CN104342370B - The biomechanical system cultivated for cell three-dimensional perfusion Compression and Expansion - Google Patents
The biomechanical system cultivated for cell three-dimensional perfusion Compression and Expansion Download PDFInfo
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Abstract
The invention provides a kind of biomechanical system cultivated for cell three-dimensional perfusion Compression and Expansion, including cell biological support, be used for simulating human body cell real three dimensional growth environment；Bioreactor, is provided with the fixture clamping described cell biological support and the cavity accommodating described cell biological support；Peristaltic pump, is used for driving cell culture fluid to enter described bioreactor and carries out perfusion；Imposed load device, the fixture in the described bioreactor of para-linkage provides periodic reverse tractive, and then cell applies stretching or Compressive Mechanical stimulation；Control module, is connected with computer, is controlled described imposed load device, regulation sample length, draw ratio, roundtrip frequency and round number of times.The present invention has imitated the mechanical environment of internal true complexity, while realizing Three-dimensional cell culture, it is achieved controlled perfusion stretching or the Quantitative study of Compressive Mechanical environment cell growth impact, explores cyto-mechanics Coupling Rule biology.
Description
Technical field
The present invention relates to the cell culture system of a kind of biomechanics, concretely, for the perfusion of cell in three dimensional biological elastic support being stretched or compression culture experiment, explore cyto-mechanics Coupling Rule biology.
Background technology
As the cell of life entity elementary cell, have been in the three-dimensional mechanical environment of complexity.Mechanical stimulation how regulating cell skeleton, cellular morphology, the mechanism of the propagation of cell, differentiation etc. is still not clear.Research shows, affects the propagation of mesenchymal stem cells MSCs, the mechanics factor of differentiation mainly has stretch stimuli, shear stress, compressive stress, gravity etc..Owing to true biological internal milieu is complicated, carrying out the separation of cell, cultivation and Experiments of Machanics the most in vitro is the important research method in biomechanics field.For investigating the mechanical stimulation impact on cell, need to apply mechanical stimulation in the incubation of three-dimensional environment, study the mechanical signal regulatory mechanism to cell.
At present, the main loading system of mechanical stimulation being generally used for biomechanics experiment has four-point bending, axial tractive, vacuum action model and Flexercell loading system, these loading systems are mainly for the culture medium of two-dimensional films structure, usually relying on loading frequency and deformation quantity regulation and control mechanical stimulation, the cell for three-dimensional material is cultivated and the method and apparatus of imposed load is the most common.The difficult point of device design is, how to cultivate cell in addition perfusion stretching/compressing in three dimension system.
Summary of the invention
It is an object of the invention to for the weak point on the cultivation of current cell and mechanical loading unit, a kind of three-dimensional perfusion stretching or the biomechanical system of compression cultivation are proposed, observe perfusion stretching or Compressive Mechanical in three-dimensional environment and stimulate the Quantitative study of cell growth impact, explore cyto-mechanics Coupling Rule biology.
In order to solve the problems referred to above, the present invention provides a kind of biomechanical system cultivated for cell three-dimensional perfusion Compression and Expansion, including:
Cell biological support, is used for simulating human body cell real three dimensional growth environment；
Bioreactor, is provided with the fixture clamping described cell biological support and the cavity accommodating described cell biological support；
Peristaltic pump, is used for driving cell culture fluid to enter described bioreactor and carries out perfusion；
Imposed load device, the fixture in the described bioreactor of para-linkage provides periodic reverse tractive, and then cell applies stretching or Compressive Mechanical stimulation；
Control module, is connected with computer, is controlled described imposed load device, regulation sample length, draw ratio, roundtrip frequency and round number of times.
Further, described cell biological support is elastic porous structure, and aperture is homogeneous, connectivity good；There is good biocompatibility or biologically inert, harmless to cytotoxic；Possess certain mechanical strength so that it is can be unlikely under force to destroy, maintain the spatial environments required for cell；Possess certain malformation and recovery capability.
Further, described bioreactor material is the rigidity plastics that biocompatibility is good, it is made up of two biopacks and elastic support fixture, solution is had to import and export on described biopack, it is connected with the blue mouth bottle equipped with cell culture fluid by corresponding silica gel tube, under described peristaltic pump effect, forms the closed circuit of perfusion.
Further, described biopack includes:
First resilient clamp, upper end is designed with the parts being bonded with force application apparatus, and lower end has female thread；
Second resilient clamp, two ends have external screw thread, and inside is cavity, and sidewall has solution gateway；
First elastic fastener and the second elastic fastener, described first elastic fastener and the second elastic fastener can be spliced into an annulus, and described annulus end face is provided with screwed hole；
3rd resilient clamp, for having the end cap shape of center hole, has female thread；
Described first elastic fastener and the second elastic fastener are fixed on the second resilient clamp end face on one side, and described first resilient clamp can screw described second resilient clamp two ends respectively with described 3rd resilient clamp.
Further, described elastic support clamp-shaped is dumbbell shaped, inner hollow, is used for placing described cell biological support；While described elastic support clamp material has favorable elasticity, there is good biologically inert, to cytotoxic；The internal cavity wall of described elastic support fixture is solidified togather with described cell biological support.
Further, described imposed load device includes motor, motor mounting flange, shaft coupling, sliding bearing, copper shoe, movable head, smooth guide, leading screw, guide supporting, gripper shoe；Described motor drives described leading screw to rotate by shaft coupling, and described movable head does horizontal motion on leading screw and smooth guide；Described imposed load device is placed in described gripper shoe by described motor mounting flange and guide supporting, empties in the middle part of described gripper shoe, it is simple to install bioreactor.
Further, described smooth guide sets a sliding bearing, be used for ensureing, in stretching or compression process, to increase the kinetic stability of horizontal direction；In drawing process, motor speed is adjustable, it is possible to achieve the tension test under different stretch frequency.
Further, by pin, described bioreactor is fixed between described movable head and guide supporting.
Further, described control module is run in laboratory virtual workbench LabVIEW, first selects the serial ports title of coupling, the most quantitatively regulates sample length, draw ratio, roundtrip frequency and round number of times；" start ", the startup of " stopping " key control device and stopping；Described peristaltic pump peristalsis frequency can control perfusion flow velocity, and flow velocity effusion meter records.
Relative to prior art, the present invention has a following technique effect:
Compared with prior art, present invention have the advantage that: imitated the mechanical environment of internal true complexity, while realizing Three-dimensional cell culture, it is achieved controlled perfusion stretching or the Quantitative study of Compressive Mechanical environment cell growth impact, explore cyto-mechanics Coupling Rule biology.
Accompanying drawing explanation
Fig. 1 is perfusion stretching or the compressor structural representation of the present invention.
Fig. 2 is the bioreactor broken away view of the present invention.
Detailed description of the invention
Below in conjunction with accompanying drawing, embodiments of the invention are described in detail.It should be noted that in the case of not conflicting, the embodiment in the application and the feature in embodiment can mutual combination in any.
Embodiment one:
The invention provides a kind of biomechanical system cultivated for cell three-dimensional perfusion Compression and Expansion as shown in accompanying drawing 1-2, including imposed load device, bioreactor, elastic cell's support and program controls portions etc..
Cell biological support (not shown), is used for simulating human body cell real three dimensional growth environment；Cell biological support is elastic porous structure, and aperture is homogeneous, connectivity good；There is good biocompatibility or biologically inert, harmless to cytotoxic；Possess certain mechanical strength so that it is can be unlikely under force to destroy, maintain the spatial environments required for cell；Possess certain malformation and recovery capability.Pore creating material is utilized uniformly to mix with organic solvent, curing molding after cast, finally remove pore creating material, be a kind of common method preparing porous cell biologic bracket material.The method is simple and practical, and porosity is high, and aperture is controlled.In the present embodiment, utilize polydimethylsiloxane (PDMS) to be combined with hydroxyapatite (HA), improve pore creating material method, be prepared for a kind of elastic cellular cell biological timbering material.Wherein PDMS is as a kind of biologically inert macromolecular material, has the most elastic and machinability.HA is the main inorganic composition of sclerous tissues in human body, plays a major role the intensity of bone.HA component makes material have good biocompatibility, improves the intensity of material.PDMS Yu HA is combined by a certain percentage and prepares three dimensional elasticity cell biological support so that prepared three dimensional elasticity cell biological timbering material is conducive to cell to stick at hole inwall while keeping favorable elasticity.
Such as Fig. 2, bioreactor, it is provided with the fixture of clamping cell biological support and accommodates the cavity of cell biological support；Bioreactor material is the rigidity plastics that biocompatibility is good, it is made up of two biopacks 10 and an elastic support fixture 11, solution is had to import and export on biopack 10, it is connected with the blue mouth bottle equipped with cell culture fluid by corresponding silica gel tube, under peristaltic pump effect, forms the closed circuit of perfusion.
Biopack 10 includes: the first resilient clamp 13, and upper end is designed with the parts 19 being bonded with force application apparatus, is fixed between movable head 9 and guide supporting 12 by bioreactor by pin, and lower end has female thread；Second resilient clamp 14, two ends have external screw thread, and inside is cavity, and sidewall has solution gateway 18, and lower end is fluted to be connected with elastic support fixture 11；First elastic fastener 15 and the second elastic fastener 16, the first elastic fastener 15 and the second elastic fastener 16 can be spliced into an annulus, and annulus end face is provided with screwed hole, is used for fixing elastic support fixture 11；3rd resilient clamp 17, for having the end cap shape of center hole, has female thread；First resilient clamp 13 and the 3rd resilient clamp 17 can screw the second resilient clamp 14 two ends respectively, and the first elastic fastener 15 and the second elastic fastener 16 are fixed between the 3rd resilient clamp 17 and the second resilient clamp 14, it is ensured that the sealing of cavity.
Elastic support fixture 11 is shaped as dumbbell shaped, inner hollow, is used for placing cell biological support；While elastic support fixture 11 material has favorable elasticity, there is good biologically inert, to cytotoxic；The internal cavity wall of elastic support fixture 11 fits together with the solidification of cell biological support.Elastic support fixture 11 and biopack 10 are fixed, and are then bonded with stretching or compressor.
Peristaltic pump, is used for driving cell culture fluid to enter bioreactor and carries out perfusion.
Imposed load device, the fixture in the bioreactor of para-linkage provides periodic reverse tractive, and then cell applies stretching or Compressive Mechanical stimulation；Imposed load device includes motor 2, motor mounting flange 1, shaft coupling 3, sliding bearing 7, copper shoe 8, movable head 9, smooth guide 4, leading screw 5, guide supporting 12, gripper shoe 6；Motor 2 drives leading screw 5 to rotate by shaft coupling 3, and movable head 9 does horizontal motion on leading screw 5 and smooth guide 4；Imposed load device is placed in gripper shoe 6 by motor mounting flange 1 and guide supporting 12, empties in the middle part of gripper shoe 6, it is simple to install bioreactor.Set a sliding bearing 7 on smooth guide 4, be used for ensureing, in stretching or compression process, to increase the kinetic stability of horizontal direction；In drawing process, motor 2 rotating speed is adjustable, it is possible to achieve the tension test under different stretch frequency.
Control module, is connected with computer, is controlled imposed load device, regulation sample length, draw ratio, roundtrip frequency and round number of times.
Control module is run in laboratory virtual workbench LabVIEW, first selects the serial ports title of coupling, the most quantitatively regulates sample length, draw ratio, roundtrip frequency and round number of times；" start ", the startup of " stopping " key control device and stopping；Peristaltic pump peristalsis frequency can control perfusion flow velocity, and flow velocity effusion meter records.
In biopack 10, cell culture fluid is connected with the solution import and export 18 of bioreactor by silica gel tube, can carry out perfusion；Under the effect of peristaltic pump, solution is entered bioreactor by entrance, carries out superfusion experiment, outlet flows out, and forms a closed circuit, and the growth for cell provides nutritional labeling, and the peristalsis frequency of peristaltic pump controls solution flow rate during perfusion.Meanwhile, by the good experiment parameter of software set, computer controls imposed load device, draws elastic support fixture 11 by movable head 9 and leading screw 5, drives timbering material and cell tensile motion.
It is embodied as: three-dimensional porous cell elasticity support is prepared as follows:
Prepared by PDMS/HAP slurry: ethyl acetate mixes with PDMS, PDMS: ethyl acetate=3 1 (mass ratio).Add ball milling mixing 5h after a certain amount of HAP；
Prepared by porous material: pour in mould by NaCl Yu the PDMS/HAP slurry after screening, apply certain pressure at 80 DEG C, solidify after 2h.Will solidification after material be removed from the molds, be soaked in ultra-pure water, supersound process, every two hours change water once, NaCl be completely dissolved after (1-2 days time) obtain required elastic porous material；Gained sample is a diameter of 6mm, the cylinder of a length of 10mm.
Solidification liquid (PDMS and firming agent DOW CORNING 184 thereof mix) coated according to the mass ratio of 10:1 by the elastic support of preparation, puts into fixture inner cavity, solidifies 2h at 80 degrees Celsius；Elastic support clamp material is medical grade silicone rubber, and matter is soft, flexible, is shaped as dumbbell shaped, inner hollow, cavity diameter 6mm, Up/down base circular diameter 18mm, and height is 10mm.
The material of biopack 10 be biocompatibility good rigidity plastics, internal cavities can accommodate 5ml 10ml liquid, solution gateway internal diameter is 2mm, external diameter is 4mm, identical with reactor material, it is threaded, fixes with biopack spiral, lower end circular groove diameter 18mm, is connected with cavity by the circular channel of diameter 6mm.
By autoclave sterilizations such as above-mentioned material and blue mouth bottle, wriggling silica gel tube, bioreactors, super-clean platform will be slowly dropped into timbering material by evacuation from timbering material top containing the suspension of rat bone marrow mesenchymal stem cells (rBMSCs), and at CO2Incubator is cultivated 2h；After plantation, spring chuck two ends are positioned in the second resilient clamp 14 groove, fixed by first elastic fastener the 15, second elastic fastener 16 and 6 screws, first resilient clamp 13 at two ends uses screw thread to screw tight with the 3rd resilient clamp 17, forming a passage sealed, the 3rd resilient clamp 17 prevents burn wound exudates for silicagel pad；Solution gateway 18 is connected with silica gel tube, and fixes with iron wire, and silica gel tube arrival end is passed through blue mouth bottle by peristaltic pump, and the port of export is passed directly in blue mouth bottle, forms perfusion loop, peristaltic pump peristalsis frequency regulation perfusion rate, is recorded by effusion meter；Bioreactor is bonded by pin with imposed load device, one end is fixed in guide supporting 12, one end is fixed on movable head 9, motor 2 drives leading screw 5 and movable head 9 horizontal motion on smooth guide 4 by shaft coupling 3, thus draw elastic support fixture 11 and drive adherent three-dimensional porous elastic cell's biological support horizontal movement, cell is applied tensile force stimulates；Imposed load device is connected with computer by control module, selects corresponding serial ports title, set parameter in stretching controls software.The a length of 10mm of this example, draw ratio 10 (10%), roundtrip frequency 1HZ, come and go number of times 21600, peristalsis frequency is 5HZ；Switch on power, start the stretching of cell three-dimensional perfusion and cultivate.
Data in Binding experiment, and compare with static culture matched group, analyzing three-dimensional dynamic fluid shear environment and stretching or compression stimulate the impact on cell activities, the most also can carry out different frequency different stretch than under stretching or compression stimulate quantitative experiment and single perfusion experiment, disclose Mechanobiology Coupling Rule.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.All within the spirit and principles in the present invention, any modification, equivalent substitution and improvement etc. made, should be included within the scope of the present invention.
Claims (6)
1. the biomechanical system cultivated for cell three-dimensional perfusion Compression and Expansion, it is characterised in that
Including:
Cell biological support, is used for simulating human body cell real three dimensional growth environment；
Bioreactor, is provided with to clamp the fixture of described cell biological support and accommodate described cell biological and props up
The cavity of frame；
Peristaltic pump, is used for driving cell culture fluid to enter described bioreactor and carries out perfusion；
Imposed load device, the fixture in the described bioreactor of para-linkage provides periodic reverse
Tractive, and then cell is applied stretching or Compressive Mechanical stimulation；
Control module, is connected with computer, is controlled described imposed load device, and regulation sample is long
Degree, draw ratio, roundtrip frequency and round number of times；
Described cell biological support is elastic porous structure, and aperture is homogeneous, connectivity good；Have good
Biocompatibility or biologically inert, harmless to cytotoxic；Possesses certain mechanical strength so that it is can
It is unlikely under force to destroy, maintains the spatial environments required for cell；Possesses certain knot
Structure deformation and recovery capability；
Described bioreactor material is the rigidity plastics that biocompatibility is good, by two biopacks and
Elastic support fixture form, described biopack has solution to import and export, by corresponding silica gel tube and equipped with
The blue mouth bottle of cell culture fluid is connected, and forms the closed circuit of perfusion under described peristaltic pump effect；
Described biopack includes:
First resilient clamp, upper end is designed with the parts being bonded with force application apparatus, and lower end has female thread；
Second resilient clamp, two ends have external screw thread, and inside is cavity, and sidewall has solution gateway；
First elastic fastener and the second elastic fastener, described first elastic fastener and the second elasticity are tight
Firmware can be spliced into an annulus, and described annulus end face is provided with screwed hole；
3rd resilient clamp, for having the end cap shape of center hole, has female thread；
Described first elastic fastener and the second elastic fastener are fixed on the second resilient clamp end face on one side
On, and described first resilient clamp and described 3rd resilient clamp can screw described second elastic clip respectively
Hold two ends.
A kind of biomechanics cultivated for cell three-dimensional perfusion Compression and Expansion
System, it is characterised in that:
Described elastic support clamp-shaped is dumbbell shaped, inner hollow, is used for placing described cell biological and props up
Frame；While described elastic support clamp material has favorable elasticity, there is good biologically inert, right
Cytotoxic；The internal cavity wall of described elastic support fixture is solidified togather with described cell biological support.
A kind of biomechanics cultivated for cell three-dimensional perfusion Compression and Expansion
System, it is characterised in that:
Described imposed load device includes that motor, motor mounting flange, shaft coupling, sliding bearing, copper are sliding
Block, movable head, smooth guide, leading screw, guide supporting, gripper shoe；Described motor passes through shaft coupling band
Dynamic described leading screw rotates, and described movable head does horizontal motion on leading screw and smooth guide；Described execute
Add load device to be placed in described gripper shoe by described motor mounting flange and guide supporting, described support
Empty in the middle part of plate, it is simple to bioreactor is installed.
A kind of Biological Strength cultivated for cell three-dimensional perfusion Compression and Expansion
System, it is characterised in that:
Set a sliding bearing on described smooth guide, be used for ensureing, in stretching or compression process, to increase water
Square to kinetic stability；In drawing process, motor speed is adjustable, it is possible to achieve different stretch frequency
Tension test under rate.
A kind of Biological Strength cultivated for cell three-dimensional perfusion Compression and Expansion
System, it is characterised in that:
By pin, described bioreactor is fixed between described movable head and guide supporting.
A kind of Biological Strength cultivated for cell three-dimensional perfusion Compression and Expansion
System, it is characterised in that:
Described control module is run in laboratory virtual workbench LabVIEW, first selects coupling
Serial ports title, the most quantitatively regulates sample length, draw ratio, roundtrip frequency and round number of times；" open
Begin ", the startup of " stopping " key control device and stopping；Described peristaltic pump peristalsis frequency can control perfusion
Flow velocity, flow velocity effusion meter records.
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| TWI672375B (en) * | 2015-12-09 | 2019-09-21 | 財團法人工業技術研究院 | Cell culture carrier module, bioreactor and cell recovery method |
| CN108018207A (en) * | 2016-10-28 | 2018-05-11 | 华东理工大学 | The biomechanical system with stretching culture is sheared for cell flow |
| CN108384717A (en) | 2017-02-03 | 2018-08-10 | 财团法人工业技术研究院 | Cell culture carrier module and cell culture system |
| CN109468225A (en) * | 2018-11-12 | 2019-03-15 | 太原理工大学 | A kind of cell mechanical tensioning devices of real time data feedback |
| CN110964637B (en) * | 2019-12-30 | 2020-11-06 | 北京航空航天大学 | In-vitro dynamic cell culture device and culture method thereof |
| CN111423983B (en) * | 2020-04-08 | 2022-03-15 | 中国科学院力学研究所 | Continuous expansion culture ware of expansible base cell |
| CN111423980B (en) * | 2020-04-08 | 2022-03-15 | 中国科学院力学研究所 | Fully-closed cell substrate stretching and flow shearing combined loading experiment system |
| CN112656557B (en) * | 2020-12-17 | 2022-09-13 | 湖北工业大学 | Peripheral nerve interface based on tissue engineering |
| CN115125122A (en) * | 2022-06-06 | 2022-09-30 | 太原理工大学 | Single cell three-dimensional dynamic mechanical stimulation device |
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| CN101372664B (en) * | 2008-05-16 | 2012-01-11 | 北京航空航天大学 | Tissue engineering reactor having tissue cultures tension-compression and rotation functions |
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