CN101298592A - Cell three-dimensional mechanical loading unit - Google Patents
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- CN101298592A CN101298592A CNA2008100698361A CN200810069836A CN101298592A CN 101298592 A CN101298592 A CN 101298592A CN A2008100698361 A CNA2008100698361 A CN A2008100698361A CN 200810069836 A CN200810069836 A CN 200810069836A CN 101298592 A CN101298592 A CN 101298592A
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Abstract
The invention discloses a drawing and loading device for cells, which relates to a cellular mechanics loading device that belongs to the medical instrument. The drawing and loading device for cells consists of a control part, a mechanical part and an electromagnetic part, which is characterized in that: the cells are cultivated on an elastic silicon membrane, and circumferential-direction drawing and compressive loading are carried out for the cells through a dynamic system, meanwhile, magnetic bead collagen with ferroferric oxide enveloped is attached to the surfaces of the cells through integrin family, then alternating magnetic field is respectively loaded at an upper and a lower bottoms of a cell cultivating box, rendering the cells to be drawn under the magnetic force while being under the circumferential stress in a base strain field, so as to control the transformation of the cells along the axis Z and control the cell morphology. By simulating the borne mechanical stress of cells under physiological conditions, the drawing and loading device for cells can realize mechanical loading with different dimensions under different mechanical forces, and has the advantages of simple structure, convenient use, wide application and excellent reproducibility, etc.
Description
Technical field
The present invention relates to the cyto-mechanics field, be specifically related to a kind of cell three-dimensional mechanical loading unit.
Background technology
The research of cyto-mechanics is a relatively more active field. it is how to conduct at cell interior that main point of interest concentrates on mechanical signal, and the change of cell 26S Proteasome Structure and Function under the mechanical signal effect.Mechanical stress plays an important role in physiological processs such as cell growth, differentiation, apoptosis and genetic expression and some pathologic process (as diseases such as myocardial hypertrophy, atherosclerosiss).There is the acceptor of counter stress sensitivity in cell surface, and these are passed to institute's induced force signal in the born of the same parents on the different structure parts by cell surface specific molecule passage by physical efficiency, the realizable force chemical transformation, thus regulate the cells physiological function.
Our current pair cell and adherent understanding of cell matrix--surface tissue of cell is regulated cell adhesion with extracellular matrix (ECM)---be based on the conclusion adhesion plaque of in vitro study and other adhesion arrangement, adhesion plaque is to integrate the structure of element, produce cell and substrate intensive adhesive power, simultaneously two-way conducts information between extracellular matrix and tenuigenin.Adhesion plaque also is the main path of the mechanical signal conduction of cell tensile.We are cell or the tissue that derives from two-dimentional substrate tension about these structures in the understanding of cell adhesion, migration, signal conduction, cytoskeleton function, yet, it is very important that three-dimensional ECM is considered to pair cell, (referring to Edna Cukierman, Roumen Pankov, Daron R.Stevens, Kenneth M.Yamada.Taking Cell-Matrix Adhesions to the Third Dimension.Science 294,1708 (2001) 1708-1712; ).Therefore, in vivo, related tissue's cell adheres to by membrane receptor and the outer adhesion molecule of born of the same parents, these adhesion molecules are connected with the collegen filament of extracellular matrix, these connections pass to cell to stress-strain. and well-known, cytoskeleton plays the mechanics conduction in cell, cytoskeleton is connected to extracellular matrix by integrin family, and extracellular matrix is three-dimensional structure.Document shows, all cells all are among the quite complicated mechanical environment in the organism, with regard to vascular endothelial cell and vascular smooth muscle cell, not only comprise shearing force, positive pressure that blood flow produces, also comprise the circumferential tension force that causes by blood pressure and the tension force and the pressure of internal blood vessel matrix and surrounding tissue self thereof.Therefore, vascular cell is subjected to dynamic substrate tension and also has been subjected to the pressure of vessel wall or bounce etc. simultaneously [referring to K.J.Gooch, C.J.Tennant.Mechanical forces:their effects on cells and tissues.First edition, New York, Springer-Verlag Berlin Heidelberg, 1997,1-15,19-55,123-147], equally, the keratinocyte of skin, inoblasts etc. are to be subjected to surperficial substrate two dimension tension force, also be subjected to simultaneously lower-hierarchy and extraneous stretching or compressive action, therefore cell be in the three-dimensional mechanical environment [, referring to Frederick H.Silver, Lorraine M.Siperko and GurinderP.Seehar.Mechanobiology offorce transduction in dermaltissue.Skin Research and Technology 2003; 9:3-23].That usually uses that pertinent instruments and device come analog cell carries out external cyto-mechanics research at the body environment, provides research model for cell in body mechanism.Typical cell stress model is to make the planar film with the material that can be out of shape to come pair cell to carry out static or periodic mechanics loading, under the mechanics loading environment, cell shows different mechanics reactions, comprise that cell rearranges, cytoskeleton is reset, cell proliferation, genetic expression, protein synthesis, cell migration and apoptosis etc.The mechanics loading of various types of cells has been reacted now understanding has preferably been arranged.
But the model that a lot of mechanics that use at present load also has many obvious defects.At first, cell mechanics on flat culture surface loads the back cell and rearranges and make cell be in minimum strain, and randomness and subsequently the cell of cell before substrate tension rearranges and shown that cell feels surperficial strained.In order to eliminate this problem, developed and waited an axle stretching device, make basilar membrane produce circumferential strain, cell can not be reset under this strain, but waits the axle stretching may not meet the physiology strain.Secondly, cell is unordered on present stretching device, the form of cell also is various, the result, and cell response may not be simulated intravital environment fully, because the tissue of cell and form influence the function of cell, so be necessary to study a new culture systems, by this culture systems, the arrangement of cell, form, mechanics loading environment can be tighter control.James H.-C.Wang etc. use little mounting technology, the basilar membrane that the new mechanics of development loads, the little groove of etching on basilar membrane, the arrangement of control cell, discovery is under same strained condition, cell and principal strain directions angle are that the a-SMA of cell expresses obviously and increases under the condition of 0 ° or 45 °, and 90 ° significantly do not change; Simultaneously, 0 ° of ratio changes obviously for 45 °, illustrate that cell deformation quantity or deformation area are big, the mechanics reaction of cell is just obvious more [referring to James H.-C.Wang et al controlling cell response to cyclic mechanicalstretching.Annals of Biomedical Engineering, vol.33, no.3,2005:337-342].The defective of this technology is that little groove is separated cell cell can not directly be exchanged, and between cell under the concrete conditions in the establishment of a specific crime and cell on every side be by integrate carry out between element or the cadherin alternative.People such as Michael Glogauer have reported a new cell tensile device, use the magnetic bead of collagen Z 250 bag quilt to adhere to cell surface by integrin family, just can carry out the mechanics loading by pair cell then, and can be placed in the electromagnetic field of alternation, cell can be subjected to the mechanical stretching of alternation in the electromagnetic field of alternation, (referring to Michael Glogauer.Jack Ferrier.A new method forapplication of force to cells via ferric oxide beads.Arch-Eur J Physiol (1998) 435:320-327), simultaneously, cell can be arranged along the direction of magnetic force, under mechanics stretched, the power that cell is subjected to was quite tangible like this.But the defective of this method is the effect that the substrate of cell can not be subjected to pulling force.
Summary of the invention
The objective of the invention is to, it is more approaching for cells in vitro is stretched at the body environment, make cell have bigger potential, overcome above-mentioned weak point of the prior art and a kind of cell three-dimensional mechanical loading unit is provided, in conjunction with existing cyto-mechanics substrate deceleration loading device and novel cell loader, with cell seeding on the silica gel substrate film, place under the three-dimensional compound force field effect, cell is in the three-dimensional field of force, improve cellular metabolism, promote its secreting function, to construct the device of tissue substituent with specific modality and function.
Technical scheme of the present invention is as follows:
A kind of cell three-dimensional mechanical loads and culture apparatus, and it has control section, mechanical part and electromagnet portion:
Described mechanical part include stepper-motor, rotary disk, connecting rod, impeller-hub, fixedly compression ring, be coated with the cell cultures dish of elastic substrates film and the stand of above-mentioned each parts be installed; Plant on the described elastic substrates film and be implanted with the cell that has carried out magnetic bead bag quilt; Described stepper-motor is by the microprocessor control of control section; The output of stepper-motor is connected with rotary disk is eccentric, connecting rod is connected on the eccentric rotary disk again, connecting rod is done periodically to-and-fro movement under the driving of stepper-motor, the upper end of connecting rod connects impeller-hub, the cell culture case that is coated with the elastic substrates film is housed on impeller-hub, the position of elastic substrates film is relative with fixedly compression ring on being fixed on stand, and when impeller-hub moved up and down, fixedly compression ring formed extrusion stretching to the cell on the elastic substrates film.
Described electromagnet portion adopts the Helmholtz coil group, described Helmholtz coil group is fixed on the position of corresponding silica gel basilar membrane on the stand, the Helmholtz coil group is driven by the start pulse signal control direct supply output pulsed current of control section output, forms electromagnetic field.
The method that the use said apparatus carries out the cell three-dimensional mechanical loading is as follows: at first by control section control step driven by motor eccentric rotary disk, the impeller-hub on the drivening rod is done periodically to-and-fro movement again; Simultaneously, control section is also controlled direct supply output pulsed current, makes the Helmholtz coil group produce pulse electromagnetic field; The elastic substrates film moves up and down under the drive of impeller-hub, the elastic substrates film contacts with fixing compression ring, fixedly compression ring makes the cell on the elastic substrates film produce a circumferential strain field, the pulse electromagnetic field of Helmholtz coil group generation simultaneously is coated with the cell of magnetic bead on Z-direction tensile elasticity basilar membrane, so just obtain the effect that three-dimensional strain field acts on cell, cell can be realized axle stretchings such as basilar membrane strained, and the Z axle stretches and three-dimensional the stretching.
The present invention combines existing cyto-mechanics substrate deceleration loading device and novel cell loader, because use substrate tension separately, have a long way to go with three-dimensional mechanical environment at body, as the research of present cell begins to turn to three-dimensional research, the substrate of independent magnetic field force stretching pair cell does not have stretching action clearly again, or be difficult to make up a three-dimensional tensile system, so the advantage of this method is to combine the mode that two kinds of mechanics load, utilize integration element and cytoskeleton pair cell to stretch, give full play to the advantage of dual mode, it is in the three-dimensional mechanics field cell, analog cell at the mechanics environment, and can simulate different directions, the mechanical environment of different angles, for example, can make the cell based base compression, its cell spaces stretches simultaneously. the model that three-dimensional culture method coupling cell three-dimensional mechanical loads, and can regulate and control the mechanics size of different angles, like this, the residing mechanics of cell field is more near the physiology field of force, can study the mechanical response of cell under the three-dimensional mechanical stretching condition, comprise the arrangement of cytoskeleton and the reaction of cell in the mechanics field.Also can make the cell orientation growth of dimensional culture by this method, improve its metabolism, promote its secreting function, to construct the device of tissue substituent with specific modality and function.This device will provide a new thinking and new research means for the research of cyto-mechanics and the structure of organizational project..
Thus, the present invention compared with prior art has following advantage:
1. by this device, can study cell and under three-dimensional mechanical loads, load different physiological responses with two dimension.
2. by this device, greatly increased the extension area of cell, be equivalent to increase the area that sticks of cell, the mechanics microenvironment of cell growth during simulation is organized in vivo effectively, increased the cytotrophy condition to a certain extent, improved and organized formation efficiency.
3. can produce axle stretchings such as varying strength substrate separately, but also pair cell carries out stretched vertically, can realize simultaneously that the three-dimensional of cell stretches.
4. cell has improved the growth conditions of cell greatly under dynamic three-dimensional mechanical function, thereby has accelerated cell growth process, and cell culture period is shortened, and metabolite content improves, and emiocytosis collegen filament amount is big, and presses certain orientation and arrange.
5. the present invention is simple in structure, easy to use, is widely used, for the function that improves cell provides new method.
Description of drawings
Fig. 1 is the structural representation of this device.
The principle framework figure of three integral parts of this device of Fig. 2.
Fig. 3 is the schematic circuit of control section.
Fig. 4 is the software workflow figure of part.
Embodiment
Describe structure of the present invention in detail below in conjunction with accompanying drawing:
Referring to Fig. 1 and Fig. 2, this cell three-dimensional mechanical loading unit specifically is made up of control section, mechanical part, electromagnet portion:
1, mechanical part include stepper-motor 2, rotary disk 4, connecting rod 8, impeller-hub 10, fixedly compression ring 7, be coated with the cell culture case 12 of elastic substrates film 9 and the support 14 of above-mentioned each parts be installed; Plant on the described elastic substrates film 9 and be implanted with the cell that has carried out magnetic bead bag quilt; Described stepper-motor 2 is by the microprocessor control of control section 1; The output of stepper-motor 2 and 4 eccentric connections of rotary disk, connecting rod 8 is connected on the eccentric rotary disk 4 again, eccentric rotary disk 4 is supported by bracing frame 5, connecting rod 8 is done periodically to-and-fro movement under the driving of stepper-motor 2, the upper end of connecting rod 8 connects impeller-hub 10, the cell cultures dish 12 that is coated with silica gel substrate film 9 is housed on impeller-hub 10, the position of elastic substrates film 9 be fixed on support 14 on stationary shaft 6 on fixedly compression ring 7 relative, when impeller-hub 10 moved up and down, fixedly the cell on 7 pairs of elastic substrates films 9 of compression ring formed extrusion stretching.The working process of mechanical part is to drive eccentric rotary disk 4 drive link 8 by stepper-motor 2, drive has the impeller-hub 10 of motion bearings 11 and does periodically to-and-fro movement, the elastic substrates film 9 that is fixed on the cell culture case 12 moves with catch bar, make the cell of planting on basilar membrane form the periodic axle that waits and stretch, be provided with the collapsing length of the disk indication material of scale.
Connecting rod 8 work reciprocating amplitudes of periodicity and frequency are by the eccentricity and the rotating speed decision of motor-driven disk, 0,2,4,6,8,10,15,20mm the eccentricity of electric motor driving garden dish:, totally 8 grades, rotating speed of motor is 0-300 rev/min, strain field intensity: 0-20%, frequency 0-5Hz, strain field intensity and frequency independent variation
2, electromagnet portion adopts Helmholtz coil group 13, described Helmholtz coil group 13 is fixed on the position of corresponding elastic substrates film 9 on the stand, Helmholtz coil group 13 is sent pulse signal control direct supply 3 output pulsed current by the microprocessor of control section and is driven, form electromagnetic field, the intensity of electromagnetic field is directly proportional with the electric current that flows through Helmholtz coil, regulate electromagnetic intensity by regulating direct supply outward current size, regulate electromagnetic field frequency by the frequency of regulating start pulse signal, simultaneously, Helmholtz coil group 13 and plant that distance is 2cm between the elastic substrates film 9 be implanted with cell.
The relation of the electromagnetic intensity B that the electric current I of the direct supply of outward current and Helmholtz coil produce is:
R, n are respectively the Helmholtz coil radius and the number of turn, R=20mm, n1=0-20000 ampere-turn, u
0=0.4 ∏ * 10
-6Ox/rice
2The special Lars (T) of electromagnetic intensity: 0-0.4, frequency: 0-1kHz, strength of electric field and frequency no dependence.
3, control section is made up of hardware components and software section:
Referring to Fig. 3, hardware components is by 8 chip microprocessors of a slice reduce power consumption, a liquid-crystal display.A keyboard monitoring interface, the photosignal testing circuit of a zero passage detection position, each phase component value latch of stepper-motor, power amplifier, and the driving computer of start pulse signal, the VMSO switching tube, direct supply and Helmholtz coil are formed
The software work principle is referring to Fig. 4, initialize routine is carried out in the start back, the original state that on LCD, shows start subsequently, wait for keyboard input machine operation pre-set parameter and electromagnetic field pre-set parameter, after this starting microprocessor sends and starts microprocessor and send starting impulse and drive stepper-motor and trigger the VMOS switching tube, motor is rotated by preset rotation speed and make direct supply output pulsed current, after the machine operation time finishes, by the zero passage photoswitch circuit.After the machine operation time finishes, send position detection pulse and desired location comparison, motor is stopped to predeterminated position by the zero passage detection photoswitch circuit; finish stop time; motor is started working again, after total working hour and the end of electromagnetic field effect time, and EP (end of program).
The magnetic bead bag of pair cell of the present invention is used following method:
1, cell: cell comprises between scleroblast, class osteocyte, osteoclast, detrusor cell, Tenocyte cell, sarcoplast, inoblast, smooth muscle cell, endotheliocyte, pulmonary epithelial cells, chondrocyte, myocardial cell, marrow towards matter stem cell or adenocarcinoma of lung cancer cells.
2, Bao Guo cell magnetic substance is selected for use:
Ferrimagnetism material: Z 250, ferric oxide etc.
Bag is by the matrix of magnetic bead: type i collagen, and the III Collagen Type VI, fiber adhesion albumen, ln mixes collagen protein, or mouse tail collagen protein.
3, process: type i collagen (U.S. Sigma company) is added in the 0.1mol/L acetic acid, stir 1h under the room temperature to dissolving fully, regulating final concentration is the collagen solution of 1g/L.Add the 0.4g magnetic bead in the 1ml collagen solution, the median size of magnetic is 0.05-0.2um, with NaOH (0.1mol/L) the accent pH to 7.4 of 100 μ l, hatches 2h under 37 ℃.Under this condition, collagen promptly is adsorbed on the magnetic bead.The magnetic bead suspension places on the magnetic force frame and washs 3 times, at last balance 24h in PBS.Use preceding with the vibrator mixing.Add 10ml magnetic bead suspension in every liter of nutrient solution,, hatch 30min under 37 ℃ for the cell of attached wall growth changes liquid.The unconjugated magnetic bead particles of PBS flush away is for cell applies vertical direction magnetic field.
4, culture condition: the culture condition of this test design can design as requested, whole device is placed in the incubator, and CO2 concentration 5%, 37 ℃ of temperature, relative humidity is more than 90%.
This device specifically make use-case:
Example 1, use this cell three-dimensional mechanical loading unit that inoblast is carried out static three-dimensional cell under the situation of no strain field (intensity is 0%) and non-electromagnetic field (field intensity is 0 tesla) to cultivate.
Example 2, to be 0.2 special Lars, frequency be used as periodic Z axle as the magnetic field force of 100Hz stretches in field intensity to the inoblast that is coated with magnetic bead to use this cell three-dimensional mechanical loading unit, do not compare the effect of getting rid of the magnetic field pair cell at adjacent cell culture case with wrapping by the inoblast kind of magnetic bead simultaneously.
Example 3, to use this cell three-dimensional mechanical loading unit be to do periodic Z axle under 0.2 special Lars, the magnetic field force effect of frequency as 100Hz to stretch to the inoblast that is coated with magnetic bead in field intensity, substrate simultaneously applies 10% strain, the circumferential periodicity of 1Hz stretches, the effect of research three dimensional strain field pair cell.
Example 4, to use this cell three-dimensional mechanical loading unit be to do periodic Z axle under 0.2 special Lars, the magnetic field force effect of frequency as 100Hz to stretch to the smooth muscle cell that is coated with magnetic bead in field intensity, substrate simultaneously applies 10% strain, the circumferential periodicity of 1Hz stretches, the effect of research three dimensional strain field pair cell.
Example 5, use this cell three-dimensional mechanical loading unit that the smooth muscle cell that is coated with magnetic bead is applied 10% strain in substrate, the circumferential periodicity of 1Hz stretches, being that 0.2 special Lars, frequency are to do periodic Z axle under the magnetic field force effect of 100Hz to stretch in field intensity simultaneously, is the effect of comparative study three dimensional strain field not have bag by the cell that magnetic bead loads same periodicity substrate strain and magnetic field simultaneously.
Claims (7)
1. cell three-dimensional mechanical loading unit, it is characterized in that: it has control section, mechanical part and electromagnet portion:
Described mechanical part include stepper-motor (2), rotary disk (4), connecting rod (8), impeller-hub (10), fixedly compression ring (7), be coated with the cell culture case (12) of elastic substrates film (9) and the support (14) of above-mentioned each parts be installed; Described elastic substrates film (9) is gone up to plant and is implanted with the cell that has carried out magnetic bead bag quilt; Described stepper-motor (2) is by the microprocessor control of control section (1); The output of stepper-motor (2) is connected with rotary disk (4) is eccentric, connecting rod (8) is connected on the eccentric rotary disk (4) again, connecting rod (8) is done periodically to-and-fro movement under the driving of stepper-motor (2), the upper end of connecting rod (8) connects impeller-hub (10), the cell cultures dish (12) that is coated with silica gel substrate film (9) is housed on impeller-hub (10), the position of elastic substrates film (9) is relative with fixedly compression ring (7) on being fixed on support (14), when moving up and down, fixedly compression ring (7) forms extrusion stretching to the cell on the elastic substrates film (9) at impeller-hub (10);
Described electromagnet portion adopts Helmholtz coil group (13), described Helmholtz coil group (13) is fixed on the position of corresponding elastic substrates film (9) on the support body, Helmholtz coil group (13) is sent pulse signal control direct supply output pulsed current by the microprocessor of control section and is driven, and forms electromagnetic field.
2, cell three-dimensional mechanical loading unit according to claim 1, it is characterized in that: described connecting rod (8) is done the eccentricity and the rotating speed decision of reciprocating amplitude of periodicity and frequency rotary disk driven by stepper motors (4), the eccentricity that stepper-motor (2) drives rotation garden dish (4) is 0,2,4,6,8,10,15,20mm, totally 8 grades, the rotating speed of stepper-motor (2) is 0-300 rev/min.
3, cell three-dimensional mechanical loading unit according to claim 1 is characterized in that: the strain field intensity of described electromagnet portion: 0-20%, frequency 0-5Hz, strain field intensity and frequency independent variation.
4, cell three-dimensional mechanical loading unit according to claim 1 is characterized in that: described Helmholtz coil group and the distance of planting between the elastic substrates film (9) that is implanted with cell are 2cm.
5, cell three-dimensional mechanical loading unit according to claim 1, it is characterized in that: the magnetic substance of the cell parcel of described magnetic bead bag quilt is ferrimagnetism materials such as Z 250 or ferric oxide, bag is pure type i collagen, III Collagen Type VI, fiber adhesion albumen or ln by the matrix of magnetic bead, mix collagen protein, or mouse tail collagen protein.
6, cell three-dimensional mechanical loading unit according to claim 1 is characterized in that: described cell comprises between scleroblast, class osteocyte, osteoclast, detrusor cell, Tenocyte cell, sarcoplast, inoblast, smooth muscle cell, endotheliocyte, pulmonary epithelial cells, chondrocyte, myocardial cell, marrow towards matter stem cell or adenocarcinoma of lung cancer cells.
7, utilize each described device of claim 1 to 6 to carry out the method that cell three-dimensional mechanical loads, at first drive eccentric rotary disk (4) by control section (1) control step motor (2), the impeller-hub (10) on the drivening rod (8) is done periodically to-and-fro movement again; Simultaneously, control section (1) is also controlled direct supply (3) output pulsed current, makes Helmholtz coil group (13) produce pulse electromagnetic field; Elastic substrates film (9) moves up and down under the drive of impeller-hub (10), elastic substrates film (9) contacts with fixing compression ring (7), fixedly compression ring (7) makes the cell on the elastic substrates film (9) produce a circumferential strain field, the pulse electromagnetic field of Helmholtz coil group (13) generation simultaneously is coated with the cell of magnetic bead on Z-direction tensile elasticity basilar membrane (9), so just obtain the effect that three-dimensional strain field acts on cell.
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101497861A (en) * | 2009-02-26 | 2009-08-05 | 中国人民解放军第三军医大学第一附属医院 | Tractor for circulating draw of anchorage-dependent cell |
| CN102109436A (en) * | 2010-12-24 | 2011-06-29 | 江苏大学 | Electromagnetic impact dynamic tensile test method and device |
| CN102533545A (en) * | 2010-12-31 | 2012-07-04 | 四川大学 | Fully adjustable electromagnetic field biological effect loading device and electromagnetic field loading method |
| CN102653719A (en) * | 2012-04-28 | 2012-09-05 | 吉林大学 | Cell culture device capable of generating multiple magnetic fields and culture method |
| CN102965286A (en) * | 2012-12-06 | 2013-03-13 | 天津理工大学 | Magnetically-driven sliding load device capable of controlling compression amount |
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