CN102382758A - Three-dimensional cell chip based on cell printing and multi-parameter sensing array integrated technology - Google Patents
Three-dimensional cell chip based on cell printing and multi-parameter sensing array integrated technology Download PDFInfo
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Abstract
The invention relates to a cell chip technology based on cell printing technology and multi-sensor integrated chip technology and application of the cell chip technology in high content medicine screening. An integrated cell MEA (Microelectrode Array) is constructed on glass or a silicon substrate by utilizing micro-electronics processing technology, and an ECIS (Electric Cell-Substrate Impedance System) comprises a disc type ECIS and an IDA (Interdigitated Array). By an arrayed chip of three sensing units, different cells and special imitated extracellular substrate material are mixed, and are positioned and printed at the assigned position in the MEA by a multi-nozzle cell printing device so as to construct the cell chip. A cell sensor on the chip is used for transmitting and recording the static and dynamic parameters such as cell action potential frequency, amplitude, wave form, network signal transmitting speed, cell attaching, moving state and the like. The invention provides a method applying the three-dimensional cell chip for medicine screening.
Description
Technical field
The invention belongs to the biology information technology field, specifically, relate to a kind of new with many cells printing technique and multiparameter sensor array integrated chip technique construction three-dimensional cell chip, construction process and in high intension drug screening, using.
Background of invention
Life science, information science combine with manufacturing science, are the important trend of 21st century development in science and technology.Make cell three-dimensional printing technique theoretical and that organizational project is theoretical based on the advanced person; And technological based on the cell chip of micro-processing technology and sensor technology, for research fields such as organizational project, drug screening, environmental monitoring have been expanded new theory and technology space.
Be based upon computingmachine 3D modeling and the advanced cell three-dimensional package technique of making on the discrete accumulation theoretical basis; Be under the anatomy digital model of histoorgan directly drives; Location assembling viable cell/material cell, the technology of the many cells precursor of manufacturing tissue or organ.Like the 3D bio-printer technology of people such as the Lee W of Harvard University exploitation, can spray cell and material, successively print fibroblast and keratinocyte and form multilayer skin texture (Lee W, et al.Biomaterials.2009; 30:1587-95).The 3D-cell printer technology of people such as Mironovf F of Univ. of South Carolina and the Boland R of Clemson university exploitation; Spray cell and special substrate material through the repacking printer head; Print activated three-dimensional cell system (Zhang C, et al.Biomaterials, 2008; 29 (28): 3781-91).The 3D bioassembly of University of Arizona Smith CM exploitation, based on direct writing technology assembling cell, and systematic study various environmental factorss to the influence of assembling cell activity (Smith CM, et al.Tissue Eng.2007; 13 (2): 373-83).The Vacanti JP of MIT and De Leipo laboratory Borenstein J etc. also are fabricated material with the cell, in conjunction with chip manufacturing process, produce capillary blood vessel system (Fidkowski C, et al.Tissue Eng.2005; 11 (1-2): 302-9).Developed the cell three-dimensional controlled group packing technique based on rapid shaping (RP) principle domestic Tsing-Hua University face Yongnian etc., assembled tissue (Yan YN, et al.Biomaterials.2005 such as can keeping 3 months active artificial livers, artificial fat; 26 (29): 5864-71).
Yet because the meticulous and complicacy of organ structure, the groups of cells packing technique also has a lot of problems to need to be resolved hurrily in the face of making complex organs such as liver, kidney, heart.Meanwhile, the groups of cells packing technique has shown important theory and using value owing to can make up complicated 3D cell culture model in other field of life science.The investigator utilizes the three-dimensional multicell of tissue engineering technique manufacturing to conduct a research in recent years, obtains remarkable achievement.People such as Basu S such as the Princeton University utilize artificial three-dimensional multicell, have studied the pattern of developmental biology and have built up problem (Basu S, et al.Nature.2005,28; 434 (7037): 1130-4).Hotary KB etc. utilizes three-dimensional multicell, finds that MT1-MMP through changing the cell spaces geometrical shape, influences propagation (Hotary KB, the et al.Cell.2003 of tumour cell in 3D matrix; 114 (1): 33-45).Basu S points out, constructs and study three-dimensional multicell, can improve the understanding to biological development and function, and can be applicable to biomaterial, in the biological sensing research.The three-dimensional multisystem of the simple technique construction of these research and utilizations has obtained effective achievement in fields such as biology and drug screenings, proves that the groups of cells packing technique has important research and using value in medicament research and development, cell chip field.
In the medicament research and development field; High flux screening technology (the High-Throughput Screening of global medicament research and development mechanism widespread use since the nineties; HTS); Face the low predicament of success ratio---the lead drug majority parmacodynamics-less activity [Bleicher KH, the et al.Nat Rev Drug Discov.2003 in integral experiment that filter out; 2 (5): 369-78.].This be since integral body be various kinds of cell at the three-dimensional space ordered arrangement, iuntercellular carries out the complex system of signal conduction, regulation and control; And the basis of HTS technology is the single drug targets molecule of research, single environment, the cultivation of cell two dimension.This species diversity pair cell 26S Proteasome Structure and Function causes remarkably influenced, and Kang X proof is compared two dimension and cultivated, form, propagation, the differentiation of cell in three-dimensional environment, and physiological properties such as genetic expression exist significant difference [Kang X, et al.Tissue Eng.2005; 11 (3-4): 458-68.].HTS develops into the high intension screening of high success rate, and (High Content Screening HCS) has become the common recognition in drug research field.The prerequisite of HCS is the influence that keeps cellularstructure, functional completeness and a plurality of Physiology and biochemistry parameters of synchronous detection sample pair cell.Be analog cell true environment in vivo, the investigator attempts making up the three-dimensional cell system and carries out drug research.W.Sun cooperates with NASA, with Multi-nozzle depositio technology assembling cell, has made up three-dimensional little hepatic tissue, is used for drug metabolism study [9].HorningJL etc. on support, set up cell cultures three-dimensional tumor model and are used for drug screening, and validity [Horning JL, the et al.Mol Pharm.2008 of proof screening; 5 (5): 849-862.].These study proof, and simple three-dimensional cell culture systems has than two-dimentional system better medicament estimates screening effect, with the development that further promotes drug screening technology.
In the biochip research field, cell and chips incorporate structure cell chip are used for disease research and drug screening, be the current research hot of research and development.Wherein carrying out the cell chip research that medicament research and development is a target to substitute laboratory animal, is the important development direction of high intension drug screening technology, and it is this area research focus that cell three-dimensional culture systems and chip technology are combined.Kloss D etc. has set up the microwell array chip that electrode is arranged at a bottom recently; After three-dimensional tumour cell microballoon combination; Become the cell chip that can carry out tumor drug screening, this cell chip possesses drug screening ability [Kloss D, the et al.Lab Chip.2008 of high intension; 8 (6): 879-84.].Minseok S.Kim has developed the chip platform of integrated micro flow control techniques and three-dimensional cell cultivation, can be used for detecting [Kim MS, et al.Biomed Microdevices.2007 based on the chemicals of cytological effect; 9:25-34.].Khetani SR etc. utilizes semiconductor microactuator processing technology and cell three-dimensional culture technique, makes up to meet the cell array that realizes the hepatic tissue functional requirement, successfully is used for drug screening [Khetani SR, et al.NatBiotechnol.2008; 26 (1): 120-126.].Khetani SR points out that the combination of chip technology and cell three-dimensional culture technique can develop complete organize models, possibly finally realize " human on a chip ".These researchs are cultivated simple three-dimensional cell system on the chip, have shown huge researching value and good prospects for application.
In cell chip research,, become the important tool of back era gene bio-science research based on the cellular electrophysiologicalsensor detection chip of microelectrode array transmitter.Classical neuroelectricity physiological detection method; Microelectrode signals collecting that adopts or confocal fluorescent imaging mode etc. carry out intracellular recording; The problem that exists mainly is: thrust, clamp down on cell with microelectrode, or adopt mode such as optical dye imaging, all can cause certain infringement by pair cell; And cause cell dead within a short period of time, limited real-time detection to action potential and ion channel current record; Simultaneously, the cell count of available acquired signal is less.And the integrated-type cell chip of integrated multiple sensors; Networking through senser element and integrated; With physiological activity information translation such as the electrical information of cell, chemical information, dynamic informations is detectable signal; And be refined as the microscopic information amount and carry out real-time analysis, realize the cell function information of fast trace and the detection of test substance character, have wide application prospects in fields such as cytobiology, environmental monitoring and drug developments.
Visible through above analysis; The modern technique of organizational engineering can be applicable to other research field and bears fruit; Particularly in cell chip and drug screening research field; If in cell chip research, can accurately control the growth position of cell on cell chip, and detect different cells physiological processes and influencing each other between them in real time; To the development and application of three-dimensional cell chip, the realization of the target that " human on a chip " is such will produce great pushing effect, and this technology is with a wide range of applications.
Summary of the invention
The purpose of this invention is to provide a kind of new cell chip and the application in high intension drug screening thereof based on cell printing technique and microelectrode array sensor technology.
Cell chip of the present invention; Comprise cellular microelectrode array (Microelectrode array; MEA) and electric impedance sensor (Electric Cell-substrate Impedance System; ECIS), and be fixed on the cell composition with three-D space structure on the transmitter, described cell composition is the blend that comprises cell and substrate material.
In the cell chip of the present invention; Microelectrode array transmitter (Microelectrode Array; MEA) be meant on glass or silicon base, metal deposition such as Au, Ir or Pt formed electrode and lead-in wire on it, adopt passivation layer protection lead-in wire with microelectronic processing technique; On electrode, expose and the cells contacting site the isoparametric cell sensor of signal velocity between transmission and record action potentials of cells frequency, amplitude, waveform and cellular network.Have make simple, biocompatibility is good, can observe advantage such as parallel use with conventional microscope, has obtained widespread use in the cell sensor research field at present.
MEA on integrated chip part can be designed to m*n array (n, m is respectively integer) arbitrarily as required, and every row's electrode diameter and interelectrode distance can be designed to 10-50 μ m, are preferably 20-30 μ m, basically and the cell size approaching.From simplified design; The present invention is designed to the 3*3 array, and every row's electrode diameter is 30 μ m, and yardstick is basic and the cell size is approaching; Interelectrode distance also is 30 μ m (removing the lower left corner electrode because of wiring reason spacing 6 μ m), is convenient to form a homogeneous, miniature test network unit.Because metal sensing point position need expose; And with cells contacting be used for sensing; Metal lead wire is then isolated with the external world, so because of under the condition that allows in manufacture craft, design the microelectrode array hole suitable with cell dia; Avoid geometrical dimension for action potentials of cells and electrophysiological influence, but lead-in wire and pad increase as far as possible because of considering impedance factors as far as possible.In this making processes, present method adopts the standard semiconductor manufacture craft, and the step summary is: four inches silicon chips (thickness 450 μ m) carry out first oxidation after standard cleaning, and thickness 1 μ m is to form one deck thin dielectric layer; Then at silicon chip surface sputter one deck Cr film, thickness 100nm, as the middle layer that adheres to Au, magnetron sputtering Au film again, thickness 500nm is as electrode layer.After making electrode pattern by lithography with AZ photoresist material S1912, the metal level etching that adopts wet etching to expose, the electrode minimum feature is 20 μ m; After etching is accomplished; Adopt PECVD each 500nm of deposit SiO2/Si3N4/SiO2 insulation layer on silicon chip; After the protection, (Reactive IonEtching RIE) etches solder joint (500 μ m) on electrode hole (minimum diameter 30 μ m) and the chip to use reactive ion etching technology more with photoresist.
The ECIS system be detect cell in the attaching of electrode surface, move the variation that isochronous cell-electrode attaches impedance, generally characterize with ac impedance spectroscopy.ECIS system in the chip of the present invention can be designed to interdigitation cell electricimpedance analysis device, and (Interdigitated array IDA), also can be designed as disc ECIS, or comprises IDA and disc ECIS simultaneously.Preferred ECIS system is for comprise IDA and disc ECIS simultaneously.
Interdigitation cell electricimpedance analysis device (Interdigitated array; IDA) but on cell migration, breeding etc. cause impedance variations tests and analyze; Belong to a kind of among the ECIS, when the cell testing impedance sensitivity higher, can monitor in real time cell state in the different condition.Cell-impedance electrical characteristic analyte sensors is the electrophysiologic study instrument that is mainly used in resistance change, membrane capacitance variation and the cellular layer-basilar membrane spatial variations of analyzing different cells.Its research object mainly is adherent type cells such as kidney cell, epithelial cell, most of tumour cell, also can be used for some and have electrophysiologic activity, can produce the adherent type cell of action potential, like myocardial cell etc.
In the design, the width of single interdigital electrode and spacing can be designed to 10-50 μ m, are preferably designed for 20-30 μ m.Interdigital length is arranged according to device cell and is designed to 2.4mm, is made up of 30 pairs of micro-strip electrodes, and single interdigital electrode active electrode test zone area is about 3mm
2, for improving contrast gradient and repeatability, two pairs of interdigital unit of design on the same chip.In order to reduce adjacent two interelectrode coupling capacitys etc., can select glass or silicon base material, if the while in order to consider the integrated chip concurrent designing of a plurality of sensing units, can be adopted silicon base to measuring the interference that causes; And, can adopt substrate of glass for ease of factors such as cell observation and raising biological coupling.Adopted two kinds of substrates to carry out processing and fabricating among the design respectively.When using multilayer photoetching process machined electrode, electrode surface interdigital to gold electrode for exposing, lead portion has all covered the insulation layer of the SiO2/Si3N4/SiO2 that is formed by the PECVD technology except pad, need expose 2 opposites simultaneously and amass and be respectively 8mm
2The effective cell adhesion zone.
In the ECIS system, another kind of effectively structure is a disc-shaped structure.Disc ECIS is a kind of electric physiology testing impedance device that can measure resistance change, membrane capacitance variation and the cellular layer basilar membrane spatial variations of multiple different cells simultaneously.Its test philosophy mainly adopts 2 telegraph circuits, i.e. working electrode and counter electrode, and the big I of electrode to the millimeter level, is used on electrode, attaching a plurality of cells from micron order.When the voltage of alternating current of certain frequency of input, measure the alternating current (size is a u A level) of output, can obtain an alternating-current impedance value, to analyze through the impedance dynamic changing process of several hrs, the physiological status of pair cell is carried out qualitative assessment.
ECIS circular electrode array chip can be designed to n group (n is an integer) as required, and each module is shared reference electrode.The big I of electrode from micron order to the millimeter level, for example 10 μ m-1mm μ m, more preferably 20 μ m-250 μ m.From optimization design, the present invention spatially carries out the ground connection isolation with four part big disk electrodes.Table 1 is a disk electrode each several part parameter, is 4 golden disk electrodes with microarray point altogether.Wherein disk electrode 1 is the gold electrode of single diameter 1.5mm, is used for the attached preliminary electric physiology testing impedance of cell and electrode paste; Disk electrode 2 diameter 1mm, the single electrode points diameter of array are that 25 μ m and ordinary cells size 10-30 μ m size are complementary; Be mainly used in the dynamicparameters such as migration and electrophysiologic activity of the adherent sexual cell of test, disk electrode 3 and 4 diameters are 1mm, and array point diameter and spacing are respectively 100um; 250um; Uniformly-spaced arrange, working electrode surface processes the site that contacts that exposes with cell, and the alternating-current impedance of transmission and record cell changes; Be mainly used in static parameter relatively such as analysis of cells form, cell attaching, and reduce the electric field interference problem between the adjacent electrode as far as possible.
Each module parameter of table 1.ECIS disk electrode array chip
Each module parameter of table 2 integrated chip
Existing cell chip technology is cultivated and is the basis with single kind cell, two dimension, and cell distribution does not in the plane have otherness, and this is that physiological simulation property, signals collecting and data processing are brought huge problem.And the cell printing technique of our exploitation, under can directly driving at the anatomy digital model of histoorgan, accurately viable cell/material cell is assembled in the location, makes the technology of the many cells precursor of tissue or organ.Therefore, with groups of cells packing technique assembling with induce various kinds of cell and control their exact position, the three-dimensional structure that makes up various kinds of cell theoretical and technical all be feasible, this makes also that cell is assembled in the location on the sensor array chip becomes possibility.
Through cell and substrate material are mixed, we obtained can with cell chip bonded cell composition.Wherein, preferred, the quantity of cell is 10
6-10
8Individual/the 1ml substrate material, preferred 1-5 * 10
7Individual/the 1ml substrate material.Preferably, cell can be myocardial cell or suprarenin pheochromocyte.Cell derives from the animal cell line that has had, and for example rat, mouse, rabbit, pig, ox or people etc. preferably derive from rat or people's clone.
Extracellular matrix constitutes the external environment of cells survival; They be in the growth course from the cell internal secretion to extracellular various macromole; Constitute the gel or the fibrous network of high degree of hydration then at cell peripheral; Play support, protection, vegetative cell, the differentiation of pair cell, motion, communication have material impact.The artificial substratum material that is applied to cell assembling should help the formation of three-dimensional group assembling structure and stable, helps cell in suitable three-dimensional open environment, to form cell mass, and transmission chemistry and mechanical signal are to cell.In addition, cell matrix materials will help the combination of cell/material composite in sensor chip surface.
The preferred substrate material of the present invention is the blend that comprises gelatin and sodium-alginate.Gelatin: the proportion optimization of sodium-alginate is 1: 0.2-1, more preferably 1: 1.Can gelatin and sodium-alginate be dissolved in respectively in the PBS damping fluid, face the time spent with two kinds of materials with 1: the even blend of 0.2-1, preferably with blend in 1: 1.Because substrate material is a blend, therefore, its gelation mode is more than the gelation mode of single-material, both can pass through controlled temperature (gelatin) and realize gelation, also can be through introducing linking agent (like Ca
2+) gelation.
Substrate material and cultured cells are mixed, obtain concentration and be about 10
6-10
8The cell composition of individual cell/mL substrate material is preferably 1-5 * 10
7Individual cell/1ml substrate material.
Can print to the cell composition that the formation of cell sensor chip surface has three-D space structure through the cell that the groups of cells packing technique will be mixed in the substrate material.The special-purpose experimental system that is used for the controlled assembling of cell three-dimensional mainly comprises the exclusive data process software; Solidworks for example; Cark delamination software etc.; Based on four-axis movement control card and stepper motor driven three-dimensional platform, material transports subsystem, and the working chamber of independent temperature and the shaping base plate of split-type design.Coordination control through total system has realized the accurate control to assembling process, obtains the cell fixed point after printing is accomplished and accurately is fixed on the cell chip.For example; Can the myocardial cell's compsn with three-dimensional structure be fixed on cellular microelectrode array (Microelectrode array; MEA) surface; Suprarenin pheochromocyte compsn with three-dimensional structure be fixed on individually or simultaneously interdigitation cell electricimpedance analysis device (Interdigitated array, IDA) and/or disc ECIS surface.
In the above-mentioned steps, gelatin/sodium-alginate/cell is through the low temperature stack shaping, again through the Ca2+ ionomer; Stability Analysis of Structures had both had certain rigidity, also had certain tensile properties; But the structure stable maintenance is more than 1 month, and the passage of design remains clear debating.
Preferably; Obtain a kind of cell chip according to above-mentioned steps; Described cell chip comprise the cellular microelectrode array (Microelectrode array, MEA) and electric impedance sensor (Electric Cell-substrate Impedance System, ECIS); The myocardial cell's compsn that wherein has three-dimensional structure is fixed on cellular microelectrode array (Microelectrode array; MEA) surface, the suprarenin pheochromocyte compsn with three-dimensional structure is fixed on electric impedance sensor (Electric Cell-substrate Impedance System, ECIS) surface.
Preferred; Obtain a kind of cell chip according to above-mentioned steps; Described cell chip comprise the cellular microelectrode array (Microelectrode array, MEA), interdigitation cell electricimpedance analysis device (Interdigitated array, IDA) with disc ECIS; The myocardial cell's compsn that wherein has three-dimensional structure is fixed on cellular microelectrode array (Microel ectrode array; MEA) and interdigitation cell electricimpedance analysis device (the suprarenin pheochromocyte compsn with three-dimensional structure is fixed on another regional interdigitation cell electricimpedance analysis device and disc ECIS surface for Interdigitated array, IDA) surface.
In the above-mentioned cell chip; Each components and parts such as cellular microelectrode array (Microelectrode array; MEA), interdigitation cell electricimpedance analysis device (Interdigitated array, IDA), the definition of disc ECIS, cell composition and substrate material as previously mentioned.
Therefore; One aspect of the present invention work is towards the cellular localization printing technique; Designed a kind of novel integrated cellular microelectrode array (Microelectrode array; MEA) and electric impedance sensor (Electric Cell-substrate Impedance System ECIS) waits the array chip of sensing unit.This integrated chip design; Adopt cellular microelectrode sensor array, electric impedance sensor (ECIS) integrated-type device; Contrast ground carries out the analysis of cellular electrophysiologicalsensor important parameter; Can detect the variations such as attaching property, transport property of action potentials of cells, cell electricimpedance and cell growth state simultaneously, realize that the real non-destructive of the multiple electric-physiology parameter of cell is measured.
Another object of the present invention has provided a kind of method for preparing three-dimensional cell chip, and this method comprises the steps:
(1) cell and substrate material mixing are obtained cell composition;
(2) cell composition is fixed on chip through the special-purpose experimental system of three-dimensional controlled assembling
Sensor surface forms the cell composition with three-dimensional structure.
In the aforesaid method, the definition of cell, substrate material, chip sensor as previously mentioned.
The special-purpose experimental system of the controlled assembling of cell three-dimensional mainly comprises the exclusive data process software in the aforesaid method; Solidworks for example; Cark delamination software etc.; Based on four-axis movement control card and stepper motor driven three-dimensional platform, material transports subsystem, and the working chamber of independent temperature and the shaping base plate of split-type design.Coordination control through total system has realized the accurate control to assembling process, obtains the cell fixed point after printing is accomplished and accurately is fixed on the cell chip.The Cell Assembly II equipment of preferred equipment such as Yin Hua company.
Another object of the present invention provides a kind of method that above-mentioned three-dimensional cell chip model is used for drug screening of using.
The present invention sets up the three-dimensional localization cell chip with cell printing technique and microelectrode array sensor technology.Through the specified location of many shower nozzles cell PRN device positioning printing in the microelectrode array sensor array, make up cell chip.Through parameters such as signal velocities between the transmission of the cell sensor on the chip and record action potentials of cells frequency, amplitude, waveform and cellular network.
It is external cardiac muscle-suprarenin regulator control system modeling framework of master that the present invention has set up with myocardial cell and adrenal pheochromocytoma.Designed the structural models that makes up cell chip in order to instruct.The application cell package technique has been accomplished the assembling of design chips.Research shows that model is incubated at the cell of chip surface than the plane can better the intravital physiological and pathological situation of emulation.
From myocardium rhythmicity and the importance of conductivity cardiovascular disorder, selected with the action potential of cardiac muscle and the signal velocity between cellular network in the MEA sensor detecting cell chip, thereby monitored myocardium automaticity and transport properties in real time.
Breeding and the importance of migration the metabolic cardiomyopathy from the suprarenin pheochromocyte; Selected impedance variations, thereby monitored growth, migration, the distortion of adrenal pheochromocytoma in real time with adrenal pheochromocytoma assembling position in the IDA sensor detecting cell chip.
After making up the cell chip of location assembling myocardial cell and adrenergic cell, add medicine to be measured, the monitoring data of detection model under normal and pathological conditions can be carried out drug screening through the pharmacologically active of analyzing medicine to be measured.
A kind of purposes of the cell chip of myocardial cell and suprarenin pheochromocyte is the pathological model that can be used as cardiovascular disorder, at the pathological characters of in-vitro simulated cardiovascular disorder.Physiological properties such as myocardial cell's shrinkability and autorhymicity, irritability, conductivity are determining the activity of heart jointly, are the important physiological bases of heart function and cardiovascular function.When a series of cardiovascular disordeies such as beating of myocardial cell takes place can to cause when unusual to quiver in arrhythmia, hypertension, atrial fibrillation, chamber, stenocardia and myocardial infarctions.Myocardial cell's function is regulated by multiple factor, and wherein adrenal pheochromocytoma excretory suprarenin plays important regulatory role, when the adrenal pheochromocytoma excretory is disorderly, can cause the generation of myocardial function disorder and a series of cardiovascular disordeies.
Myocardial cell in MEA device position assembling in the cell chip is equivalent to intravital heart, is equivalent to intravital acth secretion cell at the adrenal pheochromocytoma of IDA and/or the assembling of disk ECIS sensor site.And the various materials in the nutrient solution comprise medicine, simulate intravital normal and pathologic condition.Through myocardial cell's action potential and the signal velocity between cellular network in the detection cell chip, can monitor the automaticity and the transport properties of cardiac muscle in real time.Through detecting the impedance variations of adrenal pheochromocytoma assembling position in the cell chip, can monitor growth, migration, the distortion of adrenal pheochromocytoma in real time.This cell chip can reproduce and the real-time process of adrenal pheochromocytoma secretion suprarenin regulation and control myocardial function in the detection bodies thus, multiple cardiovascular disorder is studied and drug screening, particularly a series of cardiovascular disordeies of causing of gland pheochromocytoma.
This model and the technology of setting up model, have wide practical use in cell chip field and drug research field: wherein to the development and application of three-dimensional cell chip, the realization of the target that " human on a chip " is such will produce great pushing effect; Also will make external drug study more system, accurately near testing in the body, reduce the drug development cost and risk, accelerate the process of new drug development.This technology is with a wide range of applications.
Below in conjunction with embodiment the present invention is described further, protection scope of the present invention is not limited to embodiment, every according in disclosed by the invention perhaps principle any this area of implementing be equal to replacement, all belong to protection scope of the present invention.
Description of drawings
Fig. 1 is the cell chip assembly drawing, and wherein Fig. 1-1 is a principle process, and Fig. 1-2 is for assembling the cell chip after accomplishing.
Fig. 2 is the chip surface structure.Wherein Fig. 2-1 is metal level (adopting the electrode and the pad layer of Cr/Au material), and Fig. 2-2 is the cloudy version of insulation layer (the effective site of the electrode that insulation layer exposed and the pad area that adopt SiO2/Si3N4/SiO2 to make)
Fig. 3 is an integrated chip machining process schema
Fig. 4 gold disk electrode ac impedance spectroscopy
Fig. 5 cell impedance variations figure.The result is presented under the effect of cancer therapy drug 5-FU, and death has all taken place two kinds of cells on the chip, and wherein 5-FU is stronger to the lethal effect comparison myocardial cell's of the cell PC-12 in tumour source effect.The result proves the inspection susceptibility of chip cell growth, also shows the potentiality of screening tumour medicine.
The myocardial cell's that Fig. 6 chip detection arrives automatic rhythmicity is movable
The medicine of the different pharmacological mechanism of Fig. 7 is to printing the intrasystem myocardium jumping frequency rate influence of cell chip.Under the cardiovascular agent effect of three kinds of different mechanisms; The detected result of cell chip shows, beta-2 adrenoceptor antagonistic Proprasylyte can significantly reduce myocardial cell's jumping frequency rate, alpha-2 adrenoceptor retarding agent Phenoxybenzamine to myocardial cell's jumping frequency rate do not make significant difference, the biosynthesis inhibitor alpha-methyltyrosine of catecholamine also can significantly reduce myocardial cell's jumping frequency rate.This cell chip can filter out simultaneously, acts on myocardium beta-2 adrenoceptor and the two big types of medicines of synthetic that suppress catecholamine.The result has proved that this cell chip carries out the ability of cardiovascular agent screening.
Embodiment
Selection derives from the myocardial cell of neonate rat as one of basic seed cell of construction system.
(1) once get a suckling mouse, be placed on the sterile gauze, clean its chest belly with 70% ethanol, the extraction heart is put mark into and in 1 the beaker.Dissect not above 1 minute at every turn.
(2) reject reticular tissue and clot on the heart with mosquito forceps, change over to again and be labeled as in 2 the beaker, wash again one time, change over to and be labeled as in 3 the beaker.Gently the D-Hanks liquid sucking-off in the beaker 3, add the 0.25% pancreatin solution (37 ℃) of 2ml with suction pipe then, be broken into grains of sand size to cardiac scissors with eye scissors, add 2ml pancreatin solution, 37 ℃ digested 15 minutes behind the mixing.
(3) the digestion back is inhaled and is removed supernatant, adds the new pancreatin solution of 4ml, mixing, and 37 ℃ of digestion are abandoned supernatant after 15 minutes again.Add 2ml pancreatin solution and 2ml 0.06% collagenase (II type) solution, 37 ℃ digested 15 minutes behind the mixing.Get simultaneously in 4 15ml centrifuge tubes and to add the DMEM (Dulbecco ' s Modified Eagle ' s Medium, sigma)+) that the cold inoculation medium of 6-7ml contains 10%FBS.
(4) shift out supernatant to the above-mentioned centrifuge tube that is added with inoculation medium.Remaining tissue adds 0.03% collagenase 4ml, mixing, and 37 ℃ digested 20 minutes.Repeat with 0.03% collagenase digesting 3-5 time, until tissue block digestion fully or till remaining a little.
(5) centrifuge tube that contains substratum and cell conditioned medium centrifugal 8 minutes with 1400rpm is inhaled and is removed supernatant, all collects in the 50ml centrifuge tube with 1400rpm centrifugal 8 minutes after resuspended with the 3ml inoculation medium.After supernatant is removed in suction, add the 10ml inoculation medium, blow and beat all cells group of scattering.
(6) above-mentioned 10ml cell suspension is transferred in the glass culture dish (handling with 0.2% gelatin in advance) of 100mm specification, hatches in cell culture incubator that to carry out differential in 1.5 hours adherent.
(7) hatch the nutrient solution of back in the petridish, collect after washing petridish gently with the 10ml inoculation medium.With inoculation medium cell density is adjusted to 2 * 10
5-5 * 10
5Individual/ml, process inoculation liquid, be inoculated in the culture plate 35mm petridish.
After (8) 24 hours, change liquid to cell with preparatory warm 37 ℃ exchange substratum (DMEM+10%BS).Change the liquid continued and hatch, changed a not good liquor in later per two days.Can be observed beating of myocardial cell in inoculation after 24 hours, to about the 4th day, most cells all will be beated.The general cell of cultivating after 3 days of using experimentizes.
(1) PC-12 mouse adrenal pheochromocytoma oncocyte system (ATCC CRL-1721, American Type Culture Collection, U.S. typical case DSMZ); With high sugared DMEM (Dulbecco ' s Modified Eagle ' s Medium; Sigma)+10% the foetal calf serum substratum is incubated at general about three days of 37 ℃ of carbonic acid gas constant temperature culture, and cell reaches individual layer and converges; After cell converges, carry out the routine cultivation of going down to posterity;
(2) go down to posterity: inhale and remove nutrient solution, add 0.2% pancreatin solution 1mL, inhale behind the washing bottle wall rapidly and go; Add 0.2% pancreatin solution 1mL again; Hatch about 5min for 37 ℃, microscopic, treat 80% cell rounding after; Inhale gently and remove pancreatin solution, add the DMEM substratum that contains 15%FCS rapidly and stop digestion; Piping and druming culturing bottle wall is made into cell suspension once more; Adjustment cell concn to 1.5～3 * 105cells/mL is inoculated in the new culturing bottle.
(3) the cell cultivation of can normally going down to posterity was gone down to posterity once in per three days.
In complete processing, MEA and ECIS unit all adopt gold electrode and underlying structure all the same with protective layer structure, therefore can share complete processing.Concrete processing process is seen among Fig. 3 shown in each step.The course of processing is an example with the silicon base, and the course of processing is summarized as follows:
At first growth of device surface insulation layer and deposition adopt chemical vapour deposition (PECVD) technology to deposit the Si3N4 of one deck 100nm in the above.Process a plurality of functional units of MEA and ECIS then.On the basis of former step processing; Utilize PECVD to deposit the SiO2 layer of a layer thickness on the chip for 600nm; Metal level adopts the Au/Cr composite bed; Degree of adhesion between the silicon substrate layer that Cr is used to strengthen gold and deposit SiO2 utilizes the photoresist layer protection again, etches electrode and the lead pattern of MEA&IDE.Because lead portion needs to isolate with external insulation, so the thick Si3N4 deposition of 800nm is on it, and protection with photoresist is electropotential and pad exposure.Electrode, welding disking area with front and back adopts the oxygen plasma etching at last, and counter electrode carries out cleaning MEA this moment, ECIS two unitary three partly integrated basic technologies are accomplished.Carry out the encapsulation work of device at last, the chip surface structure is as shown in Figure 2.
Because MEA, each functional unit of ECIS on the integrated chip all belong to electricity chip category; So with the disk electrode among the ECIS is representative;, impedance maximum disk tiny array electrode minimum with diameter and spacing is example (under the chip figure left side 2) (electrod-array spacing and diameter are d=25 μ m), and disk electrode is carried out ac impedance spectroscopy test (Fig. 3).
Last figure is in each unit of ECIS chip, and the maximum disk electrode array of theoretical resistance value adopts triple electrode circuit to the electrode AC impedance collection of illustrative plates before and after electroplating (n=10, direct current biasing-0.2V load the 10mV voltage of alternating current) in 0.9% NaCl solution.When 1KHz, resistance value is about 500K Ω, has met the requirement that microelectrode can measure the outer electrical signal of cell born of the same parents.In concrete performance test, the alternating-current impedance amplitude is along with the rising of frequency descends steadily, and the impedance spectrum value difference of each electrode is different not obvious; But (1～100Hz) resistance does not have the medium-high frequency section with the slope of change of frequency and changes noticeably, so be adapted at medium-high frequency section 10 to the test of impedance magnitude sensitivity in the low-frequency range
3～10
5Hz carries out; To the phase place of disk electrode, 10
3-10
4In the Hz, be a constant value (about 80 °), therefore can judge that electrode mainly is capacitive, measurement sensitivity is not high.But the phase place of low frequency and high frequency is more obvious with change of frequency, considers that the low frequency range impedance magnitude is bigger, so when the adherent impedance of actual test cell, for the variation of the electrode surface capacitive reactances that organism such as microbial film cause, 10 are described
4-10
5Hz test preferably, and have sensitivity preferably.Therefore, can effectively increase electrode surface area to the disk electrode ac impedance measurement explanation plating before and after electroplating, thereby improve the body impedance and the sensitivity of electrode.Later stage can analyze and research in the experiment of electrode surface attaching rate for multiple vitamin H to the difference of electroplating rear electrode institute culturing cell.
(1) gelatin, sodium-alginate and the preparation of cell intermingling material: gelatin is dissolved in the PBS damping fluid, forms 20% solution; Sodium-alginate is dissolved in the PBS damping fluid, forms 5% solution, use among the NaOH respectively and remaining acetic acid; Regulate pH value to 7.1,70 ℃ of baking oven discontinuous formula sterilizations.Face the time spent with two kinds of materials with even blend in 1: 1.Material and cardiac muscle cells and adrenal pheochromocytoma are mixed respectively, obtain concentration and be about 1 * 10
7/ mL cell-matrix material intermingling material.
(2) with the disposable syringe of cell/matrix material intermingling material injection 1mL, place 4 ℃ of refrigerator precoolings more than 30 minutes;
(3) select the Cell Assembly II equipment of Yin Hua company for use, the sterilized shaping base plate of internal fixing in the working chamber is opened shaping room temperature control switch and is made working chamber's cooling, monitor temperature in real time, and when treating that temperature is reduced to 6 ℃, mounting of syringe and stationary nozzle;
(4) directly drive shower nozzle at sensor array chip surface assembling cell by Solidworks design 3D model and selected forming parameter.According to feature size and the corresponding different size shower nozzle of material rate of expansion, select the shower nozzle of diameter 120u m for use.Sweep velocity 30mm/s extrudes flow 1.2mm
3/ s.
(5) the control shower nozzle partly assembles the myocardial cell in MEA part and IDA respectively, at IDA part and disc ECIS assembling adrenal pheochromocytoma.
(6) obtain containing the cell chip of cell after shaping is accomplished.Chip is stored in the aseptic 4 ℃ environment, forms aftertreatment in 1 hour.
(7) crosslinked 1 minute of chip surface Dropwise 5 % sterilization calcium chloride solution, with DMEM washing three times;
(8) add and contain 10%FCS, 1 μ mol/L insulin, the DMEM nutrient solution of 50ng/mL EGF-1,37 ℃, 5%CO
2Environment is cultivated down 3d, the next day change liquid.
The application of embodiment 4 cell chips in drug screening
(1) the cell chip system after assembling is accomplished is with the DMEM that contains 10%FCS (Dulbecco ' sModified Eagle ' s Medium) nutrient solution, 37 ℃, 5%CO
2Environment is cultivated down 3d, the next day change liquid; Because adrenal pheochromocytoma is the knurl source, can breed secretion suprarenin, the spontaneous pathological state that develops into metabolic cardiomyopathy of model in a large number;
(2) selecting operating frequency respectively is that the Frequency point of 6kHz, 8kHz, 10kHz is tested (n=5).Pair cell is before medicine irritation, and at first (20 ℃) leave standstill 1h under room temperature, carry out the impedance sweep check;
(3) in contradistinction system, add 5-FU; The biosynthesis inhibitor of the medicine beta-2 adrenoceptor antagonistic Proprasylyte (100 μ g/mL) of 3 kinds of different mechanisms of every then interval 1h adding, alpha-2 adrenoceptor retarding agent Phenoxybenzamine (100 μ g/mL), catecholamine: alpha-methyltyrosine (α-MT) (100 μ g/mL); Carry out the action potential test of 20min, use the DMEM wash-out behind the 30min.
Two kinds of cell impedance datas that chip detection obtains, it is visible that the impedance of two kinds of cells has all descended under the effect of cancer therapy drug 5-FU, the expression necrocytosis, wherein adrenal pheochromocytoma is because have the tumour cell characteristic, and the effect of medicine is more obvious.
Know by experimental result; Native system can accurately be monitored out the pharmacological action of three kinds of different mechanisms medicines simultaneously; Experimental data comprises the propagation and the migration of myocardial cell and adrenal pheochromocytoma, and myocardial cell's automatic rhythmicity is movable, and model is accurately quick to the screening of medicine; The physiological data of gathering is comprehensive available, for follow-up deep Mechanism Study is laid a good foundation.Research has proved that fully this cell chip system carries out the potentiality of research of metabolic cardiomyopathy and drug screening, and these experiments have simultaneously also proposed performance model and carried out method for screening.
Claims (10)
1. cell chip; Comprise cellular microelectrode array (Microelectrode array; MEA) and electric impedance sensor (Electric Cell-substrate Impedance System; ECIS), and be fixed on the cell composition with three-D space structure on the transmitter, described cell composition is the blend that comprises cell and substrate material.
2. cell chip according to claim 1 is characterized in that: the MEA part on the said cell chip can be designed to m*n array (n, m is respectively integer) arbitrarily as required, and every row's electrode diameter and interelectrode distance are designed to 10-50 μ m.
3. cell chip according to claim 1; It is characterized in that: the ECIS system can be designed to interdigitation cell electricimpedance analysis device (Interdigitated array on the said cell chip; Or comprise IDA and disc ECIS simultaneously IDA) or disc ECIS.
4. cell chip according to claim 1 is characterized in that: said cell chip comprises the MEA of 1 group of 3 * 3 array, is distributed in chip center; 4 groups of ECIS circular electrode arrays are distributed in the chip left area; 2 groups of ECIS IDA array regions are distributed in zone, chip the right, up and down symmetry.
5. cell chip according to claim 1 is characterized in that: cell is myocardial cell or suprarenin pheochromocyte in the described cell composition.
6. cell chip according to claim 1 is characterized in that: described cell composition mesostroma material is the blend that comprises gelatin and sodium-alginate, gelatin: the proportioning of sodium-alginate is 1: 0.2-1.
7. each described cell chip of claim 1-6; It is characterized in that: described cell chip comprises cellular microelectrode array (Microelectrode array; MEA) and electric impedance sensor (Electric Cell-substrate Impedance System; ECIS), the myocardial cell's compsn that wherein has a three-dimensional structure is fixed on cellular microelectrode array (Microelectrode array, MEA) surface; Suprarenin pheochromocyte compsn with three-dimensional structure is fixed on electric impedance sensor (Electric Cell-substrate Impedance System, ECIS) surface.
8. cell chip according to claim 7; It is characterized in that: described cell chip comprises cellular microelectrode array (Microelectrode array; MEA), interdigitation cell electricimpedance analysis device (Interdigitated array; IDA) and disc ECIS, the myocardial cell's compsn that wherein has a three-dimensional structure is fixed on the cellular microelectrode array, and (Microelectrode array is MEA) with interdigitation cell electricimpedance analysis device (Interdigitated array; IDA) surface, the suprarenin pheochromocyte compsn with three-dimensional structure is fixed on another regional interdigitation cell electricimpedance analysis device and disc ECIS surface.
9. prepare the method for each said three-dimensional cell chip of claim 1-8, comprise the steps:
(1), cell and substrate material mixing are obtained cell composition;
(2), cell composition is fixed on the cell composition that the formation of chip sensor surface has three-dimensional structure through the special-purpose experimental system of three-dimensional controlled assembling.
10. each described cell chip application in drug screening of claim 1-8; The cell chip that is fixed with myocardial cell and suprarenin pheochromocyte can be used as the pathological model of cardiovascular disorder; Pathological characters in in-vitro simulated cardiovascular disorder; Add medicine to be measured, the monitoring data of detection model under normal and pathological conditions can be carried out drug screening through the pharmacologically active of analyzing medicine to be measured.
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