CN105420105B - Biochip and its manufacture method - Google Patents

Biochip and its manufacture method Download PDF

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Publication number
CN105420105B
CN105420105B CN201510997994.3A CN201510997994A CN105420105B CN 105420105 B CN105420105 B CN 105420105B CN 201510997994 A CN201510997994 A CN 201510997994A CN 105420105 B CN105420105 B CN 105420105B
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cell culture
liquid outlet
cell
biochip
microporous barrier
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CN105420105A (en
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陈涛
田姗姗
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Beijing University of Technology
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Beijing University of Technology
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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
    • C12M23/16—Microfluidic devices; Capillary tubes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/12—Well or multiwell plates
    • 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
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis

Abstract

The present invention relates to a kind of biochip, including cell culture microstructured layers and sealing microporous barrier, the sealing microporous barrier is covered on the cell culture microstructured layers, wherein, the cell culture microstructured layers include cell culture chamber and are respectively arranged on the inlet and liquid outlet of the cell culture chamber both sides, the cell culture chamber is by multiple microstructured bodies arrangement forms, feed pathway is provided between the inlet and cell culture chamber, liquid outlet channel is provided between the cell culture chamber and liquid outlet so that the inlet, cell culture chamber connect with liquid outlet.In accordance with the invention it is possible to provide the microenvironment for being closer to cell growth microhabitat, external controllable, the high-throughout culture of cell especially NSC is realized.

Description

Biochip and its manufacture method
Technical field
The present invention relates to biomedical engineering field, and in particular to a kind of biochip and its manufacture method.
Background technology
Central nervous system disease, such as Parkinsonism, ALS, Alzheimer disease etc. are the weights of medical domain Hang-up.Medical research shows that central nervous system can be repaired by own endogenous stem cell.Pass through neural tissue injury After discharge various chemotactic factor (CF)s, attract NSC to gather damage location, and be divided into the presence of local microenvironment Different types of cell, so as to repair and supplement the nerve cell of damage.Simultaneously NSC can strengthen nerve synapse it Between contact, establish new neural circuitry.But due to these original NSC rare numbers and remain static, lack Weary specific morphological, surface marker and differentiation antigen, thus highly purified can not separate so far and be difficult cloning.Cause This extraneous substantial amounts of culture neural stem cells transplantation is the only effective method.
Clinical and result of study shows that NSC has preferable biological characteristics, they can self-replacations and External a large amount of propagation, being implanted in NSC in host has obvious existence, migration and differentiation capability, and can significantly promote Enter neurological functional recovery.At present, the in vitro culture of NSC takes the modes such as Tissue Culture Dish, cell culture orifice plate more. Method used in neural stem cells transplantation mainly includes local injection transplanting, noted through cerebrospinal fluid injection transplantation, through blood circulation Penetrate transplanting.A large amount of zooperies and clinical practice show that conventional cell cultural method is difficult to provide Neural Stem Cells ' Growth and differentiation Required complicated multi-level microenvironment, makes result of study be differed greatly with internal truth, it is difficult to meet that stem cell is multi-party The needs of face research.Traditional implantation method efficiency is low, operation risk is big, threat to life, and cost is high, hinders nerve cord The application of cell.Therefore, a kind of altitude simulation and controllable Culture of neural stem cells microenvironment are sought, and safety can The implantation method leaned on turns into the difficult point of NSC research.
Microflow control technique is an emerging friendship for being related to chemistry, fluid physics, microelectronics, new material and biomedical engineering Pitch subject.It by the function in biological and chemical laboratory, such as the basic operation unit such as cell culture and detection be integrated in it is controllable Small chip on, analyze substantial amounts of biomolecule in a short time, the accurate bulk information obtained in sample.Micro-fluidic core Piece is dimensional culture system, can accurately control the cells such as material concentration, pH value with altitude simulation organism inner cell growing environment Microenvironment key element, can be by a variety of operating unit flexible combinations so that cell sample introduction, culture, capture, cracking and separation detection etc. Process can be completed on one chip.Micro-fluidic chip can provide the high accuracy that conventional method do not possess and controllable thin Born of the same parents' study condition, it is the leap that the in vitro culture of cell is realized.In recent years, microflow control technique is brought in cell injuring model Innovation, is successfully realized various kinds of cell and cultivates in vitro.But Neural Stem Cells ' Growth microenvironment is sufficiently complex, compare ordinary cells Environmental requirement it is more harsh.While the culture and differentiation of NSC are influenceed by a series of factors, such as cell and cell And the reaction between cell and matrix, fluid shear, microchannel flow field distribution, change liquid speed degree.Manufacture NSC preferably Culture, highly integrated, high-throughout structure is the key problem of micro-fluidic development.Meanwhile the nerve based on micro-fluidic chip Stem cell transplantation field is also blank out.
The content of the invention
Technical problem
The present invention is to propose in view of the foregoing, and it is an object of the present invention to provide a kind of biochip and its manufacture method, its The microenvironment for being closer to cell growth microhabitat can be provided, realize the external controllable of cell especially NSC , high-throughout culture.
In order to solve the above problems, biochip of the present invention is characterised by, including cell culture microstructured layers With sealing microporous barrier, wherein it is described sealing microporous barrier be covered on the cell culture microstructured layers.
Moreover, it relates to biochip manufacture method, comprise the steps：Mold Making step, using super Pure water cleaning silicon chip, when not being completely dried, cleaned using acetone ultrasound, then washed residual liquid on silicon chip with ultra-pure water It is net and dried up with nitrogen standby, chip is placed in volatilization vatting and instilled after few drops of HDMS reagents are modified, silicon chip is put It is placed in sol evenning machine center and keeps balance, after spreading negtive photoresist, the static stipulated time, then using the electric hot plate of heating, opens mercury lamp, Start photoresist spinner and carry out whirl coating, stagnate the stipulated time in a vacuum, wafer center is recovered plane, then, silicon chip is positioned over In incubator, then open exposure machine and be exposed, and developed with developer solution；Substrate making step, by silicon rubber Elastomer and curing agent are mixed evenly with the ratio of volume ratio 10: 1, and the silicone elastomer mixed and curing agent are existed Deaerated in vacuum drying chamber, be then poured over the male mold surface made, taken out after toasting the stipulated time, after cooling will The dimethyl silicone polymer of solidification is peeled off, and in precalculated position, punching forms inlet and liquid outlet respectively, is then cut into providing Size, and the successively soaked overnight in alkali lye and acid solution, with deionized water rinsing, are dried for standby；Microporous barrier manufacturing step is sealed, Polyethylene terephthalate is sealed into microporous barrier cleaning and sterilizing, is punched at corresponding gateway, cuts into given size；It is viscous Step is closed, by the cell culture microstructured layers manufactured, microporous barrier is sealed and is placed on super-clean bench, with ultra violet lamp, Then sealing microporous barrier is covered on cell culture microstructured layers, completes to bond by absorption affinity.
Moreover, it relates to biochip can be used in external efficiently cell culture, and for biological core The form of piece carries out cell transplantation.
Beneficial effect
The biochip of the present invention has cell culture microstructured layers, can realize that to be closer to cell especially neural The micro-environment in vitro of stem cell growth microhabitat, concavo-convex alternate network structure is formed in cell culture room, it is possible to achieve cell Especially NSC highdensity Growth and Differentiation in culturing room, require severe so as to solve NSC in vitro culture Carve, the low density problem of Growth and Differentiation.
Because the biochip of the present invention has sealing microporous barrier, therefore can ensure to be implanted into vivo biodistribution chip and body Inner cell nutrition base fluid is normally carried out exchanging, and ensures the survival rate of the survival rate of cell, especially NSC.In addition, also The passage that intrinsic nerve stem cell is overlapped with nerves within the body tissue can be provided, be injured nerve reparation and information transmission structure Jian Pingtai.Therefore the transplanting of cell especially NSC can in a manner of better than traditional cell transplantation, be carried out.
Brief description of the drawings
Fig. 1 is the stereogram of the biochip of the present invention.
Fig. 2 is the side view of the biochip of the present invention.
Fig. 3 is the top view of the biochip of the present invention.
Fig. 4 is the schematic diagram for the cell culture microstructured layers for diagrammatically showing the biochip of the present invention, wherein, lower half Parts of images is the enlarged drawing of the hexagon microstructured bodies of cell culture chamber.
Description of reference numerals：
1：Cell culture microstructured layers 2：Seal microporous barrier 11:Inlet 12：Feed pathway 13：Cell culture chamber, 14：Liquid outlet channel, 15：Liquid outlet
Embodiment
Hereinafter, embodiments of the present invention are described in detail with reference to accompanying drawing.
As shown in Figures 1 to 4, biochip of the present invention includes cell culture microstructured layers 1 and is covered in described Sealing microporous barrier 2 on cell culture microstructured layers.
Wherein, cell culture microstructured layers include inlet 11, liquid outlet 15, feed pathway 12, liquid outlet channel 14 and by The cell culture chamber 13 of multiple microstructured bodies arrangement forms.Wherein, inlet 11 and liquid outlet 15 are respectively arranged on cell culture chamber 13 both sides, feed pathway 12, the liquid outlet 11 and cell culture are provided between the inlet 11 and cell culture chamber 13 Liquid outlet channel 12 is provided between room 13, so so that inlet 11, cell culture chamber 13 connect with liquid outlet 15.
As cell culture microstructured layers, such as silicon, glass, quartz, polymethacrylates, poly dimethyl can be used The materials such as siloxanes, wherein, dimethyl silicone polymer (PDMS) light transmittance height, good permeability, chemically stability are high, bio-compatible Property it is high, implantation material, therefore preferably dimethyl silicone polymer (PDMS) can be used as.
To ensure that nutrient solution flows smooth and replacing nutrient solution simple operation in channel interior, cell culture microstructured layers Inlet, liquid outlet, feed pathway, the depth of liquid outlet channel and cell culture chamber are preferably identical, such as depth can be 80 μ M to 150 μm, it is preferably all 100 μm.
Inlet, liquid outlet shape can be preferably all circle with identical.The diameter of the two can with identical, for example, 0.45 To 0.7mm, 0.45mm is preferably all.On the size basis for meeting existing cell feed tube, the diameter of feed liquor and liquid outlet It is the smaller the better, so that obtained biochip size meets that implantation requires.
Feed pathway, liquid outlet channel shape can be preferably all rectangle, in this case, length can be with identical 250 to 400 μm, 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 variously-shaped, such as can be circle, polygon, ellipse etc..The present invention is excellent Cell culture chamber of the choosing with hexagonal structure.
Cell culture chamber is also provided with multiple microstructured bodies, the present invention preferably hexagon microstructured bodies.The micro- knot of hexagon The size of structure body is that inscribed circle diameter is 10 μm to 40 μm, preferably 20 μm；Depth is 10 μ to 20 μm, preferably 10 μm；Six sides Shape microstructured bodies spacing is 20 μm to 40 μm, preferably 20 μm.According to the size of cell, concavo-convex alternate net is formed in culturing room Shape structure, with the growing environment of analog cell, so as to realize cell highdensity Growth and Differentiation in culturing room.
4125 to 8625 microstructured bodies, preferably 6895 hexagon microstructured bodies can be set as needed.So, energy The complicated microhabitat of enough preferably analog cell especially Neural Stem Cells ' Growths, it is more suitable to be provided for cell growth differentiation Structure.
From the foregoing, the microstructured bodies are three-dimensional micropore structure.This three-dimensional micropore structure physical factor is to cell There is influence in growth, development, there is provided the cell growth microenvironment different from cellar culture, the two sides structure of micropore carry for cell Good space has been supplied to depend on.Under the help of such space structure, cell has two faces and material surface contact and can be more Stably grown in material surface, external controllable, the high-throughout culture of cell especially NSC can be realized.
As sealing microporous barrier, preferably using good hydrophilic property, chemical stability is high, implantable material of good biocompatibility Material, such as poly (ether sulfone) film (PES), polyethylene terephthalate film (PET), organic system nylon membrane, hydrophily polyvinylidene fluoride The materials such as alkene film (PVDF), wherein more preferably hydrophilic and stability is high, polyethylene terephthalate of good biocompatibility Ester film (PET).Also, due to the cylindrical straight through hole of passage in PET film, regular geometry and pore size is equal It is even, therefore the exchange of nutrient solution can be realized, while the synapse cell inside biochip and in-vivo tissue overlap joint are also provided Passage.
The pore density for sealing microporous barrier can be 104-1011/cm2, preferably 1.7x105/cm2.Aperture size being capable of basis Cell size is selected, as long as can ensure that cell will not be spilt.Such as it can be 8 μm to 40 μm, preferably 8 μm.
So, after in implanted chip body, sealing microporous barrier of the invention can be carried out just with internal cytotrophy base fluid Often exchange, it is ensured that the cell being implanted into vitro such as NSC in vivo can normal growth differentiation.And then seal microporous barrier and exist In the case of ensureing that chip internal NSC will not spill, the neural dendron of implantation NSC and god in vivo can be achieved Overlapped through stem cell tissue.
The present invention comprising cell culture microstructured layers and seal microporous barrier biochip thickness can be 0.3 to 0.7mm, preferably 0.5mm, width can be 1 to 5mm, and preferably 1mm, length can be 3 to 5mm, preferably 3mm.Therefore, The size of the biochip of the present invention is less than conventional size, can make experiment in vitro simple operation and implantation effect is good.
Next, the manufacture method of the biochip of the present invention is described in detail.Specifically, comprise the steps.
<The manufacturing step of cell culture microstructured layers>
It is as follows exemplified by using dimethyl silicone polymer (PDMS) material, illustrate cell culture microstructured layers Manufacturing step.
1) Mold Making.
Ultra-pure water cleaning silicon chip, when not being completely dried, it is cleaned by ultrasonic 1 minute using acetone, then with ultra-pure water by silicon chip Upper residual liquid is cleaned, and is finally dried up with nitrogen standby.
Chip is placed in volatilization vatting, 1 to 2 drop HDMS reagents is instilled, modifies 3 minutes.
Silicon chip is positioned over sol evenning machine center, balanced；Negtive photoresist SU-82015 (MICROCHEM) is spread, completes rear static 1-2 Minute；200 DEG C of electric hot plates are adjusted to 95 DEG C；Mercury lamp is opened, starts photoresist spinner and is got rid of 18 seconds in 500rpm, then got rid of in 1500rpm 30 seconds, 1-2 minutes are stagnated after terminating in a vacuum, wafer center is recovered plane, glue-line is more uniform.
Then, silicon chip is positioned over 65 DEG C 10 minutes in incubator, 95 DEG C 1 hour, then open exposure machine, exposure 310 Second.
Then, silicon chip is respectively placed in 65 DEG C 5 minutes, 95 DEG C after 20 minutes, uses developing liquid developing.
2) dimethyl silicone polymer substrate makes.
Sylgard184 silicone elastomers and Sylgard184 curing agent (Momentive companies of the U.S.) are used with volume Ratio than 10: 1 is mixed evenly.
The silicone elastomer mixed and curing agent are deaerated in vacuum drying chamber, are poured over the formpiston mould made Has a surface, 80 DEG C of bakings are taken out after 30 minutes, after cooling peel off the dimethyl silicone polymer of solidification.
Punch with exit in the entrance of design, to form inlet and liquid outlet (referring to Fig. 4) respectively, then cut Into suitable size, and the successively soaked overnight in alkali lye and acid solution, with deionized water rinsing, it is dried for standby.
<Seal the manufacturing step of microporous barrier>
Polyethylene terephthalate (PET) is sealed into microporous barrier cleaning and sterilizing, corresponding to inlet and liquid outlet Place's punching, it is standby to cut into suitable size.
<Adhesion step>
By the cell culture microstructured layers manufactured, sealing microporous barrier be placed on super-clean bench, away from uviol lamp 3 to Irradiate 3 hours, then sealing microporous barrier is covered on cell culture microstructured layers, the two is completed by absorption affinity at 4cm Bond so as to obtain the biochip of the present invention.In addition, it can also be compressed after covering with weight to promote the bonding of the two.
The biochip of the present invention can be used for the highdensity cell of in vitro culture especially NSC.
In addition, cultured cell can be transplanted in vivo by the form of chip using the biochip of the present invention.
And then using the biochip of the present invention, will can have highdensity cell to be accurately transplanted to pre- in vivo Position is determined, for example, highdensity NSC can be accurately transplanted to the predetermined position of brain.For example, in animal Such as the skull China and foreign countries side perforating of SD rats, aperture be 3 to 4mm, open endocranium, by biochip with diaphragm seal side down Mode be positioned in hole.Afterwards the hole, suture operation wound are closed with bone wax.
Embodiment
It can more understand embodiments of the present invention by the following examples.
<The manufacture of biochip>
It is 0.45mm circular inlet and liquid outlet by diameter, and length is 335 μm, width is 240 μm Rectangle feed pathway and liquid outlet channel, and hexagonal cell culturing room and inside pass through provided with 6895 hexagon microstructured bodies AutoCAD drawing softwares make printing mask plate.Wherein, the size of hexagon microstructured bodies be inscribed circle diameter be 20 μm, it is deep Spend for 10 μm, hexagon microstructured bodies spacing be 20 μm.
After Wafer Cleaning, modification, with sol evenning machine in the evenly laid out glue SU-82015 in surface.It is ultraviolet after glue-line is uniform Exposure 310 seconds, mask graph is transferred on photoresist.Again by development, unexposed photoresist is removed.
By Sylgard184 silicone elastomers and Sylgard184 curing agent (Momentive companies of the U.S.) with volume ratio 10: 1 ratio is mixed evenly.Then the silicone elastomer and curing agent that mix are deaerated in vacuum drying chamber, It is poured over the male mold surface made, 80 DEG C of bakings are taken out after 30 minutes, after cooling peel off the PDMS of solidification, so as to Cell culture chamber, inlet, liquid outlet, feed pathway, liquid outlet channel are transferred on PDMS substrates.
At the inlet selected using card punch on the PDMS substrates manufactured feed liquor is formed with punching at liquid outlet Mouth and liquid outlet.Length 3mm, width 1mm size, and the successively soaked overnight in alkali lye and acid solution are then cut into, then With deionized water rinsing, to form cell culture microstructured layers standby for drying.
Polyethylene terephthalate (PET) is sealed into microporous barrier cleaning and sterilizing, with going out liquid at corresponding to inlet Punched at mouthful, it is standby to be then cut into length 3mm, width 1mm size.
By above-mentioned manufacture good dimethyl silicone polymer (PDMS) cell culture microstructured layers, polyethylene terephthalate Ester (PET) seals microporous barrier on super-clean bench, and using uviol lamp irradiation at short distance 3 hours, then stacks gradually and uses weight Compress so that the two is bonded.So far, the manufacture of biochip is completed.
<The in vitro culture of NSC is carried out on biochip>
The biochip of above-mentioned manufacture is cleaned with distilled water, 75% alcohol, deionized water successively, to ensure channel interior Without material residues.Chip is put into 60 DEG C of insulating boxs after 121 DEG C, the autoclaving of 20 minutes again and dried.Then will be complete The biochip of white drying is cleaned 3 times with micro sample adding appliance through chip inlet injection PBS, to ensure in microchannel Liquid residue is all replaced clean by PBS.Then more Matrigel matrigels envelope chips are used, it is small to be placed in 37 DEG C of insulating boxs 2 When, then rinsed 3 times with PBS, then with laminin (laminin, LN) envelope chip, it is standby to be then placed in 4 DEG C of refrigerator overnights With.
NSC used in the present invention is taken from the ventricular zone of 0-3 days SD rats.Use 0.05% pancreas egg White enzyme (Trypsin-EDTA) digestion NSC manufacture cell suspension, then centrifuges 5 minutes under 1000rpm, discards Clearly.Then using SD neural stem cells in rats culture medium suspension cell again, by NSC Auto-regulating System of Density of Heavy Medium to 1 × 105Cells/ml is standby.
It is as follows that the SD neural stem cells in rats culture medium (purchases in Thermofisher companies) composition：
Use sterilized injector for medical purpose, internal diameter for 0.45mm capillary and connection steel syringe needle that nerve cord is thin Born of the same parents' suspension is slowly injected into chip through biochip inlet, and liquid inlet volume is about 1 μ l, to carry out loading cells.Under the microscope Observation, confirm in cell culture room, cell distribution is uniform.Then, chip is placed in 37 DEG C, 5%CO2Cultivated in incubator, 7 is small When after observe cell growth status, record cell growth condition image.Then, loading cells completion after the 24th hour again It is observed, and records cell growth condition image again.
After cell culture 24 hours, ensure to carry out implantation experiment in the case of well-grown.
<The implantation experiment of well-grown NSC biochip>
The adult SD rats 10 of upgrowth situation health are taken, using 3.6% chloraldurate, according to body weight 1ml/100g's Proportioning carries out abdomen injection method anesthesia.Then cranium is carried out out to the SD rats of anesthesia, in skull China and foreign countries side perforating, pore size For 3.5mm, endocranium is opened, chip is positioned in hole, and ensure diaphragm seal side down, then closed hole using bone wax And suture operation wound.Normally cultivate and observe rat physiological status.
Implantation biochip put to death 10 rats after 48 hours respectively, takes out biochip and carries out microexamination, and remembers Record microscopic findings.The data statistic analysis result of observation result shows that the NSC survival rate in biochip is 30%.Because normal cell growth cycling cells have 50% apoptosis rate, along with inflammatory reaction and other in vivo can not Survey factor, very important influence is had for cell growth.Therefore with the 20% of traditional direct injection NSC Survival rate is compared, and the survival rate for the stem cell transplanted using the biochip of the present invention is significantly improved.In addition, tradition culture The problem of maximum, is cell implantation with determining position, and it is big by ventricles of the brain section statining difficulty to observe cell growth.Relative to Conventional method, biochip of the invention can be it is determined that position be implanted into cell.
By foregoing description, biochip provided by the invention is used, using the teaching of the invention it is possible to provide analog cell is especially neural The cell culture microenvironment of stem cell, so as to realize the external High Density Cultivation of cell especially NSC.In addition, by In it with the sealing microporous barrier that can be normally carried out cell nutrient solution exchange, so as to realize implantation cell and internal cell Nutrition base fluid is normally exchanged, and the cell for ensureing to be implanted into vitro in vivo can normal growth differentiation.And then seal microporous barrier and exist In the case that guarantee chip internal cell will not be spilt, there is provided neural dendron and the nerves within the body for being implanted into NSC are dry thin The passage that born of the same parents' tissue is overlapped.Therefore, it can realize cell especially with relatively low cost using the biochip of the present invention Transplant to the high efficient and reliable of NSC.The present invention be better than traditional approach, solution problem at this stage, even more in CO2 laser weld and Information transfer connection proposes new method, has extensively using value.
It should be noted that embodiment disclosed above is merely illustrative of the technical solution of the present invention and is not intended to limit The present invention, it will be understood by those of skill in the art that in the objective and scope for not departing from technical solution of the present invention, can be to this The technical scheme of invention carries out various modifications or equivalent substitution, therefore these variations are also covered by the claim in the present invention Scope in.

Claims (7)

  1. A kind of 1. biochip, it is characterised in that
    Including cell culture microstructured layers and sealing microporous barrier, the sealing microporous barrier covers and to be bonded in the cell culture micro- On structure sheaf, the pore density of the sealing microporous barrier is 104~1011/cm2, aperture size is 8 μm to 40 μm, wherein,
    The cell culture microstructured layers include cell culture chamber and be respectively arranged on the cell culture chamber both sides inlet and Liquid outlet, for the cell culture chamber by multiple microstructured bodies arrangement forms, the microstructured bodies are hexagon microstructured bodies, described Feed pathway is provided between inlet and cell culture chamber, liquid outlet channel is provided between the cell culture chamber and liquid outlet, is made Obtain the inlet, cell culture chamber connects with liquid outlet.
  2. 2. biochip according to claim 1, it is characterised in that
    The material of the cell culture microstructured layers is selected from silicon, glass, quartz, polymethacrylates, dimethyl silicone polymer In one kind.
  3. 3. biochip according to claim 1, it is characterised in that
    The inlet, liquid outlet, feed pathway, the depth of liquid outlet channel and cell culture chamber are 80 μm to 150 μm.
  4. 4. biochip according to claim 1, it is characterised in that
    The size of the hexagon microstructured bodies be inscribed circle diameter be 10 μm to 40 μm, depth is 10 μ to 20 μm, hexagon is micro- Structure spacing is 20 μm to 40 μm.
  5. 5. biochip according to claim 1, it is characterised in that
    It is described sealing microporous barrier be selected from poly (ether sulfone) film (PES), polyethylene terephthalate film (PET), organic system nylon membrane, One kind in hydrophilicity kynoar film (PVDF).
  6. 6. biochip according to any one of claim 1 to 5, it is characterised in that
    The thickness of the biochip is 0.3 to 0.7mm, width is 1 to 5mm, length is 3 to 5mm.
  7. 7. the manufacture method of a kind of biochip as described in claim any one of 1-5, it is characterised in that including following steps Suddenly：
    Mold Making step, using ultra-pure water cleaning silicon chip, when not being completely dried, cleaned using acetone ultrasound, then use Ultra-pure water is clean and standby with nitrogen drying by residual liquid on silicon chip, and chip is placed in volatilization vatting and instills few drops of HDMS examinations After agent is modified, silicon chip is positioned over sol evenning machine center and keeps balance, after spreading negtive photoresist, the static stipulated time, Ran Houli With the electric hot plate of heating, mercury lamp is opened, starts photoresist spinner and carries out whirl coating, stagnate the stipulated time in a vacuum, make wafer center extensive Complex plane, then, silicon chip is positioned in incubator, then opens exposure machine and be exposed, and developed with developer solution；
    Substrate making step, silicone elastomer and curing agent are mixed evenly with the ratio of volume ratio 10: 1, will mixed Good silicone elastomer and curing agent deaerates in vacuum drying chamber, is then poured over the male mold surface made, and dries Take out after the roasting stipulated time, after cooling peel off the dimethyl silicone polymer of solidification, in precalculated position punching formed respectively into Liquid mouth and liquid outlet, are then cut into given size, and the successively soaked overnight in alkali lye and acid solution, with deionized water rinsing, It is dried for standby；
    Microporous barrier manufacturing step is sealed, polyethylene terephthalate is sealed into microporous barrier cleaning and sterilizing, corresponding to feed liquor Mouth cuts into given size with being punched at liquid outlet；
    Adhesion step, the cell culture microstructured layers manufactured, sealing microporous barrier are placed on super-clean bench, use uviol lamp Irradiation, then sealing microporous barrier is covered on cell culture microstructured layers, completes to bond by absorption affinity.
CN201510997994.3A 2015-12-25 2015-12-25 Biochip and its manufacture method Expired - Fee Related CN105420105B (en)

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