CN104931683A - Myocardial tissue sensor and myocardial tissue chip preparation method - Google Patents
Myocardial tissue sensor and myocardial tissue chip preparation method Download PDFInfo
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Abstract
The invention discloses a preparation method of myocardial tissue sensor and a myocardial tissue chip, and belongs to the technical fields of tissue engineering, biomedicine engineering and sensor. The myocardial tissue sensor comprises a myocardial tissue chip and auxiliary parts, wherein the auxiliary parts include an upper die, a lower die, an upper electrode, a lower electrode, a sensitive plate, a parallel light source and a data acquisition system. According to the invention, myocardial cells are sequentially printed on a glass sheet coated with N-isopropylacrylamide and polydimethylsiloxane by virtue of a cell printing technology so as to prepare the myocardial tissue chip; the myocardial tissue chip is kept in the auxiliary parts and is subjected to pulsatile cultivation to form myocardial tissues consistent in beating rhythm, and then the myocardial tissues are kept in a culture solution containing a medicine and undergo electrical stimulation; and a contractile change signal is collected to evaluate the effect of the medicine on cardiac muscle, so as to achieve the purposes of drug screening, testing and the like. The myocardial tissue sensor disclosed by the invention has the advantages of simple device, wide scope of testing medicine, easy detection and processing of signal, and the like.
Description
Technical field
The invention belongs to organizational project, biomedical engineering and sensing technology crossing domain, particularly the preparation method of a kind of cardiac muscular tissue sensor and cardiac muscular tissue's chip.
Background technology
Cardiac muscle cell is the main composition cell of cardiac muscular tissue, is mainly distributed in heart wall and faces on heart trunk, and cardiac muscle cell is short cylindrical shape threadiness, has obvious directivity.Compared to the main cell such as nephrocyte, liver cell based on metabolic function, the major function of cardiac muscle cell is shunk and diastole, and the blood-pumping function realizing heart with this is to promote blood circulation.About the damage of cardiac muscular tissue and pathology can cause various heart disease and cardiomyopathies, all there is very high M & M, serious threat human survival and quality of life.According to statistics, in the U.S., about have 4,800,000 patients with congestive heart failure, and an annual new patient is more than 400,000.Effective treatment of cardiomyopathies be unable to do without the exploitation of related drugs, and the screening of medicine and test are novel drugs come into operation in an indispensable link.Traditional animal or the in vivo studies of human body exist that influence factor is uncontrollable, the difficult repetition of test findings, the inherent defect such as the time-consuming effort of process of the test and relevant ethics problem.The appearance of cell (tissue) sensor then provides new thinking for addressing these problems.
Cell sensor is using cell as main functional elements, and identification and perception measured object also convert device or the device of discernible signal according to certain rules to.Because it has, volume is little, response is fast, can realize the features such as original position on-line monitoring, extensive in field application prospects such as environmental assessment, food security and drug screenings.Over nearly 30 years, the development of cell culture technology and micro electronmechanical manufacturing technology, especially for the development of cell sensor provides good platform, makes cell sensor obtain increasing concern and application in biological, chemical and field of medical analysis.Traditional cardiac muscle cell's sensor all with the electricity physiological signal of cell for detection limit, in succession there are voltage clamp cell sensor, patch-clamp cell sensor, field effect transistor cell sensor, microelectrode array cell sensor and Light Addressable Potentiometric Sensor etc. on this basis.There is complex manufacturing technology in the sensor of above-mentioned various detection cell electric signal, biocompatibility and Signal-to-Noise such as to have much room for improvement at the problem.(Feinberg AW, et.al, the Muscular thin films for building actuators and powering devices.Science2007 such as Feinberg AW; 317:1366 – 70.) in 2007, a kind of muscular thin film (muscular thin film is proposed, MTF) technology, muscle cell is planted on temperature sensitive macromolecular material and makes three-dimensional musculature, and test the drawdown deformation amount of musculature under environmental stimuli with this, the response for cardiac muscle cell detects and provides a kind of new means.On the other hand, when carrying out cell sensor system, biocompatibility is the aspect that must consider, want the physiologically active of test cell, cell and device surface must be coupled well, namely also cell can at the easy tactophily of device surface, and traditional cell seeding technology is difficult to ensure that cell is coupled with the fine of device, thus greatly have impact on accuracy of detection.
It is a kind of advanced technology constructing three-dimensional many cells system in vitro occurred in recent years that cell 3D prints (cell printing) technology, and this technology is the combination of rapid shaping technique and Biotechnology.In cell printing process, cell (or cell aggregation) and colloidal sol (presoma of hydrogel) are placed in simultaneously or celliferous nutrient solution is placed in separately the shower nozzle of printer, the deposition position containing cell drop is controlled by computing machine, print in the position pointwise of specifying, the basis having printed one deck is continued print another layer, be layering and form three-dimensional many cells-gel rubber system.Compared with conventional art, the advantage of cell printing is mainly reflected in can the different types of cell of accurate deposition over time and space, and controllability is large, can form the accumulation body with various structures.
Summary of the invention
The present invention is directed to the weak point of prior art, the preparation method of a kind of cardiac muscular tissue sensor and cardiac muscular tissue's chip is provided, aim to provide cardiac muscular tissue's sensor of a kind of novel cardiac muscular tissue detected amount of contraction, and cell printing technology is introduced in the preparation of cardiac muscular tissue's chip, make it have collection signal signal to noise ratio (S/N ratio) high, cardiac muscle cell and device can be coupled the advantage such as good.
Technical scheme of the present invention is as follows:
A kind of cardiac muscular tissue sensor, described sensor comprises cardiac muscular tissue's chip and accessory; Described accessory comprises upper die and lower die, top electrode, bottom electrode, photographic plate, parallel light source and data acquisition system (DAS); Described patrix is provided with first fluid passage, second fluid passage, guiding tongue and upper groove; Described lower film is provided with culture chamber, chip groove, low groove and direction recess; Described upper die and lower die are installed together by guiding tongue and direction recess orientation; Described cardiac muscular tissue chip is placed in chip groove; It is inner and one-body molded with upper die and lower die that described top electrode and bottom electrode lay respectively at upper die and lower die; Top electrode is connected with power supply, and bottom electrode bottom is positioned on chip groove, and with the cardiac muscular tissue's chip close contact in chip groove; Described photographic plate is placed in the upper groove of patrix, and is connected with data acquisition system (DAS); Described parallel light source is placed in the low groove of counterdie.
In technique scheme, it is characterized in that: described upper die and lower die all adopt makrolon material, described top electrode and bottom electrode adopt titanium alloy material.
Cardiac muscular tissue of the present invention chip, is characterized in that the preparation method of this cardiac muscular tissue's chip comprises the following steps:
1) one deck adhesive plaster is sticked on the surface of the glass sheet after one piece of sterilization;
2) on adhesive plaster, carry out first time cutting with laser formation machine, form at least 5 square contours, each square contour length is 6 ~ 8mm, and width is 0.6 ~ 1mm, spaced 0.1 ~ 0.3mm, and removes the square rubber pieces of cloth cut;
3) preparation quality volumetric concentration is the poly-N-isopropyl acrylamide solution of 10% ~ 50%, with spin coating method be evenly coated in glass sheet containing adhesive plaster side, remove adhesive plaster after solidification, leave square poly-N-isopropyl acrylamide layer;
4) by after dimethyl silicone polymer heating and melting, be coated onto on glass sheet with spin coating method, after solidification process, dimethyl silicone polymer to be cut into along second time cutting track (407) with laser formation machine to be middlely connected, both sides are multiple " semi-girder " (408) structure, both sides " semi-girder " spacing is 0.6 ~ 1mm;
5) preparation quality volumetric concentration be 0.1 ~ 20% hydrogel solution and mass body volume concentrations be the cross-linking agent solution of 0.1 ~ 20%, extract or inducing cardiomyocytes to make cell density be 1 × 10
7~ 2 × 10
7cardiac muscle cell's suspending liquid of individual/ml, cardiac muscle cell being mixed in hydrogel solution and making cell density is 1 × 10
6~ 5 × 10
6the hydrogel solution containing cardiac muscle cell of individual/ml; Loaded by cardiac muscle cell's suspending liquid in spray-type shower nozzle 1, cross-linking agent solution loads in spray-type shower nozzle 2, and the hydrogel solution containing cardiac muscle cell loads in squash type shower nozzle; Successively print on " semi-girder " with the composite spray jet head be made up of spray-type shower nozzle 1, spray-type shower nozzle 2 and squash type shower nozzle, the thread hydrogel that squash type shower nozzle is extruded forms water-setting collodion silk (502) after the cross-linking agent solution that spray-type shower nozzle 2 sprays is cross-linked, then cardiac muscle cell's layer (501) is formed with spray-type shower nozzle 1 at water-setting collodion silk surface sprinkling one deck cardiac muscle cell suspending liquid, repeat to extrude, be cross-linked, form cardiac muscular tissue's chip with multi-layer cellular after the operation such as sprinkling, water-setting collodion silk is between layers parallel to each other;
6) this cardiac muscular tissue's chip is placed in the chip groove of described accessory, after pulsation is cultivated, forms cardiac muscular tissue's chip of beating consistent.
Preferably, step 2) on adhesive plaster, cut 8 ~ 15 square contours with laser formation machine.
The preparation method of cardiac muscular tissue of the present invention chip, it is characterized in that, step 5) described in the solute of hydrogel solution be at least one in sodium alginate, collagen, hyaluronic acid, matrigel, dextrose, shitosan, fibronectin, gelatin and fibrinogen and matrigel, described crosslinking chemical is at least one in fibrin ferment, lime chloride or peroxophosphoric acid sodium.
The preparation method of cardiac muscular tissue of the present invention chip, it is characterized in that, step 6) described in pulsation cultivate, its process is that nutrient solution is flowed into culture chamber from the first fluid passage of accessory, flow out from second fluid passage, flowing velocity is 10 ~ 50mL/min, circulates 40 ~ 50h.
Compared with prior art, the present invention has the technique effect of following advantage and high-lighting: 1. cardiac muscular tissue of the present invention sensor can gather the contraction signal of cardiac muscular tissue, and the use of source of parallel light improves the acquisition precision of signal.2. the present invention utilizes cell printing technology to be piled up on the glass sheet by the hydrogel containing cardiac muscle cell in preparation cardiac muscular tissue chip processes, can ensure cell and device good coupling, and the size controllable precise printed, multiple accuracy requirement can be met; 3. the present invention adopts and sprays the structure of myocardial cell suspensions in the hydrogel surface containing cardiac muscle cell and pile up, and improves the density of the cardiac muscle cell in cardiac muscular tissue, can promote that iuntercellular connects formative tissue; 4. the accessory of cardiac muscular tissue of the present invention sensor can carry out pulsation cultivation to cardiac muscular tissue, and the nutrient solution of flowing with the equidirectional stimulation continued, can form the cardiac muscular tissue of directivity to cardiac muscle cell.
Accompanying drawing explanation
Tu1Shi cardiac muscular tissue sensor general illustration.
Fig. 2 is upper die structure schematic diagram.
Fig. 3 is lower die structure schematic diagram.
The schematic flow sheet of Tu4Shi cardiac muscular tissue chip fabrication processes.
Fig. 5 is that cardiac muscle cell piles up schematic diagram.
In figure: 101-patrix; 102-photographic plate; 103-data acquisition system (DAS); 104-counterdie; 105-parallel light source; 106-bottom electrode; 107-top electrode; 108-power supply; 201-upper groove, 202-first fluid passage, 203-second fluid passage, 204-leads tongue, 301-direction recess, 302-culture chamber, 303-chip groove, 304-low groove, 401-glass sheet, 402-adhesive plaster, 403-first time cutting track, 404-square rubber pieces of cloth, 405-poly-N-isopropyl acrylamide layer, 406-dimethyl silicone polymer layer, 407-second time cutting track, 408-" semi-girder ", 409-cardiac muscular tissue, 501-cardiac muscle cell's layer, 502-water-setting collodion silk.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in further details.
Fig. 1 is one provided by the invention is cardiac muscular tissue's sensor general illustration, and described accessory comprises patrix 101, counterdie 104, top electrode 107, bottom electrode 106, photographic plate 102, parallel light source 105 and data acquisition system (DAS) 103; Described patrix 101 is the right cylinders be made up of transparent polycarbonate, two through circular fluid passages are offered at top, i.e. first fluid passage 202 and second fluid passage 203, top center has a square upper groove 201, and patrix edge is provided with two guiding tongues 204; Counterdie is the right cylinder be made up of transparent polycarbonate, and upper center membrane has culture chamber 302 and chip groove 303 successively, and internal edge is provided with direction recess 301; Counterdie is the right cylinder be made up of transparent polycarbonate, and central upper portion has culture chamber 302 and chip groove 303 successively, and internal edge is provided with direction recess 301, has low groove 304 bottom counterdie; Described upper die and lower die are installed together by guiding tongue 204 and direction recess 301 orientation; Described cardiac muscular tissue chip is placed in chip groove 303; Described top electrode 107 and bottom electrode 106 lay respectively at patrix 101 and counterdie 104 is inner and one-body molded with upper die and lower die; Top electrode 107 is connected with power supply 108, and bottom electrode 106 bottom is positioned on chip groove 303, and during installation, myocardium chip is placed in chip groove 303, and with bottom electrode close contact; Described photographic plate 102 is placed in the upper groove 201 of patrix, and is connected with data acquisition system (DAS) 103; Described parallel light source 105 is placed in the low groove 304 of counterdie, upwards emitting parallel light (see Fig. 2 and Fig. 3).
The partial schematic diagram of Tu4Shi cardiac muscular tissue chip fabrication processes, the preparation method of cardiac muscular tissue's chip comprises the following steps:
1) one deck adhesive plaster 402 is sticked on the surface of the glass sheet 401 after one piece of sterilization;
2) on adhesive plaster, formation at least 5 square contours are cut along first time cutting track 403 with laser formation machine, each square contour length is 6 ~ 8mm, width is 0.6 ~ 1mm, spaced 0.1 ~ 0.3mm, and remove the square rubber pieces of cloth 404 cut, as shown in (a) in Fig. 4;
3) preparation quality volumetric concentration is the poly-N-isopropyl acrylamide solution of 10% ~ 50%, with spin coating method be evenly coated in glass sheet containing adhesive plaster side, remove adhesive plaster after solidification, leave square poly-N-isopropyl acrylamide layer 405, as shown in (b) in Fig. 4;
4) by after dimethyl silicone polymer heating and melting, be coated onto on glass sheet with spin coating method, after solidification process, dimethyl silicone polymer to be cut into along second time cutting track (407) with laser formation machine to be middlely connected, both sides are multiple " semi-girder " (408) structure, both sides " semi-girder " spacing is 0.6 ~ 1mm, as shown in (c) in Fig. 4;
5) preparation quality volumetric concentration be 0.1 ~ 20% hydrogel solution and mass body volume concentrations be the cross-linking agent solution of 0.1 ~ 20%, extract or inducing cardiomyocytes to make cell density be 1 × 10
7~ 2 × 10
7cardiac muscle cell's suspending liquid of individual/ml, cardiac muscle cell being mixed in hydrogel solution and making cell density is 1 × 10
6~ 5 × 10
6the hydrogel solution containing cardiac muscle cell of individual/ml; Loaded by cardiac muscle cell's suspending liquid in spray-type shower nozzle 1, cross-linking agent solution loads in spray-type shower nozzle 2, and the hydrogel solution containing cardiac muscle cell loads in squash type shower nozzle; Successively print on " semi-girder " with the composite spray jet head be made up of spray-type shower nozzle 1, spray-type shower nozzle 2 and squash type shower nozzle, the thread hydrogel that squash type shower nozzle is extruded forms water-setting collodion silk 502 after the cross-linking agent solution that spray-type shower nozzle 2 sprays is cross-linked, then cardiac muscle cell's layer 501 is formed with spray-type shower nozzle 1 at water-setting collodion silk surface sprinkling one deck cardiac muscle cell suspending liquid, water-setting collodion silk is between layers parallel to each other, as shown in Figure 5; Repeat to extrude, be cross-linked, make cardiac muscular tissue's chip with multi-layer cellular after spraying operation;
6) this cardiac muscular tissue's chip is placed in the chip groove of described accessory, after pulsation is cultivated, forms cardiac muscular tissue's chip of beating consistent; Described pulsation is cultivated, and its process is that nutrient solution is flowed into culture chamber 302 from the first fluid passage 202 of accessory, and flow out from second fluid passage 203, flowing velocity is 10 ~ 50mL/min, circulates 40 ~ 50h.
Enumerate several specific embodiment below, to understand the present invention further.
Embodiment 1: test cardiac glycoside medicine is to the effect of cardiac muscle:
1) to choose or to make sectional dimension be 20 × 12mm thickness is the square glass sheet of 1mm, after sterilizing one hour in 75% alcohol, stick one deck adhesive plaster on its surface;
2) on adhesive plaster, cutting out 13 length with laser formation machine is 7mm, and width is 1mm, spaced be the square contour of 0.1mm as first time cutting track, and remove the square rubber pieces of cloth cut;
3) by mass body volume concentrations be 20% the butanols of poly-N-isopropyl acrylamide solution 1:1 and 99% be by volume mixed 10% poly-N-isopropyl acrylamide solution, with spin coating method, poly-N-isopropyl acrylamide solution is coated in containing on the glass sheet of adhesive plaster side, remove remaining adhesive plaster after solidification, now glass sheet leaves the square poly-N-isopropyl acrylamide layer of 13 7 × 1mm;
4) dimethyl silicone polymer solution and hardening agent are mixed with the mass ratio of 10:1, be placed on degassed process 1h in air pump after stirring and obtain polydimethylsiloxane prepolymer thing, with spin coating method dimethyl silicone polymer covered on glass sheet and scribble poly-N-isopropyl acrylamide solution side, placing under 65 DEG C of environment after completing makes it solidify completely in 10 hours, forms dimethyl silicone polymer layer; According to long limit and first time the second time cutting track that overlaps of cutting track, dimethyl silicone polymer layer cut into 13 " semi-girders " to 3 × 1mm with laser formation machine, both sides relative distance is 1mm;
5) preparation quality volumetric concentration be 2% fibrinogen solution as hydrogel solution and 2% fibrin ferment as its crosslinking chemical, in fibrinogen solution, add fibronectin make the hydrogel solution that concentration of fibronectin is 50 μ g/mL, extraction or inducing cardiomyocytes are placed in nutrient solution and cultivate, and cell concentration is 1 × 10
7individual/ml;
6) myocardial cell suspensions and fibrin ferment are respectively charged in spray-type shower nozzle 1 and spray-type shower nozzle 2, spray-type shower nozzle aperture is 100 μm, and the hydrogel solution containing cardiac muscle cell made after myocardial cell suspensions and hydrogel solution 2:8 mixing is by volume loaded in squash type shower nozzle, nozzle inside diameter is 200 μm, with by spray-type shower nozzle 1, spray-type shower nozzle 2 and squash type shower nozzle composition composite spray jet head in step 4) in formed " semi-girder " on print, first extrude thread hydrogel with squash type shower nozzle, spray fibrin ferment to be subsequently cross-linked, then one deck myocardial cell suspensions is sprayed to the water-setting collodion silk after crosslinked and form one deck cardiac muscle cell layer outward at hydrogel, be shaped after one deck and continued to print lower one deck, water-setting collodion silk is between layers parallel to each other, repeatedly pile up the cardiac muscular tissue's chip being formed and there are 10 confluent monolayer cells,
7) cardiac muscular tissue's chip is placed in the chip groove of counterdie, and with bottom electrode close contact, patrix is arranged on counterdie according to guiding tongue and direction recess, now top electrode effectively contacts with bottom electrode, parallel light source is placed in counterdie low groove, photographic plate is placed in patrix upper groove, and be connected with data acquisition system (DAS), top electrode is linked on power supply by wire, whole device is placed in constant temperature oven, cell culture fluid is slowly injected counterdie culture chamber from the first fluid passage of patrix, and flow out from second fluid passage, with the flow velocity of 20mL/min perfusion 48h repeatedly,
8) opening power and parallel light source, after data acquisition system (DAS) can collect and has obvious contraction change signal, cardiac glycoside is dissolved in cell culture fluid, stop perfusion, after this, change a solution every 6h, Real-time Collection contraction signal, test after 60 hours, quit work, the signal before and after analyzing passes judgment on the effect of cardiac glycoside drugs of cardiomyocyte accordingly.
Embodiment 2: test Fufang Danshen Pian is to the therapeutic action of pathology cardiac muscle:
1) to choose or to make sectional dimension be 20 × 12mm thickness is the square glass sheet of 1mm, after sterilizing one hour in 75% alcohol, stick one deck adhesive plaster on its surface;
2) on adhesive plaster, cutting out 15 length with laser formation machine is 6mm, and width is 0.6mm, spaced be the square contour of 0.2mm as first time cutting track, and remove the square rubber pieces of cloth cut;
3) by mass body volume concentrations be 40% the butanols of poly-N-isopropyl acrylamide solution 1:1 and 99% be by volume mixed 20% poly-N-isopropyl acrylamide solution, with spin coating method, poly-N-isopropyl acrylamide solution is coated in containing on the glass sheet of adhesive plaster side, remove remaining adhesive plaster after solidification, now glass sheet leaves the square poly-N-isopropyl acrylamide layer of 15 6 × 0.6mm;
4) dimethyl silicone polymer solution and hardening agent are mixed with the mass ratio of 10:1, be placed on degassed process 1h in air pump after stirring and obtain polydimethylsiloxane prepolymer thing, with spin coating method dimethyl silicone polymer covered on glass sheet and scribble poly-N-isopropyl acrylamide solution side, placing under 65 DEG C of environment after completing makes it solidify completely in 10 hours, forms dimethyl silicone polymer layer; According to long limit and first time the second time cutting track that overlaps of cutting track, dimethyl silicone polymer layer cut into 15 " semi-girders " to 2.6 × 0.6mm with laser formation machine, both sides relative distance is 0.8mm;
5) preparation quality volumetric concentration be 5% sodium alginate soln as hydrogel solution and 3% calcium chloride solution as its crosslinking chemical, extract the cardiac muscle cell of pathology and be placed in nutrient solution and cultivate, cell concentration is 2 × 10
7individual/ml;
6) myocardial cell suspensions and calcium chloride solution are respectively charged in spray-type shower nozzle 1 and spray-type shower nozzle 2, spray-type shower nozzle aperture is 100 μm, and the hydrogel solution containing cardiac muscle cell made after myocardial cell suspensions and hydrogel solution 1:9 mixing is by volume loaded in squash type shower nozzle, nozzle inside diameter is 200 μm, with by spray-type shower nozzle 1, spray-type shower nozzle 2 and squash type shower nozzle composition composite spray jet head in step 4) in formed " semi-girder " on print, first extrude thread hydrogel with squash type shower nozzle, spray calcium chloride solution to be subsequently cross-linked, then one deck myocardial cell suspensions is sprayed to the water-setting collodion silk after crosslinked and form one deck cardiac muscle cell layer outward at hydrogel, be shaped after one deck and continued to print lower one deck, water-setting collodion silk is between layers parallel to each other, repeatedly pile up the cardiac muscular tissue's chip being formed and there are 12 confluent monolayer cells,
7) cardiac muscular tissue's chip is placed in the chip groove of counterdie, and with bottom electrode close contact, patrix is arranged on counterdie according to guiding tongue and direction recess, now top electrode effectively contacts with bottom electrode, parallel light source is placed in counterdie low groove, photographic plate is placed in patrix upper groove, and be connected with data acquisition system (DAS), top electrode is linked on power supply by wire, whole device is placed in constant temperature oven, cell culture fluid is slowly injected counterdie culture chamber from the first fluid passage of patrix, and flow out from second fluid passage, with the flow velocity of 10mL/min perfusion 50h repeatedly,
8) opening power and parallel light source, after data acquisition system (DAS) can collect and has obvious contraction change signal, Fufang Danshen Pian is dissolved in cell culture fluid, stop perfusion, after this, change a solution every 6h, Real-time Collection contraction signal, test after 60 hours, quit work, the signal before and after analyzing passes judgment on the result for the treatment of of Fufang Danshen Pian to pathology cardiac muscle accordingly.
Embodiment 3: test Nifedipine Tablets is to the effect of pathology cardiac muscle:
1) to choose or to make sectional dimension be 20 × 12mm thickness is the square glass sheet of 1mm, after sterilizing one hour in 75% alcohol, stick one deck adhesive plaster on its surface;
2) on adhesive plaster, cutting out 8 length with laser formation machine is 7mm, and width is 1mm, spaced be the square contour of 0.3mm as first time cutting track, and remove the square rubber pieces of cloth cut;
3) by mass body volume concentrations be 60% the butanols of poly-N-isopropyl acrylamide solution 1:1 and 99% be by volume mixed 30% poly-N-isopropyl acrylamide solution, with spin coating method, poly-N-isopropyl acrylamide solution is coated in containing on the glass sheet of adhesive plaster side, remove remaining adhesive plaster after solidification, now glass sheet leaves the square poly-N-isopropyl acrylamide layer of 87 × 1mm;
4) dimethyl silicone polymer solution and hardening agent are mixed with the mass ratio of 10:1, be placed on degassed process 1h in air pump after stirring and obtain polydimethylsiloxane prepolymer thing, with spin coating method dimethyl silicone polymer covered on glass sheet and scribble poly-N-isopropyl acrylamide solution side, placing under 65 DEG C of environment after completing makes it solidify completely in 10 hours, forms dimethyl silicone polymer layer; According to long limit and first time the second time cutting track that overlaps of cutting track, dimethyl silicone polymer layer cut into 8 " semi-girders " to 3.2 × 1mm with laser formation machine, both sides relative distance is 0.6mm;
5) natural macromolecular material powder is dissolved in DMEM nutrient solution, obtain mass body volume concentrations be 20% gelatin solution, mass body volume concentrations be the fibrinogen solution of 5%, above gelatine solution, fibrinogen solution equal-volume are mixed to get hydrogel solution, the thrombin solution of 2% is as its crosslinking chemical, the cardiac muscle cell extracting pathology is placed in nutrient solution and cultivates, and cell concentration is 1.5 × 10
7individual/ml;
6) myocardial cell suspensions and thrombin solution are respectively charged in spray-type shower nozzle 1 and spray-type shower nozzle 2, spray-type shower nozzle aperture is 100 μm, and the hydrogel solution containing cardiac muscle cell made after myocardial cell suspensions and hydrogel solution 1:9 mixing is by volume loaded in squash type shower nozzle, nozzle inside diameter is 200 μm, with by spray-type shower nozzle 1, spray-type shower nozzle 2 and squash type shower nozzle composition composite spray jet head in step 4) in formed " semi-girder " on print, first extrude thread hydrogel with squash type shower nozzle, spray thrombin solution to be subsequently cross-linked, then one deck myocardial cell suspensions is sprayed to the water-setting collodion silk after crosslinked and form one deck cardiac muscle cell layer outward at hydrogel, be shaped after one deck and continued to print lower one deck, water-setting collodion silk is between layers parallel to each other, repeatedly pile up the cardiac muscular tissue's chip being formed and there are 15 confluent monolayer cells,
7) cardiac muscular tissue's chip is placed in the chip groove of counterdie, and with bottom electrode close contact, patrix is arranged on counterdie according to guiding tongue and direction recess, now top electrode effectively contacts with bottom electrode, parallel light source is placed in counterdie low groove, photographic plate is placed in patrix upper groove, and be connected with data acquisition system (DAS), top electrode is linked on power supply by wire, whole device is placed in constant temperature oven, cell culture fluid is slowly injected counterdie culture chamber from the first fluid passage of patrix, and flow out from second fluid passage, with the flow velocity of 15mL/min perfusion 45h repeatedly,
8) opening power and parallel light source, after data acquisition system (DAS) can collect and has obvious contraction change signal, Nifedipine Tablets is dissolved in cell culture fluid, stop perfusion, after this, change a solution every 6h, Real-time Collection contraction signal, test after 60 hours, quit work, the signal before and after analyzing passes judgment on the effect of Nifedipine Tablets to pathology cardiac muscle accordingly.
Embodiment 4: test cardiotoxin is to the effect of cardiac muscle:
1) to choose or to make sectional dimension be 20 × 12mm thickness is the square glass sheet of 1mm, after sterilizing one hour in 75% alcohol, stick one deck adhesive plaster on its surface;
2) on adhesive plaster, cutting out 10 length with laser formation machine is 8mm, and width is 0.9mm, spaced be the square contour of 0.3mm as first time cutting track, and remove the square rubber pieces of cloth cut;
3) by mass body volume concentrations be 50% the butanols of poly-N-isopropyl acrylamide solution 1:1 and 99% be by volume mixed 25% poly-N-isopropyl acrylamide solution, with spin coating method, poly-N-isopropyl acrylamide solution is coated in containing on the glass sheet of adhesive plaster side, remove remaining adhesive plaster after solidification, now glass sheet leaves the square poly-N-isopropyl acrylamide layer of 10 8 × 0.9mm;
4) dimethyl silicone polymer solution and hardening agent are mixed with the mass ratio of 10:1, be placed on degassed process 1h in air pump after stirring and obtain polydimethylsiloxane prepolymer thing, with spin coating method dimethyl silicone polymer covered on glass sheet and scribble poly-N-isopropyl acrylamide solution side, placing under 65 DEG C of environment after completing makes it solidify completely in 10 hours, forms dimethyl silicone polymer layer; According to long limit and first time the second time cutting track that overlaps of cutting track, dimethyl silicone polymer layer cut into 8 " semi-girders " to 3.5 × 0.9mm with laser formation machine, both sides relative distance is 1mm;
5) natural macromolecular material powder is dissolved in DMEM nutrient solution, obtain mass body volume concentrations be 20% hyaluronic acid solution, mass body volume concentrations be the fibrinogen solution of 6%, hydrogel solution is mixed to get with above-mentioned hyaluronic acid solution, fibrinogen solution equal-volume, the thrombin solution of 2% is as its crosslinking chemical, extraction cardiac muscle cell is placed in nutrient solution and cultivates, and cell concentration is 2 × 10
7individual/ml;
6) myocardial cell suspensions and thrombin solution are respectively charged in spray-type shower nozzle 1 and spray-type shower nozzle 2, spray-type shower nozzle aperture is 100 μm, and the hydrogel solution containing cardiac muscle cell made after myocardial cell suspensions and hydrogel solution 1:9 mixing is by volume loaded in squash type shower nozzle, nozzle inside diameter is 200 μm, with by spray-type shower nozzle 1, spray-type shower nozzle 2 and squash type shower nozzle composition composite spray jet head in step 4) in formed " semi-girder " on print, first extrude thread hydrogel with squash type shower nozzle, spray thrombin solution to be subsequently cross-linked, then one deck myocardial cell suspensions is sprayed to the water-setting collodion silk after crosslinked and form one deck cardiac muscle cell layer outward at hydrogel, be shaped after one deck and continued to print lower one deck, water-setting collodion silk is between layers parallel to each other, repeatedly pile up the cardiac muscular tissue's chip being formed and there are 10 confluent monolayer cells,
7) cardiac muscular tissue's chip is placed in the chip groove of counterdie, and with bottom electrode close contact, patrix is arranged on counterdie according to guiding tongue and direction recess, now top electrode effectively contacts with bottom electrode, parallel light source is placed in counterdie low groove, photographic plate is placed in patrix upper groove, and be connected with data acquisition system (DAS), top electrode is linked on power supply by wire, whole device is placed in constant temperature oven, cell culture fluid is slowly injected counterdie culture chamber from the first fluid passage of patrix, and flow out from second fluid passage, with the flow velocity of 12mL/min perfusion 48h repeatedly,
8) opening power and parallel light source, after data acquisition system (DAS) can collect and has obvious contraction change signal, stop perfusion, cardiotoxin is dissolved in cell culture fluid, after this, change a solution every 6h, Real-time Collection contraction signal, test after 50 hours, quit work, the signal before and after analyzing passes judgment on the effect of cardiotoxin to cardiac muscle accordingly.
Claims (6)
1. cardiac muscular tissue's sensor, is characterized in that: described sensor comprises cardiac muscular tissue's chip and accessory; Described accessory comprises patrix (101), counterdie (104), top electrode (107), bottom electrode (106), photographic plate (102), parallel light source (105) and data acquisition system (DAS) (103); Described patrix (101) is provided with first fluid passage (202), second fluid passage (203), guiding tongue (204) and upper groove (201); Described lower film is provided with culture chamber (302), chip groove (303), low groove (304) and direction recess (301); Described upper die and lower die are installed together by guiding tongue (204) and direction recess (301) orientation; Described cardiac muscular tissue chip is placed in chip groove (303); Described top electrode (107) and bottom electrode (106) lay respectively at patrix (101) and counterdie (104) is inner and one-body molded with upper die and lower die; Top electrode (107) is connected with power supply (108), and bottom electrode (106) bottom is positioned on chip groove (303), and with the cardiac muscular tissue's chip close contact in chip groove; Described photographic plate (102) is placed in the upper groove (201) of patrix, and is connected with data acquisition system (DAS) (103); Described parallel light source (105) is placed in the low groove (304) of counterdie.
2. according to a kind of cardiac muscular tissue according to claim 1 sensor, it is characterized in that: described upper die and lower die all adopt makrolon material, described top electrode and bottom electrode adopt titanium alloy material.
3. a preparation method for cardiac muscular tissue's chip as claimed in claim 1, is characterized in that the method comprises the following steps:
1) one deck adhesive plaster (402) is sticked on the surface of the glass sheet (401) after one piece of sterilization;
2) cut along first time cutting track (403) on adhesive plaster with laser formation machine, form at least 5 square contours, each square contour length is 6 ~ 8mm, width is 0.6 ~ 1mm, spaced 0.1 ~ 0.3mm, and remove the square rubber pieces of cloth (404) cut;
3) preparation quality volumetric concentration is the poly-N-isopropyl acrylamide solution of 10% ~ 50%, with spin coating method be evenly coated in glass sheet containing adhesive plaster side, remove adhesive plaster after solidification, leave square poly-N-isopropyl acrylamide layer (405);
4) by after dimethyl silicone polymer heating and melting, be coated onto on glass sheet with spin coating method, after solidification process, dimethyl silicone polymer to be cut into along second time cutting track (407) with laser formation machine to be middlely connected, both sides are multiple " semi-girder " (408) structure, both sides " semi-girder " spacing is 0.6 ~ 1mm;
5) preparation quality volumetric concentration be 0.1 ~ 20% hydrogel solution and mass body volume concentrations be the cross-linking agent solution of 0.1 ~ 20%, extract or inducing cardiomyocytes to make cell density be 1 × 10
7~ 2 × 10
7cardiac muscle cell's suspending liquid of individual/ml, cardiac muscle cell being mixed in hydrogel solution and making cell density is 1 × 10
6~ 5 × 10
6the hydrogel solution containing cardiac muscle cell of individual/ml; Loaded by cardiac muscle cell's suspending liquid in the first spray-type shower nozzle, cross-linking agent solution loads in the second spray-type shower nozzle, and the hydrogel solution containing cardiac muscle cell loads in squash type shower nozzle; Successively print on " semi-girder " with the composite spray jet head be made up of the first spray-type shower nozzle, the second spray-type shower nozzle and squash type shower nozzle, the thread hydrogel that squash type shower nozzle is extruded forms water-setting collodion silk (502) after the cross-linking agent solution that the second spray-type shower nozzle sprays is cross-linked, and then forms cardiac muscle cell's layer (501) with the first spray-type shower nozzle at water-setting collodion silk surface sprinkling one deck cardiac muscle cell suspending liquid; Repeat to extrude, be cross-linked, form cardiac muscular tissue's chip with multi-layer cellular after the operation such as sprinkling, water-setting collodion silk is between layers parallel to each other;
6) this cardiac muscular tissue's chip is placed in the chip groove of described accessory, after pulsation is cultivated, forms cardiac muscular tissue's chip of beating consistent.
4. the preparation method of a kind of cardiac muscular tissue chip as claimed in claim 3, is characterized in that: step 2) on adhesive plaster, cut 8 ~ 15 square contours with laser formation machine.
5. the preparation method of a kind of cardiac muscular tissue chip as claimed in claim 3, it is characterized in that, step 5) described in the solute of hydrogel solution be at least one in sodium alginate, collagen, hyaluronic acid, matrigel, dextrose, shitosan, fibronectin, gelatin and fibrinogen and matrigel, described crosslinking chemical is at least one in fibrin ferment, lime chloride or peroxophosphoric acid sodium.
6. the preparation method of a kind of cardiac muscular tissue chip as claimed in claim 3, it is characterized in that, step 6) described in pulsation cultivate, its process is that nutrient solution is flowed into culture chamber (302) from the first fluid passage (202) of accessory, flow out from second fluid passage (203), flowing velocity is 10 ~ 50mL/min, circulates 40 ~ 50h.
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