CN105586249A - Circulating perfusion biological reactor device capable of achieving circulating perfusion of three-dimensional support - Google Patents
Circulating perfusion biological reactor device capable of achieving circulating perfusion of three-dimensional support Download PDFInfo
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Abstract
The invention discloses a circulating perfusion biological reactor device capable of achieving circulating perfusion of a three-dimensional support. The device comprises a biological reactor and a perfusion device. The perfusion device comprises a peristaltic pump and a pipeline system arranged on the peristaltic pump. The biological reactor comprises a sterile culture chamber and a closed matching cover. The three-dimensional support can be connected into the biological reactor, the requirement of a cultivation space needed by the three-dimensional support in the biological reactor can be met, waste of lots of culture liquid is avoided, the three-dimensional support in the sterile chamber can be conveniently operated, culture liquid is stored in the sterile culture chamber, the three-dimensional support is placed in the sterile culture chamber, the peristaltic pump can be adjusted and controlled by setting a perfusion mode, perfusion speed, perfusion flow and the like, and different researching requirements and structure building requirements are met. The whole perfusion system is simple, operation is convenient, and perfusion of the culture liquid in the support can be controlled by adjusting the perfusion mode, the perfusion speed and the perfusion pressure arranged on the peristaltic pump.
Description
Technical field
The invention belongs to clinical medicine, organizational project and regenerative medicine field, specifically, is that one can be realIn existing organizational project three-dimensional rack, seed cell oxygen divides and the new equipment of nutriment sustainable supply.
Background technology
Organizational project is principle and the method for incorporation engineering and life science, under normal and pathology two statesMammal institutional framework and the relation of function carry out the most basic research, and exploitation has biologyThe substitute of energy is for repairing, maintain or improve the subject of damaged tissues or organ dysfunction. Its general principle is:Normal tissue cell is adsorbed on the biomaterial that biocompatibility is good and forms compound, through after a whileCultivation, cell amplification biomaterial absorptions of degrading gradually simultaneously, thus formation have specific modality, structure andThe respective organization of function, organ, reach the object that promotes regeneration, repairs wound and Reconstruction of The Function. TissueThe core of engineering is to set up the three dimensions complex being made up of Cell and organism material. As carrying of organizational projectBody, support is a key content of Tissue Engineering Study, and the three-dimensional environment of Growth of Cells and newly old is not only providedThe place of metabolism, also determines shape and the size of cambium organ. Wherein, de-cytoskeleton has retained because of itWhole blood vessel network structure and extracellular matrix components and make it to become the study hotspot of field of tissue engineering technology. OrderBefore, along with the development of tissue engineering technique and materialogy, the histoorgan that some are simple in structure, as skin, bloodPipe, bone, cartilage etc. can be by artificial constructed, for the treatment of Organ Transplantation Patients has brought dawn. But for knotThe organ-tissue of structure and function relative complex, is still faced with huge challenge. Wherein, thin in bulk three-dimensional rackBorn of the same parents' oxygen divides and lasting, the effective supply of nutriment are a great problems. How to realize cell long in three-dimensional rackPhase cultivates and maintains with function is that our emphasis considers a problem. Organizational project bioreactor appear as organizational projectThe structure of organ provides technical support.
In the evolution of organizational project bioreactor, plate bioreactor, revolving bottle are successively there isType bioreactor, hollow fiber bioreactor, perfusion bedrest type bioreactor, seed cell bagWrap up in/suspension type biological reactor etc. But above-mentioned reactor cannot solve bulk three-dimensional tissue inside modules cellThe supply problem of oxygen and nutriment, its applicability is comparatively limited to, and is mainly cell, micro-capsule, small groupKnit module etc. For this reason, in the time that being applicable to the bioreactor of bulk three-dimensional rack, structure need to consider: 1)Perfusate can have influence on the seed cell of three-dimensional rack deep inside; 2) provide physiological condition in analogue bodyContinous perfusion; 3) provide the biology under physiological environment in analogue body, mechanical environment and sufficient nutrition exchange.By regulation and control perfusion rate, and under the support of pressure negative-feedback regulating system, realize continuing of the interior perfusate of supportCircumfusion.
The present invention is based on existing circulation perfusion bioreactor basis and improved, is in vitro culture organizational projectOrgan provides a kind of easy circulating perfusion device, for the research of Stem cell differentiation under three-dimensional condition and Growth of Cells is carriedFor technical support.
Summary of the invention
The present invention is directed to current organizational project three-dimensional rack inner cell because of the keeping of oxygen and nutriment not enough notCan long-term surviving, the problem such as motility rate is low and proposing, a kind of circulation perfusion bioreactor device is disclosed, be threeDimensional scaffold inner cell provides lasting nutriment supply, for Stem cell differentiation under three-dimensional condition and Growth of CellsResearch provides technical support, overcomes the defect of existing static culture, is built with the organizational project organ of functional activity.Maintain thereby realize long-term cultivation and the function of cell in three-dimensional rack, realize the structure of organizational project organ,And ensureing to realize cell support long-term cultivation in vitro again cytoactive in the situation that.
The present invention is achieved through the following technical solutions:
The invention discloses a kind of circulation perfusion bioreactor device, device comprises bioreactor and perfusion dressPut, perfusion device comprises peristaltic pump and be arranged at the pipe-line system on peristaltic pump, bioreactor comprises aseptic trainingSupport chamber and airtight identical lid. In bioreactor, three-dimensional rack can be connected, the required training of three-dimensional rack in it can be metSupport space, avoid a large amount of wastes of nutrient solution, conveniently three-dimensional rack in desinfection chamber is operated sterile cultureChamber stores nutrient solution and places three-dimensional rack, and peristaltic pump can be by arranging perfusion pattern, perfusion rate, perfusion flowDeng regulating control, adapt to different Research Requirements and tissue construction demand.
As improving further, on the sidewall of bioreactor of the present invention, offer liquid on reactorInput, airtight identical covering offers liquid input and gas feed on reactor, and on reactor, liquid is defeatedGoing out end is placed in below the indoor fluid level of sterile culture.
As improving further, pipe-line system of the present invention is silicone tube spline structure, comprises that silica gel flows intoSection and silica gel flow out section, and the pipe-line system arrival end on silica gel inflow segment is connected with liquid output on reactor,The pipe-line system port of export that silica gel flows out in section is connected with liquid input on reactor. Thereby make it to form oneAirtight circulation perfusion bioreactor device;
As improving further, in gas feed of the present invention, be connected with pneumatic filter. Gas filtrationIt is aseptic that device can ensure that reactor enters the indoor gas such as oxygen, carbon dioxide of sterile culture outward, to ensure nothingGerm-free condition in bacterium culturing room, meets nutrient solution top off simultaneously and supports.
As improving further, liquid output on liquid input and reactor on reactor of the present inventionEnd is the independent tubulose spline structure of detachable displacement. This design can avoid airtight identical lid too heavy, simultaneously itsGood with the anastomose property of bioreactor, can ensure the seal of whole reactor assembly.
As improving further, airtight identical lid of the present invention and Sterile culture room body anastomosis arrangeFor frosted. Ensure the seal of reactor assembly.
As improving further, on reactor of the present invention, liquid input two ends are tiny, respectively with siliconSebific duct and trochar are connected, and mid diameter increases, and present and drip kettle spline structure, liquid input on described reactorEnd is 70-80 ° of angle with sidewall of reactor. And non-perpendicular entering, this design can make silica gel pipeline system on the one handMore than the gas producing in system is positioned at input intraluminal fluid plane, thereby effectively avoid bubble in silica gel pipe-line systemEnter in support. On the other hand, can make the inflow entrance of three-dimensional rack take advantage of a situation and be connected with the liquid input on sidewall,And do not cause that pulling of support draw distortion.
As improving further, bioreactor of the present invention is by transparent sample material or disposable sterilizedMaterial is made. Described transparent sample material is heat-resisting, high temperature resistant, can tolerate the high pressure steam sterilization of 120-200 degree,The repeated multiple times use of realization response apparatus, and the transparent design of container can more conveniently be observed. DescribedDisposable sterilized material is disposable material transparent, nontoxic, stable performance, and it is easy to use, efficient, keep awayExempt from cross pollution, under cell culture environment, can not discharge toxicant and affect Growth of Cells, the transparent sample of containerDesign can conveniently be observed equally.
As improving further, transparent sample material of the present invention is transparent sample Pyrex or quartzy glassGlass.
As improving further, pipe-line system of the present invention is the high steam that can tolerate 120-200 degreeThe silicone tube of sterilizing. Silicone tube is high temperature resistant, stable performance.
The present invention has following advantage compared with already present liver tissue engineering bioreactor:
The first, the indoor nutrient solution of bioreactor sterile culture enters closed conduit through the liquid output of reactorSystem, under the control of peristaltic pump, through liquid reactor input, flows in three-dimensional rack again, thereby realThe completely circulation of existing nutrient solution in whole three-dimensional rack, for support inner cell provides effective nutrition supply, gasBody exchange, the survival rate of raising internal stent cell, and three-dimensional rack cell efficiency again, in addition, wriggleThe pulsation of pump stimulates, blood flow in analogue body.
The second, reactor is provided with gas feed, is equipped with pneumatic filter on it, can ensure that reactor enters outwardEnter the indoor gas such as oxygen, carbon dioxide of sterile culture aseptic, support to meet nutrient solution top off.
The 3rd, on reactor, liquid input is used for reference the design principle of one-time infusion tube " Mo Feishi dropper ",Be designed to two ends tiny, be connected respectively with silicone tube and trochar, mid diameter increases, and presents and drips kettle spline structure.Described liquid input and sidewall of reactor are a 70-80 ° angle, and non-perpendicular entering, this design is on the one handMore than the gas that can make to produce in silica gel pipe-line system is positioned at input intraluminal fluid plane, thereby effectively avoid silica gelIn pipe-line system, bubble enters in support. On the other hand, this design can make three-dimensional rack with sidewall on liquidInput is taken advantage of a situation connected, and does not cause that pulling of support draw distortion.
The 4th, whole perfusion system is simple, easy to operate, by regulate, arrange perfusion pattern on peristaltic pump,Perfusion rate, perfusion pressure etc. can be controlled the nutrient solution perfusion in support.
Brief description of the drawings
Fig. 1 is the theory structure sketch of extracorporal circulatory system perfusion bioreactor device;
In figure, 1 is bioreactor, and 2 is perfusion device, and 3 is Sterile culture room, and 4 is pipe-line system, 5For liquid input on reactor, 6 is liquid output on reactor, and 7 is gas feed, 8 pipe-line systemsArrival end, 9 is the pipe-line system port of export, and 10 is pneumatic filter, and 11 is airtight identical lid.
Detailed description of the invention
Fig. 1 is the theory structure sketch of extracorporal circulatory system perfusion bioreactor device; The invention discloses oneCirculation perfusion bioreactor device, device comprises bioreactor 1 and perfusion device 2, perfusion device 2 wrapsDraw together peristaltic pump and be arranged at the pipe-line system 4 on peristaltic pump, bioreactor 1 comprises Sterile culture room 3 and closeClosing coincide covers 11. On the sidewall of bioreactor 1, offer liquid input 5 on reactor, airtight identical lidOn 11, offer liquid input 5 and gas feed 7 on reactor. Pipe-line system 4 is silicone tube spline structure,Comprise that silica gel inflow segment and silica gel flow out section, liquid on the pipe-line system arrival end 8 on silica gel inflow segment and reactorBody output 6 is connected, liquid input 5 on the pipe-line system port of export 9 in silica gel outflow section and reactorBe connected, in gas feed 7, be connected with pneumatic filter 10, on reactor on liquid input 5 and reactorLiquid output 6 is the independent tubulose spline structure of detachable displacement, airtight identical lid 11 and Sterile culture room 3Body anastomosis is set to frosted, and on reactor, liquid input 5 two ends are tiny, respectively with silicone tube and sleeve pipePin be connected, mid diameter increase, present drip a kettle spline structure, on described reactor liquid input 5 with reactDevice sidewall is 70-80 ° of angle, and bioreactor 1 is made up of transparent sample material or disposable sterilized material,Transparent sample material is transparent sample Pyrex or quartz glass, and pipe-line system 4 is for tolerating the height of 120-200 degreeThe silicone tube of pressing steam sterilizing, disposable sterilized material is transparent, nontoxic, stable performance, at cell culture environmentProperty can not discharge toxicant.
Blood flow situation in circulation perfusion bioreactor 1 system simulation body disclosed by the invention, provides thin in supportThe necessary nutritional labeling of born of the same parents' long-term cultivation. Circulating perfusion device 2 and bioreactor 1 are all positioned over titanium dioxideIn carbon incubator. On reactor, liquid output 6 gos deep into below fluid level, the work of the peristaltic pump outside reactorWith lower, liquid in Sterile culture room 3 is pumped, through airtight silica gel pipe-line system 4 and be attached thereto and connectLiquid input flows in three-dimensional rack, then flows out and enters aseptic in reactor through the flow export of three-dimensional rackCulturing room 3, thus the lasting circulation perfusion of realizing three-dimensional rack is cultivated. Three-dimensional rack is for having liquid inflow entranceA kind of biocompatible materials with flow export, retains complete vascular system, the liquid stream of trochar and supportEnter muzzle and prick connection, form the feed tube of this three-dimensional rack, liquid input on described feed tube and reactor5 connect, and the liquid flowing outlet of three-dimensional rack is directly opened on Sterile culture room 3.
The present invention, by taking the de-cytoskeleton of full liver as example, takes off cytoskeleton cell and external training again from full liverSupport two stages and carry out the enforcement of concrete case row, technical scheme of the present invention is further described:
The de-cytoskeleton cell stage again of full liver, i.e. the cell seeding stage. To fill with to rinse through detergent order and obtainThe de-cytoskeleton of full liver be placed in aseptic ware, seed cell is suspended from 5ml nutrient solution, slowly pushes away through portal veinIn injection support, in incubator, leave standstill and cultivate after a period of time, can carry out tissue perfusion cultivation.
In the in vitro culture stage, circulating perfusion device 2 and bioreactor 1 are all placed in CO2gas incubator.The de-cytoskeleton of full liver is at the interior cultivation of Sterile culture room 3 of bioreactor 1, the interior nutrient solution of Sterile culture room 3On reactor through going deep into fluid level, liquid output 6 enters closed conduit system 4, under peristaltic pump control,Nutrient solution liquid input 5 on reactor carries out in full liver three-dimensional rack. Nutrient solution is in full liver three-dimensional rackAfter circulation, enter Sterile culture room 3, repeat above-mentioned cyclic process, thereby realize complete at three-dimensional rack of nutrient solutionComplete alternation.
Embodiment 1: seed cell is cultivated in the de-cytoskeleton of full liver
Under aseptic condition, open abdomen through rat abdomen center and see portal vein, trochar is inserted to carry out through portal veinThe method of follow-up continous pouring detergent is obtained the de-cytoskeleton of the full liver of rat, and this support retains complete arteries and veinsTubular construction, and extracellular matrix components. Seed cell is suspended from the nutrient solution of 5ml, through pricking with portal vein coverThe trochar connecting is injected in support, carries out the circulation perfusion of full liver in culture dish after standing cultivation a period of timeCultivate. In the Sterile culture room 3 of bioreactor 1, add nutrient solution, on full rami hepatici frame, be connected with portal veinTrochar is connected with liquid input 5 on reactor, wherein liquid input 5 and sidewall of reactor on reactorBe 75 ° of angles, the liquid flowing outlet of full rami hepatici frame is directly opened on Sterile culture room 3. On reactor, liquid is defeatedGo out end and 6 go deep under the interior fluid level of Sterile culture room 3, the pipe-line system arrival end 8 on silica gel inflow segment with reactOn device, liquid output 6 is connected, liquid input on the pipe-line system port of export 9 in silica gel outflow section and reactorEnd 5 is connected, thereby makes it to form an airtight circulation perfusion bioreactor device. Airtight identical lid 11On pneumatic filter 10 to ensure to enter gas in bioreactor 1 aseptic. Under the regulation and control of peristaltic pump,Realize the circulation perfusion of nutrient solution in full rami hepatici frame, ensure that the oxygen of seed cell in de-cytoskeleton divides, seeksSupport material supply. A whole set of circulation perfusion bioreactor 1 device is placed in CO2gas incubator.
Embodiment 2: the impact of different perfusion rate on seed cell in full rami hepatici frame
Under aseptic condition, open abdomen through rat abdomen center and see portal vein, trochar is inserted to carry out through portal veinThe method of follow-up continous pouring detergent is obtained the de-cytoskeleton of the full liver of rat, and this support retains complete arteries and veinsTubular construction, and extracellular matrix components. Seed cell is suspended from the nutrient solution of 5ml, through pricking with portal vein coverThe trochar connecting is injected in support, carries out the circulation perfusion of full liver in culture dish after standing cultivation a period of timeCultivate. In the Sterile culture room 3 of bioreactor 1, add nutrient solution, on full rami hepatici frame, be connected with portal veinTrochar is connected with liquid input 5 on reactor, wherein liquid input 5 and sidewall of reactor on reactorBe 75 ° of angles, the liquid flowing outlet of full rami hepatici frame is directly opened on Sterile culture room 3. On reactor, liquid is defeatedGo out end and 6 go deep under the interior fluid level of Sterile culture room 3, the pipe-line system arrival end 8 on silica gel inflow segment with reactOn device, liquid output 6 is connected, liquid input on the pipe-line system port of export 9 in silica gel outflow section and reactorEnd 5 is connected, thereby makes it to form an airtight circulation perfusion bioreactor device. Airtight identical lid 11On pneumatic filter 10 to ensure to enter gas in bioreactor 1 aseptic. By adjusting the filling of peristaltic pumpFlow Velocity, the continous perfusion carrying out under different perfusion rate is cultivated. Cultivate after one week the different perfusions of observation and comparisonThe impact of speed on seed cell in de-cytoskeleton.
Embodiment 3: the differentiation of stem cell in de-cytoskeleton cultivated
Under aseptic condition, open abdomen through rat abdomen center and see portal vein, trochar is inserted to carry out through portal veinThe method of follow-up continous pouring detergent is obtained the de-cytoskeleton of the full liver of rat, and this support retains complete arteries and veinsTubular construction, and extracellular matrix components. Mesenchymal stem cells MSCs (BMSCs) is suspended from 5ml containing 10%FBSDMEM in, carry out full liver and take off cytoskeletal cell again through pricking with portal vein cover the trochar being connected. TrainingSupport the circulation perfusion cultivation of carrying out full liver in ware after standing cultivation a period of time. The sterile culture of bioreactor 1In chamber 3, add nutrient solution, liquid input 5 on the trochar being connected with portal vein on full rami hepatici frame and reactorConnect, wherein on reactor, liquid input 5 is 75 ° of angles with sidewall of reactor, the liquid of full rami hepatici frameFlow export is directly opened on Sterile culture room 3. On reactor, liquid output 6 gos deep into the interior liquid of Sterile culture room 3Under plane, the pipe-line system arrival end 8 on silica gel inflow segment is connected with liquid output 6 on reactor, silica gelThe pipe-line system port of export 9 flowing out in section is connected with liquid input 5 on reactor, thereby makes it to form oneAirtight circulation perfusion bioreactor device. Pneumatic filter 10 on airtight identical lid 11 ensures to enter lifeGas in thing reactor 1 is aseptic. Under the regulation and control of peristaltic pump, realize the circulation of nutrient solution in full rami hepatici framePerfusion, ensures that the oxygen of seed cell in de-cytoskeleton divides, nutriment supply. Treat BMSCs amplification cultivationLatter some days, use serum-free IMDM nutrient solution instead and cultivate 2 days, carry out afterwards in the de-cytoskeleton of full liver dry thinBorn of the same parents' liver property differentiation is cultivated, after Cyclic culture 3 days, and 1,2,3 week collection organization and culture supernatant are carried outStem cell differentiation effect in the de-cytoskeleton of Analysis and Identification, the impact of the de-cytoskeleton of assessment on Stem cell differentiation.
Continuing the nutriment of circulation perfusion to seed cell and the supply of nutrient and play critical effect, is to adjustControl Growth of Cells and the key factor that maintains functional activity. Bioreactor of the present invention is simple and easy, easy to operate, canFor the seed cell in the de-cytoskeleton of full liver provides the extracorporal circulatory system of lasting simulation physiological condition, careful for plantingIntracellular growth provides sufficient gas and nutriment, for de-cytoskeleton again cell technical support is provided, for touchingThe best tissue culture technique supplying method of rope.
What more than exemplify is only the preferred embodiment of the present invention, and the present invention is not limited to above embodiment, abilityField technique personnel directly derive without departing from the spirit and concept in the present invention or associate other changeEnter and change, all should think and be included in protection scope of the present invention.
Claims (10)
1. a circulation perfusion bioreactor device, is characterized in that, described device comprises bioreactor (1)And perfusion device (2), described perfusion device (2) comprise peristaltic pump and are arranged at the pipeline system on peristaltic pumpSystem (4), described bioreactor (1) comprises Sterile culture room (3) and airtight identical lid (11).
2. circulation perfusion bioreactor device according to claim 1, is characterized in that described biologyOn the sidewall of reactor (1), offer liquid input (5) on reactor, described airtight identical lid (11)On offer liquid input (5) and gas feed (7) on reactor.
3. circulation perfusion bioreactor device according to claim 2, is characterized in that described pipelineSystem (4) is silicone tube spline structure, comprises that silica gel inflow segment and silica gel flow out section, described silica gel inflow segmentOn pipe-line system arrival end (8) be connected with liquid output (6) on reactor, described silica gel flows out sectionOn the pipe-line system port of export (9) be connected with liquid input (5) on reactor.
4. circulation perfusion bioreactor device according to claim 2, is characterized in that described gasIn import (7), be connected with pneumatic filter (10).
5. according to the circulation perfusion bioreactor device described in claim 1 or 2 or 3 or 4, it is characterized in that,On described reactor, on liquid input (5) and reactor, liquid output (6) is the only of detachable displacementStandpipe shape spline structure.
6. according to the circulation perfusion bioreactor device described in claim 1 or 2 or 3 or 4, it is characterized in that,Described airtight identical lid (11) is set to frosted with Sterile culture room (3) body anastomosis.
7. circulation perfusion bioreactor device according to claim 1 and 2, is characterized in that, describedOn reactor, liquid input (5) two ends are tiny, are connected respectively with silicone tube and trochar, and mid diameter increasesGreatly, present and drip kettle spline structure, on described reactor, liquid input (5) is 10-20 ° with sidewall of reactorAngle.
8. according to the circulation perfusion bioreactor device described in claim 1 or 2 or 3 or 4, it is characterized in that,Described bioreactor (1) is made up of transparent sample material or disposable sterilized material.
9. according to the circulation perfusion bioreactor device described in claim 1 or 2 or 3 or 4, it is characterized in that,Described transparent sample material is transparent sample Pyrex or quartz glass.
10. according to the circulation perfusion bioreactor device described in claim 1 or 2 or 3 or 4, it is characterized in that,Described pipe-line system (4) is the silicone tube that can tolerate the high pressure steam sterilization of 120-200 degree.
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| CN105950468A (en) * | 2016-05-23 | 2016-09-21 | 西安交通大学 | Bioreactor for perfusion and bioreactor for permeation |
| CN108753686A (en) * | 2018-06-22 | 2018-11-06 | 北京达博威迎医药技术有限公司 | Organizational project hepatic model, its construction method and its application |
| CN109055216A (en) * | 2018-09-21 | 2018-12-21 | 中国科学院苏州生物医学工程技术研究所 | High-throughput 3D cell, class loading and organoid dynamic cultivation system |
| CN109477051A (en) * | 2016-07-19 | 2019-03-15 | 自动化合作关系（剑桥）有限公司 | Fluid filter system with integrated discharge function |
| CN110106085A (en) * | 2019-05-24 | 2019-08-09 | 中山大学 | A kind of integrated bioreactor for attached cell culture |
| CN110511870A (en) * | 2019-08-30 | 2019-11-29 | 英诺维尔智能科技（苏州）有限公司 | A kind of biological products large-scale production is without impact liquid sedimentation conduit |
| CN110669669A (en) * | 2019-11-05 | 2020-01-10 | 西安交通大学医学院第一附属医院 | Modular three-dimensional perfusion cell culture system |
| CN110684642A (en) * | 2019-10-29 | 2020-01-14 | 康珞生物科技（武汉）有限公司 | Three-dimensional perfusion type cell culture instrument |
| CN112625902A (en) * | 2020-12-03 | 2021-04-09 | 广州迈普再生医学科技股份有限公司 | Bioreactor and have its biological reaction system |
| CN112708557A (en) * | 2021-01-18 | 2021-04-27 | 英诺维尔智能科技（苏州）有限公司 | Perfusion culture device |
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