CN104931683B - A kind of cardiac muscular tissue sensor and the preparation method of cardiac muscular tissue's chip - Google Patents
A kind of cardiac muscular tissue sensor and the preparation method of cardiac muscular tissue's chip Download PDFInfo
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Abstract
A kind of cardiac muscular tissue sensor and the preparation method of cardiac muscular tissue's chip, belong to organizational project, biomedical engineering and sensor technical field.This cardiac muscular tissue's sensor includes cardiac muscular tissue's chip and accessory；Described accessory includes upper die and lower die, upper electrode, bottom electrode, photographic plate, parallel light source and data collecting system.The present invention utilizes cell printing technology to print myocardial cell successively on the sheet glass scribbling poly-N N-isopropylacrylamide and polydimethylsiloxane and makes cardiac muscular tissue's chip, it is placed in accessory after pulsation is cultivated and forms consistent cardiac muscular tissue of beating, then insert in the culture fluid of drug containing and apply electricity irritation, gather its contraction change signal, and evaluate the medicine action effect to cardiac muscle with this, the purpose such as drug screening and test can be realized.It is simple that cardiac muscular tissue's sensor of the present invention has device, and testable medicine scope is wide, and signal easily detects and the advantage such as process.
Description
Technical field
The invention belongs to organizational project, biomedical engineering and sensing technology crossing domain, particularly to a kind of cardiac muscular tissue sensing
Device and the preparation method of cardiac muscular tissue's chip.
Background technology
Myocardial cell is the main composition cell of cardiac muscular tissue, is distributed mainly on heart wall and faces on the big blood vessel of the heart, and myocardial cell is short
Cylindrical fibre shape, has obvious directivity.Compared to cells such as main nephrocyte based on metabolic function, hepatocyte,
The major function of myocardial cell is to shrink and diastole, realizes the blood-pumping function of heart to promote blood circulation with this.Relevant cardiac muscle
Damage and the pathological changes of tissue can cause various heart disease and cardiomyopathy, are respectively provided with the highest M & M, serious threat
Human survival and quality of life.According to statistics, in the U.S., there are about 4,800,000 patients with congestive heart failure, and annual new sending out is suffered from
Person is more than 400,000.The effectively treatment of cardiomyopathy be unable to do without the exploitation of related drugs, and the screening of medicine and test are novel drugs throwings
Enter a link indispensable in using.Traditional animal or the in vivo test of human body exist influence factor uncontrollable, test
The inherent defects such as the repetition of result difficulty, the time-consuming effort of process of the test and relevant ethical issues.And cell (tissue) sensor
Occur then providing new thinking for these problems of solution.
Cell sensor is as main functional elements, identification and perception measured object using cell to be also converted into according to certain rules and can know
The device of level signal or device.Because it has that volume is little, response is fast, can realize the feature such as on-line monitoring in situ, environmental assessment,
The field such as food safety and drug screening application prospect is extensive.Over nearly 30 years, cell culture technology and micro electronmechanical manufacturing technology
Development is that the development of cell sensor provides good platform especially so that cell sensor is at biological, chemistry and medical analysis
Field has obtained increasing concern and application.Traditional myocardial cell sensor all with the electricity physiological signal of cell as detection limit,
The most in succession occur in that voltage clamp cell sensor, patch-clamp cell sensor, field-effect transistor cell sensor,
Microelectrode array cell sensor and LAPS etc..There is system in the sensor of the above-mentioned various detection cell signal of telecommunication
Make the problems such as complex process, biocompatibility and Signal-to-Noise have much room for improvement.Feinberg AW etc. (Feinberg AW,
et.al,Muscular thin films for building actuators and powering devices.Science
2007；317:1366 70.) in 2007, a kind of muscular thin film (muscular thin film, MTF) technology is proposed, will
Muscle cell is planted on temperature sensitive macromolecular material and makes three-dimensional muscular tissue, and tests muscular tissue in the external world with this
Contraction distortion amount under Ci Jiing, the response detection for myocardial cell provides a kind of new means.On the other hand, cell is being carried out
During sensing system design, biocompatibility is the aspect that have to consider, the physiologically active of test cell, cell with
Device surface have to couple well, namely cell can be at the easy tactophily of device surface, and traditional cell seeding technology
It is difficult to ensure that cell couples with the fine of device, thus greatly have impact on accuracy of detection.
It is a kind of three-dimensional many cells system of structure in vitro occurred in recent years that cell 3D prints (cell printing) technology
Advanced technology, this technology is the combination of rapid shaping technique and Biotechnology.During cell printing, cell (or
Cell aggregation) it is concurrently placed at colloidal sol (presoma of hydrogel) or celliferous culture fluid is individually placed in the spray of printer
In Tou, computer control the deposition position containing cell drop, print, on the basis having printed a layer in the position pointwise specified
Upper continuation prints another layer, the three-dimensional many cells-gel rubber system of the formation that is layering.Compared with conventional art, the advantage of cell printing
Be mainly reflected in can the different types of cell of accurate deposition over time and space, controllability is big, can be formed and have various structures
Accumulation body.
Summary of the invention
The present invention is directed to the weak point of prior art, it is provided that a kind of cardiac muscular tissue sensor and the preparation method of cardiac muscular tissue's chip,
Aim to provide cardiac muscular tissue's sensor of a kind of novel cardiac muscular tissue's detected amount of contraction, and cell printing technology is introduced cardiac muscle
In the preparation of organization chip so that it is having collection Signal-to-Noise high, myocardial cell can couple the advantages such as good with device.
Technical scheme is as follows:
A kind of cardiac muscular tissue sensor, described sensor includes cardiac muscular tissue's chip and accessory；Described accessory includes
Mould, lower mold, upper electrode, bottom electrode, photographic plate, parallel light source and data collecting system；Described upper mold is provided with first-class
Body passage, second fluid passage, guiding tongue and upper groove；Described lower film be provided with culture chamber, chip slot, 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 put
In chip slot；It is internal and one-body molded with upper die and lower die that described upper electrode and bottom electrode lay respectively at upper die and lower die；Power on
Pole is connected with power supply, and bottom electrode bottom is positioned on chip slot, and is in close contact with the cardiac muscular tissue's chip in chip slot；Described sense
Tabula rasa is placed in the upper groove of upper mold, and is connected with data collecting system；Described parallel light source is placed in the low groove of lower mold.
In technique scheme, it is characterised in that: described upper die and lower die all use makrolon material, described upper electrode and under
Electrode uses titanium alloy material.
Cardiac muscular tissue of the present invention chip, it is characterised in that the preparation method of this cardiac muscular tissue's chip comprises the following steps:
1) the surface patch last layer adhesive plaster of the sheet glass after one piece of sterilization；
2) carry out cutting for the first time on adhesive plaster with laser formation machine, form at least 5 square contours, each square contour
A length of 6～8mm, width is 0.6～1mm, spaced 0.1～0.3mm, and removes the square rubber pieces of cloth cut；
3) the poly-N-isopropyl acrylamide solution that mass body volume concentrations is 10%～50% is prepared, with spin coating method, it is equal
Even be coated in sheet glass containing adhesive plaster side, remove adhesive plaster after solidification, leave square poly-N-isopropyl acrylamide layer；
4) by after polydimethylsiloxane heating and melting, it is coated onto on sheet glass with spin coating method, after cured,
Polydimethylsiloxane cut into along second time cutting track (407) with laser formation machine middle be connected, both sides are multiple " outstanding
Arm beam " (408) structure, both sides " cantilever beam " spacing is 0.6～1mm；
5) preparing mass body volume concentrations is 0.1～the hydrogel solution of 20% and cross-linking agent that mass body volume concentrations is 0.1～20%
Solution, it is 1 × 10 that extraction or inducing cardiomyocytes make cell density7～2 × 107The myocardial cell suspension of individual/ml, by the heart
Myocyte is mixed in hydrogel solution and makes cell density is 1 × 106～5 × 106The hydrogel solution containing myocardial cell of individual/ml；
Being loaded by myocardial cell suspension in spray-type shower nozzle 1, cross-linking agent solution loads in spray-type shower nozzle 2, the water containing myocardial cell
Gel solution loads in squash type shower nozzle；With the compound spray being made up of spray-type shower nozzle 1, spray-type shower nozzle 2 and squash type shower nozzle
Head successively prints on " cantilever beam ", the crosslinking that the thread hydrogel of squash type shower nozzle extrusion sprays through spray-type shower nozzle 2
Water-setting collodion silk (502) is formed, then with spray-type shower nozzle 1 at one layer of myocardial cell of water-setting collodion silk surface sprinkling after agent solution crosslinking
Suspension forms myocardial cell layer (501), repeat extrude, cross-link, after the operation such as sprinkling formation there is myocardium group of multi-layer cellular
Knitting chip, water-setting collodion silk between layers is parallel to each other；
6) this cardiac muscular tissue's chip is placed in the chip slot in described accessory, is formed after pulsation is cultivated and beat unanimously
Cardiac muscular tissue's chip.
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 characterised in that step 5) described in hydrogel solution molten
Matter is sodium alginate, collagen, hyaluronic acid, matrigel, dextrose, chitosan, fibronectin, gelatin and fiber egg
At least one in the most former and matrigel, described cross-linking agent is at least one in thrombin, calcium chloride or peroxophosphoric acid sodium.
The preparation method of cardiac muscular tissue of the present invention chip, it is characterised in that step 6) described in pulsation cultivate, its mistake
Journey is that from the first fluid passage of accessory, culture fluid is flowed into culture chamber, flows out from second fluid passage, flowing velocity be 10～
50mL/min, circulates 40～50h.
Compared with prior art, the invention have the advantages that and the technique effect of salience: 1. cardiac muscular tissue's sensor of the present invention
The contraction signal of cardiac muscular tissue can be gathered, and the use of source of parallel light improves the acquisition precision of signal.2. the present invention is in preparation
Cardiac muscular tissue's chip processes utilize cell printing technology piled up on the glass sheet by the hydrogel containing myocardial cell, it is ensured that thin
Born of the same parents and device good coupling, and the size controllable precise printed, can meet multiple required precision；3. the present invention uses containing cardiac muscle
The hydrogel surface of cell is sprayed the structure of myocardial cell suspensions and is piled up, and improves the density of myocardial cell in cardiac muscular tissue,
Can promote that iuntercellular is set up to connect and form tissue；4. cardiac muscular tissue can be carried out by the accessory of cardiac muscular tissue's sensor of the present invention
Pulsation is cultivated, and the culture fluid of flowing with lasting equidirectional stimulation, can form the cardiac muscular tissue of directivity to myocardial 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 myocardial cell piles up schematic diagram.
In figure: 101-upper mold；102-photographic plate；103-data collecting system；104-lower mold；105-parallel light source；106-
Bottom electrode；The upper electrode of 107-；108-power supply；201-upper groove, 202-first fluid passage, 203-second fluid passage, 204-
Guiding tongue, 301-direction recess, 302-culture chamber, 303-chip slot, 304-low groove, 401-sheet glass, 402-adhesive plaster,
403-cutting track, 404-square rubber pieces of cloth, 405-poly-N-isopropyl acrylamide layer, 406-polydimethylsiloxane for the first time
Layer, 407-second time cutting track, 408-" cantilever beam ", 409-cardiac muscular tissue, 501-myocardial cell layer, 502-water-setting collodion silk.
Detailed description of the invention
Below in conjunction with the accompanying drawings and the present invention is described in further details by embodiment.
Fig. 1 be the one that the present invention provides be cardiac muscular tissue's sensor general illustration, described accessory include upper mold 101,
Lower mold 104, upper electrode 107, bottom electrode 106, photographic plate 102, parallel light source 105 and data collecting system 103；
The cylinder that described upper mold 101 is made up of transparent Merlon, top offers two through circular fluid passages, and i.e. first
Fluid passage 202 and second fluid passage 203, top center has a square upper groove 201, and upper mold edge is provided with two and leads
To tongue 204；The cylinder that lower mold is made up of transparent Merlon, upper center membrane has culture chamber 302 and chip successively
Groove 303, internal edge is provided with direction recess 301；The cylinder that lower mold is made up of transparent Merlon, central upper portion is successively
Having culture chamber 302 and chip slot 303, internal edge is provided with direction recess 301, has low groove 304 bottom lower mold；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
In groove 303；Described upper electrode 107 and bottom electrode 106 lay respectively at upper mold 101 and lower mold 104 is internal and and upper die and lower die
One-body molded；Upper electrode 107 is connected with power supply 108, and bottom electrode 106 bottom is positioned on chip slot 303, during installation, and cardiac muscle
Chip is placed in chip slot 303, and is in close contact with bottom electrode；Described photographic plate 102 is placed in the upper groove 201 of upper mold,
And be connected with data collecting system 103；Described parallel light source 105 is placed in the low groove 304 of lower mold, upwards launches parallel
Light (sees 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) the surface patch last layer adhesive plaster 402 of the sheet glass 401 after one piece of sterilization；
2) on adhesive plaster, at least 5 square contours, Mei Gefang are formed along first time cutting track 403 cutting with laser formation machine
Shape profile length is 6～8mm, and width is 0.6～1mm, spaced 0.1～0.3mm, and removes the square rubber pieces of cloth cut
404, as shown in (a) in Fig. 4；
3) the poly-N-isopropyl acrylamide solution that mass body volume concentrations is 10%～50% is prepared, with spin coating method, it is uniform
Be coated in sheet glass containing adhesive plaster side, remove adhesive plaster after solidification, leave square poly-N-isopropyl acrylamide layer 405, such as Fig. 4
In (b) shown in；
4) by after polydimethylsiloxane heating and melting, it is coated onto on sheet glass with spin coating method, after cured, with swashing
Seterolithography machine along second time cutting track (407) polydimethylsiloxane cut into middle be connected, both sides are multiple " cantilevers
Beam " (408) structure, both sides " cantilever beam " spacing is 0.6～1mm, as shown in (c) in Fig. 4；
5) prepare mass body volume concentrations be 0.1～the hydrogel solution of 20% and mass body volume concentrations be 0.1～20% cross-linking agent molten
Liquid, it is 1 × 10 that extraction or inducing cardiomyocytes make cell density7～2 × 107The myocardial cell suspension of individual/ml, by cardiac muscle
Cell is mixed in hydrogel solution and makes cell density is 1 × 106～5 × 106The hydrogel solution containing myocardial cell of individual/ml；Will
Myocardial cell suspension loads in spray-type shower nozzle 1, and cross-linking agent solution loads in spray-type shower nozzle 2, the water-setting containing myocardial cell
Sol solution loads in squash type shower nozzle；With the composite spray jet head being made up of spray-type shower nozzle 1, spray-type shower nozzle 2 and squash type shower nozzle
" cantilever beam " successively prints, the cross-linking agent that the thread hydrogel of squash type shower nozzle extrusion sprays through spray-type shower nozzle 2
Form water-setting collodion silk 502 after solution crosslinking, then suspend at one layer of myocardial cell of water-setting collodion silk surface sprinkling with spray-type shower nozzle 1
Liquid forms myocardial cell layer 501, and water-setting collodion silk between layers is parallel to each other, as shown in Figure 5；Repeat extrusion, crosslinking,
Cardiac muscular tissue's chip with multi-layer cellular is made after spraying operation；
6) being placed in the chip slot in described accessory by this cardiac muscular tissue's chip, after pulsation is cultivated, formation is beated consistent
Cardiac muscular tissue's chip；Described pulsation is cultivated, and its process is that from the first fluid passage 202 of accessory, culture fluid is flowed into training
Supporting chamber 302, flow out from second fluid passage 203, flowing velocity is 10～50mL/min, circulates 40～50h.
Enumerate several specific embodiment below, to be further appreciated by the present invention.
Embodiment 1: the test cardiac glycoside medicine effect to cardiac muscle:
1) choose or make sectional dimension be 20 × 12mm thickness be the square glass sheet of 1mm, sterilize in 75% ethanol one little
Paste last layer adhesive plaster on its surface time after；
2) cutting out 13 a length of 7mm on adhesive plaster with laser formation machine, width is 1mm, spaced square for 0.1mm
Profile is as first time cutting track, and removes the square rubber pieces of cloth cut；
3) the butanol mixing of the poly-N-isopropyl acrylamide solution 1:1 by volume and 99% that mass body volume concentrations is 20% is made
Become the poly-N-isopropyl acrylamide solution of 10%, with spin coating method, poly-N-isopropyl acrylamide solution is coated in containing glue
On the sheet glass of cloth side, remove remaining adhesive plaster after solidification, now on sheet glass, leave the square poly-N-isopropyl of 13 7 × 1mm
Acrilamide layer；
4) polydimethylsiloxane solution and firming agent are mixed with the mass ratio of 10:1, be placed in air exhauster de-after stirring
Gas disposal 1h obtains polydimethylsiloxane prepolymer thing, is covered by polydimethylsiloxane with spin coating method and scribbles on sheet glass
Poly-N-isopropyl acrylamide solution side, places under 65 DEG C of environment after completing and makes it be fully cured in 10 hours, form poly-diformazan
Radical siloxane layer；According to long limit and the second time cutting track of cutting track coincidence for the first time, with laser formation machine by poly dimethyl
Siloxane layer cuts into 13 " cantilever beams " to 3 × 1mm, and both sides relative distance is 1mm；
5) prepare fibrinogen solution that mass body volume concentrations is 2% to cross-link as it as the thrombin of hydrogel solution and 2%
Agent, adds fibronectin in fibrinogen solution and makes the water-setting peptization that concentration of fibronectin is 50 μ g/mL
Liquid, extracts or inducing cardiomyocytes is placed in culture fluid and cultivates, and cell concentration is 1 × 107Individual/ml；
6) myocardial cell suspensions and thrombin are respectively charged in spray-type shower nozzle 1 and spray-type shower nozzle 2, spray-type shower nozzle aperture
It is 100 μm, and the water-setting containing myocardial cell will made after myocardial cell suspensions and hydrogel solution 2:8 by volume mixing
Sol solution loads in squash type shower nozzle, and 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 " cantilever beam " on print, first with squash type shower nozzle extrusion thread
Hydrogel, sprays thrombin subsequently and cross-links, then the water-setting collodion silk after crosslinking is sprayed one layer of myocardial cell suspensions at water-setting
Forming one layer of myocardial cell layer outside glue, continue to print next layer after having shaped one layer, water-setting collodion silk between layers is parallel to each other,
Repeatedly pile up and form cardiac muscular tissue's chip with 10 confluent monolayer cells；
7) being placed in the chip slot of lower mold by cardiac muscular tissue's chip, and be in close contact with bottom electrode, upper mold is according to guiding tongue and leading
It is arranged in lower mold to groove, now goes up electrode and effectively contact with bottom electrode, parallel light source is placed in lower mold low groove, sense
Tabula rasa is placed in upper mold upper groove, and is connected with data collecting system, and upper electrode is linked on power supply by wire, and whole device is put
In calorstat, cell culture fluid is slowly injected lower mold culture chamber from the first fluid passage of upper mold, and lead to from second fluid
Road flows out, with the flow velocity perfusion 48h repeatedly of 20mL/min；
8) turn on the power and parallel light source, after data collecting system can collect and have obvious contraction change signal, by heart tonifying
Glycosides dissolves in cell culture fluid, stops perfusion, hereafter, changes a solution, Real-time Collection contraction signal every 6h, test 60
After hour, quitting work, the signal before and after analysis passes judgment on the effect of cardiac glycoside drugs of cardiomyocyte accordingly.
Embodiment 2: the test FUFANG DANSHEN PIAN therapeutical effect to pathological changes cardiac muscle:
1) choose or make sectional dimension be 20 × 12mm thickness be the square glass sheet of 1mm, sterilize in 75% ethanol one little
Paste last layer adhesive plaster on its surface time after；
2) cutting out 15 a length of 6mm on adhesive plaster with laser formation machine, width is 0.6mm, the spaced side for 0.2mm
Shape profile is as first time cutting track, and removes the square rubber pieces of cloth cut；
3) the butanol mixing of the poly-N-isopropyl acrylamide solution 1:1 by volume and 99% that mass body volume concentrations is 40% is made
Become the poly-N-isopropyl acrylamide solution of 20%, with spin coating method, poly-N-isopropyl acrylamide solution is coated in containing glue
On the sheet glass of cloth side, after solidification, remove remaining adhesive plaster, now on sheet glass, leave the square poly-N-isopropyl of 15 6 × 0.6mm
Base acrilamide layer；
4) polydimethylsiloxane solution and firming agent are mixed with the mass ratio of 10:1, be placed in air exhauster de-after stirring
Gas disposal 1h obtains polydimethylsiloxane prepolymer thing, is covered by polydimethylsiloxane with spin coating method and scribbles on sheet glass
Poly-N-isopropyl acrylamide solution side, places under 65 DEG C of environment after completing and makes it be fully cured in 10 hours, form poly-diformazan
Radical siloxane layer；According to long limit and the second time cutting track of cutting track coincidence for the first time, with laser formation machine by poly dimethyl
Siloxane layer cuts into 15 " cantilever beams " to 2.6 × 0.6mm, and both sides relative distance is 0.8mm；
5) prepare sodium alginate soln that mass body volume concentrations is 5% to hand over as it as the calcium chloride solution of hydrogel solution and 3%
Connection agent, the myocardial cell of extraction pathological changes is placed in culture fluid to be cultivated, and cell concentration is 2 × 107Individual/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 make after myocardial cell suspensions and hydrogel solution 1:9 by volume are mixed containing myocardial cell
Hydrogel solution loads in squash type shower nozzle, and nozzle inside diameter is 200 μm, with by spray-type shower nozzle 1, spray-type shower nozzle 2 with squeeze
Pressure type shower nozzle composition composite spray jet head in step 4) in formed " cantilever beam " on print, first with squash type shower nozzle extrude
Thread hydrogel, sprays calcium chloride solution subsequently and cross-links, then the water-setting collodion silk after crosslinking is sprayed one layer of myocardial cell and hang
Liquid forms one layer of myocardial cell layer outside hydrogel, continues to print next layer, water-setting collodion silk between layers after having shaped one layer
It is parallel to each other, repeatedly piles up and form cardiac muscular tissue's chip with 12 confluent monolayer cells；
7) being placed in the chip slot of lower mold by cardiac muscular tissue's chip, and be in close contact with bottom electrode, upper mold is according to guiding tongue and leading
It is arranged in lower mold to groove, now goes up electrode and effectively contact with bottom electrode, parallel light source is placed in lower mold low groove, sense
Tabula rasa is placed in upper mold upper groove, and is connected with data collecting system, and upper electrode is linked on power supply by wire, and whole device is put
In calorstat, cell culture fluid is slowly injected lower mold culture chamber from the first fluid passage of upper mold, and lead to from second fluid
Road flows out, with the flow velocity perfusion 50h repeatedly of 10mL/min；
8) turn on the power and parallel light source, after data collecting system can collect and have obvious contraction change signal, by compound recipe
Radix Salviae Miltiorrhizae Tabellae dissolves in cell culture fluid, stops perfusion, hereafter, changes a solution, Real-time Collection contraction signal, test every 6h
After 60 hours, quitting work, the signal before and after analysis passes judgment on the FUFANG DANSHEN PIAN therapeutic effect to pathological changes cardiac muscle accordingly.
Embodiment 3: the test Nifedipine Tablets effect to pathological changes cardiac muscle:
1) choose or make sectional dimension be 20 × 12mm thickness be the square glass sheet of 1mm, sterilize in 75% ethanol one little
Paste last layer adhesive plaster on its surface time after；
2) cutting out 8 a length of 7mm on adhesive plaster with laser formation machine, width is 1mm, spaced square for 0.3mm
Profile is as first time cutting track, and removes the square rubber pieces of cloth cut；
3) the butanol mixing of the poly-N-isopropyl acrylamide solution 1:1 by volume and 99% that mass body volume concentrations is 60% is made
Become the poly-N-isopropyl acrylamide solution of 30%, with spin coating method, poly-N-isopropyl acrylamide solution is coated in containing glue
On the sheet glass of cloth side, remove remaining adhesive plaster after solidification, now on sheet glass, leave the square poly-N-isopropyl of 87 × 1mm
Acrilamide layer；
4) polydimethylsiloxane solution and firming agent are mixed with the mass ratio of 10:1, be placed in air exhauster de-after stirring
Gas disposal 1h obtains polydimethylsiloxane prepolymer thing, is covered by polydimethylsiloxane with spin coating method and scribbles on sheet glass
Poly-N-isopropyl acrylamide solution side, places under 65 DEG C of environment after completing and makes it be fully cured in 10 hours, form poly-diformazan
Radical siloxane layer；According to long limit and the second time cutting track of cutting track coincidence for the first time, with laser formation machine by poly dimethyl
Siloxane layer cuts into 8 " cantilever beams " to 3.2 × 1mm, and both sides relative distance is 0.6mm；
5) natural macromolecular material powder is dissolved in DMEM culture fluid, obtains gelatin solution, matter that mass body volume concentrations is 20%
Amount volumetric concentration is the fibrinogen solution of 5%, and above gelatine solution, fibrinogen solution equal-volume are mixed to get water-setting
Sol solution, the thrombin solution of 2% is as its cross-linking agent, and the myocardial cell of extraction pathological changes is placed in culture fluid to be cultivated, cell
Concentration is 1.5 × 107Individual/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 make after myocardial cell suspensions and hydrogel solution 1:9 by volume are mixed containing myocardial cell
Hydrogel solution loads in squash type shower nozzle, and nozzle inside diameter is 200 μm, with by spray-type shower nozzle 1, spray-type shower nozzle 2 with squeeze
Pressure type shower nozzle composition composite spray jet head in step 4) in formed " cantilever beam " on print, first with squash type shower nozzle extrude
Thread hydrogel, sprays thrombin solution subsequently and cross-links, then the water-setting collodion silk after crosslinking is sprayed one layer of myocardial cell and hang
Liquid forms one layer of myocardial cell layer outside hydrogel, continues to print next layer, water-setting collodion silk between layers after having shaped one layer
It is parallel to each other, repeatedly piles up and form cardiac muscular tissue's chip with 15 confluent monolayer cells；
7) being placed in the chip slot of lower mold by cardiac muscular tissue's chip, and be in close contact with bottom electrode, upper mold is according to guiding tongue and leading
It is arranged in lower mold to groove, now goes up electrode and effectively contact with bottom electrode, parallel light source is placed in lower mold low groove, sense
Tabula rasa is placed in upper mold upper groove, and is connected with data collecting system, and upper electrode is linked on power supply by wire, and whole device is put
In calorstat, cell culture fluid is slowly injected lower mold culture chamber from the first fluid passage of upper mold, and lead to from second fluid
Road flows out, with the flow velocity perfusion 45h repeatedly of 15mL/min；
8) turn on the power and parallel light source, after data collecting system can collect and have obvious contraction change signal, by nitre benzene
Ground plain film dissolves in cell culture fluid, stops perfusion, hereafter, changes a solution, Real-time Collection contraction signal, test every 6h
After 60 hours, quitting work, the signal before and after analysis passes judgment on the Nifedipine Tablets effect to pathological changes cardiac muscle accordingly.
Embodiment 4: the test cardiotoxin effect to cardiac muscle:
1) choose or make sectional dimension be 20 × 12mm thickness be the square glass sheet of 1mm, sterilize in 75% ethanol one little
Paste last layer adhesive plaster on its surface time after；
2) cutting out 10 a length of 8mm on adhesive plaster with laser formation machine, width is 0.9mm, the spaced side for 0.3mm
Shape profile is as first time cutting track, and removes the square rubber pieces of cloth cut；
3) the butanol mixing of the poly-N-isopropyl acrylamide solution 1:1 by volume and 99% that mass body volume concentrations is 50% is made
Become the poly-N-isopropyl acrylamide solution of 25%, with spin coating method, poly-N-isopropyl acrylamide solution is coated in containing glue
On the sheet glass of cloth side, after solidification, remove remaining adhesive plaster, now on sheet glass, leave the square poly-N-isopropyl of 10 8 × 0.9mm
Base acrilamide layer；
4) polydimethylsiloxane solution and firming agent are mixed with the mass ratio of 10:1, be placed in air exhauster de-after stirring
Gas disposal 1h obtains polydimethylsiloxane prepolymer thing, is covered by polydimethylsiloxane with spin coating method and scribbles on sheet glass
Poly-N-isopropyl acrylamide solution side, places under 65 DEG C of environment after completing and makes it be fully cured in 10 hours, form poly-diformazan
Radical siloxane layer；According to long limit and the second time cutting track of cutting track coincidence for the first time, with laser formation machine by poly dimethyl
Siloxane layer cuts into 8 " cantilever beams " to 3.5 × 0.9mm, and both sides relative distance is 1mm；
5) natural macromolecular material powder is dissolved in DMEM culture fluid, obtain hyaluronic acid solution that mass body volume concentrations is 20%,
Mass body volume concentrations is the fibrinogen solution of 6%, mixes with above-mentioned hyaluronic acid solution, fibrinogen solution equal-volume
To hydrogel solution, the thrombin solution of 2% is as its cross-linking agent, and extraction myocardial cell is placed in culture fluid to be cultivated, cell
Concentration is 2 × 107Individual/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 make after myocardial cell suspensions and hydrogel solution 1:9 by volume are mixed containing myocardial cell
Hydrogel solution loads in squash type shower nozzle, and nozzle inside diameter is 200 μm, with by spray-type shower nozzle 1, spray-type shower nozzle 2 with squeeze
Pressure type shower nozzle composition composite spray jet head in step 4) in formed " cantilever beam " on print, first with squash type shower nozzle extrude
Thread hydrogel, sprays thrombin solution subsequently and cross-links, then the water-setting collodion silk after crosslinking is sprayed one layer of myocardial cell and hang
Liquid forms one layer of myocardial cell layer outside hydrogel, continues to print next layer, water-setting collodion silk between layers after having shaped one layer
It is parallel to each other, repeatedly piles up and form cardiac muscular tissue's chip with 10 confluent monolayer cells；
7) being placed in the chip slot of lower mold by cardiac muscular tissue's chip, and be in close contact with bottom electrode, upper mold is according to guiding tongue and leading
It is arranged in lower mold to groove, now goes up electrode and effectively contact with bottom electrode, parallel light source is placed in lower mold low groove, sense
Tabula rasa is placed in upper mold upper groove, and is connected with data collecting system, and upper electrode is linked on power supply by wire, and whole device is put
In calorstat, cell culture fluid is slowly injected lower mold culture chamber from the first fluid passage of upper mold, and lead to from second fluid
Road flows out, with the flow velocity perfusion 48h repeatedly of 12mL/min；
8) turn on the power and parallel light source, after data collecting system can collect and have obvious contraction change signal, stop filling
Stream, dissolves in cardiotoxin in cell culture fluid, hereafter, changes a solution, Real-time Collection contraction signal, test every 6h
After 50 hours, quitting work, the signal before and after analysis passes judgment on the cardiotoxin effect to cardiac muscle accordingly.
Claims (6)
1. cardiac muscular tissue's sensor, it is characterised in that: described sensor includes cardiac muscular tissue's chip and accessory；Described accessory includes upper mold (101), lower mold (104), upper electrode (107), bottom electrode (106), photographic plate (102), parallel light source (105) and data collecting system (103)；Described upper mold (101) is provided with first fluid passage (202), second fluid passage (203), guides tongue (204) and upper groove (201)；Described lower mold is provided with culture chamber (302), chip slot (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 slot (303)；Described upper electrode (107) and bottom electrode (106) lay respectively at upper mold (101) and lower mold (104) is internal and one-body molded with upper die and lower die；Upper electrode (107) is connected with power supply (108), and bottom electrode (106) bottom is positioned on chip slot (303), and is in close contact with the cardiac muscular tissue's chip in chip slot；Described photographic plate (102) is placed in the upper groove (201) of upper mold, and is connected with data collecting system (103)；Described parallel light source (105) is placed in the low groove (304) of lower mold.
2. according to a kind of cardiac muscular tissue sensor described in claim 1, it is characterised in that: described upper die and lower die all use makrolon material, described upper electrode and bottom electrode to use titanium alloy material.
3. the preparation method of a cardiac muscular tissue as claimed in claim 1 chip, it is characterised in that the method comprises the following steps:
1) surface patch last layer adhesive plaster (402) of the sheet glass (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 a length of 6～8mm, width is 0.6～1mm, spaced 0.1～0.3mm, and remove the square rubber pieces of cloth (404) cut；
3) prepare the poly-N-isopropyl acrylamide solution that mass body volume concentrations is 10%～50%, with spin coating method be evenly coated in sheet glass containing adhesive plaster side, remove adhesive plaster after solidification, leave square poly-N-isopropyl acrylamide layer (405)；
4) by after polydimethylsiloxane heating and melting, it is coated onto on sheet glass with spin coating method, after cured, polydimethylsiloxane cut into along second time cutting track (407) with laser formation machine middle be connected, both sides are multiple " cantilever beam " (408) structure, both sides " cantilever beam " spacing is 0.6～1mm；
5) preparing mass body volume concentrations is 0.1～the hydrogel solution of 20% and cross-linking agent solution that mass body volume concentrations is 0.1～20%, extract or inducing cardiomyocytes to make cell density be 1 × 107～2 × 107The myocardial cell suspension of individual/ml, being mixed in hydrogel solution by myocardial cell and making cell density is 1 × 106～5 × 106The hydrogel solution containing myocardial cell of individual/ml；Being loaded by myocardial cell suspension in the first spray-type shower nozzle, cross-linking agent solution loads in the second spray-type shower nozzle, and the hydrogel solution containing myocardial cell loads in squash type shower nozzle；Successively print on " cantilever beam " with the composite spray jet head being made up of the first spray-type shower nozzle, the second spray-type shower nozzle and squash type shower nozzle, the thread hydrogel of squash type shower nozzle extrusion forms water-setting collodion silk (502) after the cross-linking agent solution crosslinking that the second spray-type shower nozzle sprays, and then forms myocardial cell layer (501) with the first spray-type shower nozzle at one layer of myocardial cell suspension of water-setting collodion silk surface sprinkling；Forming cardiac muscular tissue's chip with multi-layer cellular after repeating extrusion, crosslinking, spraying operation, water-setting collodion silk between layers is parallel to each other；
6) this cardiac muscular tissue's chip is placed in the chip slot in the accessory described in claim 1, after pulsation is cultivated, forms the cardiac muscular tissue's chip beating consistent.
The preparation method of a kind of cardiac muscular tissue the most as claimed in claim 3 chip, it is characterised in that: step 2) on adhesive plaster, cut 8～15 square contours with laser formation machine.
The preparation method of a kind of cardiac muscular tissue the most as claimed in claim 3 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, chitosan, fibronectin, gelatin and Fibrinogen, described cross-linking agent is at least one in thrombin, calcium chloride or peroxophosphoric acid sodium.
The preparation method of a kind of cardiac muscular tissue the most as claimed in claim 3 chip, it is characterized in that, step 6) described in pulsation cultivate, its process is that from the first fluid passage (202) of accessory, culture fluid is flowed into culture chamber (302), flow out from second fluid passage (203), flowing velocity is 10～50mL/min, circulates 40～50h.
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| CN110954249B (en) * | 2019-12-18 | 2021-04-06 | 东南大学 | Method for measuring in-vitro myocardial tissue contractility based on protein wire spring |
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| CN113908333A (en) * | 2020-07-10 | 2022-01-11 | 国家纳米科学中心 | Flexible electric conduction chip, preparation method and application thereof |
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