CN106754355A - A kind of organ vitro culture system - Google Patents

A kind of organ vitro culture system Download PDF

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Publication number
CN106754355A
CN106754355A CN201611037060.6A CN201611037060A CN106754355A CN 106754355 A CN106754355 A CN 106754355A CN 201611037060 A CN201611037060 A CN 201611037060A CN 106754355 A CN106754355 A CN 106754355A
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culture
organ
pipe
pump
inner chamber
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CN201611037060.6A
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齐忠权
仝彩玲
李明晖
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Xiamen University
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Xiamen University
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    • C—CHEMISTRY; METALLURGY
    • 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
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/08—Flask, bottle or test tube
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/10—Perfusion
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C—CHEMISTRY; METALLURGY
    • 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
    • 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
    • C12M41/18—Heat exchange systems, e.g. heat jackets or outer envelopes
    • C12M41/22—Heat exchange systems, e.g. heat jackets or outer envelopes in contact with the bioreactor walls
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/48—Automatic or computerized control

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
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Abstract

The invention discloses a kind of organ vitro culture system, including a mixed gas tank, a medium container, a disposable infusion tube heating device, an organ culture set bottle, a thermostatic bath and a titration pump.Pump is introduced culture systems by the present invention using the operation principle of pump, and culture medium changes problem in can solving organ culture so that the organ of culture can obtain enough nutrients, beneficial to its growth.

Description

A kind of organ vitro culture system
Technical field
The invention belongs to organ Vitro Culture Techniques field, and in particular to a kind of organ vitro culture system.
Background technology
Daily global thousands of organ failure patients wait for organ transplant, with extending life, however, the shortage of organ It is a great problem that the world today faces.Neomorph has become the focus of research.Neomorph refers to by organ transplant Make the organ function of dysfunction with artificial organs or operation.Due to the organ critical shortage for transplanting, artificial device Official turns into the emphasis of neomorph research, and the in vitro culture of organ turns into the most important thing of research.In 18th century, people begin to Research to organ culture,《Organ culture》One book was published in Paris in 1964 in the form of French, 1970 years Published again in the form of English after J.Thomas reviseds, the book is constantly revised, increase newest organ research new Progress, great impetus has been played to organ research.The book also describes the main method of some organ cultures：1) solidify Plasma matrix cultivation.It is initially to be founded by Fell, H.B and Robison, organ fragment or organ is placed on and is coated with Cultivated on the blood plasma of solidification and the surface plate of chick embryo extract1.2) Agar substrate cultivation.Nineteen fifty-two E.Wolff et al. is containing Having the method that organ is directly placed on the agar medium of embryo Extract carries out organ culture.3) floating method 4) grid cultivation 5) It is alternately exposed to nutrient solution and gas phase cultivation.People constantly explore vitro in organ culture method and influence organ culture because Element, and obtain prominent achievement.Early in the sixties in 19th century, people explore the method for people's small intestine in vitro culture, and obtain Experience.The culture of organ first is generally also received with the in vitro culture of the 3D cultural methods and organ similar to internal cell growth To the regulation of hormone；Secondly, the component of gas is also the key factor for influenceing organ culture.Cell in incubation, typically Need 5% CO2Concentration and 95% air, and organ is to O2Demand then greatly increase, in heart3And small intestine2Cultivated O in journey2Concentration reach more than 90%.3D cultural methods are widely used in man-made organ, and artificial skelecton is using raw bone Cell inoculation 3D supports carry out the culture of bone.2D culture of the cell 3D growing environments in vivo with cell in vitro is compared, Cell has very big difference in terms of migration, adhesion, propagation and gene expression.It has been found that mescenchymal stem cell is cultivated in 3D Expression with albumen in 2D cultures has differences, and 3D cultures can more accurately simulate form, propagation and the differentiation of normal cell, institute The structure of organizational project organ is widely applied to 3D cultures.Recently, the hydrogel containing magnetic iron oxide extensively should For the 3D cultures of cell, cell can be suspended in the middle of culture medium and magnetisable material, break away from cell because of the effect of gravity The aggregation of generation.The structure that 3D cell culture technologies are applied to organizational project has the advantage that：1) 3D cell culture systems make Setting up for cell growth microenvironment is simpler；2) 3D cell culture systems are closer to internal cell growth environment, cell Growth and Differentiation is closer to internal, so that the function of engineered tissue organ is closer to normal organ；3) natural three-dimensional branch Frame material has unique advantage in 3D cultures, and 3D natural scaffolds immunogenicity first is less than non-natural synthesis support, its Secondary, 3D natural scaffolds permeability is good, reduces oxygen-deficient generation, and the existence differentiation to cell provides basic attachment base Matter.
The content of the invention
It is an object of the invention to provide a kind of organ vitro culture system.
Technical scheme is as follows：
A kind of organ vitro culture system, including：
One mixed gas tank, is used to the mixed gas of the oxygen and carbon dioxide required for supplying organ culture；
One medium container, is loaded with culture medium, and go out with the air inlet pipe stretched into below culture medium liquid level, one in it Liquid pipe and a liquid-in pipe,；
One disposable infusion tube heating device；
One organ culture covers bottle, including an exocoel and an inner chamber, and exocoel parcel inner chamber is so as to keep constant 37 in inner chamber DEG C and with inner chamber isolation, the inner chamber in have two organ culture fixing pipes, be used to by de- cytoskeleton fixation in the lumen；
With a titration pump；
Mixed gas tank connects the air inlet pipe of medium container, and the drain pipe of medium container is by woven hose and disposable Infusion tube heating device is connected with the liquid feeding end of titration pump, and disposable infusion pipe heating device is used to heat and flows through above-mentioned woven hose Interior culture medium, the outlet end connection organ culture for titrating pump covers an organ culture fixing pipe of bottle, and organ culture covers the another of bottle One organ culture fixing pipe is connected with the liquid-in pipe of medium container.
In a preferred embodiment of the invention, also including a thermostatic bath, thermostatic bath connection organ culture Cover the exocoel of bottle so that full of constant 37 DEG C of heating liquid in the exocoel.
In a preferred embodiment of the invention, the titration pump is card Gadamer multifunctional intellectual speed governing measuring pump KSP-F01A。
Beneficial effects of the present invention：
1st, pump is introduced culture systems by the present invention using the operation principle of pump, and culture medium is more in can solving organ culture Problem is changed so that the organ of culture can obtain enough nutrients, beneficial to its growth.
2nd, organ culture is different from cell culture, and it needs more gas supplies.The present invention is utilized to have and stretches into culture The medium container of an air inlet pipe, a drain pipe and a liquid-in pipe below base fluid face solves gas supply problem, makes organ Can be developed in sufficient gaseous environment.
3rd, the present invention is solved using the culture medium circulatory system, culture medium is circulated in organ, and controllable flow Dynamic speed and flowing time, make organ obtain enough nutrients.
4th, for solving 37 DEG C of temperature conditionss of the organ of culture, the present invention is flowed using 37 DEG C of water-bath pump circulations, to protect Temperature maintains 37 DEG C all the time in holding blake bottle so that cell at this temperature can in organ proliferation.
Brief description of the drawings
Fig. 1 is structural representation of the invention.
Fig. 2 is the de- cytoskeleton of heart in treatment, and A is de- state photo of the cytoskeleton in PBS perfusates of heart； B is state photo of the de- cytoskeleton of heart in DMEM culture mediums balance.
Fig. 3 is the state photo of the de- cytoskeleton inoculating cell of heart.
Fig. 4 is the HE stained photographs of the de- cytoskeleton of heart, and wherein A is the state before inoculating cell；B is for after inoculating cell Cultivate the state of a week.
Specific embodiment
The present invention is further detailed and described below by way of specific embodiment combination accompanying drawing.
As shown in figure 1, a kind of organ vitro culture system, including a mixed gas tank 1, a medium container 2, are once Property infusion tube heating device 3, organ culture set bottle 4, a thermostatic bath 5 and a titration pump 6.
Mixed gas tank 1, is used to the mixed gas of the oxygen and carbon dioxide required for supplying organ culture, and it can lead to The air inflow of valve regulated mixed gas is crossed, it is ensured that the content of oxygen in culture medium, so that the supply by culture medium ensures device It is sufficient that official supplies oxygen in incubation；
Medium container 2, is loaded with culture medium in it, and with the air inlet pipe 21, stretched into below culture medium liquid level The liquid-in pipe 23 of drain pipe 22 and, can recycle the culture medium containing saturated gas；
Disposable infusion pipe heating device 3, is chosen as infusion thermostat or transfusion heating rod, can be with heated at constant temperature from culture The culture medium pumped out in based containers 2, so that the culture medium into organ keeps 37 DEG C of optimum temperature；
Organ culture covers bottle 4, including an exocoel 41 and an inner chamber 42, and the parcel inner chamber 42 of exocoel 41 in inner chamber 42 so as to keep Constant 37 DEG C and completely cut off with inner chamber 42, there are two organ culture fixing pipes 43 in inner chamber 42, be used to fix de- cytoskeleton In inner chamber 42；
Thermostatic bath 5, connection organ culture cover bottle 4 exocoel 41 so that in the exocoel 41 full of constant 37 DEG C plus Hot liquid, for the organ in organ culture set bottle 4 provides environmental optima temperature；
Titration pump 6, preferably card Gadamer multifunctional intellectual speed governing measuring pump KSP-F01A, can continue at regular time and quantity from Draw culture medium in medium container 2 to enter in organ culture set bottle 4, so as to ensure organ in well-fed fresh culture In cultivated；
Mixed gas tank 1 connects the air inlet pipe 21 of medium container 2, and the drain pipe 22 of medium container 2 passes through woven hose Connected with disposable infusion pipe heating device 3 with the liquid feeding end of titration pump 6, disposable infusion pipe heating device 3 is used to heat stream Through the culture medium in above-mentioned woven hose, the outlet end connection organ culture for titrating pump 6 covers an organ culture fixing pipe 43 of bottle 4, Another organ culture fixing pipe 43 of organ culture set bottle 4 is connected with the liquid-in pipe 23 of medium container 2.
Process with present invention culture rat heart is as follows：
1) after rat heart is won, it is placed in the inner chamber 42 of organ culture set bottle 4, pulmonary artery and the organ culture set of heart One organ culture fixing pipe 43 of bottle 4 connects and ligatures solid, and the organ culture fixing pipe 43 is digestive juice or culture medium Flow export, the arch of aorta be connected with another organ culture fixing pipe 43 and ligature it is solid, the organ culture fixing pipe 43 for digestion The inflow entrance of liquid or culture medium.
2) 1% SDS solution is placed in medium container 2, open titration pump 6, perfusion 500ml 1% SDS, with The distilled water of perfusion 500ml afterwards, then the Triton X-100 solution 100ml of perfusion 0.5%, form the de- cytoskeleton of heart, Again with the aseptic PBS perfusions of 1L, the de- cytoskeleton (as shown in Figure 2 A) of cleaning balance heart.
3) the de- cytoskeleton (as shown in Figure 2 B) of heart is balanced with the DMEM of 1L.
4) mixed gas tank 1 is opened, being passed through culture medium by flexible pipe with 95% oxygen and 5% carbon dioxide holds In culture medium in device 2, make culture medium oxygen saturation, property infusion tube heating device 3 opened once again and thermostatic bath 5 so that Culture medium and the de- cytoskeleton temperature of heart are maintained at 37 DEG C, and perfusion 200ml DMEM/FBS balances heart is de- thin in such circumstances Born of the same parents' support, is that cell inoculation is prepared.
5) regulation titration pump 6, the speed for making pump is 1 minute DMEM cardiac muscle cell's complete medium of injection 500ul.
6) cell 1 × 10 is prepared7Individual cell, the heart is injected into by cell from the organ culture fixing pipe 43 of the connection arch of aorta It is dirty, continue to cultivate one week, you can see that cell is colonized growth (as shown in Figures 2 to 4, Fig. 2A and B on the de- cytoskeleton of heart In the de- cytoskeleton of heart in acellular presence, after organ culture system cultivates, as shown in figure 3, observe it can be seen that Heart matrix becomes opaque, has cell to be present in the de- cytoskeleton of heart, and as shown in Figure 4 A, heart is de- thin before inoculating cell Acellular presence in born of the same parents' support, after being cultivated through organ culture system, as shown in Figure 4 B, has cell to be colonized in matrix and is present in the heart Dirty de- cytoskeleton).
The above, only presently preferred embodiments of the present invention, therefore can not according to this limit the scope of present invention implementation, i.e., The equivalence changes made according to the scope of the claims of the present invention and description and modification, all should still belong in the range of the present invention covers.

Claims (3)

1. a kind of organ vitro culture system, it is characterised in that：Including：
One mixed gas tank, is used to the mixed gas of the oxygen and carbon dioxide required for supplying organ culture；
One medium container, is loaded with culture medium in it, and with the air inlet pipe, a drain pipe stretched into below culture medium liquid level With a liquid-in pipe,；
One disposable infusion tube heating device；
One organ culture covers bottle, including an exocoel and an inner chamber, and exocoel parcel inner chamber is so as to keep constant culture temperature in inner chamber Spend and completely cut off with inner chamber, there are two organ culture fixing pipes in inner chamber, be used to fix de- cytoskeleton in the lumen；
With a titration pump；
Mixed gas tank connects the air inlet pipe of medium container, and the drain pipe of medium container passes through woven hose and disposable infusion Pipe heating device is connected with the liquid feeding end of titration pump, and disposable infusion pipe heating device is used to heat and flows through in above-mentioned woven hose Culture medium, the outlet end connection organ culture for titrating pump covers an organ culture fixing pipe of bottle, and organ culture covers another device of bottle Official's culture fixing pipe is connected with the liquid-in pipe of medium container.
2. a kind of organ vitro culture system as claimed in claim 1, it is characterised in that：Also include a thermostatic bath, the perseverance Warm bath groove connection organ culture covers the exocoel of bottle so that the heating liquid full of steady temperature in the exocoel.
3. a kind of organ vitro culture system as claimed in claim 1, it is characterised in that：The titration pump is many work(of card Gadamer Can intelligent speed-regulating measuring pump KSP-F01A.
CN201611037060.6A 2016-11-23 2016-11-23 A kind of organ vitro culture system Pending CN106754355A (en)

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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10348746A1 (en) * 2003-08-13 2005-03-17 Volker Dr. Kraus Perfusion device for isolated organs, useful for performing biochemical and biomedical investigations, comprises a storage vessel for perfusate, a gasification vessel, a vessel for the organ and a circulation pump
US20050147958A1 (en) * 1997-09-23 2005-07-07 Waleed Hassanein Compositions, method and devices for maintaining an organ
CN2738750Y (en) * 2003-12-22 2005-11-09 冯滨 Heart-tissue engineering valve pulsating-fluid cultivation device
WO2006118990A2 (en) * 2005-04-29 2006-11-09 Transplan, Inc. Method and device for organ perfusion
CN101322861A (en) * 2007-06-13 2008-12-17 宋文彤 Transplanted organ in vitro preservation and vigor monitoring device as well as method
CN103194390A (en) * 2013-04-19 2013-07-10 南方医科大学南方医院 Double-loop oscillation perfusion-type biological reaction system
CN204763020U (en) * 2015-02-16 2015-11-18 杭州电子科技大学 External intelligent support system of subnormal temperature transplant organ
CN205213967U (en) * 2015-11-24 2016-05-11 中国人民解放军第四军医大学 Novel separation tissue constant temperature perfusion groove
US20160227762A1 (en) * 2012-07-06 2016-08-11 Arigos Biomedical, Inc. Method and Apparatus for Prevention of Thermo-Mechanical Fracturing in Vitrified Tissue Using Rapid Cooling and Warming by Persufflation

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050147958A1 (en) * 1997-09-23 2005-07-07 Waleed Hassanein Compositions, method and devices for maintaining an organ
DE10348746A1 (en) * 2003-08-13 2005-03-17 Volker Dr. Kraus Perfusion device for isolated organs, useful for performing biochemical and biomedical investigations, comprises a storage vessel for perfusate, a gasification vessel, a vessel for the organ and a circulation pump
CN2738750Y (en) * 2003-12-22 2005-11-09 冯滨 Heart-tissue engineering valve pulsating-fluid cultivation device
WO2006118990A2 (en) * 2005-04-29 2006-11-09 Transplan, Inc. Method and device for organ perfusion
CN101322861A (en) * 2007-06-13 2008-12-17 宋文彤 Transplanted organ in vitro preservation and vigor monitoring device as well as method
US20160227762A1 (en) * 2012-07-06 2016-08-11 Arigos Biomedical, Inc. Method and Apparatus for Prevention of Thermo-Mechanical Fracturing in Vitrified Tissue Using Rapid Cooling and Warming by Persufflation
CN103194390A (en) * 2013-04-19 2013-07-10 南方医科大学南方医院 Double-loop oscillation perfusion-type biological reaction system
CN204763020U (en) * 2015-02-16 2015-11-18 杭州电子科技大学 External intelligent support system of subnormal temperature transplant organ
CN205213967U (en) * 2015-11-24 2016-05-11 中国人民解放军第四军医大学 Novel separation tissue constant temperature perfusion groove

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Application publication date: 20170531