CN101148324A - Preparation method for cell cultivation chip based on ITO glass substance and application thereof - Google Patents
Preparation method for cell cultivation chip based on ITO glass substance and application thereof Download PDFInfo
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- CN101148324A CN101148324A CNA2007100459972A CN200710045997A CN101148324A CN 101148324 A CN101148324 A CN 101148324A CN A2007100459972 A CNA2007100459972 A CN A2007100459972A CN 200710045997 A CN200710045997 A CN 200710045997A CN 101148324 A CN101148324 A CN 101148324A
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Abstract
The present invention relates to one kind of ITO glass substrate-based cell culture chip and its preparation process and application. The cell culture chip is prepared through the following steps: adopting ITO glass as substrate material, painting AZ4620 photoresist to the side without sputtered ITO film layer, exposing and developing; mixing PDMS monomer and curing agent, pouring the mixture to the side with ITO film and heating to cure; etching the ITO glass in etching solution; stripping PDMS from ITO film; bonding the PDMS film and ITO glass with etched micro pipeline under the action of oxygen plasma to obtain cell culture chip; and connecting external temperature control system to the side without bonded PDMS film of the ITO glass. The ITO glass substrate-based cell culture chip has no hydrophobic problem, simplified preparation process and direct chip temperature control. It may be applied widely for cell research.
Description
Technical field
The invention belongs to the cell cultivation chip field, particularly relate to a kind of preparation method and application thereof of glass cell cultivation chip.
Background technology
Utilize CO
2The conventional cultural method of incubator culturing cell is a kind of good method of studying viable cell, has all obtained using widely in every field such as virusology, immunology, genetics, oncology, but still has existed obviously not enough.At first be that the cell manipulation method is loaded down with trivial details, the microenvironment of wayward cell growth is difficult to the true environment of comprehensive simulated cell life; Be exactly that the cell desired number is huge in addition, be difficult for carrying out single celled control and manipulation, observe not directly, expend century.With micro-fluidic chip and the cell culture technology resulting cell cultivation chip that combines, has obvious superiority.Say that from device size the chip features size can be comparable with the cell size, helps to realize single celled Biochemistry Experiment; On structure, interior mass transfer of chip and thermal conduction are very fast, help the fast and stable of cell culture environment; Say from integration, can integrated multiple analysis means on the chip, realize the microminiaturization of cell cultures.Because the existence of these advantages, cell cultivation chip plays an increasingly important role in a plurality of fields such as cell migration, cytodifferentiation, drug screenings.
When design chips, must consider the bio-compatibility of chip material, the influence of the mobile mechanical force pair cell that causes of nutrient solution and the factors such as transmission conveying of effective constituent.Wherein, how processing the good material of bio-compatibility by simple fine process fast is the matter of utmost importance that needs solution.The material that is used to process cell cultivation chip at present mainly contains silicon, glass and polydimethylsiloxane polymer materialss such as (PDMS).The PDMS chip adopts the replica stamper fabrication techniques usually, can realize that the high-fidelity of micrometre-grade pattern is duplicated, and has good bio-compatibility, can directly cultivate various types of cells.But, be subject to the organic reagent effect and produce phenomenons such as microchannel distortion, swelling because PDMS is a resilient material.And the PDMS pipe surface is a hydrophobicity, is difficult to wetting hydrophilic solution, is easy to generate bubble and damaging cells in nutrient solution sample introduction process.
Silicon materials are hydrophilic, can tolerate liquid such as sulfuric acid, hydrochloric acid, and character is relatively stable, the processing technology maturation.Its shortcoming mainly be frangible, price is higher, light tight, electrical insulating property is good inadequately, the surface chemistry behavior is complicated.Compare with silicon, PDMS material, the glass cheapness, be easy to get, and have good hydrophilicity energy, mechanical property, making equipment is compatible mutually with traditional IC (unicircuit) processing unit.Yet, the method of making glass-chip now is: thin-film materials such as sputter layer of metal or polysilicon carry out wet method or dry etching then as etch mask layer on substrate of glass, utilize high-voltage or high temperature to carry out bonding at last in super-clean environment, whole complete processing complexity costs an arm and a leg.
Cells in vitro is cultivated also needs certain isoperibol and suitable gas supply.Under conventional the cultivation, CO
2Incubator can provide 37 ℃ isoperibol and suitable O for cell cultures
2, CO
2Supply.Therefore, when making cell cultivation chip, need the integrated temperature control device, and satisfy the demand of cell gas.
Summary of the invention
The purpose of this invention is to provide a kind of based on ITO (Indium-Tin-Oxide, tin indium oxide) the cell cultivation chip preparation method and the application thereof of substrate of glass, overcome the hydrophobicity problem of existing polydimethylsiloxane (PDMS) chip and the complete processing challenge of glass material chip, for cell provides good condition in external stable cultivation.
The preparation method of the cell cultivation chip based on the ito glass substrate of the present invention comprises step:
With the ito glass is the substrate material of cell cultivation chip; With ito glass not a side of sputtering ITO film get rid of and be coated with the mask layer of AZ4620 photoresist material as glass corrosion, expose, develop according to the mask of design; With PDMS monomer, solidifying agent uniform mixing in proportion, be cast in ito thin film one side, be heating and curing; Ito glass is placed corrosive fluid, and etching gets microchannel and " dam type " structure; Peel off the PDMS on the ito thin film; PDMS monomer, solidifying agent in proportion behind the uniform mixing, are tiled on the slide glass, be heating and curing the PDMS film; With the PDMS film and ito glass one side bonding under the oxygen plasma effect of etching microchannel obtain cell cultivation chip; Again ito glass not a side of bonding PDMS film be connected into the outside temperature Controlling System by sticking plain conductor.
Described microchannel divides two zones, and one of them zone is the cell cultures zone, and the another one zone is nutrient solution sample introduction zone, and two zones connect by " dam type " structure;
Described " dam type " structure height is less than the microchannel degree of depth;
Described corrosive fluid is BOE (Buffered Oxide Etch);
Described solidifying agent is the sylgard184 product that Dow Coming company is provided;
The weight ratio of mixture of described PDMS monomer and solidifying agent is 10: 1;
Described being heating and curing is 80 ℃ and was heating and curing 1 hour;
The described plain conductor that sticks is by conductive resin plain conductor to be attached on the ito thin film;
Described outside temperature Controlling System, its controlled target temperature is the temperature of liquid in the chip microchannel, its temperature sensor is the Pt1000 thermo-sensitive resistor, and its heating means are the direct heating mode of ito thin film conductive exothermal, and its control center is an AD μ C812 micro-chip.
The application of a kind of cell cultivation chip based on the ito glass substrate of the present invention is repercussion study between cell migration, cytodifferentiation, the cell.
Described cell is zooblast or vegetable cell, or normal somatic cell or tumour cell;
The nutrient solution that uses in the cultivation of described cell adds Leibovitz ' s L-15 substratum.
Beneficial effect of the present invention:
(1) utilize the undercutting effect in the glass wet etching course to etch the structure of dam type that highly is lower than the microchannel degree of depth, isolated cell is cultivated zone and nutrient solution sample introduction zone, has reduced the influence of the hydrodynamic shear pair cell cultivation that the nutrient solution sample introduction produced;
(2) utilize the ito thin film conductive exothermal character of ito glass, integrated temperature control system on chip;
(3) utilize the ventilation property characteristics of PDMS material, need not aerating oxygen in experimentation, reduced the complexity of cell cultures micro-system design;
(4) nutrient solution that uses in cell cultivation process has added Leibovitz ' s L-15 substratum, can keep the stable of medium pH value, thereby removes CO
2Supply.
Description of drawings
Fig. 1 is an ito glass chip fabrication technique process synoptic diagram;
Fig. 2 is the mask design diagram of chip;
Fig. 3 is the microchannel synoptic diagram behind the glass etching;
Fig. 4 is cell cultures and observes the micro-system synoptic diagram;
Fig. 5 is the adherent expansion synoptic diagram of PIEC endotheliocyte in chip;
Fig. 6 is the relative movement synoptic diagram of 3T3 cell in chip.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Cell cultures glass-chip manufacture craft flow process is as shown in Figure 1, and is specific as follows:
1. clean ITO (Indium-Tin-Oxide, tin indium oxide) glass, use acetone and alcohol each ultrasonic 11 minutes earlier, rinse well with deionized water again;
Ito glass not a side of sputtering ITO film get rid of and be coated with AZ4620 photoresist material (2000 revolutions per seconds, 30 seconds), baking is 3 minutes before 80 ℃;
3. expose according to the mask (as shown in Figure 2) of design;
4. develop, baking after 130 ℃ obtains and the pairing graphic structure of chip design;
5. polydimethylsiloxane (PDMS) monomer and solidifying agent (the sylgard184 product that Dow Coming company is provided) were mixed by weight 10: 1, the degassing is handled after 30 minutes and is cast in ito thin film one side, and 80 ℃ solidified 1 hour;
6. ito glass is placed the BOE corrosive fluid of dilution in 1: 6 to corrode 50 minutes, with figure transfer to ito glass, the microchannel degree of depth that etching obtains is about 39 microns, and the structure of dam type maximum height that obtains is about 31 microns, and the microchannel after etching is finished as shown in Figure 3;
7. remove the PDMS material that covers on the ito glass, glass is cleaned up with acetone, each ultrasonic 5min of alcohol;
8. after PDMS monomer and solidifying agent being mixed by 10: 1 weight ratios, the degassing was handled 30 minutes, was cast on the slide glass of horizontal positioned, and 80 ℃ solidified one hour, and obtaining thickness is the PDMS film of 1mm;
With the PDMS film and ito glass one side of etching microchannel in the plasma etching instrument, handled 45 seconds, finish both irreversible bondings;
10. in ito glass bonding one side not, be that sputter has the ito thin film place, utilize conductive resin to stick plain conductor, be connected into the outside temperature Controlling System, its controlled target temperature is the temperature of liquid in the chip microchannel, its temperature sensor is the Pt1000 thermo-sensitive resistor, and its heating means are the direct heating mode of ito thin film conductive exothermal, and its control center is an AD μ C812 micro-chip.
Embodiment 2
Nutrient solution adopts 1640 substratum, and it with after the L-15 substratum mixed with 1: 1, is replenished 10% foetal calf serum, 100Unit/ml Streptomycin sulphate.With the conventional PIEC endotheliocyte of cultivating of trysinization liquid (containing 0.25% trypsinase and 0.02%EDTA) digestion, centrifugal to go supernatant, nutrient solution to disperse resuspended, and the concentration of cell suspension is adjusted to 4 * 10
5Cells/mm
3, suck cell suspension with syringe then, slowly inject in the chip by syringe pump.With this chip of transfered cell link to each other with temperature-control device, nutrient solution sampling device and carry out the cultivation of cell.Control target temperature is arranged on 37 ± 0.5 ℃, and nutrient solution sample introduction speed is 8 μ L/s.Chip places the upgrowth situation of the cell of Real Time Observation under the inverted microscope.Cell cultures and observation micro-system are as shown in Figure 4.Fig. 5 is the PIEC endotheliocyte in importing chip 4 hours, the cell attachment stretch-out view of utilizing video system to note.
Nutrient solution adopts DMEM (high sugar) substratum, with it with after the L-15 substratum mixed with 1: 1, additional 10% foetal calf serum, 100Unit/ml Streptomycin sulphate.With the conventional 3T3 cell of cultivating of trysinization liquid (containing 0.25% trypsinase and 0.02%EDTA) digestion, centrifugal to go supernatant, nutrient solution to disperse resuspended, and the concentration of cell suspension is adjusted to 4 * 10
5Cells/mm
3,, suck cell suspension with syringe then, slowly inject in the chip by syringe pump.With this chip of transfered cell link to each other with temperature-control device, nutrient solution sampling device and carry out the cultivation of cell.Control target temperature is arranged on 37 ± 0.5 ℃, and nutrient solution sample introduction speed is 8 μ L/s.Chip places the upgrowth situation of the cell of Real Time Observation under the inverted microscope.Cell cultures and observation micro-system are as shown in Figure 4.
Fig. 6 be the 3T3 cell in the chip culturing process, two cells that utilize video system to note process that in 2 hours, relatively moves.
Claims (12)
1. preparation method based on the cell cultivation chip of ito glass substrate comprises step:
With the tin indium oxide ito glass is the substrate material of cell cultivation chip; With ito glass not a side of sputtering ITO film get rid of and be coated with the mask layer of AZ4620 photoresist material as glass corrosion, expose, develop according to the mask of design; With polydimethylsiloxane PDMS monomer, solidifying agent uniform mixing in proportion, be cast in ito thin film one side, be heating and curing; Ito glass is placed corrosive fluid, and etching gets microchannel and " dam type " structure; Peel off the PDMS on the ito thin film; With PDMS monomer, solidifying agent in proportion behind the uniform mixing, be tiled in be heating and curing on the slide glass the PDMS film; With the PDMS film and ito glass one side bonding under the oxygen plasma effect of etching microchannel obtain cell cultivation chip; Again ito glass not a side of bonding PDMS film be connected into the outside temperature Controlling System by sticking plain conductor.
2. the preparation method of the cell cultivation chip based on the ito glass substrate according to claim 1, it is characterized in that: described microchannel divides two zones, one of them zone is the cell cultures zone, the another one zone is nutrient solution sample introduction zone, and two zones connect by " dam type " structure.
3. the preparation method of the cell cultivation chip based on the ito glass substrate according to claim 1 and 2, it is characterized in that: described " dam type " structure height is less than the microchannel degree of depth.
4. the preparation method of the cell cultivation chip based on the ito glass substrate according to claim 1, it is characterized in that: described corrosive fluid is Buffered Oxide Etch.
5. the preparation method of the cell cultivation chip based on the ito glass substrate according to claim 1, it is characterized in that: the weight ratio of mixture of described PDMS monomer and solidifying agent is 10: 1.
6. the preparation method of the cell cultivation chip based on the ito glass substrate according to claim 1 is characterized in that: described being heating and curing is 80 ℃ and was heating and curing 1 hour.
7. the preparation method of the cell cultivation chip based on the ito glass substrate according to claim 1, it is characterized in that: the described plain conductor that sticks is by conductive resin plain conductor to be attached on the ito thin film.
8. the preparation method of the cell cultivation chip based on the ito glass substrate according to claim 1, it is characterized in that: described outside temperature Controlling System, its controlled target temperature is the temperature of liquid in the chip microchannel, its temperature sensor is the Pt1000 thermo-sensitive resistor, its heating means are the direct heating mode of ito thin film conductive exothermal, and its control center is an AD μ C812 micro-chip.
9. repercussion study between cell migration, cytodifferentiation, the cell that is applied in based on the cell cultivation chip of ito glass substrate.
10. the application of the cell cultivation chip based on the ito glass substrate according to claim 10, it is characterized in that: described cell is zooblast or vegetable cell.
11. the application of the cell cultivation chip based on the ito glass substrate according to claim 10, it is characterized in that: described cell is normal somatic cell or tumour cell.
12. the application according to claim 10,11 or 12 described cell cultivation chips based on the ito glass substrate is characterized in that: the nutrient solution that uses in the cultivation of described cell adds Leibovitz ' s L-15 substratum.
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| CN102530833A (en) * | 2011-12-02 | 2012-07-04 | 江苏大学 | Closed-type microfluidic channel etching method and automatic etching device |
| CN103278643A (en) * | 2013-05-16 | 2013-09-04 | 中国科学院上海微系统与信息技术研究所 | Preparation method of microchip for microprotein detection |
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| CN102530833B (en) * | 2011-12-02 | 2014-10-29 | 江苏大学 | Closed-type microfluidic channel etching method and automatic etching device |
| CN103278643A (en) * | 2013-05-16 | 2013-09-04 | 中国科学院上海微系统与信息技术研究所 | Preparation method of microchip for microprotein detection |
| CN103278643B (en) * | 2013-05-16 | 2015-05-27 | 中国科学院上海微系统与信息技术研究所 | Preparation method of microchip for microprotein detection |
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| CN105602846A (en) * | 2016-02-01 | 2016-05-25 | 北京理工大学 | Cell culture device for miniaturized space experiments |
| CN105602846B (en) * | 2016-02-01 | 2018-05-11 | 北京理工大学 | One kind miniaturization Space Experiments cell culture apparatus |
| CN107858289A (en) * | 2017-12-25 | 2018-03-30 | 上海大学 | A kind of cell cut chip, device and method |
| CN107858289B (en) * | 2017-12-25 | 2019-06-21 | 上海大学 | A kind of cell scratch chip, device and method |
| CN113980794A (en) * | 2021-09-22 | 2022-01-28 | 中国科学院合肥物质科学研究院 | Multi-channel micro-fluidic chip suitable for cell migration analysis and application thereof |
| CN113980794B (en) * | 2021-09-22 | 2024-04-16 | 中国科学院合肥物质科学研究院 | Multichannel microfluidic chip suitable for cell migration analysis and application thereof |
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