CN109016275A - Micropore mold and its preparation method and application - Google Patents
Micropore mold and its preparation method and application Download PDFInfo
- Publication number
- CN109016275A CN109016275A CN201811096910.9A CN201811096910A CN109016275A CN 109016275 A CN109016275 A CN 109016275A CN 201811096910 A CN201811096910 A CN 201811096910A CN 109016275 A CN109016275 A CN 109016275A
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- bottom plate
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- pull ring
- micropore mold
- cell
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- 238000002360 preparation method Methods 0.000 title claims abstract description 23
- 238000004113 cell culture Methods 0.000 claims description 13
- 239000000463 material Substances 0.000 claims description 9
- 229920000642 polymer Polymers 0.000 claims description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical group O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 7
- 239000000741 silica gel Substances 0.000 claims description 7
- 229910002027 silica gel Inorganic materials 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 5
- 229920002725 thermoplastic elastomer Polymers 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 3
- 241000894006 Bacteria Species 0.000 claims 1
- 230000001954 sterilising effect Effects 0.000 abstract description 6
- 238000004659 sterilization and disinfection Methods 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 2
- 231100000252 nontoxic Toxicity 0.000 abstract description 2
- 230000003000 nontoxic effect Effects 0.000 abstract description 2
- 230000009967 tasteless effect Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 52
- 210000002242 embryoid body Anatomy 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 9
- 229920001817 Agar Polymers 0.000 description 7
- 239000008272 agar Substances 0.000 description 7
- 230000001580 bacterial effect Effects 0.000 description 7
- 210000005036 nerve Anatomy 0.000 description 5
- 229920000936 Agarose Polymers 0.000 description 4
- 102000008763 Neurofilament Proteins Human genes 0.000 description 3
- 108010088373 Neurofilament Proteins Proteins 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 210000005044 neurofilament Anatomy 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000011712 cell development Effects 0.000 description 2
- 230000024245 cell differentiation Effects 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 229910052710 silicon Inorganic materials 0.000 description 2
- 239000010703 silicon Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 238000000608 laser ablation Methods 0.000 description 1
- 230000000873 masking effect Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 238000007639 printing Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000004381 surface treatment Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 229920001169 thermoplastic Polymers 0.000 description 1
- 239000004416 thermosoftening plastic Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000005406 washing Methods 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
A kind of micropore mold and its preparation method and application, the micropore mold includes bottom plate, hand-held stick and pull ring, wherein there are multiple bulge-structures of proper alignment in the lower surface of the bottom plate, there is vertical hand-held stick and pull ring in the upper surface of the bottom plate, the hand-held stick and the pull ring are closely connect with the bottom plate.Micropore mold of the invention does not crack with wear-resistant, long service life；It is non-toxic and tasteless, it has no effect to cell；Autoclave sterilization can be carried out, Reusability is facilitated；Cost is relatively low；The reproducible advantage of Bioexperiment.
Description
Technical field
The invention belongs to Cell Biology Experiment equipment preparation technical fields, and in particular to a kind of micropore mold and its preparation
Methods and applications.
Background technique
Cell Biology Experiment mold has application in various procedures, and main assistant experiment is carried out and obtains stable number
According to.It especially at present prepared by cell three-dimensional structure, more and more in fundamental biological knowledge research.It is thin with conventional two dimension
Born of the same parents structure is different, Three-dimensional cell culture because its construct in vitro with it is internal similar in cell development structural system, become thin
The hot spot of born of the same parents' biological study.And the preparation needs of three-dimensional cell structure are completed by micropore mold.Currently, this use is thin
Micropore mold in born of the same parents' biological experiment, which is all monopolized by foreign biotech firm, to be produced.This kind of micropore mold of foreign corporation's production is equal
Adopt made of plastic, needing to be carried out special surface treatment using complicated technology just can be used, and not reproducible utilization.It is using
A series of matched reagents are further related in the process, greatly increase experimental cost.To sum up, such micropore mold of foreign-made not only valence
Lattice are expensive, many and diverse using program, require the operating technology of experimenter very high.
Summary of the invention
Regarding the issue above, the present invention provides a kind of micropore mold and its preparation method and application, it can be significant
Reduce experimental cost.
In order to achieve the above object, the invention adopts the following technical scheme:
On the one hand, the present invention provides a kind of micropore mold, including bottom plate, hand-held stick and pull ring, wherein the bottom plate
There are multiple bulge-structures of proper alignment in lower surface, has vertical hand-held stick and pull ring in the upper surface of the bottom plate, described
Hand-held stick and the pull ring are closely connect with the bottom plate.
Preferably, the bottom plate is round, rectangle or square.
Preferably, the bottom plate is circle, it is preferable that diameter 20-50mm, with a thickness of 3-5mm.
Preferably, the bulge-structure is frustum of a pyramid shape or truncated cone-shaped.Preferably, the height of the bulge-structure is
0.1-0.5mm, floor space 0.5-1.0mm2。
Preferably, the hand-held stick is located at the center of the bottom plate, and length is preferably 35mm to 45mm.
Preferably, the diameter of the hand-held stick is gradually reduced from the bottom up, and the floor space diameter of the hand-held stick is preferred
For 7mm to 12mm.
Preferably, the pull ring is located at the edge of the bottom plate, and shape is annulus or elliptical ring.
Preferably, the pull ring shape is elliptical ring, it is preferable that outer diameter long axis is 17mm to 20mm, short axle 10mm
To 13mm, internal diameter long axis is 13mm to 16mm, and short axle is 6mm to 9mm.
Preferably, the material of the bottom plate, the hand-held stick and the pull ring is high molecular polymer, it is preferable that institute
The material of bottom plate, the hand-held stick and the pull ring is stated independently selected from silica gel, soft PVC or thermoplastic elastomer (TPE).
On the other hand, the present invention provides a kind of preparation methods of micropore mold, comprising:
A plate is provided, the surface of the plate has sunk structure；
It is coated with the high molecular polymer of one layer of liquid on the plate, and solidifies the high molecular polymer；
The high molecular polymer is separated with the plate, obtains the bottom plate of the micropore mold；
Hand-held stick and pull ring are adhesively fixed on the bottom plate.
Preferably, the method also includes sterilizing to the micropore mold of preparation.
Another aspect, the present invention provides a kind of micropore molds to prepare the application in Three-dimensional cell culture cell.
Compared with prior art, the invention has the following advantages:
Porous silica gel mold of the invention is wear-resistant not to crack, long service life；It is non-toxic and tasteless, to cell without any shadow
It rings；Autoclave sterilization can be carried out, Reusability is facilitated；Cost is relatively low for opposite foreign brand name；And Bioexperiment is reproducible；
Porous silica gel mold of the invention can improve the inhomogeneities that in cell cultivation process prepared by three-dimensional cell structure；
Improve the efficiency and yield of the preparation of three-dimensional cell structure；It can control the size of three-dimensional cell structure by changing initial cell volume；
Improve activity and survival rate of the cell during dimensional culture；It is particularly useful for embryoid in embryonic stem cell development Analytical Chemical Experiment
The preparation of body three-dimensional structure, improves cell differentiation efficiency.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of micropore mold in the embodiment of the present invention；
Fig. 2 is the pictorial diagram of micropore mold in the embodiment of the present invention；
Fig. 3 is the pictorial diagram of lower surface bulge-structure in the embodiment of the present invention；
Fig. 4 is the microstructure photograph of lower surface bulge-structure in the embodiment of the present invention；
Fig. 5 is the flow chart that micropore mold is made in the embodiment of the present invention；
Fig. 6 is the flow chart for preparing agar bacterial culture cell in the embodiment of the present invention using micropore mold；
Fig. 7 be the embryoid body three-dimensional structure microphoto prepared in the embodiment of the present invention and its size statistics (1000 cells/
It is a), wherein (a) is with the cell culture cell for having formed three-dimensional cell group；(b) three-dimensional to be collected in cell culture cell
Cell mass (c) rolls into a ball size distribution situation for same batch three-dimensional cell；
Fig. 8 be the embryoid body three-dimensional structure microphoto prepared in the embodiment of the present invention and its size statistics (2000 cells/
It is a), wherein (a) is with the cell culture cell for having formed three-dimensional cell group；(b) three-dimensional to be collected in cell culture cell
Cell mass (c) rolls into a ball size distribution situation for same batch three-dimensional cell；
Fig. 9 be the embryoid body three-dimensional structure microphoto prepared in the embodiment of the present invention and its size statistics (5000 cells/
It is a), wherein (a) is with the cell culture cell for having formed three-dimensional cell group；(b) three-dimensional to be collected in cell culture cell
Cell mass (c) rolls into a ball size distribution situation for same batch three-dimensional cell；
Figure 10 is the aobvious of three-dimensional cell cavity (human embryo stem cell embryoid body) structure prepared in the embodiment of the present invention
Micro- photo；
Figure 11 is the adherent microphoto for growing neurofilament of nerve ball prepared in the embodiment of the present invention；
In figure, appended drawing reference meaning is as follows: 1- holds stick, 2- pull ring, 3- bottom plate.
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with specific embodiment, and reference
Attached drawing, the present invention is described in further detail.
Micropore mold of the invention can be used as the experiment mould of cell three-dimensional structure cultivating system preparation.Such as Fig. 1 and 2 institute
Show, in one embodiment, micropore mold includes hand-held stick 1, pull ring 2 and bottom plate 3.There is proper alignment in the lower surface of bottom plate 3
Bulge-structure, the upper surface of bottom plate 3 have vertical hand-held stick 1 and pull ring 2, and hand-held stick 1 and pull ring 2 are fixedly connected with bottom plate 3.
As shown in Figures 3 and 4, in this embodiment, bottom plate 3 is circle, and basal diameter 35mm is highly 4mm.Protrusion knot
Structure is truncated cone, is highly 0.24mm, floor space 0.66mm2.Bulge-structure can completely inscription rubbing on soft medium, and
It washing and sterilizing can use repeatedly.
Hand-held stick 1 is located at the center point of bottom plate 3, length 40mm.Its material is identical as bottom plate 3 and is completely embedded, convenient
It takes micropore mold, avoids close contact bottom surface microcellular structure and lower dielectric, guarantee that the sterile working in manufacturing process reaches
To requirement, be conducive to going on smoothly for subsequent cell culture experiment.
The diameter of hand-held stick 1 is gradually reduced from the bottom up, is conoid structure, and the structure and bottom plate 3 adhere to securely, no
It is easy to fall off, mold is placed in culture dish conducive to experimenter, and be conducive to repeatedly use.Its floor space diameter is
10mm。
Pull ring 2 is located at the edge of bottom plate 3, and shape is elliptical ring.Outer diameter long axis is 20mm, short axle 13mm, internal diameter long axis
For 16mm, short axle 9mm.Its material is identical as bottom plate 3 and is completely embedded, and divides during being easy to use with the medium below bottom plate
From.
The material of hand-held stick 1, pull ring 2 and bottom plate 3 can be independently selected from silica gel, soft PVC, thermoplastic elastic
The high molecular polymers such as body.
Hand-held stick 1 and pull ring 2 can be adhesively fixed with the integrated molding of bottom plate 3 or hand-held stick 1 and pull ring 2 with bottom plate 3.
As shown in figure 5, in one embodiment, the manufacturing process of micropore mold includes: to utilize 3 D-printing or laser ablation
Etc. technologies obtain the hard plate with sunk structure, sunk structure is chamfered edge taper type, and the hard plate material is unlimited.It will be pre-
Mixed liquid-state silicon gel is started the hard plate that the uniform collapse structure with marshalling is uniformly poured into another side from one side
In, pay attention to having avoided air from being mixed into generation bubble during pouring into.It is incubated in 37 DEG C of baking ovens to being fully cured.It will be cured
Silica gel is separated with hard plate.It is cut into the size almost the same with Tissue Culture Dish, obtains die bottom plate, is bonded on bottom plate
It is fixed to shift to an earlier date ready-made hand-held stick and pull ring, it is cleaned with deionized water, then wrap masking foil and carry out autoclave sterilization, after drying
It is spare.
Liquid-state silicon gel in the embodiment has good mobility, and filling can be come into full contact with hard plate, forms wheel
Clean up clear, the uniform bulge-structure of the high-precision of marshalling.Such silica gel material can carry out autoclave sterilization, guarantee subsequent
The sterility requirements of cell culture experiments.
As shown in fig. 6, in one embodiment, the system of agar bacterial culture cell is carried out using sterilized micropore mold
It is standby.The 2.5% liquid agarose solution that volume is 5mL is added in the culture dish of 60mm.Hand-held stick 1 is held, vertically by micropore
Mold is put into rapidly in not solidified agarose solution, and gently concussion discharge gas, avoids bubble from retaining.Wait agarose solution
After cooled and solidified, pull ring 2 is pulled, micropore mold and agarose are slowly separated.
The agar bacterial culture cell of preparation can be used for the formation of cell mass required by various Three-dimensional cell cultures with
Preparation, such as embryoid body preparation, nerve ball preparation etc..
In one embodiment, in agar bacterial culture cell, it is spare that the submergence of DMEM/F-12 culture medium is added.DMEM/
After F-12 culture medium infiltrates 12 hours, single cell suspension is directly added into agar bacterial culture cell, carries out three-dimensional cell group
Formation.
As Figure 7-9, using the three-dimensional cell group of agar bacterial culture cell preparation, not only dead cell is few, and size
It is uniform, yield is high, illustrate the uniform three-dimensional cell group the present invention can obtain high quality and high quantity.By controlling initial cell
Cell total amount (1000 cells/cell, 2000 cells/cell, 5000 cells/cell) in suspension can achieve control preparation
The big acquisition three-dimensional dimension of the purpose of three-dimensional cell group's size, i.e. initial cell total amount is big, the small acquisition three-dimensional dimension of cell total amount is small,
Illustrate that the present invention has preferable applicability, the three-dimensional cell group for producing a variety of scales can be required according to experimenter.
In one embodiment, embryoid body three-dimensional structure is prepared for using agar bacterial culture cell of the invention.Figure
10 be the microphoto for the three-dimensional cell cavity body structure prepared in the embodiment of the present invention.The present invention is suitable in embryonic stem cell
Embryoid body three-dimensional structure (human embryo stem cell embryoid body) preparation in Development And Differentiation experiment, and can obtain with cavity body structure
Embryoid body, illustrate that its cell differentiation efficiency is guaranteed.
Figure 11 is the adherent microphoto for growing neurofilament of nerve ball prepared in the embodiment of the present invention, illustrates the present invention
Outwardly extending neurofilament is capable of forming after the nerve ball re-posted wall for being equally applicable to the preparation of nerve ball, and preparing.
Particular embodiments described above has carried out further in detail the purpose of the present invention, technical scheme and beneficial effects
Describe in detail bright, it should be understood that the above is only a specific embodiment of the present invention, is not intended to restrict the invention, it is all
Within the spirit and principles in the present invention, any modification, equivalent substitution, improvement and etc. done should be included in protection of the invention
Within the scope of.
Claims (10)
1. a kind of micropore mold, which is characterized in that including bottom plate, hand-held stick and pull ring, wherein the lower surface of the bottom plate have it is whole
The multiple bulge-structures arranged together have vertical hand-held stick and pull ring, the hand-held stick and institute in the upper surface of the bottom plate
Pull ring is stated closely to connect with the bottom plate.
2. micropore mold according to claim 1, wherein the bottom plate is round, rectangle or square.
3. micropore mold according to claim 1, wherein the bulge-structure is frustum of a pyramid shape or truncated cone-shaped.
4. micropore mold according to claim 1, wherein the hand-held stick is located at the center of the bottom plate, it is preferable that institute
The diameter for stating hand-held stick is gradually reduced from the bottom up.
5. micropore mold according to claim 1, wherein the pull ring is located at the edge of the bottom plate, and shape is annulus
Or elliptical ring.
6. micropore mold according to claim 1, wherein the material of the bottom plate, the hand-held stick and the pull ring is
High molecular polymer.
7. micropore mold according to claim 6, wherein the material of the bottom plate, the hand-held stick and the pull ring is only
On the spot it is selected from silica gel, soft PVC or thermoplastic elastomer (TPE).
8. the preparation method of micropore mold described in a kind of any one of claim 1-7, comprising:
A plate is provided, the surface of the plate has sunk structure；
It is coated with the high molecular polymer of one layer of liquid on the plate, and solidifies the high molecular polymer；
The high molecular polymer is separated with the plate, obtains the bottom plate of the micropore mold；
Hand-held stick and pull ring are adhesively fixed on the bottom plate.
9. preparation method according to claim 8, wherein the method also includes going out to the micropore mold of preparation
Bacterium.
10. micropore mold described in a kind of any one of claim 1-7 is preparing the application in Three-dimensional cell culture cell.
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 |
Applications Claiming Priority (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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| CN109016275A true CN109016275A (en) | 2018-12-18 |
| CN109016275B CN109016275B (en) | 2024-01-30 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI766266B (en) * | 2019-04-17 | 2022-06-01 | 中央研究院 | Microwell device and method of manufacturing the same |
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