CN102796659A - Porous single cell observation plate and use thereof - Google Patents
Porous single cell observation plate and use thereof Download PDFInfo
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- CN102796659A CN102796659A CN2012102991758A CN201210299175A CN102796659A CN 102796659 A CN102796659 A CN 102796659A CN 2012102991758 A CN2012102991758 A CN 2012102991758A CN 201210299175 A CN201210299175 A CN 201210299175A CN 102796659 A CN102796659 A CN 102796659A
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- 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
- C12M23/04—Flat or tray type, drawers
Abstract
The invention provides a porous single cell observation plate and a use thereof. The porous single cell observation plate comprises multiple small-hole units. Each one of the small-hole units comprises a small hole and multiple repetitive cell culture rooms and diffusion paths, and the repetitive cell culture rooms are communicated with the small hole by the diffusion paths. The porous single cell observation plate is mainly used for cell biology and high-throughput gene phenotype screening. The porous single cell observation plate adopts a small hole-cell microculture chamber combined technology. According to different high-throughput yeast cell microfloras, the porous single cell observation plate utilizes small holes and a culture chamber having a specific height and used for yeast cell monolayer growth observation so that yeast cell monolayer growth can be realized and observed well. A hole distance matching with a multi-channel pipettor and a liquid automatic microarrayer is designed so that high throughput and automation of a system are realized really. Only 10 microliters of a liquid can cover the porous single cell observation plate and an observation process can be carried out for about 5-10 hours.
Description
Technical field
Micro-fluidic chip (Microfluidic chip) technology is the field of develop rapidly in recent years, its characteristic mainly be its hold fluidic resulting structure (passage, reaction chamber and some other functional component) at least on a latitude for the micron order yardstick.Micro-fluidic chip is integrated into basic operation units such as the specimen preparation of biological, chemistry, medical analysis process, reaction, separation, detection on the chip of a centimeter scale; Because it can realize high-throughout cellular array; Accurate control cell culture environment; Analyze fast, the consumption of a large amount of minimizing reagent is made the strongest instrument that cell is analyzed now and become.But most of research work before all need be by automatic syringe pump system, for example people (C.Luo, X.Zhu such as Luo; T.Yu, et al., A fast cell loading and high-throughput microfluidic system for long-term cell culture in zero-flow environments; Biotechnology and Bioengineering, 2008,101; 190-195.) and people (L.Wang, X.Ni, C.Luo such as Wang; Et al., Self-loading and cell culture in one layer microfluidic devices, Biomedical Microdevices; 2009,11, research 679-684.); Itself and the existing volley of rifle fire or the some liquid system can not be compatible automatically are difficult to accomplish high-throughput (48 different samples and more than) truly, and existing commercial porous plate and be not suitable for doing real-time yeast single cell and follow the tracks of.
Recently, report (G.Yang, Quan are arranged; Y., Wang, W.; Et al., Dynamic equilibrium between cancer stem cells and non-stem cancer cells in human SW620 and MCF-7 cancer cell populations, BRITISH JOURNAL OF CANCER; 2012,106,1512-1519.) utilize aperture and fluid channel bonded mode; Utilize the special air-breathing character of polymer P DMS material, import cancer cells in the back of degassing, the cancer cells solution that then substitutes in the aperture is nutrient solution; The influence that traditional porous plate changes liquid operation pair cell has been stopped in this operation, is fit to dyeing and real-time monitored in order to make cancer cells.This technology is still inapplicable for high-throughout yeast cell research.
Summary of the invention
To the weak point of research cell field, the present invention proposes the unicellular observation board of a kind of porous.
Another purpose of the present invention is the method that proposes to use the unicellular observation board of said porous.
The concrete technical scheme that realizes above-mentioned purpose of the present invention is:
The unicellular observation board of a kind of porous comprises a plurality of apertures unit, and each aperture unit comprises an aperture, a plurality of multiple cell culture chamber and diffusion admittance, and each cell culture chamber is connected with aperture through diffusion admittance.
Wherein, the unitary number of aperture is 1-1000, and the diameter of aperture is 2-4mm, and the pitch of holes of aperture is that 4-10mm and pitch of holes all equate.Pitch of holes is impartial, make the unicellular observation board of this porous can with this area volley of rifle fire commonly used with point sample instrument institute is adaptive automatically.
The height of said diffusion admittance, width or diameter are all greater than the characteristic dimension of cell.The size of passage can let cell or bacterium pass through greater than the diameter of cell.For example, the budding yeast cell is 3-7 μ m for the general magnitude range of different mutant strains, and the fission yeast diameter is 3.5 μ m, bacillus yardstick 1-3 μ m.
Wherein, the height of cell culture chamber be the cell minimum feature size 1-2 doubly.The height of cell culture chamber is no more than the twice of the cell minimum feature size of being studied, and cell is a monolayer growth in culturing room.
The unicellular observation board of described porous is to process with YSR 3286 (PDMS).
A kind of method for preparing the unicellular observation board of porous of the present invention's proposition is to use the method for soft lithographic and punching to prepare the unicellular observation board of said porous.
Use the method for the unicellular observation board observation of the porous cell of the present invention's proposition, comprise step:
1) the unicellular observation board of the said porous of plasma treatment places the unicellular observation board of said porous on one plane then;
2) will contain in the said a plurality of apertures of the even adding of solution of cell;
3) after solution that said aperture, cell culture chamber, diffusion admittance are all contained cell is full of, remove the residual solution that contains cell in the aperture.
Because to the wellability of liquid with to the characteristic of gas absorption, cell solution is absorbed into cell (mikrobe) culturing room to PDMS automatically after plasma treatment, when cell culture chamber is accomplished by liquid filling, the indoor flow rate of liquid of cell cultures reduces to zero.Because the cell cultures chamber is filled the back for the dead band, when substituting in the aperture solution, the cell culture chamber liquid that can not flows, only through diffusional interchange nutrition.
Wherein, in the said step 3), behind the residual solution that contains cell in the removal aperture, also comprise the step that is full of aperture with not celliferous solution.Liquid through the volley of rifle fire or liquid dot model machine substitute in the aperture realizes that the environment of removing the cell solution in the aperture and realizing pair cell culturing room inner cell (mikrobe) substitutes.Said not celliferous solution preferably uses cell culture fluid.
Wherein, in the said step 3), also comprise the step that covers the unicellular observation board of said porous.Can prevent liquid evaporation like this.
The said solution that contains cell can be various kinds of cell solution, for example, adopts the volley of rifle fire or point sample instrument to add respectively in the different apertures different mutant bacterial bacterial strains.
Beneficial effect of the present invention is:
The direction that the unicellular observation board of porous that the present invention proposes is mainly used is cytobiology, the screening of high-throughput gene phenotype etc.Device of the present invention adopts aperture and cell culture chamber bonded mode; Can carry out high-throughout research to different yeast cell floras; 1) adopts aperture and the cultivation chamber of the certain height observed for the yeast cell monolayer growth; Promptly cultivate 2 times (optimal value are 1.5 times) of chamber height, make the cell monolayer growth, help observational study less than yeast cell; 2) pitch of holes of the automatic point sample instrument coupling of the design and the volley of rifle fire and liquid, the system that makes really can realize high Tonghua and robotization, is applicable to the research work to yeast genes involved function more; 3) cover the unicellular observation board of this porous, can carry out for up to about 5-10 hour observation only with the liquid of 10 μ l.The design of the different scale through cell cultures chamber and diffusion admittance can also be carried out high-throughput research to multiple mikrobe.
Description of drawings
Fig. 1 is the mask a of the embodiment of the invention 1 Computer Aided Drawing.
Fig. 2 is the mask b that area of computer aided is drawn in the embodiment of the invention 1.
Fig. 3 is the vertical view of the unicellular observation board of porous in the embodiment of the invention 1.
Fig. 4 is a unitary vertical view of aperture.
Fig. 5 is the partial enlarged drawing of circle part among Fig. 4.
Among the figure, 1 is aperture, and 2 is cell culture chamber, and 3 is diffusion admittance.
Embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.
Wherein PDMS preformed body is available from GE company, and model is RTV615.
The micro-fluidic chip that the present invention proposes can be used for the research of human body cell, vegetable cell, zooblast, bacterium, and its cell dimension is 10
-4-10
-6M.
Embodiment 1: the unicellular observation board of preparation porous
1) draw with L-Edit, like mask a and the mask b of Fig. 1 and Fig. 2, the hole diameter of mask a is 3mm, and pitch of holes is 4.5mm, and this pitch of holes and the volley of rifle fire (DragonMed, the DR27506 0.5 μ l-10 μ l 8 passage volley of rifle fires) are suitable.Mask b is cell culture chamber and diffuser figure layer, and different according to the mikrobe of research, the cross-sectional area of diffusion admittance can be designed to the 2-80% of cell culture chamber area.In the present embodiment, cell culture chamber is that diameter is 250 microns a roundlet, highly is 8 μ m, and through one section 40 microns wide, 40 microns high, 60 microns long diffuser link to each other with aperture.Repeat the aperture unit and can be 4 * 2 to 36 * 16.In the present embodiment, multiple aperture unit is 12 * 4.
2) adopt photoresist material SU83005, through photoresist spinner at the photoresist material (rotating speed 2000rpm, the time is 30s) that is coated with 7 microns on the silicon chip; Through 65 degrees centigrade of bakings 1 minute; 95 degrees centigrade of bakings after the baking, were put in exposure machine under exposure before 5 minutes, the mask of employing for the moon edition of design Fig. 2 figure; Time shutter is 50 seconds, and light intensity is 20mW/cm
2Exposure back sample be put in 65 degrees centigrade 1 minute, baking is 5 minutes after 95 degrees centigrade; Through behind the developing liquid developing, obtain mould like Fig. 2.Through utilizing SU83025, step is the same again, preceding baking condition be 65 degrees centigrade 1 minute, 95 degrees centigrade 10 minutes, adopt corresponding mask, the time shutter is 90 seconds, light intensity 20mW/cm
2, back baking condition be 65 degrees centigrade 1 minute, 95 degrees centigrade 10 minutes.The figure layer of alignment Fig. 1 makes the mould like Fig. 3.
3) be poured on the mould with liquid PDMS preformed body (A:B=8:1), the height of PDMS is approximately 1-2mm, and 1h makes PDMS solidify in 75 degree baking ovens.Downcut the back after corresponding aperture place adopts the punch tool of 3mm diameter to punch, clean, for use with 70% alcohol ultrasonic clean.
Embodiment 2: the unicellular observation board research of the porous of application implementation example 1 cell
The unicellular observation board of porous of embodiment 1 preparation is through (plasma treatment machine after the plasma treatment; Harrick company; PDC-002; Handled one minute) picture surface is put in downwards on the clean slide, adopts the volley of rifle fire in different apertures, to add budding yeast flora 1 μ l solution (the flora density 3 * 10 that identical scale size is 4-5 μ m
7Individual/as ml) to wait for 1 minute, cell solution can be inhaled into whole cell chamber.Adopt the cell solution in the volley of rifle fire sucking-off aperture; Wash twice aperture (each 10 μ l) with fresh nutrient solution; Then with aperture replace with hope the residing solution of cell (YPDA nutrient solution) environment after, cover above the unicellular observation board of PDMS porous with onesize slide.
This device is put on the microscopic system that has automatic translation stage and focusing automatically, can carries out the unicellular record of sequential different cells/bacteria types.Cover the unicellular observation board of this porous, can carry out for up to 10 hours observation.
Embodiment 3: with the unicellular observation board research of 4 * 2 porous cell
Have 4 * 2 unicellular observation boards of the unitary porous of aperture through plasma treatment after picture surface be put in downwards on the clean slide; Adopt automatic point sample instrument (beckman company, biomek FX) to add 4 kinds of different budding yeast floras 1 μ l solution (flora density 2 * 10 at different apertures
7Individual/ml, 3 * 10
7Individual/ml, 4 * 10
7Individual/ml, 5 * 10
7Individual/as ml), to wait for 2-3 minute, cell solution can be filled whole cell chamber.Adopt the cell solution in the automatic point sample instrument sucking-off aperture, wash twice aperture (each 10ul) with fresh nutrient solution, then aperture is replaced with YPDA nutrient solution environment after, cover above the unicellular observation board of PDMS porous with onesize slide.
This device is put on the microscopic system that has automatic translation stage and focusing automatically, can carries out the unicellular record of sequential different cells/bacteria types.Cover the unicellular observation board of this porous, can carry out for up to 10 hours observation.
Embodiment 4
The aperture that has 4 * 2 unicellular observation boards of the unitary porous of aperture is the square of the 400 μ m length of sides, and other is with embodiment 1.
Embodiment 5
The unicellular observation board of porous is used for fission yeast or colibacillary experiment.In the present embodiment, (Escherichia coli, E.coli) the cell minimum feature size is 1 μ m (shaft-like, diameter is 1 μ m, and length is 3-8 μ m) to the intestinal bacteria of studying.The unicellular observation board of employed porous is totally 36 * 16 multiple aperture unit.The cell culture chamber height is 1.5 μ m.The diffusion admittance height and width are 4 μ m.
Embodiment 6
The unicellular observation board of 4 * 2 porous is used for the parallel comparison of different strains under varying environment, for example various mutant strains, nutrition, microbiotic etc.In the present embodiment, use the mutant strain of budding yeast, totally four kinds, join respectively in the different apertures unit, walk abreast relatively.
Above embodiment describes preferred implementation of the present invention; Be not that scope of the present invention is limited; Design under the prerequisite of spirit not breaking away from the present invention; Various modification and improvement that the common engineering technical personnel in this area make technical scheme of the present invention all should fall in the definite protection domain of claims of the present invention.
Claims (9)
1. the unicellular observation board of porous is characterized in that, comprises a plurality of apertures unit, and each aperture unit comprises an aperture, a plurality of multiple cell culture chamber and diffusion admittance, and each cell culture chamber is connected with aperture through diffusion admittance.
2. the unicellular observation board of porous as claimed in claim 1 is characterized in that, the unitary number of aperture is 1-1000, and the diameter of aperture is 2-4mm, and the pitch of holes of aperture is that 4-10mm and pitch of holes all equate.
3. the unicellular observation board of porous as claimed in claim 1 is characterized in that the height of said diffusion admittance, width or diameter are all greater than the cell characteristic size.
4. the unicellular observation board of porous as claimed in claim 1 is characterized in that, the height of said cell culture chamber is 1-2 a times of cell minimum feature size.
5. the unicellular observation board of porous as claimed in claim 1 is characterized in that it is to process with YSR 3286.
6. a method for preparing the unicellular observation board of the arbitrary described porous of claim 1-5 is characterized in that, is to use the method preparation of soft lithographic and punching.
7. application rights requires the method for the unicellular observation board observation of the arbitrary described porous of 1-5 cell, it is characterized in that, comprises step:
1) the unicellular observation board of the said porous of plasma treatment places the unicellular observation board of said porous on one plane then;
2) will contain in the said aperture of the even adding of solution of cell;
3) after solution that said aperture, cell culture chamber, diffusion admittance are all contained cell is full of, remove the residual solution that contains cell in the aperture.
8. method as claimed in claim 7 is characterized in that, in the said step 3), behind the residual solution that contains cell in the removal aperture, also comprises the step that is full of aperture with not celliferous solution.
9. like claim 7 or 8 described methods, it is characterized in that, in the said step 3), also comprise the step that covers the unicellular observation board of said porous.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210299175.8A CN102796659B (en) | 2012-08-21 | 2012-08-21 | Porous single cell observation plate and use thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210299175.8A CN102796659B (en) | 2012-08-21 | 2012-08-21 | Porous single cell observation plate and use thereof |
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| Publication Number | Publication Date |
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| CN102796659A true CN102796659A (en) | 2012-11-28 |
| CN102796659B CN102796659B (en) | 2014-06-11 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015051678A1 (en) * | 2013-10-09 | 2015-04-16 | 上海交通大学 | Micropore plate for high-throughput detection and application thereof |
| CN106047665A (en) * | 2016-07-29 | 2016-10-26 | 北京大学 | Method for high-throughput cell migration research and cell migration research system |
| CN107533218A (en) * | 2015-01-22 | 2018-01-02 | Idea生物医学有限公司 | Automatically the method and apparatus focused on |
| CN111257206A (en) * | 2018-11-30 | 2020-06-09 | 山东大学 | Method for manufacturing cell distribution analysis device |
| CN114650884A (en) * | 2019-09-10 | 2022-06-21 | 浩康生物系统公司 | Mesh for cell layer preparation |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101275114A (en) * | 2008-04-22 | 2008-10-01 | 北京大学 | Microflow cell culture array and application thereof |
-
2012
- 2012-08-21 CN CN201210299175.8A patent/CN102796659B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101275114A (en) * | 2008-04-22 | 2008-10-01 | 北京大学 | Microflow cell culture array and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| 《British Journal of Cancer》 20120403 G Yang等 Dynamic equilibrium between cancer stem cells and non-stem cancer cells in human SW620 and MCF-7 cancer cell populations 第1512-1519页 1-9 第106卷, 第9期 * |
| CHUNXIONG LUO等: "A Fast Cell Loading and High-Throughput Microfluidic System for Long-Term Cell Culture in Zero-Flow Environments", 《BIOTECHNOLOGY AND BIOENGINEERING》 * |
| G YANG等: "Dynamic equilibrium between cancer stem cells and non-stem cancer cells in human SW620 and MCF-7 cancer cell populations", 《BRITISH JOURNAL OF CANCER》 * |
| 叶雄英等: "PDMS 氧等离子体表面改性工艺参数优化", 《清华大学学报( 自然科学版)》 * |
| 薛向尧等: "PDMS氧等离子体长效活性表面处理及与S i的键合", 《功能材料与器件学》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015051678A1 (en) * | 2013-10-09 | 2015-04-16 | 上海交通大学 | Micropore plate for high-throughput detection and application thereof |
| CN107533218A (en) * | 2015-01-22 | 2018-01-02 | Idea生物医学有限公司 | Automatically the method and apparatus focused on |
| CN107533218B (en) * | 2015-01-22 | 2020-06-12 | Idea生物医学有限公司 | Method and apparatus for auto-focusing |
| CN106047665A (en) * | 2016-07-29 | 2016-10-26 | 北京大学 | Method for high-throughput cell migration research and cell migration research system |
| CN106047665B (en) * | 2016-07-29 | 2018-07-06 | 北京大学 | It is a kind of for the method for high-flux cell migration research and cell migration research system |
| CN111257206A (en) * | 2018-11-30 | 2020-06-09 | 山东大学 | Method for manufacturing cell distribution analysis device |
| CN114650884A (en) * | 2019-09-10 | 2022-06-21 | 浩康生物系统公司 | Mesh for cell layer preparation |
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| CN102796659B (en) | 2014-06-11 |
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