CN110628618A - Method for accurately controlling shape of single cell by using PDMS stamp - Google Patents
Method for accurately controlling shape of single cell by using PDMS stamp Download PDFInfo
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- CN110628618A CN110628618A CN201910921374.XA CN201910921374A CN110628618A CN 110628618 A CN110628618 A CN 110628618A CN 201910921374 A CN201910921374 A CN 201910921374A CN 110628618 A CN110628618 A CN 110628618A
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- pdms
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- 239000004205 dimethyl polysiloxane Substances 0.000 title claims abstract description 48
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 title claims abstract description 48
- 238000000034 method Methods 0.000 title claims abstract description 26
- 235000013870 dimethyl polysiloxane Nutrition 0.000 title claims description 45
- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 title claims description 35
- 238000004987 plasma desorption mass spectroscopy Methods 0.000 title claims description 35
- 102000016359 Fibronectins Human genes 0.000 claims abstract description 22
- 108010067306 Fibronectins Proteins 0.000 claims abstract description 22
- 239000000758 substrate Substances 0.000 claims abstract description 19
- 239000011521 glass Substances 0.000 claims abstract description 15
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims abstract description 12
- -1 dimethyl siloxane Chemical class 0.000 claims abstract description 12
- 229910052710 silicon Inorganic materials 0.000 claims abstract description 12
- 239000010703 silicon Substances 0.000 claims abstract description 12
- 230000012010 growth Effects 0.000 claims abstract description 7
- 238000001312 dry etching Methods 0.000 claims abstract description 6
- 230000010261 cell growth Effects 0.000 claims abstract description 5
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 28
- 239000007787 solid Substances 0.000 claims description 17
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 16
- RVGRUAULSDPKGF-UHFFFAOYSA-N Poloxamer Chemical compound C1CO1.CC1CO1 RVGRUAULSDPKGF-UHFFFAOYSA-N 0.000 claims description 12
- 238000010438 heat treatment Methods 0.000 claims description 12
- 229960000502 poloxamer Drugs 0.000 claims description 12
- 229920001983 poloxamer Polymers 0.000 claims description 12
- 239000002953 phosphate buffered saline Substances 0.000 claims description 11
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 10
- 238000002444 silanisation Methods 0.000 claims description 9
- 239000011259 mixed solution Substances 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- 238000004140 cleaning Methods 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 238000005520 cutting process Methods 0.000 claims description 3
- 238000012545 processing Methods 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 2
- 238000012546 transfer Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 31
- 238000000059 patterning Methods 0.000 description 3
- 238000003491 array Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 210000004292 cytoskeleton Anatomy 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J7/00—Chemical treatment or coating of shaped articles made of macromolecular substances
- C08J7/12—Chemical modification
-
- 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
-
- 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
- C12N5/0068—General culture methods using substrates
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2383/00—Characterised by the use of macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing silicon with or without sulfur, nitrogen, oxygen, or carbon only; Derivatives of such polymers
- C08J2383/04—Polysiloxanes
-
- 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
- C12N2533/30—Synthetic polymers
-
- 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
- C12N2533/50—Proteins
- C12N2533/52—Fibronectin; Laminin
Abstract
The invention provides a method for accurately controlling the shape of a single cell by using a PDMS stamp, belongs to the technical field of cell biology, and relates to a method for accurately controlling the boundary of a single cell, which can change the shape of a cell and is used for specific cell experiments. The method comprises the steps of firstly designing a micro-pattern array with a specific size, manufacturing a corresponding silicon plate through dry etching, pouring dimethyl siloxane PDMS (polydimethylsiloxane) by using the silicon plate to obtain a stamp, dripping fibronectin on the surface of the micro-pattern of the stamp, rubbing the fibronectin on the surface of a glass substrate subjected to hydrophobic treatment, and forming a hydrophilic micro-pattern area for cell growth. The cells are planted on the glass substrate, so that the adhesion boundary of the cells can be restrained, and the growth shape of the cells can be controlled.
Description
Technical Field
The invention belongs to the technical field of cell biology, and relates to a method for artificially designing a micro pattern to change a cell growth boundary based on an adhesion spreading principle of cells and a microfluidic technology so as to accurately control the shape of the cells.
Background
Most cells must adhere to the substrate to grow. For a single cell, the growth environment is very wide, so that the cell can grow into different shapes randomly. By preparing a substrate with a specific micro-pattern and adhering cells on the substrate to grow into cells with specific shapes, partial structures of the cells, such as growth areas of local adhesion spots and arrangement of cytoskeletons, can be changed. Such a cell patterning technique can be widely used in various research fields such as cell biology, tissue engineering, drug screening, and wound treatment. In the current cell patterning technology, the shape of a cell population is mostly controlled, the patterning aiming at single cells is rarely carried out, the operation is complicated, and the use efficiency is not high. There is a need for a method that is simple to operate and can accurately control the shape of the growth of single cells.
Disclosure of Invention
The invention aims to provide a method for accurately controlling the shape of a single cell by utilizing a PDMS stamp, which controls the growth shape of the single cell by designing a PDMS stamp containing a micro-pattern array with a specific shape and a specific size, and mainly aims to artificially change the partial structure of the cell so as to provide a tool for better researching the function of the partial structure of the cell.
The technical scheme of the invention is as follows:
a method for accurately controlling the shape of a single cell by using a PDMS stamp comprises the following specific processes:
designing a micro-pattern array for cell growth, and obtaining a silicon plate 1-2 through dry etching processing;
pouring the polydimethylsiloxane PDMS mixed solution 1-1 on a silicon plate 1-2, heating and cutting the silicon plate 1-2 to obtain a solid PDMS stamp 1-3, and performing silanization treatment on the surface of the micro-pattern of the solid PDMS stamp 1-3;
thirdly, dripping fibronectin solution on the surface of the micro-pattern of the solid PDMS stamp 1-3 and drying;
step four, performing hydrophobization treatment on the glass substrates 1-5 by using poloxamer solution;
step five, pasting the micro-pattern surface of the solid PDMS stamp 1-3 on the surface of the glass substrate 1-5, and transferring fibronectin 1-4 to the glass substrate 1-5;
and step six, cell planting is carried out, and the planted cells can only grow in the fibronectin 1-4 area with the preset shape, so that the growth boundary of the cells is accurately controlled.
In the first step, the side length of the micro-pattern in the micro-pattern array is not more than 20 μm, the distance between the micro-patterns is not less than 100 μm, and the dry etching depth is 10 μm.
In the second step, the polydimethylsiloxane PDMS mixed solution 1-1 is prepared by mixing a prepolymer of polydimethylsiloxane PDMS and a curing agent according to a mass ratio of 10: 1; the heating temperature is 70-80 deg.C, and the heating time is 60-90 min.
In the second step, the silanization treatment process specifically comprises the following steps: mixing 3-aminopropyltriethoxysilane and an acetone solution according to the proportion of 1:15-1:20 to prepare a silanization solution; dropping the silanized solution on the surface of the micro-pattern array of the solid PDMS stamp 1-3 for 20-30min, sequentially cleaning with acetone, absolute ethyl alcohol and pure water, and heating at 110-120 ℃ for 60-90 min.
In the third step, the fibronectin solution is prepared by dissolving fibronectin in phosphate buffered saline at a ratio of 2% w/v.
In the fourth step, the poloxamer solution is prepared by dissolving poloxamer into phosphate buffered saline solution according to the proportion of 0.5% w/v.
The invention has the beneficial effects that: the invention can culture cells with specific shapes by self-designing the micro-patterns of the cell growth area, and purposefully change the cell structure for corresponding scientific experimental research. The technology is simple and easy to operate, and the stamp can be repeatedly used.
Drawings
FIG. 1 is a schematic flow chart of PDMS stamp method.
FIG. 2 is a schematic diagram of a PDMS stamp structure.
In the figure: 1-1 polydimethylsiloxane PDMS mixed solution; 1-2 silicon plates; 1-3 solid PDMS stamp; 1-4 fibronectin; 1-5 glass substrates; 2-1 micropattern array.
Detailed Description
The technical solution of the present invention will be further described with reference to specific examples.
1. Design and fabrication of micro-patterned silicon plates
Each micro pattern was designed to have a side length of 20 μm and a pitch of 100 μm in order to obtain a specific shape of individual cells. The micropattern was designed using Auto-CAD software to form a 30 x 30 array as shown in figure 2. And processing the micro-pattern array by dry etching, wherein the depth is 10 mu m, and obtaining the silicon plate of the micro-pattern array.
2. Preparation of a polydimethylsiloxane stamp
As shown in figure 1, the prepolymer of polydimethylsiloxane PDMS and the curing agent are mixed and stirred evenly according to the proportion of 10:1, poured on a silicon plate 1-2 made of a micro-pattern array and placed on a heating plate at 80 ℃ for baking for 1h until the polydimethylsiloxane is solid. And (3) removing the micro-pattern arrays, and cutting the micro-pattern arrays to obtain solid PDMS stamps 1 to 3.
3. Hydrophobic modified glass substrate
Poloxamer was dissolved in PBS buffer at a ratio of 0.5% (w/v) to prepare a poloxamer solution. Dropping the poloxamer solution on a glass substrate 1-5, sucking off the redundant poloxamer solution after 1h, standing for 20min and air-drying.
4. Transfer of fibronectin to a substrate
3-Aminopropyl-Triethoxysilane and acetone solution are mixed according to the proportion of 1:19 to prepare a silanization solution. Dropping the silanized solution on the surface of the micro-pattern array of the solid PDMS stamp 1-3 for 30min, sequentially cleaning with acetone, absolute ethyl alcohol and pure water, and heating on a heating plate at 120 ℃ for 1 h. Fibronectin (fibronectin dissolved in phosphate buffered saline at a ratio of 2% (w/v)) 1-4 drops were applied to the surface of the solid PDMS stamp 1-3 microarray, left to stand for 40min and dried. The surface of the micro-pattern array of the solid PDMS stamp 1-3 was attached to the glass substrate 1-5 which had been subjected to hydrophobic treatment, and a load of 0.06N was applied for 10 s. The stamp is removed and fibronectin 1-4 is transferred to the glass substrate 1-5 in a designed micro-pattern array.
5. Cell planting
Cells were seeded on this glass substrate by conventional culture techniques. Cells can only grow on the micro-patterned areas of the glass substrate containing hydrophilic fibronectin, resulting in cells of a specific shape.
Claims (10)
1. A method for accurately controlling the shape of a single cell by using a PDMS stamp is characterized by comprising the following specific steps:
step one, designing a micro-pattern array for cell growth, and obtaining a silicon plate (1-2) through dry etching processing;
pouring the polydimethylsiloxane PDMS mixed solution (1-1) on a silicon plate (1-2), heating the silicon plate (1-2), cutting to obtain a solid PDMS stamp (1-3), and performing silanization treatment on the surface of the micro-pattern of the solid PDMS stamp (1-3);
thirdly, dripping fibronectin solution on the micro-pattern surface of the solid PDMS stamp (1-3) and drying;
step four, performing hydrophobic treatment on the glass substrate (1-5) by using poloxamer solution;
step five, pasting the micro-pattern surface of the solid PDMS stamp (1-3) on the surface of the glass substrate (1-5) to transfer the fibronectin (1-4) to the glass substrate (1-5);
and step six, cell planting is carried out, and the planted cells can only grow in the fibronectin (1-4) area with a preset shape, so that the growth boundary of the cells is accurately controlled.
2. The method as claimed in claim 1, wherein in the first step, the side length of the micro-patterns in the micro-pattern array is not more than 20 μm, the micro-pattern pitch is not less than 100 μm, and the dry etching depth is 10 μm.
3. The method for precisely controlling the shape of a single cell by using a PDMS stamp according to claim 1 or 2, wherein in the second step, the polydimethylsiloxane PDMS mixed solution (1-1) is prepared by mixing a prepolymer of polydimethylsiloxane PDMS and a curing agent according to a mass ratio of 10: 1; the heating temperature is 70-80 deg.C, and the heating time is 60-90 min.
4. The method according to claim 1 or 2, wherein the silanization process in the second step is specifically: mixing 3-aminopropyltriethoxysilane and an acetone solution according to the proportion of 1:15-1:20 to prepare a silanization solution; dropping the silanized solution on the surface of the micro-pattern array of the solid PDMS stamp (1-3) for 20-30min, sequentially cleaning with acetone, absolute ethyl alcohol and pure water, and heating at 110-120 ℃ for 60-90 min.
5. The method of claim 3, wherein in the second step, the silanization process comprises: mixing 3-aminopropyltriethoxysilane and an acetone solution according to the proportion of 1:15-1:20 to prepare a silanization solution; dropping the silanized solution on the surface of the micro-pattern array of the solid PDMS stamp (1-3) for 20-30min, sequentially cleaning with acetone, absolute ethyl alcohol and pure water, and heating at 110-120 ℃ for 60-90 min.
6. A method for accurately controlling the shape of individual cells using PDMS stamps according to claim 1, 2 or 5, wherein in step three, the fibronectin solution is prepared by dissolving fibronectin in phosphate buffered saline at a ratio of 2% w/v.
7. The method of claim 3, wherein the fibronectin solution is prepared by dissolving fibronectin in Phosphate Buffered Saline (PBS) at a ratio of 2% w/v.
8. The method of claim 4, wherein the fibronectin solution is prepared by dissolving fibronectin in Phosphate Buffered Saline (PBS) at a ratio of 2% w/v.
9. A method for accurately controlling the shape of individual cells using PDMS stamps according to claim 1, 2, 5, 7 or 8, wherein in step four the poloxamer solution is prepared by dissolving poloxamer in phosphate buffered saline at a ratio of 0.5% w/v.
10. The method of claim 6, wherein in the fourth step, the poloxamer solution is prepared by dissolving poloxamer in phosphate buffered saline at a ratio of 0.5% w/v.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910921374.XA CN110628618A (en) | 2019-09-26 | 2019-09-26 | Method for accurately controlling shape of single cell by using PDMS stamp |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910921374.XA CN110628618A (en) | 2019-09-26 | 2019-09-26 | Method for accurately controlling shape of single cell by using PDMS stamp |
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| Publication Number | Publication Date |
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| CN110628618A true CN110628618A (en) | 2019-12-31 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910921374.XA Pending CN110628618A (en) | 2019-09-26 | 2019-09-26 | Method for accurately controlling shape of single cell by using PDMS stamp |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102465119A (en) * | 2010-11-12 | 2012-05-23 | 国家纳米科学中心 | Substrate for cell micropatterning growth as well as preparation method and application thereof |
| JP2012187072A (en) * | 2011-03-14 | 2012-10-04 | National Institute Of Advanced Industrial Science & Technology | Method for producing cell-immobilized substrate having micropattern of cell adhesion region |
| CN108384744A (en) * | 2018-01-17 | 2018-08-10 | 北京航空航天大学 | A kind of curved surface albumen micro-patterning production method |
| CN108641892A (en) * | 2018-04-17 | 2018-10-12 | 广州波奇亚标准及检测技术有限公司 | One kind being used for the patterned novel micro-contact printing system of cell |
| CN109082155A (en) * | 2017-08-24 | 2018-12-25 | 中山大学 | A kind of application of tannic acid as micro-contact printing ink in cell patterning |
-
2019
- 2019-09-26 CN CN201910921374.XA patent/CN110628618A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102465119A (en) * | 2010-11-12 | 2012-05-23 | 国家纳米科学中心 | Substrate for cell micropatterning growth as well as preparation method and application thereof |
| JP2012187072A (en) * | 2011-03-14 | 2012-10-04 | National Institute Of Advanced Industrial Science & Technology | Method for producing cell-immobilized substrate having micropattern of cell adhesion region |
| CN109082155A (en) * | 2017-08-24 | 2018-12-25 | 中山大学 | A kind of application of tannic acid as micro-contact printing ink in cell patterning |
| CN108384744A (en) * | 2018-01-17 | 2018-08-10 | 北京航空航天大学 | A kind of curved surface albumen micro-patterning production method |
| CN108641892A (en) * | 2018-04-17 | 2018-10-12 | 广州波奇亚标准及检测技术有限公司 | One kind being used for the patterned novel micro-contact printing system of cell |
Non-Patent Citations (1)
| Title |
|---|
| 邱昌俊等: "细胞外基质蛋白模式对成肌细胞分化的影响", 《中国生物医学工程学报》 * |
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Application publication date: 20191231 |