CN106434346A - Chip for sieving medicines under oxygen gradient and preparation method and application of chip - Google Patents
Chip for sieving medicines under oxygen gradient and preparation method and application of chip Download PDFInfo
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Abstract
The invention discloses a chip for sieving medicines under an oxygen gradient and a preparation method and application of the chip. The chip device comprises a cell culture channel positioned on the upper layer, a chemical oxygen consumption channel positioned on the lower layer and a PDMS film positioned between the upper layer and the lower layer; the chemical oxygen consumption channel is successively provided with a reactant inlet, a mixing region, a reaction region and a reactant outlet; and the reaction region is vertically aligned with the cell culture region in the cell culture channel. Experiments prove that by the chip device provided by the invention, cytotoxicity and gene damage of to-be-detected medicines to tumor cells under the condition of different oxygen concentrations can be detected on the chip, and the chip plays an important role in sieving of anti-tumor medicines with effects on tissues of anoxic solid tumors.
Description
Technical field
The invention belongs to biological technical field is and in particular to a kind of chip for drug screening under oxygen gradient and its system
Preparation Method and application.
Background technology
The generally existing of low-oxygen environment is one of key property of solid tumor, due to the tumor cell within solid tumor away from
Blood vessel has certain distance, and the speed of oxygen expansion slows down gradually, and oxygen tension declines, and leads to the oxygen of this region cell under-supply, that is,
Tumor cell fast breeding and oxygen supply relatively lag behind and define the internal distinctive hypoxia microenvironment of solid tumor tissue.This
Significantly changing of tumor microenvironment, can make tumor such as intracellular growth stagnate, after tumor cell is to the adaptation of low-oxygen environment around in early days
Start fast-growth, meanwhile, its biological characteristics also occurs accordingly to change again.Research shows, progressively adapts to low in tumor cell
During oxygen environment, many signal paths change, and promote and generate and malignant invasion with the development of tumor, new vesselses
The expression of the related genes such as transfer, makes the solid tumor cell under low-oxygen environment that the sensitivity of radiation and chemotherapy is declined, it has also become
Impact oncotherapy and the major obstacles of prognosis.Due to tumor cell under the hypoxia microenvironment of solid tumor mass to multiple anti-swollen
Tumor medicine all shows drug resistance, and conventional external drug screening acquired results cannot be made accurately to the anti-tumor activity of medicine
Evaluate, cytotoxicity under the conditions of normal oxygen for the medicine and genotoxicity can only be reflected.Additionally, solid tumor cell is not only in hypoxia
In microenvironment, oxygen concentration is not homogeneous numerical value about, but exists gradiently.Therefore, in tumor drug screening
When, investigate drug effect under different oxygen concentrations for the tumour medicine, effect feelings in solid tumor mass for the medicine can be objectively responded
Condition, thus further provide for theoretical foundation for the research and development of cancer therapy drug.
How to build external high-flux cell model and the microenvironment of cells in vivo can be simulated to greatest extent, from
And it is preferably consistent to ensure that results of laboratory based on in-vitro screening and internal real life process and biological effect have
Property, it is the great demand that current life scientific research and drug screening are faced.The appearance of biochip technology, breaches tradition
Channel dimension not only can be narrowed down to a micron order of magnitude by the restriction of biochemical analysises means, also can set up cell by various means
Controlled on yardstick and accurate microenvironment, the true survival condition of analog cell；Also provide a new research to regard simultaneously
Angle, that is, under minute yardstick, studies behavior and the growth of cell, increased verity and the credibility of result of study.But at present still
There are not the cell analysis that can simulate under tumor hypoxia microenvironment and cytotoxicity and the biochip of genotoxicity analysis.
Content of the invention
It is an object of the invention to provide a kind of chip for drug screening under oxygen gradient and preparation method and application.
The technical problem to be solved is to screen under oxygen concentration in realization to have effect to aim cell
Medicine.For solving the above problems, present invention firstly provides a kind of micro-fluidic chip model generating oxygen concentration gradients, with
When load and can be used for carrying out the micro-array chip of single cell culture, be applied to evaluation and the sieve of drug cytotoxicity and genotoxicity
Choosing.
The chip apparatus that the present invention provides, including the cell culture passage positioned at upper strata, lead to positioned at the biochemical oxygen demand of lower floor
Road and the PDMS film being located between levels；
Reactant inlet port, mixed zone, reaction zone and reaction stream outlet are set gradually on biochemical oxygen demand passage；
Cell culture area consistency from top to bottom in described reaction zone and cell culture passage.
In said chip device, described biochemical oxygen demand passage is provided with reactant and enters passage and reactant flow pass, its
Opening is respectively positioned on one end of described cell culture passage, and described reactant enters passage and reactant flow pass runs through upper and lower
Layer.
The opening that described reactant enters passage is reactant inlet port；The opening of described reactant flow pass is reaction
Stream outlet；
Described reaction zone is concretely perfectly aligned up and down with the cell culture area in cell culture passage, in order to oxygen
The foundation of gas Concentraton gradient.
Described chip apparatus also include cover plate；Described cover plate is placed in above described cell culture passage.
Described cell culture passage be shaped as square or rectangular；Width is 1-1.5cm；It is highly 0.4-0.8cm；
Unicellular dimensional culture can be carried out in this cell culture passage.
The width of described biochemical oxygen demand passage is 100-120 μm；Highly it is 80-100 μm；
The thickness of described PDMS film is 60-120 μm.Too thin PDMS is easily rupturable, makes hypoxia response liquid enter cell culture area
Domain, too thick, it is unfavorable for that oxygen spreads.
The number of described reactant inlet port is 2, and accordingly, the number that described reactant enters passage is also 2；Instead
The number answering stream outlet is 1；Accordingly, the number of described reactant flow pass is also 1；
Described mixed zone is S type passage；Both reactants can be made to be sufficiently mixed and react；
Described conversion zone is long S type passage, positioned at lower section one side region of cell culture insert；
Above-mentioned mixed zone and conversion zone all can be passed through reagent and carry out oxygen consumption reaction, and above it, oxygen is saturating through diffusion
Cross PDMS film and can enter lower channel, after reaching dynamic equilibrium, up can form oxygen gradient by cell culture pool area.Oxygen
Concentration range is from being gradually increased to away from reaction side, its oxygen concentration range concretely 1%- close to reaction side
12%.
Described reactant entrance and outlet are spherical；Radius is 200-400 μm.
It is mounted with unicellular micro-array chip, specially agarose unicellular microarray core in described cell culture passage
Piece；This chip can be used for single cell gel electrophoresis, detects DNA damage.
In the unicellular micro-array chip of described agarose, agarose matrix is the aqueous solution of 1% normal melting point agarose, with
Fibronectin soaks to be modified, and obtains so that 0.6% low melting-point agarose covers after unicellular loading again.Can be according to cell culture insert
The unicellular micro-array chip of cutting is make it suitable for be loaded in the middle of cell culture insert.
In addition, the application in screening anti-tumor medicine for the chip apparatus that provides of the invention described above and this chip apparatus are anti-
Application in the screening of tumour medicine and this chip apparatus application in antitumor cell screening, falls within the guarantor of the present invention
Shield scope.
Present invention also offers a kind of method of antitumor medicine screening, the method comprises the steps：
1) tumor cell and the medicine to be measured containing certain concentration are added in the cell culture passage in described chip apparatus
The cell culture medium of thing, enters passage to the reactant in described biochemical oxygen demand passage and each leads into oxygen consumption reaction reagent；
2) after described chip apparatus being cultivated, measure respectively in described cell culture passage the survival rate of cell and
DNA damage index, can screen to antitumor drug according to gained cell survival rate and DNA damage index.
Specifically, the method for this antitumor medicine screening may include following steps：
A, the aforementioned present invention provide chip apparatus in cell culture passage in add tumor cell so as to by weight
Power effect is loaded in the middle of described unicellular micro-array chip, then with wash buffer；
B, the low melting-point agarose aqueous solution of use fusing cover cell so as to form unicellular array, obtain can be used for medicine
The unicellular chip of thing screening analysis；
C, add containing certain concentration in the cell culture passage of the upper cell culture pond in described chip apparatus
The cell culture medium of medicine to be measured, each leads into oxygen consumption reaction examination to two reaction-ure inlets in described biochemical oxygen demand passage
Agent；
D, described chip apparatus are cultivated after, respectively measure oxygen concentration gradients under cell survival rate and DNA damage
Hinder index, antitumor drug can be screened according to gained cell survival rate and DNA damage index.
In step a of said method, described buffer for pH be 7.4, the PBS of 0.01M.
Described step 1) and step c in, oxygen consumption reaction reagent be pyrogallol and sodium hydroxide.
Described step 2) and step d in, cell survival rate using live extremely double dye methods detected, fluoroscopic image collection finish
Carry out comet electrophoresis immediately afterwards, to detect DNA Damage situation.
In addition, application in genescreen for the chip apparatus of the invention described above offer, fall within the protection model of the present invention
Enclose.
Above-mentioned micro-fluidic chip can be obtained according to various conventional methods, is obtained such as by following steps：
(1) with the microchannel of computer aided design software design and drafting micro-fluidic chip；
(2) bear in the SU-8 setting thickness, in the silicon chip template of optical cement, lower layer chip microchannel is formed by uv-exposure
Pattern；
(3) take described mould, be placed in exsiccator, add 20 μ L trichlorine (1H, 1H, 2H, 2H- perfluoro capryl) silane, take out true
Sky, places 3h.The purpose carrying out this step is in order to increase the hydrophobicity of mould.
(4) by PDMS prepolymer and cross-linking agent mix homogeneously, there is the SU-8 mould in cofferdam in the surrounding being poured into horizontal positioned
On, vacuum outgass post-heating takes out to after solidify, cuts and peel off PDMS；
(5) PDMS being configured is poured on circular silicon chip, being placed in rotation on sol evenning machine makes PDMS evenly laid out in silicon
On piece, it is heated to solidifying；
(6) the PDMS sheet after punching is bonded on the silicon chip being covered with PDMS film with oxygen gas plasma, forms envelope
Close microchannel, the chip being bonded with PDMS film is taken off after thin film is cut from silicon chip；
(7) PDMS being configured is poured on special upper channel silicon chip, makes and include koilocyte culture pond area
The upper strata chip in domain；
(8) levels chip is carried out plasma bonding, then respectively at entrance and exit punch, by its again with glass
Glass piece carries out permanent bonding, that is, complete the making of oxygen gradient micro-fluidic chip.
Present invention also offers a kind of be loaded in the system that chip upper cell cultivates the unicellular micro-array chip of pool area
Preparation Method, is carried out with reference to patented technology disclosed in Application No. 201610330403.1, through the following steps that complete：
(1) with the microchannel of computer aided design software design and drafting micro-fluidic chip；(2) setting thickness
SU-8 bears and forms lower layer chip microchannel pattern by uv-exposure in the silicon chip template of optical cement；(3) take described mould, be placed in
Exsiccator, adds 20 μ L trichlorine (1H, 1H, 2H, 2H- perfluoro capryl) silane, evacuation, places 3h.The purpose carrying out step 3 is
Increase the hydrophobicity of mould.(4), after completing step 3, described mould, 1% (mass volume ratio) normal melting point of pouring molten are taken
Agarose solution (about 85 DEG C), and add a cover GelBond film, room temperature is cooled to 25 DEG C；Then GelBond film is shelled from mould
From getting off, as micro-array chip.(5) take described micro-array chip, be dipped to the fibronectin that concentration is 200 μ g/mL water-soluble
Liquid processes 30min, promotes the unicellular adhesion on agarose gel surface and the growing multiplication being loaded in subsequent step.(6) press
The size that photo cell cultivates pool area cuts to chip, and unicellular array chip is placed in micro fluidic device upper strata
Culture pool area, the chip apparatus for pharmaceutical analysiss under oxygen gradient complete.
It is demonstrated experimentally that the oxygen concentration in cell culture insert is to can be utilized oxygen to diffuse to form, logical in lower layer chip passage
Enter reagent and carry out oxygen consumption reaction, in upper cell culture pond, oxygen spreads in lower channel, after balance, form oxygen gradient.
The sign of oxygen concentration gradients is to be realized by measuring the fluorescent material to oxygen sensitive.Meanwhile, under oxygen concentration gradients
Culture of tumor cell, sensitive to oxygen and tumour medicine, by measuring survival rate, DNA damage and the protein expression of tumor cell
Can be with the realization of reaction chip oxygen concentration gradients etc. index.
The method that the present invention provides has the advantage that：
One is that the micro fluidic device that the present invention provides can preferably simulate internal tumor tissues oxygen atmosphere, compared to biography
The method that the liquid crossed by gas mixing, gas balance in system method or chemical reagent and electrochemistry produce oxygen, this
The method that invention provides avoids the use of exogenous gas, has that making is simple, is easy to observe and little to cytotoxic
Advantage.
Two is that the method that the present invention provides can carry out precise control to oxygen gradient over time and space, internal to simulate
The microenvironment of tumor cell, provides the data closer to internal truth for pharmaceutically active analysis, can be more accurately anti-
Reflect the drug effect of tumour medicine, provide foundation for setting up medicaments sifting model in vitro.
Three is that the method that the present invention provides has stronger Multifunctional centralized and becomes second nature, and can be used for the quick of various lead compound
Screening and optimization, such as to optimize lead compound usually through synthesized micromolecule organic compound storehouse in new drug development, can pass through
The method that the present invention provides replaces the cytotoxicity of being repeated several times property of traditional method and the independent detection of gene damage, thus
Complete the high throughput testing of the cytotoxicity to medicine and gene damage in once testing, be that drug screening analysis research obtains greatly
Amount effective information.
Four is that chip apparatus provided by the present invention provide for single celled biological nature under the different oxygen atmospheres of research
High-throughout analysis approach, can encode to it according to cell position, provide the specificity information of individual cells.This device is also
By designing various sizes of micro- trap, variety classes cell can be carried out with unicellular capture and dimensional culture, in conjunction with various unicellular
Analytical technology, can make people deeper into acquisition and parsing oxygen for the impact of unicellular internal vital movement every letter
Breath.
Brief description
Fig. 1 is chip apparatus involved in the present invention, and wherein, (A) is chip structure schematic diagram, and 1 is cell culture passage,
2 is biochemical oxygen demand passage, and 3 is PDMS film, and 4 is reactant inlet port, and 5 is mixed zone, and 6 is reaction zone, and 7 flow out for reactant
Mouthful, 8 is cover plate, and 9 is substrate of glass；(B) it is chip pictorial diagram；
Fig. 2 is that in cell culture insert, oxygen concentration gradients form schematic diagram, and wherein, (A) is fluorescence intensity profile, (B)
For diverse location oxygen concentration scattergram；
Fig. 3 is that detection tirapazamine processes the cytotoxicity result after A549 cell 6h under oxygen gradient.
Fig. 4 is that detection tirapazamine processes the gene damage result after A549 cell 6h under oxygen gradient.
Fig. 5 is that detection bleomycin processes the cytotoxicity result after SF767 cell 12h under oxygen gradient.
Fig. 6 is that detection bleomycin processes the gene damage result after SF767 cell 12h under oxygen gradient.
Specific embodiment
Experimental technique in following embodiments, if no special instructions, is conventional method.
Material used, reagent etc. in following embodiments, if no special instructions, all commercially obtain.
Quantitative test in following examples, is respectively provided with three times and repeats to test, results averaged.Human glioma cell
SF767 and human lung cancer cell A549 are the Chinese Academy of Medical Sciences and coordinate the product of cell resource center.Hereinafter, human brain glue
Matter oncocyte SF767 abbreviation SF767 cell, human lung cancer cell A549's abbreviation A549 cell.
Silicon chip is the product of Beijing Chemical Plant.SU-8 2050 negative photoresist is MicroChem Products, product mesh
Record number is 15040268.SU-8 developer solution is MicroChem Products, and catalog number is 15030226.Trichlorine (1H, 1H,
2H, 2H- perfluoro capryl) silane be Sigma Products, catalog number be 448931.Normal melting point agarose is Biowest
Products, catalog number is 111860.GelBondFilm is Lonza Products, and alternatively referred to as GelBond gel props up
Hold film, purposes is to support agarose gel.Fibronectin is Corning Products, and catalog number is 354008.Low melting point
Agarose is HydraGene Products, and catalog number is 15120601.GelRed is Biotium Products, product mesh
Record number is 13G0307.Pancreatin, hyclone, Penicillin-Streptomycin-Glutamine (100 ×), MEM culture
Base, RM1640 culture medium and pH7.4,0.01MPBS buffer are Gibco Products, and catalog number is followed successively by
25200072nd, 16000069,10378016,11095072 and 10010049.Cytoactive test kit is that ThermoFisher is public
Department's product, catalog number is L3224.Chromium plate glass is Shenzhen Qing Yi Opto-electrical Co., Ltd's product.Tirapazamine is
Sigma Products, catalog number is SML0552.Bleomycin is lark prestige company product, and catalog number is
BIZ0413.Micro-injection pump used by experiment is HARVARD Products.Fluorescence microscope model IX81, is OLYMPUS company
Product.Pure water needed for solution allocation, by Milli-Q ultrapure water system, is Millipore Products.
In the specific embodiment of the invention, the component of related solution or compound method are as follows：
The preparation of (a) tumour medicine
Tirapazamine solution：Weigh tirapazamine, be dissolved in normal saline, prepare the storing solution of 20mol/L, be stored in-
20℃.During use, it is diluted to 100 μM with the culture medium containing 10% serum, now with the current.
Bleomycin solution：Weigh bleomycin, be dissolved in normal saline, prepare the storing solution of 20mol/L, be stored in-
20℃.During use, it is diluted to 200 μM with the culture medium containing 10% serum, now with the current.
B () is lived dead cell is double to contaminate solution
Calcein-AM and Ethd-1 storing solution is returned to after room temperature, plus 5 μ LCalcein-AM storing solutions, 20 μ
To 10mlPBS buffer, Calcein-AM ultimate density is 2 μm of ol/L to LEthd-1 storing solution, and EthD-1 ultimate density is 4 μ
mol/L.
(c) MEM resistance culture base：
Containing 10% (percent by volume) hyclone and 1% (percent by volume) Penicillin-Streptomycin-
The MEM culture medium of Glutamine (100 ×).
(d) RM1640 culture medium：
Containing 10% (percent by volume) hyclone and 1% (percent by volume) Penicillin-Streptomycin-
The RM1640 culture medium of Glutamine (100 ×).
(e) SF767 cell suspending liquid：The SF767 cell pancreatin being in exponential phase is digested, with the training of MEM resistance
Foster base terminates digestion, and 1000rpm is centrifuged, and adds MEM culture medium to obtain SF767 cell suspending liquid, SF767 cell hangs in precipitation
In supernatant liquid, the concentration of SF767 cell is 5 × 106Individual/mL.
(f) A549 cell suspending liquid：The A549 cell pancreatin being in exponential phase is digested, uses MEM resistance culture
Base terminates digestion, and 1000rpm is centrifuged, and adds RM1640 culture medium to obtain A549 cell suspending liquid, A549 cell hangs in precipitation
In supernatant liquid, the concentration of A549 cell is 5 × 106Individual/mL.
The specifically used method of (g) trypsin：Sop up the culture medium in culture bottle, plus 2mLPBS solution, repeated washing two
Secondary, remove PBS solution in bottle, add 1mL0.25% trypsin solution, jiggle culture bottle, so that trypsin solution is fully covered
The all cells in lid bottom, outwell trypsin solution, subsequently culture bottle are put into incubation 8min in incubator；Add 2mL culture medium eventually
Only digest, draw culture in glassware base with liquid-transfering gun, piping and druming culture bottle bottom is to all cell detachments repeatedly；Cell suspension is shifted
To in the PV pipe of 1.5mL, use centrifuge 5min；The culture medium on upper strata, piping and druming centrifugation after centrifugation terminates, is removed with liquid-transfering gun
Rear cell is so as to become uniform cell suspension standby.
H the solute of () lysate and its concentration are 1% (percent by volume) TritonX-100,10% (percent by volume)
DMSO, 2.5MNaCl, 100mMEDTA, 10mMTris and 1% (mass volume ratio) sodium sarcosinate；Solvent is water；PH value is
10.0.
The solute of (i) electrophoresis liquid and its concentration 0.3M sodium hydroxide and 1mMNa2EDTA；Solvent is water；PH value is 13.0.
Embodiment 1, a kind of making of oxygen gradient micro flow control chip device
The preparation method of oxygen gradient micro flow control chip device is completed by following steps：(1) drawn with AutoCAD2010
Microchannel pattern, then pattern is printed on transparent film, then transfer the pattern onto setting thickness using standard photolithography techniques
SU-8 bear optical cement silicon chip template on, form the pattern being made up of raised SU-8 after post bake；(2) by PDMS prepolymer and
Cross-linking agent is according to mass ratio 10:1 mix homogeneously, the surrounding being poured into horizontal positioned has on the SU-8 mould in cofferdam, empty degassing 15
After minute, heat 2 hours in 70 DEG C of baking ovens, until take out after just solidifying, cutting and peel off PDMS, then with hollow stainless
Steel micropin punches respectively at entrance and exit；(3) PDMS being configured is poured on circular silicon chip, is placed on sol evenning machine
1000rpm rotation 30s makes it evenly laid out on silicon chip, is heated to solidifying；(4) by the PDMS sheet after punching with plasma
It is bonded on the silicon chip being covered with PDMS film, form closing microchannel, key is taken off after thin film is cut from silicon chip
Close the chip having PDMS film；(5) PDMS being configured is poured on special upper channel silicon chip, it is thin that making includes hollow
Born of the same parents cultivate the upper strata chip of pool area；(6) levels chip is carried out plasma bonding, then respectively at entrance and exit
Punching, it is carried out permanent bonding with sheet glass again, that is, completes the making of oxygen gradient micro-fluidic chip.
Made oxygen gradient microfluidic chip structure refers to Fig. 1, is mainly made up of upper, middle and lower three part, upper strata is thin
Born of the same parents' culture channel 1, for cell culture and agent-feeding treatment；Intermediate layer is 60-120 μm of PDMS film 3, makes in levels chip
Oxygen can carry out free diffusing and exchange；Lower floor is biochemical oxygen demand passage 2, is mainly used in oxygen consumption.Lead to positioned at cell culture
Cover plate 8 above road is unable to reach compared with low oxygen concentration, intermediate layer PDMS in order to prevent gas in pond from quickly being exchanged with air
Film 3 is bonded by Plasma plasma with levels chip, so that oxygen in levels chip can be effectively handed within the range
Change to reach dynamic equilibrium；Lower layer chip is provided with reactant and enters passage (its opening is reactant inlet port 4) and reaction stream
Go out passage (its opening exports 7 for reaction stream), its opening is respectively positioned on one end of cell culture passage 1, and reactant enters and leads to
Road and reactant flow pass run through levels, are spherical, and reactant enters passage and is 2, accordingly, has two and enters
Enter the spherical opening (reactant inlet port 4) of passage；Enter in reactant and be sequentially provided with S between passage and reactant flow pass
Type mixed zone 5 and S type reaction zone 6；Reaction zone 6 is perfectly aligned up and down with the cell culture area in cell culture passage 1.To two
Individual reactant inlet port 4 each leads into pyrogallol and sodium hydroxide solution, can consume the oxygen in surrounding, oxygen after mixing
Gas gradient is formed on cell culture passage 1, and oxygen concentration range is from being gradually increased to away from reaction close to reaction side
Side.
The mensure of oxygen concentration gradients and sign on embodiment 2, chip
The oxygen concentration of upper strata chip cell culture area using oxygen be quenched type fluorescence indicator three (4,7- biphenyl -1,
10 phenanthroline) ruthenous chloride (II) complex characterized, and the fluorescence intensity of this kind of fluorescence indicator can be dynamically sudden by molecular oxygen
Go out and weaken, be the effective oxygen probe based on intensity or die-away time measurement.This fluorescent dye has under different oxygen concentrations
Different fluorescence intensities, and oxygen concentration is lower, and fluorescence intensity is bigger, a length of 455nm of maximum absorption wave, maximum emission wavelength
For 613nm.The computational methods of oxygen concentration refer to patented technology disclosed in Application No. 201510009968.5 and carry out, and pass through
The fluorescence intensity of detection and Stern-Volmer formula are calculating：I0/ I=1+Kq[O2].The fluorescence that wherein I represents during aerobic is strong
Degree, I0Representing during anaerobic is fluorescence intensity in pure nitrogen gas environment, and Kq represents and coefficient is quenched.Making I represent oxygen concentration is 21%
When corresponding fluorescence intensity, from above formula, as long as recording I0, I air just can get Kq, KqValue is brought above formula into and can be obtained
Relation equation between fluorescence intensity and oxygen concentration.
Choose 300 μM of fluorescence indicator concentration (being dissolved in dehydrated alcohol), add in upper cell culture pond, when lower floor's oxygen consumption
When reaction starts, the oxygen that pool area is cultivated on upper strata can spread to lower floor through middle PDMS film, chooses 100 μm of PDMS film, hydrogen
Sodium oxide and pyrogallol concentration are respectively 1M and 200mg/mL, and flow speed control is 0.05mL/h, the upper strata chip now recording
Oxygen concentration gradients distribution as shown in Figure 2 it can be seen that under the reaction of lower layer chip oxygen consumption and gas diffusion effect, chip upper strata
The foundation of oxygen concentration gradients can be realized, oxygen concentration range is from being gradually increased close to reaction side in cell culture passage
To away from reaction side, its oxygen concentration range is 1%-12%.
Embodiment 3, oxygen gradient chip detection tirapazamine act on to the cytotoxicity of A549 and genotoxicity
Preparation A549 unicellular microarray agar carbohydrate chip, concrete grammar application reference number is 201610330403.1 disclosures
Patented technology carry out, a diameter of 25 μm of chip, depth be 40 μm, chip matrix be 1% (w/v) normal melting point agarose molten
Liquid, makes micro- trap using mould in agarose layer, removes mould, micro-array chip makes and finishes after its solidification, adds fine
Even protein solution carries out being surface-treated 30min, adds a cover bottomless porous culture plate above chip, using clip, agar carbohydrate chip is solid
Between glass plate and culture plate, adjustment cell suspension density to 5 × 106Individual/mL carries out unicellular loading, stands 10min,
Residual cells are rinsed after 3 times with 25 DEG C of PBS, cover surface using 0.6% (w/v) low melting-point agarose, fixing thin
Born of the same parents form it into the unicellular array chip of A549.Chip is cut into suitable size, is placed in oxygen gradient micro flow control chip device
In cell culture insert, add the RM1640 cell culture medium containing 100 μM of tirapazamines prepared to be processed, arrange blank simultaneously
Matched group.Micro-fluidic chip upper cell culture pond length and width are 1cm, highly for 0.8cm.The oxygen consumption reaction channel choosing of chip lower floor
Take naoh concentration 1M, pyrogallol concentration is 200mg/mL, flow velocity is 0.1mL/h, now oxygen concentration is 1%-12%.
Tirapazamine is a kind of new biological reductant class antineoplastic agent, has special cytotoxicity to anoxic cell.At 37 DEG C
Reason 6h after abandon pharmaceutical culture medium, unicellular to chip carry out extremely double dyes of living, under fluorescence microscope after 20min observation of cell survival feelings
Condition, calculates cell survival rate.Row single cell gel electrophoresis analyzes the variation tendency that DNA single-strand break damages afterwards, is placed in cracking
Overnight crack 12h in liquid, add PBS solution to wash away the lysate on surface afterwards, then be placed in unwindase 12 0min in electrophoresis liquid, electrophoresis
Time is set to 30min, afterwards with GelRed dyestuff (1:3000 preparations) carry out dyeing and be placed under fluorescence microscope carrying out picture
Collection, and using comet image processing software, statistical analysiss are carried out to the data obtained.
Fig. 3 is dead dyeing gained fluoroscopic image alive after cell culture 6h, cell survival rate can be observed with oxygen concentration
Increase and raise, show that tirapazamine has higher cytotoxicity under oxygen concentration is compared with low environment to A549.
Fig. 4 carries out, for after cell survival rate detection, the DNA Damage situation that comet electrophoresis obtain, with cytotoxicity inspection
Survey result consistent, DNA single-strand break damages and weakens with the increase of oxygen concentration, show under anoxic conditions medicine have higher
Genotoxicity effect.
Using the detection of conventional CCK-8 method, obtain 1%O2Under concentration, the RM1640 through 100 μM of tirapazamines for the A549 cell cultivates
After based sols process 12h, cell mortality is 88.1%, and 21%O2Under concentration, A549 cell is through 100 μM of tirapazamines
Mortality rate after RM1640 culture medium solution processes 12h reaches 55.6%.Result table using the detection of conventional comet electrophoresis method
Bright, A549 through 100 μM of tirapazamines RM1640 culture medium solution process 12h after, 1%O2And 21%O2Under oxygen concentration, carefully
Born of the same parents' DNA single-strand break damages meansigma methodss respectively 97.8% and 72.5%, consistent with chip detection variation tendency.
Embodiment 4, oxygen gradient chip detection bleomycin act on to the cytotoxicity of SF767 and genotoxicity
Method according to embodiment 3 makes human glioma cell SF767 micro-array chip, only replaces with A549 cell
SF767 cell, a diameter of 25 μm of micro- trap replaces with a diameter of 20 μm of micro- trap, and other steps are all constant, obtain the unicellular core of SF767
Piece.Load to the cell culture insert of oxygen gradient micro flow control chip device, after 37 DEG C of process 12h, carry out medicine with reference to embodiment 3
Thing is processed and oxicity analysis.It is blank right to add containing the MEM culture medium solution of 200 μM of bleomycin in cell culture insert and arrange
According to group, this medicine can lead to DNA damage and inducing cell apoptosis under conditions of oxygen abundance.Chip lower floor oxygen consumption reaction channel
Choose naoh concentration 1M, pyrogallol concentration is 200mg/mL, flow velocity is 0.2mL/h, now oxygen concentration is 4%-
21%.
Fig. 5 show the unicellular array of SF767 and processes the survival rate testing result after 6h through bleomycin, and result shows,
Increase with oxygen concentration, cell survival rate is in reduction trend.
Fig. 6 carries out, for after cell survival rate detection, the DNA Damage situation that comet electrophoresis obtain, with cytotoxicity inspection
Survey result consistent, it is in increase trend that DNA single-strand break damages with the rising of oxygen concentration, show to win under anoxic conditions mould
Element is suppressed to the toxic action of SF767, and cells show goes out certain drug resistance, and under the conditions of normal oxygen, medicine has higher
Cytotoxicity and genotoxicity effect.
Using the detection of conventional CCK-8 method, obtain 1%O2Under concentration, the MEM culture medium through 100 μM of bleomycin for the SF767 cell
After solution processes 6h, cell mortality is 45.8%, and 21%O2Under concentration, the MEM through 100 μM of bleomycin for the SF767 cell
Mortality rate after culture medium solution processes 6h reaches 75.7%.Result using the detection of conventional comet electrophoresis method shows, SF767
After the MEM culture medium solution of 100 μM of bleomycin processes 6h, 1%O2And 21%O2Under oxygen concentration, DNA Single-Strand Breakage in Cells
Damage meansigma methodss respectively 52.7% and 93.2%, consistent with chip detection variation tendency.
Claims (10)
1. a kind of chip apparatus, including the cell culture passage positioned at upper strata, be located at lower floor biochemical oxygen demand passage and be located at upper
PDMS film between lower floor；
Reactant inlet port, mixed zone, reaction zone and reaction stream outlet are set gradually on biochemical oxygen demand passage；
Cell culture area consistency from top to bottom in described reaction zone and cell culture passage.
2. chip apparatus according to claim 1 it is characterised in that：Described biochemical oxygen demand passage is provided with reactant and enters and leads to
Road and reactant flow pass, its opening is respectively positioned on one end of described cell culture passage, and described reactant enter passage and
Reactant flow pass runs through levels.
3. chip apparatus according to claim 1 and 2 it is characterised in that：Described cell culture passage be shaped as pros
Shape or rectangle；Width is 1-1.5cm；It is highly 0.4-0.8cm；
The width of described biochemical oxygen demand passage is 100-120 μm；Highly it is 80-100 μm；
The thickness of described PDMS film is 60-120 μm；
The number of described reactant inlet port is 2, and the number of reaction stream outlet is 1；
Described mixed zone and reaction zone are S type passage；
Described reactant inlet port and reactant flow out button be spherical；Radius is 200-400 μm.
4. according to described chip apparatus arbitrary in claim 1-3 it is characterised in that：It is mounted with described cell culture passage
Unicellular micro-array chip or the unicellular micro-array chip of agarose；
In the unicellular micro-array chip of described agarose, agarose matrix is the aqueous solution of 1% normal melting point agarose, with fine even
Albumen soaks to be modified, and obtains so that 0.6% low melting-point agarose covers after unicellular loading again.
5. application in screening anti-tumor medicine for arbitrary described chip apparatus in claim 1-4；Or,
Application in the screening of antitumor drug for arbitrary described chip apparatus in claim 1-4；
Application in screening anti-tumor medicine for arbitrary described chip apparatus in claim 1-4.
6. a kind of method of antitumor medicine screening, comprises the steps：
1) add tumor cell in the cell culture passage in arbitrary described chip apparatus in claim 1-4 and containing specific
The cell culture medium of the medicine to be measured of concentration, each leads into oxygen consumption to the reactant entrance passage in described biochemical oxygen demand passage anti-
Answer reagent；
2), after described chip apparatus being cultivated, measure the survival rate of cell and DNA in described cell culture passage respectively and damage
Hinder index, antitumor drug can be screened according to gained cell survival rate and DNA damage index.
7. a kind of method of antitumor medicine screening, comprises the steps：
Tumor cell is added so as to pass through in cell culture passage in a, arbitrary described chip apparatus in claim 1-4
Action of gravity is loaded in the middle of described unicellular micro-array chip, then with wash buffer；
B, the low melting-point agarose aqueous solution of use fusing cover cell so as to form unicellular array, obtain can be used for drug sieve
The unicellular chip of choosing analysis；
C, add the cell culture medium of the medicine to be measured containing certain concentration in the cell culture passage in described chip apparatus,
Each lead into oxygen consumption reaction reagent to two reactant inlet ports in described biochemical oxygen demand passage；
D, described chip apparatus are cultivated after, respectively measure oxygen concentration gradients under cell survival rate and DNA damage refer to
Mark, can screen to antitumor drug according to gained cell survival rate and DNA damage index.
8. method according to claim 7 it is characterised in that：In described step a, described buffer for pH be 7.4,
The PBS of 0.01M.
9. the method according to claim 6 or 7 it is characterised in that：Described step 1) and step c in, oxygen consumption reaction reagent
For pyrogallol and sodium hydroxide.
10. application in genescreen for arbitrary described chip apparatus in claim 1-4.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114317269A (en) * | 2022-03-09 | 2022-04-12 | 苏州大学 | Multi-organ chip and application thereof in drug evaluation |
| CN116254180A (en) * | 2023-02-06 | 2023-06-13 | 苏州大学 | Cerebral ischemia reperfusion injury chip and application thereof in developing new medicine |
| CN117482770A (en) * | 2023-12-29 | 2024-02-02 | 赛悟德半导体科技(上海)股份有限公司 | Concentration-based gas mixing control method and system |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104498331A (en) * | 2015-01-08 | 2015-04-08 | 青岛大学 | Preparation method and application of single-channel double-concentration-gradient microfluidic chip |
| CN105950467A (en) * | 2016-05-18 | 2016-09-21 | 清华大学 | Method for detecting cytotoxicity of to-be-tested medicine to target cell and cell chip specially used by method |
-
2016
- 2016-11-30 CN CN201611088991.9A patent/CN106434346A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104498331A (en) * | 2015-01-08 | 2015-04-08 | 青岛大学 | Preparation method and application of single-channel double-concentration-gradient microfluidic chip |
| CN105950467A (en) * | 2016-05-18 | 2016-09-21 | 清华大学 | Method for detecting cytotoxicity of to-be-tested medicine to target cell and cell chip specially used by method |
Non-Patent Citations (2)
| Title |
|---|
| CHIA-WEN CHANG ET AL: "A polydimethylsiloxane–polycarbonate hybrid microfluidic device capable of generating perpendicular chemical and oxygen gradients for cell culture studies", 《LAB ON A CHIP》 * |
| 谢晓冬等: "《肿瘤分子靶向治疗不良反应防治》", 31 October 2015 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114317269A (en) * | 2022-03-09 | 2022-04-12 | 苏州大学 | Multi-organ chip and application thereof in drug evaluation |
| CN114317269B (en) * | 2022-03-09 | 2022-05-31 | 苏州大学 | Multi-organ chip and application thereof in drug evaluation |
| WO2023168857A1 (en) * | 2022-03-09 | 2023-09-14 | 苏州大学 | Multi-organ-on-chip and application thereof in drug evaluation |
| CN116254180A (en) * | 2023-02-06 | 2023-06-13 | 苏州大学 | Cerebral ischemia reperfusion injury chip and application thereof in developing new medicine |
| CN117482770A (en) * | 2023-12-29 | 2024-02-02 | 赛悟德半导体科技(上海)股份有限公司 | Concentration-based gas mixing control method and system |
| CN117482770B (en) * | 2023-12-29 | 2024-03-19 | 赛悟德半导体科技(上海)股份有限公司 | Concentration-based gas mixing control method and system |
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