 cells/cell and 5000 cells/cell) in the initial cell suspension, namely, the initial cell quantity is large to obtain the three-dimensional size, and the cell quantity is small to obtain the three-dimensional size, so that the method has better applicability and can produce the three-dimensional cell mass with various scales according to the requirements of experimenters.
In one embodiment, embryoid body three-dimensional structures are prepared using agarose cell culture chambers of the present invention. FIG. 10 is a photomicrograph of a three-dimensional cell cavity structure prepared in an example of the present invention. The invention is suitable for preparing the embryoid body three-dimensional structure (embryoid body of human embryonic stem cells) in embryonic stem cell development and differentiation experiments, and can obtain embryoid bodies with cavity structures, which shows that the cell differentiation efficiency is ensured.
Fig. 11 is a photomicrograph of a nerve filament grown from a nerve ball attachment prepared in an example of the present invention, illustrating that the present invention is equally applicable to the preparation of nerve balls, and that the prepared nerve balls are capable of forming outwardly extending nerve filaments after being attached.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.
Claims (2)
1. Use of a microwell die for preparing a three-dimensional cell culture cell, comprising:
adding liquid agarose solution into a culture dish, putting the sterilized microporous mould into the non-coagulated agarose solution, and oscillating to discharge gas;
after the agarose solution is cooled and solidified, pulling a pull ring on the microporous mould, and separating the microporous mould from agarose to obtain a three-dimensional cell culture chamber;
the micropore mould comprises a bottom plate, a handheld rod and a pull ring, wherein the lower surface of the bottom plate is provided with a plurality of protruding structures which are orderly arranged;
the convex structure is a frustum of a pyramid and can be fully developed and printed on a soft medium;
the upper surface of the bottom plate is provided with a vertical handheld rod and a pull ring, the handheld rod and the pull ring are tightly connected with the bottom plate, and the bottom plate, the handheld rod and the pull ring are made of any one of silica gel, soft polyvinyl chloride or thermoplastic elastomer;
the pull ring is positioned at the edge of the bottom plate and is in an elliptical ring shape;
the handheld rod is positioned in the center of the bottom plate, and the diameter of the handheld rod is gradually reduced from bottom to top and is of a conical-like structure;
the bottom plate is round, and the diameter of the bottom surface is 20-50 mm;
the length of the handheld rod is 35-45 mm, and the diameter of the bottom area is 7-12 mm.
2. The use according to claim 1, wherein,
the preparation method of the micropore mould comprises the following steps:
providing a flat plate, wherein the surface of the flat plate is provided with a concave structure;
coating a layer of liquid high molecular polymer on the flat plate, and solidifying the high molecular polymer;
separating the high molecular polymer from the flat plate to obtain a bottom plate of the microporous die;
and the handheld rod and the pull ring are fixedly bonded on the bottom plate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811096910.9A CN109016275B (en) | 2018-09-19 | 2018-09-19 | Micropore mould and preparation method and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811096910.9A CN109016275B (en) | 2018-09-19 | 2018-09-19 | Micropore mould and preparation method and application thereof |
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| Publication Number | Publication Date |
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| CN109016275A CN109016275A (en) | 2018-12-18 |
| CN109016275B true CN109016275B (en) | 2024-01-30 |
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| US11492581B2 (en) | 2019-04-17 | 2022-11-08 | Academia Sinica | Microwell device and method of manufacturing the same |
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