CN101265466B - Cultivating method used for coronary camber tissue under composite load and bioreactor thereof - Google Patents
Cultivating method used for coronary camber tissue under composite load and bioreactor thereof Download PDFInfo
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- CN101265466B CN101265466B CN2008100529522A CN200810052952A CN101265466B CN 101265466 B CN101265466 B CN 101265466B CN 2008100529522 A CN2008100529522 A CN 2008100529522A CN 200810052952 A CN200810052952 A CN 200810052952A CN 101265466 B CN101265466 B CN 101265466B
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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
- C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
- C12M41/14—Incubators; Climatic chambers
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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
- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
- C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
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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
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
Abstract
The invention relates to a culture method and a bioreactor used for coronary curved surface tissue under a combined load, and comprises a tri-gas incubator with O2, CO2 and thermoregulation, conditions for keeping culture fluid fresh, such as perfusion, and active factors added into the culture fluid. A culture blank with coronary curved surface is fixed on a bottom plate of a culture room. In the culture period, the culture is subjected to the action of a rolling load or a sliding load, and/or a twisting and shearing load, and/or an oscillating load so as to culture a culture with coronary curved surface (including flat shape), and the final culture comprises complexes of cell-scaffold, or keeps in vitro tissue. The mechanical condition of combined load and/ or the hypoxia environment provided by the invention are close to the mechanical environment of the growth and the development of the tissue, so as to be favorable for the growth and the development of cells and the tissue under the condition of load. The final culture with coronary curved surface can be applied to the restoration of special shape defects.
Description
[technical field]:
The invention belongs to the biomedical engineering technology field.Be specifically related to field of tissue engineering technology, particularly the mechanical condition of combined load such as roll extrusion load or slip load and/or torsional shear load and/or oscillating load and low-oxygen environment can be used for having the method and the bio-reactor of crown curved surface tissue culture.
[background technology]:
Organizational project is a new subject that is rising in recent years, be after Celluar and Molecular Biology, another new milestone on the life science development history indicates that medical science will walk out the category of organ transplantation, steps into the New Times of making tissue and organ.Organizational project will become the new high-tech industry that has potentiality this century, will produce huge social and economic benefit.The basic skills of organizational project be with from body or variant cell after vitro culture, amplification, be inoculated on the good biomaterial of a kind of histocompatibility, carry out vitro culture, form certain class tissue, the implant defect merges with host tissue then, reaches the purpose of repair deficiency.Wherein cartilage tissue engineered all is to adopt the complex body of different seed cells, timbering material and active factor to cultivate respectively with bone tissue engineer, and cartilaginous tissue also may adopt identical seed cell with osseous tissue, as mesenchymal stem cells MSCs.
Tissue defect betides the different sites of human body, and repair deficiency needs the graft of difference, suitable shape, currently makes difform artificial organ according to clinical shape need and also is very important, and helps the reparation at tissue defect position like this.Wherein crown tissue, as the femoral head cartilage, eye portion of tissue etc. all have heavy demand.These crown organizing also need to cultivate the artificial organ that formation has the load function under the mechanical environment effect, can satisfy clinical demand better.With the femoral head cartilage is example, and crown cartilaginous tissue is compressed, is out of shape the mechanical environment of shearing, vibration.Therefore we will set up this kind mechanical condition, make up to have the coronal tissue, will obtain structure during this tissue culture and function is grown simultaneously.
Current, along with the development of organizational project, how deepening continuously of seed cell, timbering material and active factor research is cultivated graft and is subjected to more concern in bio-reactor, wherein the mechanical environment of graft culture condition is more and more paid attention to.With the cartilage is example, and for cartilage, its live body mechanical environment is very complicated.Suitable mechanical condition be turn out structure, function all with the important factor of the close graft of natural cartilage.Recent years, correlative study person was to different mechanical stimulations, had done big quantity research with the 26S Proteasome Structure and Function of optimizing culture.The domestic investigator of having adopts quiet tension stress that the rat condylar cell propagation effect is studied, and experiment shows that cell proliferation quantity increases with quiet tension stress and raise [West China stomatology magazine, 2003,21 (1), 57-60]; The influence [dissection journal, 2001,32 (4), 385-387] of research air stress to vitro culture chondrocyte secrete cytokines also arranged, effect [the Chinese experimental surgery magazine of centrifugal force in the external structure tissue engineering bone/cartilage.2005,22 (3), 281-283], the periodic current body stress stimulates the experimental study of tissue engineering bone/cartilage differentiation influence [B﹠J damage magazine, 2003,18 (9), 620-622].These loading methods can promote the performance of cell proliferation or function, but and real cartilage stand under load be differentiated.[J Biomech Eng, 2000 such as Mauck; 122 (3): 252-260] chondrocyte being seeded in sepharose dynamically directly exerts pressure, magnitude of load is to make complex body distortion 10%, frequency is 1Hz, explore the cartilage tissue engineered cultivation of functionalization, 4 week back discovery GAG (glycosaminoglycan) raise 45%, collagen content increases by 37%, apparently higher than not loading group.Buschmann etc. apply 1Hz, the dynamic compressive load of strain 3% is carried out cartilage and is made up, and experiment shows that chondroitin sulfate increases 15%-25%, and proline(Pro) increases 10%-25%, simultaneously, the variation of the different incubation time culture Young's modulus of research under this mechanical condition.
Current bio-reactor pair cell cultivation, tissue and cell-scaffold culture are cultivated, are kept, normally used have four kinds of reactors: i.e. static state, rotating and culturing bottle, rotating wall vessel bioreactor and perfusion type bio-reactor [Advanced Drug DeliveryReviews33 (1998) 15-30, Tissue Engineering.2003,9 (3), 549-553, different seed cells, New-support material, somatomedin etc. constitute culture, cultivating in these several bio-reactors respectively, mainly is the growth that promotes culture form, structure.Although the growth of culture has been optimized in the use of various bio-reactors in the prior art, the growth of culture, growth still do not reach clinical requirement.In engineered cultivation, also comprise the application of various mechanical environments as bio-reactor, the artificial cartilage tissue development is bad, problems such as poor mechanical property, cell degradation, death.The solution of these problems need use current bio-reactor further to test on the one hand, and the development of other association area, as RESEARCH ON CELL-BIOLOGY, and the development of timbering material, the manufacturing of active factor; Need the optimization of bioreactor culture condition on the other hand.Mechanical environment occupies critical positions in the development of bio-reactor, mechanics factor influences the integral body of organism, the biological procedures of organ, tissue, cell and each level of molecular water equality [Guilak et al.Functional Tissue Engineering, 2003, Springer].The optimization of mechanical condition is very important.
[summary of the invention]:
Purpose of the present invention aims to provide cultural method and the bio-reactor that is used to have crown curved surface tissue under a kind of combined load of simulating the live body mechanical environment.
Described combined load is: in three gas incubators, apply roll extrusion load or slip load, and/or torsional shear load, and/or the mechanical condition of oscillating load, and/or low-oxygen environment, form the physical environment that artificial organ or cytoskeleton complex body are grown, can cultivate the method and the bio-reactor of crown curved surface (comprising planeform) tissue, there is the culture of crown curved surface (comprising planeform) under this condition, to grow, can optimizes the structure and the function of culture.
Being used under the combined load mechanical condition provided by the invention that the cultural method of crown curved surface tissue culture is arranged is to carry out in bio-reactor, comprises possessing O
2, CO
2With 37 degree temperature regulation, three gas incubators of cell cultures, and keep adding active factor (as rhIGF-1 (IGF) in fresh perfusion condition of nutrient solution and the nutrient solution, transforming growth factor-beta (TGF-β), Basic Fibroblast Growth Factor (bFGF), bone morphogenetic protein (BMP), platelet-derived growth factor (PDGF), cartilage regulin (ChM)), it is characterized in that: will have the culture blank of crown curved surface tissue to be fixed in culturing room's base plate, in the training period, apply the mechanical environment and/or the low-oxygen environment of roll extrusion load or slip load and/or torsional shear load and territory oscillating load to culture, several load and low-oxygen environment can act on separately or be used in combination mutually, difference applies order and is different culture environment, makes culture grow into the implantable artificial tissue; The time that loads is loading in per 24 hours 1-3 time, each 10min-360min, or load 4-6 10min-20min, cultivated 1-60 days.
The above load and low-oxygen environment include but not limited to:
Culture environment 1: apply roll extrusion load separately; Culture environment 2: apply roll extrusion load, apply torsional shear load simultaneously; Culture environment 3: apply roll extrusion load, apply torsional shear load again; Culture environment 4: apply roll extrusion load, apply torsional shear load and oscillating load simultaneously; Culture environment 5: apply roll extrusion load, apply torsional shear load again, apply oscillating load again; Culture environment 6: apply roll extrusion load, apply oscillating load again, apply torsional shear load again; Culture environment 7: apply roll extrusion load, apply oscillating load, torsional shear load and low-oxygen environment etc. simultaneously.
Detailed process is as follows: perfusion system guarantees the replacing of nutrient solution in the culturing room, oxygen content is 1%-10% in the three gas incubators, culture is under the coenocorrelation that cell can grow, by computer system control, by the roll extrusion load maintainer culture is rolled back and forth by roller, be subjected to roll extrusion load, wherein: rolling speed is 0.001-15cm/s, adding the carrier frequency of back and forth rolling is 0.0001-3Hz, roller rolled contact position cartilage culture distortion 0%-60%; Described slip load is to use roll body to slide through the process of culture, and sliding velocity is 0.001-15cm/s, and the frequency that reciprocatingly slides is 0.0001-3Hz, sliding contact place culture distortion 0-60%; Roll body is a rotator, and its rotation baseline is camber line or straight line.In addition by reverse with vibrating mechanism culture is applied reverse, oscillating load, described torsional load, be with the relative roller rotation with culturing room of culture, make culture be subjected to the torsional shear load of relative roller, the circular frequency that culture is reversed is 0.001-3.14rad/s.Described oscillating load is to make the roller position altitude hold, and culture is compressed by relative dynamic, is subjected to oscillating load, and the oscillating load range of frequency is 0.0001-100Hz, and the strain amplitude is 0.000001-0.6; The oscillating load waveform is square wave, sine wave, choppy sea.The time that loads is loading in per 24 hours 1-3 time, every 10min-360min, or load 4-6 10min-20min, cultivated 1-60 days.Make tissue growth, growth at this kind mechanical environment, finish the cultivation of culture.
Described culture is following portable body: chondrocyte, tissue, chondrocyte and support complex body, transplanted tissue, intraocular culture or cell-scaffold complex body; Its shape includes in vitro tissue or the cell and the support complex body of crown curved surface, also comprises the culture of planeform.
Said crown culture is a kind of tissue or two kinds of tissues, or two species complexs that stack.
Described roller is that stainless material is made, and the surface is for the pellosil of uniform thickness 0.1-4mm or do not have.
Nutrient solution of the present invention is that biological cell is cultivated conventional nutrient solution, can buy from the market.Described three gas incubators are that biological cell is cultivated conventional the use, can buy from the market.
The employed bio-reactor of a kind of above-mentioned cultural method, comprise three gas incubators, the nutrient solution perfusion system, culturing room and computer control system, nutrient solution perfusion system and culturing room all are placed in the three gas incubators, the roll extrusion of this bio-reactor and slip load maintainer comprise the stepper-motor that is installed on the upper plate, with motor shaft fixed leading screw and pull bar, the roller two ends are hingedly mounted on the pull bar, the upper end of roller axle closely contacts with the lower surface of press strip on being fixed on support, support is installed on the upper plate, equal-sized two and tooth bar meshed gears are installed with one heart in the both sides of roller simultaneously, tooth bar is fixed on the support by screw rod, and roller is positioned over realizes on the culture in the culturing room that pure roll extrusion loads; The screw rod that unclamps stationary rack slides tooth bar, and relative fixed between the wheel and rack loads the slip of culture to realize roller;
Reverse-vibrating mechanism comprises the pilot bearing that is installed in below the upper plate, the loading head upper end is connected with culturing room's base plate, the lower end is passed and place in the pilot bearing by the open holes on the upper plate, the pilot bearing key connects loading head, and be connected by key with motor shaft, be connected a piezoelectric ceramics shifter between loading head and the motor shaft, between loading head and the pilot bearing return spring is installed, motor is installed on the lower shoe, fixes by pillar between upper plate and the lower shoe.
Regulating oxygen content in the three gas incubators is 1%-10%, and described perfusion system comprises the nutrient solution storage bottle, and storage bottle is connected with culturing room by silicone tube, provides mobilization dynamic by peristaltic pump; Described computer control system is controlled automatically to stepper-motor, piezoelectric ceramics shifter and peristaltic pump.
Advantage of the present invention and positively effect:
Combined load provided by the invention, be the mechanical condition of roll extrusion load or slip load and/or torsional shear load and/or oscillating load and/or the mechanical environment that low-oxygen environment is more grown near tissue growth, help growth, the growth of cell, tissue at this load-up condition.It is damaged that culture after the cultivation can be used for repair tissue, has the culture of crown curved surface to can be applicable to the damaged reparation of special shape in addition.
[description of drawings]:
Fig. 1 is the load maintainer schematic perspective view in the bio-reactor of the present invention;
Fig. 2 is the vertical view in the bio-reactor of the present invention;
Fig. 3 is the diagrammatic cross-section of Fig. 2;
Fig. 4 is two kinds of different rotary body roll body synoptic diagram, and the rotation baseline of A roller is a camber line, and the rotation baseline of B roller is a straight line;
Fig. 5 is the culture diagrammatic cross-section, A crown curved surface culture, two kinds of crown curved surface stack cultures of B, C planeform culture, two kinds of planeform stacks of D culture;
Fig. 6 is the perfusion system synoptic diagram in the bio-reactor of the present invention.
Concrete label is as follows among the figure:
1 roller; 2 gears; 3 press strips; 4 culturing room; 5 pull bars; 6 leading screws; 7 stepper-motors; 8 perfusion interfaces; 9 supports; 10 upper plates; 11 tooth bars; 12 pillars; 13 pilot bearings; 14 motor shafts; 15 motors; 16 lower shoes; 17 loading heads; 18 piezoelectric ceramic actuators; 19 cultures; 19-1 culture bed die; 20 return springs; 21 shaft adapters; 22 slipper seal pads; 23 peristaltic pumps; 24 cultivate liquid pool.
[embodiment]:
Embodiment 1:
Mechanical condition and/or the low-oxygen environment that a kind of roll extrusion load or slip load and/or torsional shear load and/or oscillating load are provided of the present invention, can cultivate the method for crown curved surface (comprising planeform) tissue, be to carry out in bio-reactor: it comprises possessing hypoxemia and the thermoregulator cell cultures incubator of 37 degree, reach conditions such as perfusion, under aseptic condition, culture is transferred in the culturing room of sterilizing, press cell, the operation of tissue culture standard procedure, culture is fixed in culturing room's base plate, drive the nutrient solution perfusion by peristaltic pump, keep nutrient solution fresh, add active factor in the nutrient solution (as rhIGF-1 (IGF), transforming growth factor-beta (TGF-β), Basic Fibroblast Growth Factor (bFGF), bone morphogenetic protein (BMP), platelet-derived growth factor (PDGF), cartilage regulin (ChM)).Oxygen content can be 1%-10% in the incubator of culture, regulates by three gas incubators and realizes.Whole process is finished by computer system control.
In the training period, applying the mechanical environment of roll extrusion load or slip load and/or torsional shear load and/or oscillating load and/or several load of low-oxygen environment and low-oxygen environment to culture can act on separately or be used in combination mutually, and difference applies order and is different culture environment.
The loading of roll extrusion load: roller is rolled back and forth by the roll extrusion load maintainer, culture is subjected to roll extrusion load, wherein: rolling speed load is 0.001-15cm/s, and adding the carrier frequency of back and forth rolling is 0.001-3Hz, roller rolled contact position cartilage culture distortion 1%-60%; Roller is a stainless material, and the surface is the pellosil of uniform thickness 0.1-4mm, or does not have.Roll body is a rotator, and its rotation baseline is camber line or straight line.
The slip loading procedure is that roller does not roll, and slides through culture, and sliding velocity is 0.001-15cm/s, and the frequency that reciprocatingly slides is 0.0001-3Hz, sliding contact place culture distortion 0-60%.
Torsional load is that the relative roller of culture is reversed, and velocity of rotation is 0.001-3.14rad/s.
Oscillating load is impacted culture, and range of frequency is 0.001-100Hz, and the strain amplitude is 0.000001-0.6.
The time that loads is loading in per 24 hours 1-3 time, every 10min-360min, or load 4-6 10min-20min, cultivated 1-60 days.Make tissue growth, growth at this kind mechanical environment, finish the cultivation of culture.
Nutrient solution of the present invention is that biological cell is cultivated conventional nutrient solution, can buy from the market.Described incubator is that biological cell is cultivated the conventional three gas incubators that use, and can buy from the market.
Culture of the present invention is: cell, tissue, cell and support complex body or transplanted tissue.The culture of crown curved surface is also arranged, and is a kind of tissue or two kinds of tissues, or cell-scaffold complex body or two species complexs that stack
Perfusion condition of the present invention is formed by the perfusion system that can buy on the market.
At first make the culture of crown curved surface.The chondrocyte is seeded on the crown collagen scaffold; Scleroblast is seeded on the segment shape cancellous bone scaffolds simultaneously, chondrocyte-collagen composite is superimposed upon on the complex body of segment shape again, forms culture.
Roller is a rotator, and its rotation baseline conforms to the cartilage profile.
Culture environment is: under the 5% oxygen level condition, the loading of first roll extrusion load slide to load again, torsional load again, and oscillating load again, every kind of load acts on 10 minutes respectively, in 24 hours 3 times respectively.Below every kind of load specifically introduce.
The loading of roll extrusion load: roller rolling speed back and forth is 1cm/s, and the frequency of back and forth rolling is 0.1Hz, roller rolled contact position cartilage culture distortion 20%; Roller is a stainless material, and the surface does not have pellosil.
The slip loading procedure is that roller does not roll, and slides through culture, and sliding velocity is 0.1cm/s, and the frequency that reciprocatingly slides is 0.1Hz, sliding contact place culture distortion 20%.
Torsional load is that the relative roller of culture is reversed, and velocity of rotation is 0.01rad/s.
Oscillating load is impacted culture, and range of frequency is 1Hz, and the strain amplitude is 0.1.
The bio-reactor that the mechanical condition of a kind of roll extrusion load provided by the invention or slip load and/or torsional shear load and/or oscillating load and/or low-oxygen environment can be used for having crown curved surface tissue culture.Mainly be to design at method proposed by the invention, extremely shown in Figure 6 as Fig. 1, this bio-reactor comprises three gas incubators, nutrient solution perfusion system, roll extrusion and slip load maintainer, reverses-vibrating mechanism, culturing room 4, upper plate 10, lower shoe 16, pillar 12 and computer system etc.
Described upper plate 10 and lower shoe 16 are fixed into a skeleton construction by four pillars 12, and described roll extrusion and slip load maintainer and culturing room 4 are fixed on the upper plate 10 by two vertical slab-like support 9.
Described roll extrusion load maintainer is the mechanism that makes the pure rolling of a roller roll extrusion culture, comprises the roller 1 of surface of revolution, and its both sides are equipped with equal-sized two gears 2 with one heart; Two tooth bars 11 are fixed on the support 9 along support 9 inside bottom; Gear 2 moves on tooth bar 11, and gear 2 is coaxial with roller 1, and leading screw 6 is by the axle of U-shaped pull bar 5 connection rollers 1, and the stepper-motor 7 that is installed on the upper plate 10 is connected with leading screw 6.
Described slip load maintainer is the mechanism that the relative culture 19 of roller 1 is slided, with the roll extrusion load maintainer be same mechanism.During the load maintainer that just slides operation fixed two tooth bars 11 are unclamped, can in support 9, slide, gear 2 and tooth bar 11 relative fixed.Roller 1 slips at culture 19 upper surfaces.
Described reversing-vibrating mechanism mainly comprises pilot bearing 13, motor shaft 14, motor 15 and piezoelectric ceramic actuator 18, shaft adapter 21 etc.; Motor shaft 14 is connected with shaft adapter 21, and shaft adapter 21 is connected with piezoelectric ceramic actuator 18 again, and piezoelectric ceramic actuator 18 the other ends connect loading head 17.Return spring 20 is enclosed within on the loading head 17, loading head is resetted, as shown in Figure 3.
Culturing room's 4 inner bottom surface fixed cultures 19, cultivating on the locular wall has perfusion interface 8, and culturing room 4 is connected with perfusion system, wherein cultivates locular wall by slipper seal pad 22 separated into two parts, and relative top, lower section is rotatable.
During this bio-reactor work, by computer system control, oxygen content is 1%-10% in the three gas incubators of culture; For the roll extrusion load maintainer, roller 1 is fixed on the same axle with the equal-sized gear 2 in its both sides, and gear 2 is controlled to-and-fro movements by stepper-motor 7 by leading screw 6 on tooth bar 11, drives roller reciprocal pure rolling in culturing room like this.Simultaneously press strip 3 is pressed on the axle of roller, regulates press strip 3 downwards, gives the axially following power of roller, and roller 1 moves downward, to culture 19 certain draughts.
When using, unclamp the fixing of tooth bar 11, the relative position of stationary rack 11 and gear 2 for the slip load maintainer.Other process loads identical with roll extrusion.
For reversing-vibrating mechanism, there are shaft adapter 21, piezoelectric ceramic actuator 18, loading head 17 to interconnect successively in the pilot bearing 13, wherein shaft adapter 21 is connected with loading head 17 splines, can transmit moment of torsion.Loading head 17 directly contacts, fixes with culturing room 4 base plate bottom surfaces.Motor 15 drives motor shaft 14 rotations, loading head 17, culturing room's 4 base plates rotation, culture 19 relative roller 1 rotations; Piezoelectric ceramic actuator 18 is transfused to varying voltage signal simultaneously, cause piezoelectric ceramic actuator 18 different telescopic displacements, cause loading head 17 and culturing room's 4 up-down vibration, the altitude hold of roller 1, make culture 19 be subjected to oscillating load, return spring 20 resets the vibration load maintainer.Realize can be used for having roll extrusion load or the mechanical condition of slip load and/or torsional shear load and/or oscillating load and/or the cultivation under the low-oxygen environment of crown curved surface culture like this.
Claims (7)
1. one kind causes the cultural method that is used to have crown curved surface tissue culture under the combined load mechanical condition by roll body, comprises possessing O
2, CO
2And temperature regulation, three gas incubators of cell cultures, and keep adding active factor in fresh perfusion condition of nutrient solution and the nutrient solution, it is characterized in that: will have the blank of crown curved surface tissue culture to be fixed in culturing room's base plate, in the training period, roll body applies the mechanical environment and/or the low-oxygen environment of roll extrusion load or slip load and/or torsional shear load and/or oscillating load to culture, wherein roll body is a rotator, its rotation baseline is a camber line, several load and low-oxygen environment act on separately or are used in combination mutually, make culture grow into the implantable artificial tissue; The time that loads is loading in per 24 hours 1-3 time, each 10min-360 min, or load 4-6 time, each 10min-20min cultivated 1-60 days.
2. cultural method according to claim 1 is characterized in that described crown curved surface tissue culture is arranged is following portable body: transplanted tissue, intraocular culture or cell-scaffold complex body.
3. cultural method according to claim 2 is characterized in that said crown curved surface tissue culture is arranged is a kind of tissue or two species complexs that stack.
4. cultural method according to claim 1 is characterized in that oxygen level is 1%-10% in the three gas incubators.
5. according to claim 1,2,3 or 4 described cultural methods, it is characterized in that described rolling load is to use the process of roll body roll extrusion culture, rolling speed is 0.001-15cm/s, and the frequency of back and forth rolling is 0.0001-3Hz, rolling contact position culture distortion 0-60%; Described slip load is to use roll body to slide through the process of culture, and sliding velocity is 0.001-15cm/s, and the frequency that reciprocatingly slides is 0.0001-3Hz, sliding contact place culture distortion 0-60%; Described torsional load is with the relative roller rotation with culturing room of culture, makes culture be subjected to the torsional shear load of relative roller, and rotational angular is 0.001-3.14rad/s; Described oscillating load is to make the roller position altitude hold, and culture is compressed by relative dynamic, is subjected to oscillating load, and range of frequency is 0.001-100Hz, and the strain amplitude is 0.000001-0.6.
6. be used to have the bio-reactor of crown curved surface tissue culture cultivation under the combined load that the described cultural method of claim 1 uses, comprise three gas incubators, the nutrient solution perfusion system, culturing room and computer control system, nutrient solution perfusion system and culturing room all are placed in the three gas incubators, it is characterized in that: the roll extrusion of this bio-reactor and slip load maintainer comprise the stepper-motor that is installed on the upper plate, with motor shaft fixed leading screw and pull bar, the roller two ends are hingedly mounted on the pull bar, the upper end of roller axle closely contacts with the lower surface of press strip on being fixed on support, support is installed on the upper plate, equal-sized two and tooth bar meshed gears are installed with one heart in the both sides of roller simultaneously, tooth bar is fixed on the support by screw rod, and roller is positioned over realizes on the culture in the culturing room that pure roll extrusion loads; The screw that unclamps stationary rack makes tooth bar slidably, and relative fixed between the wheel and rack loads the slip of culture to realize roller;
Reverse-vibrating mechanism comprises the pilot bearing that is installed in below the upper plate, the loading head upper end is connected with culturing room's base plate, the lower end is passed and place in the pilot bearing by the open holes on the upper plate, the pilot bearing key connects loading head, and be connected by key with motor shaft, be connected a piezoelectric ceramics shifter between loading head and the motor shaft, between loading head and the pilot bearing return spring is installed, motor is installed on the lower shoe, fixes by pillar between upper plate and the lower shoe.
7. bio-reactor according to claim 6 is characterized in that regulating oxygen content in the three gas incubators is 1%-10%, and described perfusion system comprises the nutrient solution storage bottle, and storage bottle is connected with culturing room by silicone tube, provides mobilization dynamic by peristaltic pump; Described computer control system is controlled automatically to stepper-motor, piezoelectric ceramics shifter and peristaltic pump.
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| CZ21539U1 (en) * | 2010-11-08 | 2010-12-13 | Ceské@vysoké@ucení@technické@v@Praze@@Fakulta@strojní | Bioreactor for functional tissue engineering |
| CN106190834B (en) * | 2016-07-08 | 2018-10-02 | 天津理工大学 | A kind of single roll is synchronous with unidirectional sliding to compare the bioreactor loaded |
| CN106840921B (en) * | 2017-01-23 | 2019-04-05 | 浙江科技学院（浙江中德科技促进中心） | The cementing area's torsional strength of sand particle nanogel and shearing strain test device and method |
| CN106940333A (en) * | 2017-04-17 | 2017-07-11 | 山西医科大学 | The outer Electrophysiology research perfusion system of isolated cells |
| CN108795756B (en) * | 2018-05-25 | 2023-01-24 | 南京理工大学 | Dynamic rolling culture system for tissue engineering articular cartilage |
| CN109401963A (en) * | 2018-10-25 | 2019-03-01 | 江苏久腾医学科技有限公司 | A kind of concussion frame of stem cell culture |
| CN110055175B (en) * | 2019-03-22 | 2022-09-16 | 天津理工大学 | Rolling type bioreactor for in vitro cartilage culture |
| CN110484426A (en) * | 2019-09-16 | 2019-11-22 | 常州市第一人民医院 | A kind of power stimulation loading device and its working method |
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2008
- 2008-04-30 CN CN2008100529522A patent/CN101265466B/en not_active Expired - Fee Related
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