CN201648396U - In-vitro cellular pressure loading device - Google Patents
In-vitro cellular pressure loading device Download PDFInfo
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- CN201648396U CN201648396U CN 200920243788 CN200920243788U CN201648396U CN 201648396 U CN201648396 U CN 201648396U CN 200920243788 CN200920243788 CN 200920243788 CN 200920243788 U CN200920243788 U CN 200920243788U CN 201648396 U CN201648396 U CN 201648396U
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/12—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by pressure
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/40—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pressure
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/48—Automatic or computerized control
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Abstract
The utility model discloses an in-vitro cellular pressure loading device which is characterized by comprising an air pressure source (1), an air inlet electromagnetic valve (2), a filter (3), a pressure reducing valve (4), a pressure chamber body (5) and a digital regulation and control system; the air pressure source is connected with the pressure chamber body through the air inlet electromagnetic valve, the filter and the pressure reducing valve; one end of the pressure chamber body (5) is connected with an air outlet electromagnetic valve (6); the air outlet electromagnetic valve (6) is connected with a computer (14) through a drive circuit (7) and a multifunctional data collection card (8); the other side of the drive circuit (7) is connected with the air inlet electromagnetic valve (2); the other end of the pressure chamber body (5) is connected with a pressure sensor (9); the pressure sensor (9) is connected with the computer (14) through a programmable gain amplifier (10) and the multifunctional data collection card (8); and the pressure chamber body (5) is connected with the computer (14) through a standard air cylinder (11), a linear stepper motor (12), a power amplifier (13) and the multifunctional data collection card (8).
Description
Technical field
The utility model relates to a kind of cell in vitro pressure-loaded device, belongs to organizational project and cell engineering field.
Background technology
Cyto-mechanics is one of basis of organizational project and cell engineering, usually will study the influence of mechanics factor pair cell metamorphosis and growth.Because the structure of bio-tissue and organ is extremely complicated, also have bigger difference between the biont, cause in somatic mechanical environment complexity various, thereby be increased in the difficulty of somatocyte mechanical behavior research.At body tissue, cell pressurized is the common a kind of bearing mode of organism, and in musculoskeletal system, bone is the main bearing structure of human body, and it provides support framework for human body, protects internal organs, and finishes the transmission of power; The human body cartilage of joint part often bears the pressure load that transmits through bone, and the effect of pressure is most important to the growth of joint cartilage, metabolism etc.Therefore, the mechanical behavior of cell has crucial meaning under the research pressure load.The basis of cyto-mechanics research and key are the cell loading techniques, because the size of human body cell is between tens to tens microns, the thickness of cytolemma only has several nanometers to tens nanometer, conventional macromechanics loading method and experimental technique can't directly be used, therefore, the in-vitro separation cell is the matter of utmost importance that cyto-mechanics faces with setting up suitable loading culture model.
When cells in vitro was cultivated, by the ambient stress of simulation cell in body tissue, the experimental technique of the mechanical characteristic of cell can be traced back to early stage Glucksmann (Anatomical Record, 1939 under the research pressure load; Research work 73:39-56): chicken embryo shin bone endo cell is cultivated on intercostal muscle matrix, and along with the atrophy of muscle, rib is close to each other, the cell effect of being under pressure.Rodan (Science, 1975; 189:467-469; Calcifid Tissue Research, 1975; 18:125-131; Journal of Cellular Physiology, 1976; 88 (3): 353-361) research of Denging has epoch making significance, and the static pressure that they utilize gas or liquid to produce makes cell be subjected to corresponding action of compressive stress, and research enters quantization stage.Through improving for many years and developing, the augmenter of multiple cultured cell in vitro is developed out.Be broadly divided into the centrifugal force deceleration loading device, the fluid shearing force loading device, air liquid static pressure deceleration loading device, substrate deformation deceleration loading device (brachmorphy substrate tension deceleration loading device, static membrane type cell distraction force deceleration loading device, Flexercell cell loader (Bone Miner Res, 1998,13:218-28), Petriperm elastic force film culture dish deceleration loading device (Am J Orthod Dentofac Orthop, 1991,99:226-40)) etc., the commercialization of part deceleration loading device, but all these devices all respectively have relative merits, and are applicable to the cell type that will study separately.As the real physiological status of reacting cells of centrifugal force deceleration loading device, Flexercell cell loader, Petriperm elastic force film culture dish deceleration loading device complex structure, cost an arm and a leg, the fluid shearing force loading device is only applicable to external loading experiment of studying vascular endothelial cell etc.
Summary of the invention
The purpose of this utility model is to provide a kind of cell in vitro pressure-loaded device at the deficiencies in the prior art, be characterized in culturing cell being carried out pressure-loaded under specified culture condition, size, effect frequency and the acting duration etc. of on-load pressure are quantitatively controlled external.It has friendly interface, stable performance, advantage simple to operate.
The used cell loader of the utility model is the controllable pressure cell loader, is used for simulating the ambient stress of cell in body tissue.The principle of work of system is: culture dish and the cultured cells thereof that will keep cells in vitro existence and growth are put into pressure chamber, the high pressure gas of high-pressure gas indoor (steel cylinder) are entered in the pressure chamber with stable pressure by the pressure maintaining valve regulation and control after the two-stage decompression device reduces pressure certain pressure intensity, when the pressure in the pressure chamber reaches preset value, close the air inlet magnetic valve, form stable closed environment in the pressure chamber, pressure-loaded part principle drives the cylinder pressurization for utilizing linear stepping motor, make and form certain frequency in the pressure chamber, the periodicity pressure variation of size, thereby make the cell in the pressure chamber be subjected to corresponding pressure effect, experiment finishes, open exhaust solenoid valve, gas is discharged through it.The homo(io)thermism of environment guaranteed the biological activity of cell in the experimentation at 37 ℃ in system's use Water Tank with Temp.-controlled control device made.
The purpose of this utility model is realized by following technical measures:
Cell in vitro pressure-loaded device comprises pneumatic supply, air inlet magnetic valve, strainer, reducing valve, pressure cavity and digital regulator control system, and pneumatic supply is connected with pressure cavity by air inlet magnetic valve, strainer, reducing valve; One end of pressure cavity is connected with the magnetic valve of giving vent to anger, and the magnetic valve of giving vent to anger is connected with computer by driving circuit, multifunctional data acquisition card; The driving circuit opposite side is connected with the air inlet magnetic valve; The other end of pressure cavity is connected with pressure transmitter; Pressure transmitter is connected with computer by program-controlled gain amplifier, multifunctional data acquisition card; Pressure cavity is connected with computer by standard cylinder, linear stepping motor, power amplifier, multifunctional data acquisition card.
Establish multilayer culture dish dish in the pressure cavity, every layer of culture dish dish is provided with a plurality of sample grooves, the culture dish layer is fixing on the cover board, cover plate is fastening by sealing-ring and pressure cavity, cover plate is provided with inlet mouth, air outlet and pressure transmitter import, pressure cavity is supported by mount holder, and cover plate pushes away, spur and make the culture dish layer free in and out pressure cavity.
Driving circuit is by phase inverter, and current-limiting resistance, solid state relay, fly-wheel diode are formed and the magnetic valve composition.The control signal of multifunctional data acquisition card output is logic level U1N, when U1N=5V, the phase inverter output low level, make lumination of light emitting diode in the solid state relay, the inner silicon controlled rectifier conducting of solid state relay, magnetic valve gets electric work, and when U1N=0V, phase inverter is output as high level, the solid state relay photodiode is not luminous, silicon controlled rectifier ends in the solid state relay, and magnetic valve ends, and computer receives the pressure variation signal by multifunctional data acquisition card, after program calculating, instruction is fed back to multifunctional data acquisition card, give vent to anger and the air inlet magnetic valve, reach the purpose of regulating the pressure intracavity gas by driving circuit control.
Stress loads the pneumatic supply of flow system and selects 5%CO
2With 95% Air mixing gas, utilize the air filter Purge gas, guarantee the gas gnotobasis; In view of the singularity of cell cultures, pressure chamber is selected for use, and pollution-free, avirulent medical acid-resistant stainless steel materials processing forms.
Numeral regulation and control part comprises four processes: multifunctional data acquisition card is finished the information transmission between computer and the observing and controlling passage, just the mutual conversion between analog quantity and the digital quantity; Program-controlled gain amplifier cooperates pressure transmitter to constitute measurement passage, the pressure in the test pressure chamber; Power amplifier and topworks's stepper-motor constitute control channel, drive steam cylinder piston; Computer is finished demonstration, storage and the processing of data.
Software design is the core of numeral regulation and control, uses Borland C++builder5 to write the pressure-loaded computer control software, and its function is as follows:
Solenoid control function: any time, open or close the respective electrical magnet valve at the magnetic valve of selecting to regulate.
Step motor control function: set pulse-repetition (unit is Hz) or motor and promote the operation of piston range ability (unit is mm) dual mode control step motor.Detect the displacement of motor piston simultaneously, monitor the running status of linear stepping motor.According to detected situation,, make system be in perfect condition by the pressure parameter of computer sampling pilot circuit automatic regulating device.
Data gathering: point sampling modes such as sample mode employing, finish the A/D conversion of pressure signal.
Data storage function: for manually storing and timing acquiring deposit dual mode.Under the manual mode, any time presses the button, and program is deposited automatically at 10 all after dates of collection, under the timing acquiring deposit mode, by menu item is set the pitch time of depositing earlier, starts the timing acquiring memory function then.
Data processing function: the cycle that drives with linear stepping motor is the timed interval, after the data gathering of finishing one-period, calculates maximum value, mean value and the minimum value of this cycle pressure, and shows in corresponding display box.
The data presentation function: data acquisition module is finished the data collection task to pressure in pressure cavity, and the data that the collect mode with digital oscilloscope is shown in real time.
The utlity model has following advantage:
1. this device is divided into four parts with system, connects with lead or airway mutually, accomplishes remote control, can experiment be regulated and control in good time not influencing under the successional prerequisite of whole culture systems.
2. adopt computer to control force value, the clamping time of loading, have convenience, accurate.
3. frequency control program COM amplifies the opening-closing valve of back regulation and control magnetic valve again by the little electric current of serial ports by direct current, makes the CYCLIC LOADING that reaches comparatively objective, credible.
4. friendly control panel also makes easy and simple to handle; Furthermore applying pressure transmitter, the effect of the size of control pressure value, so pressure is exactly studied the variation characteristics of cell under the effect of different pressures value accurately and reliably, and clamping time is adjustable, and experimentation can be set as required.
Description of drawings
Fig. 1 is cell in vitro pressure stowage unit figure
1, steel cylinder, 2, the air inlet magnetic valve, 3, air filter, 4, reducing valve, 5, pressure chamber, 6, the magnetic valve of giving vent to anger, 7, vibrating circuit, 8, multifunctional data acquisition card, 9, transmitter, 10, program-controlled gain amplifier, 11, standard cylinder, 12, linear stepping motor, 13, power amplifier, 14, computer, 15, housing.
Fig. 2 is a stress LOADED CAVITY structural representation
16, mount holder, 17, the culture dish dish, 18, the sample groove, 19, sealing-ring, 20, cover plate, 21, the air outlet, 22, pressure transmitter, 23, inlet mouth.
Fig. 3 is the driving circuit schematic diagram
24, phase inverter, 25, current-limiting resistance, 26, solid state relay, 27, fly-wheel diode, 28, magnetic valve.
Fig. 4 loads the computer control interface for gaseous tension
Fig. 5 changes oscillogram for pressure in pressure cavity
A) static pressure oscillogram
B) dynamic pressure waveform figure
Fig. 6 is the influence of dynamic and static stress to the early stage morphocytology of MSCs Osteoblast Differentiation
A) bone was induced 0 day, no pressure
B) bone was induced 0 day, executed dynamic pressure 5 days
C) bone was induced 0 day, executed static pressure 5 days
D) bone was induced 3 days, no pressure
E) bone was induced 3 days, executed dynamic pressure 5 days
F) bone was induced 3 days, executed static pressure 5 days
G) bone was induced 7 days, no pressure
H) bone was induced 7 days, executed dynamic pressure 5 days
I) bone was induced 7 days, executed static pressure 5 days
Embodiment
Below by embodiment the utility model is carried out concrete description; be necessary to be pointed out that at this present embodiment only is used for the utility model is further specified; but can not be interpreted as the restriction to the utility model protection domain, the person skilled in the art in this field can make some nonessential improvement and adjustment according to above-mentioned content of the present utility model.
Embodiment:
Cell in vitro pressure-loaded device as shown in Figure 1, cell in vitro pressure-loaded device comprises pneumatic supply 1, air inlet magnetic valve 2, strainer 3, reducing valve 4, pressure cavity 5 and digital regulator control system, pneumatic supply is connected with pressure cavity by air inlet magnetic valve, strainer, reducing valve; One end of pressure cavity 5 is connected with the magnetic valve 6 of giving vent to anger, and the magnetic valve of giving vent to anger is by driving circuit 7.Multifunctional data acquisition card 8 is connected with computer 14; Driving circuit 7 opposite sides are connected with air inlet magnetic valve 2; The other end of pressure cavity 5 is connected with pressure transmitter 9; Pressure transmitter is connected with computer 14 by program-controlled gain amplifier 10, multifunctional data acquisition card 8; Pressure cavity 5 is connected with computer 14 by standard cylinder 11, linear stepping motor 12, power amplifier 13, multifunctional data acquisition card 8.
Establish multilayer culture dish dish 17 in the pressure cavity 5, every layer of culture dish dish is provided with a plurality of sample grooves 18, the culture dish layer is fixed on the cover plate 20, cover plate is fastening by sealing-ring 19 and pressure cavity, cover plate is provided with inlet mouth 23, air outlet 21 and pressure transmitter import 22, pressure cavity is supported by mount holder 16, and cover plate pushes away, spur and make the culture dish layer free in and out pressure cavity.
Driving circuit 7 is by phase inverter 24, current-limiting resistance 25, solid state relay 26, fly-wheel diode form 27 and magnetic valve 28 form.The control signal of multifunctional data acquisition card output is logic level U1N, and when U1N=5V, the phase inverter output low level makes lumination of light emitting diode in the solid state relay, the inner silicon controlled rectifier conducting of solid state relay, and magnetic valve gets electric work.When U1N=0V, phase inverter is output as high level, and the solid state relay photodiode is not luminous, and silicon controlled rectifier ends in the solid state relay, and magnetic valve ends.Computer receives the pressure variation signal by multifunctional data acquisition card, after calculating through program, instruction is fed back to multifunctional data acquisition card, gives vent to anger and the air inlet magnetic valve by driving circuit control, reaches the purpose of regulating the pressure intracavity gas.
Below be of the influence of the utility model deceleration loading device in mesenchymal stem cells MSCs (MSCs) the Osteoblast Differentiation commitment cell phychology and the active power of rising in value.
1. experimental cell
Well-grown 2～4 generation 2-3 male rat mescenchymal stem cell in age in week
Above cell is provided by key lab of West China College of Stomatology Sichuan University
2. pressure chamber culture condition
Temperature: 37 waters bath with thermostatic control
Atmosphere surrounding: 5%CO2 and 95% air gas mixture
Pressure: static pressure stress 23Kpa,, the stress size is dynamic compressive stress 10～35Kpa, the frequency of dynamic compressive stress is selected 0.25Hz.
3. experiment flow
The inferior back osteogenic induction 3 days (OS-3d) and 7 days (OS-7d) respectively that merges of cell.Apply dynamic compressive stress and static pressure stress stimulation respectively, one hour afterburning time of every day, continue 1,3,5 day.Other puts culture dish at CO
2The constant temperature incubator is organized in contrast.
4. experimental result example
A. experiment grouping
According to difference, be divided into following each group to pair cell on-load pressure in the pressure chamber:
Control group: do not have external pressure-loaded
Static pressure group cell: 23Kpa pressure in addition
Dynamic pressure group cell: 10～35Kpa in addition, frequency 0.25Hz
B. cellular form variation after the various pressure-loaded in the pressure chamber
The equal adherent growth of cell is good.Corresponding time point dynamic pressure group and control group are relatively, do not see that tangible cell is arranged, morphocytology changes (Fig. 5), and some static pressure group cellular form can " expansions " become circle (the typical change that loads cellular form generation behind the static pressure shown in the figure f) by spindle shape.
Experiment adopts 0.25% trypsinase and 0.02%EDTA to digest the MSCs of osteogenic induction not or osteogenic induction 3d and 7d, ends with the serum nutrient solution that does not contain the osteogenic induction agent, and counting is also adjusted cell density, with 4 * 10
3/ hole is inoculated in 96 orifice plates, behind the cultivation 24h, culture dish is put into pressure chamber begin to carry out pressure-loaded, and afterburning condition is the same.
Experiment sample is put into methyl thiazolyl tetrazolium 20 μ L mixings, and incubator is hatched 3.5h, inhales and removes liquid.Add 200 μ L dimethyl sulfoxide (DMSO) and place shaking table concussion 15min behind the incubator 0.5h, (PE USA), sets the 570nm wavelength to use HTS 7000Plus porous plate efficient analysis instrument.As can be seen, dynamic and static pressure group MSCs Osteoblast Differentiation commitment proliferation activity is to very similar with the not afterburning control group of time point.Have only static pressure to load not induce that the MSCs proliferation activity has tangible enhancing (P＜0.05) after MSCs5 days.
Claims (3)
1. cell in vitro pressure-loaded device, it is characterized in that this device comprises pneumatic supply (1), air inlet magnetic valve (2), strainer (3), reducing valve (4), pressure cavity (5) and digital regulator control system, pneumatic supply (1) is connected with pressure cavity by air inlet magnetic valve, strainer, reducing valve; One end of pressure cavity (5) is connected with the magnetic valve of giving vent to anger (6), and the magnetic valve of giving vent to anger is by driving circuit (7), and multifunctional data acquisition card (8) is connected with computer (14); Driving circuit (7) opposite side is connected with air inlet magnetic valve (2); The other end of pressure cavity (5) is connected with pressure transmitter (9); Pressure transmitter is by program-controlled gain amplifier (10), and multifunctional data acquisition card (8) is connected with computer (14); Pressure cavity (5) is connected with computer (14) by standard cylinder (11), linear stepping motor (12), power amplifier (13), multifunctional data acquisition card (8).
2. cell in vitro pressure-loaded device according to claim 1, it is characterized in that pressure cavity (5) establishes multilayer culture dish dish (17), every layer of culture dish dish is provided with a plurality of sample grooves (18), the culture dish layer is fixed on the cover plate (20), cover plate is fastening by sealing-ring (19) and pressure cavity, and cover plate is provided with inlet mouth (23), air outlet (21) and pressure transmitter import (23), pressure cavity is supported by mount holder (16), and the cover plate push-and-pull is moving to make the culture dish layer free in and out pressure cavity.
3. cell in vitro pressure-loaded device according to claim 1, it is characterized in that driving circuit is by phase inverter (24), current-limiting resistance (25), solid state relay (26), fly-wheel diode forms (27) and magnetic valve (28) is formed, the control signal of multifunctional data acquisition card output is logic level U1N, when U1N=5V, the phase inverter output low level, make lumination of light emitting diode in the solid state relay, the inner silicon controlled rectifier conducting of solid state relay, magnetic valve gets electric work, when U1N=0V, phase inverter is output as high level, and the solid state relay photodiode is not luminous, and silicon controlled rectifier ends in the solid state relay, magnetic valve ends, computer receives the pressure variation signal by multifunctional data acquisition card, after calculating through program, instruction is fed back to multifunctional data acquisition card, give vent to anger and the air inlet magnetic valve by driving circuit control, reach the purpose of regulating the pressure intracavity gas.
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| CN 200920243788 CN201648396U (en) | 2009-12-08 | 2009-12-08 | In-vitro cellular pressure loading device |
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| CN 200920243788 CN201648396U (en) | 2009-12-08 | 2009-12-08 | In-vitro cellular pressure loading device |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102759481A (en) * | 2012-06-26 | 2012-10-31 | 上海中医药大学附属岳阳中西医结合医院 | Multi-cell mechanical simulation experiment platform |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102759481A (en) * | 2012-06-26 | 2012-10-31 | 上海中医药大学附属岳阳中西医结合医院 | Multi-cell mechanical simulation experiment platform |
| CN102759481B (en) * | 2012-06-26 | 2014-12-10 | 上海中医药大学附属岳阳中西医结合医院 | Multi-cell mechanical simulation experiment platform |
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Granted publication date: 20101124 Termination date: 20121208 |