CN105420105A - Biochip and manufacturing method thereof - Google Patents
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- CN105420105A CN105420105A CN201510997994.3A CN201510997994A CN105420105A CN 105420105 A CN105420105 A CN 105420105A CN 201510997994 A CN201510997994 A CN 201510997994A CN 105420105 A CN105420105 A CN 105420105A
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Abstract
The invention relates to a biochip, which includes a cell culture microstructure layer and a seal microporous membrane. The microporous membrane covers the cell culture microstructure layer. Specifically, the cell culture microstructure layer includes a cell culture chamber and a liquid inlet and a liquid outlet that are respectively arranged on both sides of the cell culture chamber. The cell culture chamber is formed by arrangement of a plurality of microstructures, a liquid inlet channel is arranged between the liquid inlet and the cell culture chamber, and a liquid outlet channel is arranged between the cell culture chamber and the liquid outlet, so that the liquid inlet, the cell culture chamber and the liquid outlet can be communicated. According to the invention, the microenvironment more close to the ecological niche of cell growth can be provided, thus realizing in-vitro controllable and high throughput culture of cells, especially neural stem cells.
Description
Technical field
The present invention relates to biomedical engineering field, be specifically related to a kind of biochip and manufacture method thereof.
Background technology
Central nervous system disease, such as Parkinsonism, ALS, alzheimer's disease etc. are the great difficult problems of medical field.Medical research shows that central nervous system is repaired by own endogenous stem cell.By discharging various chemokine after neural tissue injury, attracting neural stem cell to gather damage location, and be divided into different types of cell under the effect of local microenvironment, thus repairing and supplement the neurocyte of damage.Neural stem cell can strengthen the contact between nerve synapse simultaneously, sets up new neural circuitry.But remain static due to these original neural stem cell rare numbers, lack specific morphological, surface marker and differentiation antigen, therefore highly purifiedly can not to be separated so far and to be difficult to cloning.Therefore extraneous a large amount of cultivation neural stem cells transplantation is unique effective means.
Clinical and result of study shows, neural stem cell has good biological characteristics, they can also breed in vitro in a large number in self-replacation, are implanted in neural stem cell in host and have obvious existence, migration and differentiation capability, and significantly can promote neurological functional recovery.At present, the vitro culture of neural stem cell takes the mode such as Tissue Culture Dish, cell cultures orifice plate more.The method that neural stem cells transplantation uses mainly comprises local injection and transplants, through cerebrospinal fluid injection transplantation, through blood circulation injection transplantation.A large amount of experimentation on animals and clinical application show that conventional cell cultural method is difficult to the microenvironment providing Neural Stem Cells ' Growth multi-level with the complexity needed for differentiation, truth in result of study and body is differed greatly, is difficult to the needs meeting the research of stem cell many-side.Traditional implantation method efficiency is low, operation risk large, threat to life, and cost is high, hinders the application of neural stem cell.Therefore, seek a kind of altitude simulation and the Culture of neural stem cells microenvironment that can control, and safe and reliable implantation method becomes the difficult point of neural stem cell research.
Microflow control technique is an emerging cross discipline relating to chemistry, fluid physics, microelectronics, novel material and biomedical engineering.It is by the function in biological and chemical laboratory, and the such as basic operation unit such as cell cultures and detection is integrated on controlled small chip, analyzes a large amount of biomolecules at short notice, the bulk information in Obtaining Accurate sample.Micro-fluidic chip is dimensional culture system, can altitude simulation organism inner cell growing environment, the cell micro-environment key elements such as accurate control material concentration, pH value, multiple operating unit flexible combination can be made cell sample introduction, cultivates, catches, the process such as cracking and separation detection can complete on one chip.The high precision that micro-fluidic chip can provide traditional method not possess and controllable cell research condition, the leap that the vitro culture for cell realizes.In recent years, microflow control technique brings the innovation at cell injuring model, successfully achieves various kinds of cell and cultivates in vitro.But Neural Stem Cells ' Growth microenvironment is very complicated, more harsher than the environmental requirement of ordinary cells.The simultaneously cultivation of neural stem cell and differentiation, by the impact of a series of factor, as cell and cell and the reaction between cell and matrix, the distribution of fluid shear, microchannel flow field, change liquid speed.Manufacture Culture of neural stem cells preferably, highly integrated, high-throughout structure is the key problem of micro-fluidic development.Meanwhile, the neural stem cells transplantation field based on micro-fluidic chip is also blank out.
Summary of the invention
Technical problem
The present invention proposes in view of the foregoing, object is to provide a kind of biochip and manufacture method thereof, it can provide more close to the microenvironment of Growth of Cells microhabitat, realizes external controlled, the high-throughout cultivation of cell especially neural stem cell.
In order to solve the problem, the feature of the biochip that the present invention relates to is, comprise cell cultures microstructured layers and sealing microporous membrane, wherein said sealing microporous membrane covers on described cell cultures microstructured layers.
In addition, the manufacture method of the biochip that the present invention relates to, comprise the steps: Mold Making step, use ultrapure water cleaning silicon chip, when non-complete drying, adopt that acetone is ultrasonic cleans, again with ultrapure water by residual liquid on silicon chip clean and dry up for subsequent use with nitrogen, chip being placed in volatilization vatting instills after several HDMS reagent modify, silicon chip is positioned over sol evenning machine center and keeps balance, after the negative glue of paving, the static specified time, then the hot plate of heating is utilized, open mercury lamp, start photoresist spinner and carry out whirl coating, stagnate the specified time in a vacuum, wafer center is made to recover plane, then, silicon chip is positioned in insulation can, then open exposure machine to expose, and develop with developing solution, substrate making step, by silicone elastomer and solidifying agent with the ratio mixing and stirring of volume ratio 10: 1, by the silicone elastomer that mixes and solidifying agent degassed in vacuum drying oven, then the male mold surface made is poured over, take out after the baking specified time, after cooling the polydimethylsiloxane of solidification is peeled off, in predetermined position, punching forms fluid inlet and liquid outlet respectively, then specified dimension is cut into, and priority soaked overnight in alkali lye and acid solution, by deionized water rinsing, dry for standby, sealing microporous membrane manufacturing step, seals microporous membrane cleaning and sterilizing by polyethylene terephthalate, in the punching of corresponding gangway place, cut into specified dimension, adhesion step, is placed on super clean bench by the cell cultures microstructured layers manufactured, sealing microporous membrane, with ultra violet lamp, then covers on cell cultures microstructured layers by sealing microporous membrane, rely on adsorptive power to complete bonding.
In addition, the biochip that the present invention relates to can be used in external cell cultures efficiently, and for carrying out Transplanted cells with the form of biochip.
Beneficial effect
Biochip of the present invention has cell cultures microstructured layers, can realize more close to the micro-environment in vitro of cell especially Neural Stem Cells ' Growth microhabitat, at the concavo-convex alternate reticulated structure of the indoor formation of cell cultures, cell especially neural stem cell highdensity Growth and Differentiation in culturing room can be realized, thus it is harsh to solve the requirement of neural stem cell vitro culture, the low density problem of Growth and Differentiation.
Because biochip of the present invention has sealing microporous membrane, therefore, it is possible to ensure that being implanted into biochip and cells in vivo nutrition base fluid in body normally exchanges, and ensures the survival rate of cell, the especially survival rate of neural stem cell.In addition, nervous tissue in intrinsic nerve stem cell and body can also be provided to carry out the passage overlapped, for injured nerve reparation and information transmit construction platform.Therefore, it is possible to be better than traditional Transplanted cells mode, carry out the transplanting of cell especially neural stem cell.
Accompanying drawing explanation
Fig. 1 is the stereographic map of biochip of the present invention.
Fig. 2 is the side-view of biochip of the present invention.
Fig. 3 is the vertical view of biochip of the present invention.
Fig. 4 is the schematic diagram of the cell cultures microstructured layers that biochip of the present invention is diagrammatically shown, wherein, lower part image is the enlarged view of the hexagon microstructure of cell culture chamber.
Description of reference numerals:
1: cell cultures microstructured layers 2: sealing microporous membrane 11: fluid inlet 12: feed pathway 13: cell culture chamber, 14: liquid outlet channel, 15: liquid outlet
Embodiment
Below, by reference to the accompanying drawings embodiments of the present invention are described in detail.
As shown in Figures 1 to 4, the sealing microporous membrane 2 that the biochip that the present invention relates to comprises cell cultures microstructured layers 1 and covers on described cell cultures microstructured layers.
Wherein, cell cultures microstructured layers comprises fluid inlet 11, liquid outlet 15, feed pathway 12, liquid outlet channel 14 and is arranged the cell culture chamber 13 formed by multiple microstructure.Wherein, fluid inlet 11 and liquid outlet 15 are located at the both sides of cell culture chamber 13 respectively, feed pathway 12 is provided with between described fluid inlet 11 and cell culture chamber 13, liquid outlet channel 12 is provided with between described liquid outlet 11 and cell culture chamber 13, like this, make fluid inlet 11, cell culture chamber 13 is communicated with liquid outlet 15.
As cell cultures microstructured layers, the materials such as such as silicon, glass, quartz, polymethacrylate, polydimethylsiloxane can be used, wherein, polydimethylsiloxane (PDMS) transmittance is high, good permeability, chemically stability are high, biocompatibility is high, can be used as embedded material, therefore preferred polydimethylsiloxane (PDMS).
For ensureing nutrient solution smooth and easy and replacing nutrient solution simple operation in channel interior flowing, the degree of depth of the fluid inlet of cell cultures microstructured layers, liquid outlet, feed pathway, liquid outlet channel and cell culture chamber is preferably identical, such as the degree of depth can be 80 μm to 150 μm, is preferably 100 μm.
Fluid inlet, liquid outlet shape can be identical, are preferably circle.The diameter of the two can be identical, such as, be 0.45 to 0.7mm, be preferably 0.45mm.On the size basis meeting existing cell liquid-inlet pipe, the diameter of feed liquor and liquid outlet is the smaller the better, implants requirement to make the biochip size obtained meet.
Feed pathway, liquid outlet channel shape can be identical, are preferably rectangle, and in this case, length can be 250 to 400 μm, and width can be 200 to 300 μm.Wherein, preferred length is 335 μm, and width is 240 μm.
Cell culture chamber can be designed to different shape, such as, can be circle, Polygons, ellipse etc.The present invention preferably has the cell culture chamber of hexagonal structure.
Cell culture chamber can also be provided with multiple microstructure, the preferred hexagon microstructure of the present invention.It is 10 μm to 40 μm that hexagon microstructure is of a size of inscribed circle diameter, is preferably 20 μm; The degree of depth is 10 μ to 20 μm, is preferably 10 μm; Hexagon microstructure spacing is 20 μm to 40 μm, is preferably 20 μm.According to the size of cell, in culturing room, form concavo-convex alternate reticulated structure, with the growing environment of analog cell, thus cell highdensity Growth and Differentiation in culturing room can be realized.
4125 to 8625 microstructure can be set as required, preferably 6895 hexagon microstructure.Like this, can the microhabitat of the analog cell especially complexity of Neural Stem Cells ' Growth better, for Growth of Cells differentiation provides more suitable structure.
From the above, described microstructure is three-dimensional micropore structure.This three-dimensional micropore structure physical factor cell growth, grow and there is impact, provide the Growth of Cells microenvironment different from cellar culture, the two sides structure of micropore is that cell provides good space and depends on.Under such space structure helps, cell has two faces and material surface contact also can more stably grow at material surface, can realize external controlled, the high-throughout cultivation of cell especially neural stem cell.
As sealing microporous membrane, preferred use good hydrophilic property, chemical stability are high, the implantable material of good biocompatibility, the materials such as such as poly (ether sulfone) film (PES), polyethylene terephthalate film (PET), organic system nylon membrane, hydrophilicity kynoar film (PVDF), are wherein more preferably hydrophilic and stability is high, the polyethylene terephthalate film of good biocompatibility (PET).Further, due to the cylindrical straight shape through hole of passage in PET film, regular geometry and pore size are even, therefore, it is possible to realize the exchange of nutrient solution, also provide the passage that the synapse cell of biochip inside and in-vivo tissue overlap simultaneously.
The pore density of sealing microporous membrane can be 10
4-10
11/ cm
2, preferred 1.7x10
5/ cm
2.Aperture size can be selected according to cell size, as long as can ensure that cell can not spill.Can be such as 8 μm to 40 μm, be preferably 8 μm.
Like this, after in implanted chip body, sealing microporous membrane of the present invention normally can exchange with cells in vivo nutrition base fluid, guarantees that the cell such as neural stem cell of external implantation in vivo can normal growth differentiation.And then sealing microporous membrane, when ensureing that chip internal neural stem cell can not spill, can realize neural stem cell tissue in the neural dendron of implantable neural stem cell and body and overlap.
The thickness comprising the biochip of cell cultures microstructured layers and sealing microporous membrane of the present invention can be 0.3 to 0.7mm, and be preferably 0.5mm, width can be 1 to 5mm, and be preferably 1mm, length can be 3 to 5mm, is preferably 3mm.Therefore, the size of biochip of the present invention is less than conventional size, can make experiment in vitro simple operation and implant effective.
Next, the manufacture method of biochip of the present invention is described in detail.Particularly, comprise the steps.
The manufacturing step > of < cell cultures microstructured layers
To adopt polydimethylsiloxane (PDMS) material, as follows, illustrate the manufacturing step of cell cultures microstructured layers.
1) Mold Making.
Ultrapure water cleaning silicon chip, when non-complete drying, adopts acetone ultrasonic cleaning 1 minute, then is cleaned by residual liquid on silicon chip with ultrapure water, finally dry up with nitrogen for subsequent use.
Chip is placed in volatilization vatting, instills 1 to 2 HDMS reagent, modify 3 minutes.
Silicon chip is positioned over sol evenning machine center, balances; The negative glue SU-82015 (MICROCHEM) of paving, completes rear static 1-2 minute; 200 DEG C of hot plates are adjusted to 95 DEG C; Open mercury lamp, start photoresist spinner and get rid of 18 seconds at 500rpm, then get rid of 30 seconds at 1500rpm, stagnate 1-2 minute in a vacuum after terminating, make wafer center recover plane, glue-line is more even.
Then, silicon chip to be positioned in insulation can 65 DEG C 10 minutes, 95 DEG C 1 hour, then open exposure machine, expose 310 seconds.
Then, silicon chip is positioned over respectively 65 DEG C 5 minutes, 95 DEG C after 20 minutes, uses developing liquid developing.
2) polydimethylsiloxane substrate makes.
Adopt Sylgard184 silicone elastomer and Sylgard184 solidifying agent (Momentive company of the U.S.) with the ratio mixing and stirring of volume ratio 10: 1.
By the silicone elastomer that mixes and solidifying agent degassed in vacuum drying oven, be poured over the male mold surface made, 80 DEG C of bakings were taken out after 30 minutes, were peeled off the polydimethylsiloxane of solidification after cooling.
Punch in the ingress designed and exit, to form fluid inlet and liquid outlet (see Fig. 4) respectively, then cut into suitable size, and priority soaked overnight in alkali lye and acid solution, by deionized water rinsing, dry for standby.
< seals the manufacturing step > of microporous membrane
Polyethylene terephthalate (PET) being sealed microporous membrane cleaning and sterilizing, punching corresponding to fluid inlet and liquid outlet place, cutting into suitable size for subsequent use.
< adhesion step >
The cell cultures microstructured layers manufactured, sealing microporous membrane are placed on super clean bench, irradiation 3 hours are located apart from ultraviolet lamp 3 to 4cm, then cover on cell cultures microstructured layers by sealing microporous membrane, the two relies on adsorptive power complete bonding thus obtain biochip of the present invention.In addition, also can compress with weight with the bonding of both promotions after covering.
Biochip of the present invention can be used for the highdensity cell of vitro culture especially neural stem cell.
In addition, biochip of the present invention is used can be transplanted in body by cultured cell by the form of chip.
And then, using biochip of the present invention, can being accurately transplanted to predetermined position in body by having highdensity cell, such as, highdensity neural stem cell can be transplanted to accurately the preposition of brain.Such as, at the skull China and foreign countries side perforating of animal such as SD rat, aperture is 3 to 4mm, opens endocranium, is positioned in hole by biochip in sealing membrane side mode down.This hole is closed afterwards, suture operation wound with bone wax.
Embodiment
More embodiments of the present invention can be understood by the following examples.
The manufacture > of < biochip
Diameter is circular fluid inlet and the liquid outlet of 0.45mm, be 335 μm with length, width is rectangle feed pathway and the liquid outlet channel of 240 μm, and hexagonal cell culturing room and inside are provided with 6895 hexagon microstructure and are made by AutoCAD drawing software and print mask plate.Wherein, hexagon microstructure is of a size of that inscribed circle diameter is 20 μm, the degree of depth is 10 μm, hexagon microstructure spacing is 20 μm.
After Wafer Cleaning, modification, with sol evenning machine at surface uniform tiling glue SU-82015.After glue-line is even, uv-exposure 310 seconds, is transferred to mask graph on photoresist material.Again through development, remove unexposed photoresist material.
By Sylgard184 silicone elastomer and Sylgard184 solidifying agent (Momentive company of the U.S.) with the ratio mixing and stirring of volume ratio 10: 1.Then the silicone elastomer mixed and solidifying agent degassed in vacuum drying oven, be poured over the male mold surface made, 80 DEG C of bakings were taken out after 30 minutes, after cooling the PDMS of solidification is peeled off, thus cell culture chamber, fluid inlet, liquid outlet, feed pathway, liquid outlet channel are transferred on PDMS substrate.
Punch tool fluid inlet place selected on the PDMS substrate manufactured and liquid outlet place is adopted to punch to form fluid inlet and liquid outlet.Then the size of length 3mm, width 1mm is cut into, and priority soaked overnight in alkali lye and acid solution, then use deionized water rinsing, oven dry formation cell cultures microstructured layers for subsequent use.
Polyethylene terephthalate (PET) is sealed microporous membrane cleaning and sterilizing, is punching corresponding to fluid inlet place and liquid outlet place, then cut into length 3mm, width 1mm size for subsequent use.
Polydimethylsiloxane (PDMS) cell cultures microstructured layers good for above-mentioned manufacture, polyethylene terephthalate (PET) are sealed microporous membrane on super clean bench, and use ultraviolet lamp irradiation at short distance 3 hours, then stack gradually and compress to make the two bonding with weight.So far, the manufacture of biochip is completed.
< carries out the vitro culture > of neural stem cell on biochip
The biochip of above-mentioned manufacture is used successively distilled water, 75% alcohol, washed with de-ionized water, to ensure that channel interior is without material residues.Chip is put into 60 DEG C of thermostat containers again after the autoclaving of 121 DEG C, 20 minutes to dry.Then the biochip micro sample adding appliance of complete drying is injected PBS buffer solution for cleaning 3 times through chip fluid inlet, all clean by PBS displacement to ensure the liquid residue in microchannel.Then adopt many Matrigel matrigel envelope chip, be placed in 37 DEG C of thermostat containers 2 hours, then rinse 3 times with PBS, then use ln (laminin, LN) envelope chip, then put into 4 DEG C of refrigerator overnight for subsequent use.
Neural stem cell used in the present invention is taken from the ventricular zone of 0-3 days SD rats.Use the trypsin Trypsin-EDTA of 0.05%) digest neural stem cell and manufacture cell suspension, then under 1000rpm centrifugal 5 minutes, discard supernatant.Then SD neural stem cells in rats substratum Eddy diffusion cell is used, by neural stem cell Auto-regulating System of Density of Heavy Medium to 1 × 10
5cells/ml is for subsequent use.
Described SD neural stem cells in rats substratum (purchasing in Thermofisher company) composition is as follows:
Adopt injector for medical purpose, the internal diameter of having sterilized to be the kapillary of 0.45mm and connect steel syringe needle neural stem cell suspension is slowly injected chip through biochip fluid inlet, liquid inlet volume is about 1 μ l, to carry out loading cells.Examine under a microscope, confirm that cell cultures is indoor, cell distribution is even.Then, chip is placed in 37 DEG C, 5%CO
2cultivate in incubator, observation of cell growing state after 7 hours, record cell growth condition image.Then, within the 24th hour after loading cells completes, observe again, and again record cell growth condition image.
After cell cultures 24 hours, under ensureing well-grown situation, carry out implantation experiment.
< well-grown neural stem cell biochip implantation experiment >
Get the adult SD rats 10 of upgrowth situation health, adopt the Chloral Hydrate of 3.6%, carry out the anesthesia of abdomen injection method according to the proportioning of body weight 1ml/100g.Then open cranium to the SD rat of anesthesia, at skull China and foreign countries side perforating, pore size is 3.5mm, opens endocranium, is positioned in hole by chip, and ensures sealing membrane side down, then uses bone wax to be closed and suture operation wound in hole.Normal cultivation also observes rat physiological situation.
10 rats were put to death after 48 hours by implantable bioartificial chip respectively, took out biochip and carried out microscopic examination, and record microscopic findings.Neural stem cell survival rate in the data statistic analysis result display biochip of observations is 30%.Because normal cell growth cycling cells has the apoptosis rate of 50%, add in inflammatory reaction and other bodies and can not survey factor, very important impact is all had for Growth of Cells.Therefore, compared with the survival rate of 20% of traditional direct injection neural stem cell, the survival rate adopting biochip of the present invention to carry out the stem cell transplanted significantly improves.In addition, tradition is cultivated maximum problem and is that cell is implanted and determines position, and observation Growth of Cells relies on ventricles of the brain section statining difficulty large.Relative to traditional method, biochip of the present invention can determine that cell is implanted in position.
Known by foregoing description, use biochip provided by the invention, the cell cultures microenvironment of analog cell especially neural stem cell can be provided, thus realize the external high-density culture of cell especially neural stem cell.In addition, because it has the sealing microporous membrane that normally can carry out cell nutrient solution exchange, thus can realize implanting cell and cells in vivo nutrition base fluid and normally exchange, ensure that the cell of external implantation can break up by normal growth in vivo.And then sealing microporous membrane, when ensureing that chip internal cell can not spill, provides neural stem cell tissue in the neural dendron of implantable neural stem cell and body and carries out the passage overlapped.Therefore, use biochip of the present invention can with lower cost, realize cell especially neural stem cell high efficient and reliable transplant.The present invention is better than traditional way, solves a present stage difficult problem, proposes novel method especially at CO2 laser weld and information transfer connection, has widespread use and is worth.
It should be noted that, disclosed embodiment is only not intended in order to technical scheme of the present invention to be described limit the present invention above, those skilled in the art is to be understood that, in the aim not departing from technical solution of the present invention and scope, can carry out various amendment or equivalent replacement to technical scheme of the present invention, therefore these variation are also encompassed in comprising in scope of claim of the present invention.
Claims (8)
1. a biochip, is characterized in that,
Comprise cell cultures microstructured layers and sealing microporous membrane, described sealing microporous membrane covers on described cell cultures microstructured layers, wherein,
Described cell cultures microstructured layers comprises cell culture chamber and is located at fluid inlet and the liquid outlet of described cell culture chamber both sides respectively, described cell culture chamber is arranged by multiple microstructure and is formed, feed pathway is provided with between described fluid inlet and cell culture chamber, be provided with liquid outlet channel between described cell culture chamber and liquid outlet, make described fluid inlet, cell culture chamber is communicated with liquid outlet.
2. biochip according to claim 1, is characterized in that,
The material of described cell cultures microstructured layers is selected from the one in silicon, glass, quartz, polymethacrylate, polydimethylsiloxane.
3. biochip according to claim 1, is characterized in that,
The degree of depth of described fluid inlet, liquid outlet, feed pathway, liquid outlet channel and cell culture chamber is 80 μm to 150 μm.
4. biochip according to claim 1, is characterized in that,
Described microstructure is hexagon microstructure, and this hexagon microstructure is of a size of that inscribed circle diameter is 10 μm to 40 μm, the degree of depth is 10 μ to 20 μm, hexagon microstructure spacing is 20 μm to 40 μm.
5. biochip according to claim 1, is characterized in that,
Described sealing microporous membrane is selected from the one in poly (ether sulfone) film (PES), polyethylene terephthalate film (PET), organic system nylon membrane, hydrophilicity kynoar film (PVDF).
6. biochip according to any one of claim 1 to 5, is characterized in that,
The pore density of described sealing microporous membrane is 10
4~ 10
11/ cm
2, aperture size is 8 μm to 40 μm.
7. biochip according to any one of claim 1 to 6, is characterized in that,
The thickness of described biochip is 0.3 to 0.7mm, width is 1 to 5mm, length is 3 to 5mm.
8. a manufacture method for biochip, is characterized in that, comprises the steps:
Mold Making step, use ultrapure water cleaning silicon chip, when non-complete drying, adopt that acetone is ultrasonic cleans, again with ultrapure water by residual liquid on silicon chip clean and dry up for subsequent use with nitrogen, chip being placed in volatilization vatting instills after several HDMS reagent modify, silicon chip is positioned over sol evenning machine center and keeps balance, after the negative glue of paving, the static specified time, then the hot plate of heating is utilized, open mercury lamp, start photoresist spinner and carry out whirl coating, stagnate the specified time in a vacuum, wafer center is made to recover plane, then, silicon chip is positioned in insulation can, then open exposure machine to expose, and develop with developing solution,
Substrate making step, by silicone elastomer and solidifying agent with the ratio mixing and stirring of volume ratio 10: 1, by the silicone elastomer that mixes and solidifying agent degassed in vacuum drying oven, then the male mold surface made is poured over, take out after the baking specified time, after cooling the polydimethylsiloxane of solidification is peeled off, in predetermined position, punching forms fluid inlet and liquid outlet respectively, then specified dimension is cut into, and priority soaked overnight in alkali lye and acid solution, by deionized water rinsing, dry for standby;
Sealing microporous membrane manufacturing step, sealing microporous membrane cleaning and sterilizing, punching corresponding to fluid inlet and liquid outlet place, cutting into specified dimension by polyethylene terephthalate;
Adhesion step, is placed on super clean bench by the cell cultures microstructured layers manufactured, sealing microporous membrane, with ultra violet lamp, then covers on cell cultures microstructured layers by sealing microporous membrane, rely on adsorptive power to complete bonding.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110669665A (en) * | 2018-09-21 | 2020-01-10 | 浙江大学 | Microfluidic chip for culturing liver cancer slices and application method thereof |
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