CN105087380A - System for massively culturing animal cells - Google Patents

System for massively culturing animal cells Download PDF

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Publication number
CN105087380A
CN105087380A CN201510468943.1A CN201510468943A CN105087380A CN 105087380 A CN105087380 A CN 105087380A CN 201510468943 A CN201510468943 A CN 201510468943A CN 105087380 A CN105087380 A CN 105087380A
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branch pipeline
reactor
bio
cell
interface
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高毅
傅超毅
李阳
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Southern Medical University Zhujiang Hospital
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Southern Medical University Zhujiang Hospital
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Priority to CN201510468943.1A priority Critical patent/CN105087380A/en
Priority to PCT/CN2015/087917 priority patent/WO2017020365A1/en
Publication of CN105087380A publication Critical patent/CN105087380A/en
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS 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
    • C12M1/00Apparatus for enzymology or microbiology
    • C12M1/34Measuring or testing with condition measuring or sensing means, e.g. colony counters
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS 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
    • C12M41/00Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/26Means for regulation, monitoring, measurement or control, e.g. flow regulation of pH
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS 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
    • C12M29/00Means for introduction, extraction or recirculation of materials, e.g. pumps
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS 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
    • C12M29/00Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/10Perfusion
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    • C12MAPPARATUS 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
    • C12M41/00Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/34Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of gas

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Abstract

The invention discloses a system for massively culturing animal cells. The system comprises a cell factory bioreactor, an oxygen supplier, a liquid storage bottle, a peristaltic pump and circulating pipelines, wherein the cell factory bioreactor, the oxygen supplier, the liquid storage bottle and the peristaltic pump are communicated and connected by the circulating pipelines. The circulating pipelines include a first branch pipeline, a second branch pipeline, a third branch pipeline, a fourth branch pipeline and a fifth branch pipeline, wherein the first branch pipeline is provided with two pipe clamping valves and a first connector between the two pipe clamping valves, and the second branch pipeline is provided with two pipe clamping valves and a second connector between the two pipe clamping valves; the left ends of the first branch pipeline and the second branch pipeline are connected to form a third connector; the right ends of the first branch pipeline and the second branch pipeline are connected to form a fourth connector; the left end and the right end of the third branch pipeline are respectively connected with the first connector and the second connector, and the oxygen supplier, the liquid storage bottle and the peristaltic pump are serially connected through the third branch pipeline; the first connector is connected with the cell factory bioreactor through the fourth branch pipeline; the second connector is connected with the cell factory bioreactor through the fifth branch pipeline.

Description

The system of large scale culturing zooblast
Technical field
The present invention relates to a kind of animal cell culture technology, particularly relate to a kind of system of large scale culturing zooblast.
Background technology
Add up according to WHO: the whole world has 6.5 hundred million to infect various liver problem sufferer, and recent statistics about has 2,000,000 people to die from various hepatopathy every year.China's acute and chronic liver failure is very common, and domestic this type of patient annual reaches 300,000 according to statistics.Case fatality rate is up to 80%.Current treatment liver failure method mainly contains: (1) medical treatment.(2) liver transplantation treatment.(3) artificial liver in treatment.And medical treatment poor effect, in surgical intervention, liver transplantation is the most effective methods for the treatment of of generally acknowledging at present, but due to reasons such as donor shortage, technical difficulty height, significantly limit extensively carrying out of orthotopic liver transplantation, most patient is dead in the process waiting for liver transplantation.Bioartificial liver has the functions such as liver cell removing toxic substances, conversion, synthesis, can replace liver major part function.Bio-reactor is the core of whole bioartificial liver, and its performance will be directly connected to the support effect of bioartificial liver.Desirable bioartificial liver's bio-reactor should reach following standard: (1) cell density reaches 1 × 10 8-10individual cell/ml level; (2) reaction unit can arbitrarily increase-volume as required, and cell cultures amount can reach several liter; (3) realize the two-way mass transfer of effective nutritive substance, oxygen and meta-bolites, simulate liver cell microenvironment in vivo as far as possible; (4) aspects such as automatic online cell state, medium pH value, oxygen concn can be carried out detect and regulatory function, so that the supervision of medical personnel and operation; (5) liver cell metabolic function at least reaches the level of monolayer culture, and at least keeps more than 2 weeks; (6) be convenient to frozen, transport and assembling.
The key problem of current bioartificial liver development is the new-type bioreactor that design is best, realizes effective maintenance of hepatocyte function and motility rate in the large scale culturing of hepatocyte and culturing process thereof, while can meet the needs of clinical application.Domestic and international in the structure of bio-reactor at present, following 4 large classes can be divided into up to now:
The bio-reactor of 1, monolayer culture/flat: such reactor is directly planted on flat board by liver cell, surface to volume ratio is declined, and reactor cell is monolayer culture, can not permanently effective survival keep function with active, and not easily amplify, cannot clinical requirement be reached.
2, hollow fiber bioreactor: the research of this reactor at present also achieves the following progress: 1) integrate independently pipeline oxygen for system, as mixed type LSS-MELS system; 2) find new mould material or existing mould material is improved; 3) on the basis of carrying out liver cell biotherapy, increase gac etc. physics antidotal therapy is integrated, as LIVERaid system; 4) new training mode is used, as LSS system improves hepatocellular activity and function by Dual culture mode.But this reactor still exists following problem: (1) limited volume, loading cells amount is little, nutrient solution and liver cell exchange area limited, be unfavorable for that external mass-producing is increased; (2) side opening of semi-permeable membranes is easily blocked by cell mass, affects exchange efficiency, is also unfavorable for the permanently effective maintenance of hepatocellular function and vigor.
3, bed/support bio-reactor is poured into: this reactor makes cell and timbering material reach suspended state by stirring, this bio-reactor mechanical stirring can produce certain shearing force, easily the damage of going up largely is caused to cell, thus limit it and further use.
4, wrap by suspended biological reactor: this bio-reactor is wrapped up at a kind of semipermeable membrane material of liver cell, make porous microcapsule, then perfusion culture is carried out. its shortcoming is due to the mutual gathering between the existence of semi-permeable membranes and liver cell, causes the exchange of matter energy inside and outside capsule limited.Liver cell is anchorage-dependent cell, as lost the attaching to timbering material, can inspire apoptosis.
Making a general survey of above prior art, generally there is reactor exchange of substance inefficiency and the technical problem such as pipeline structure is numerous and diverse in it.
Summary of the invention
The object of this invention is to provide a kind of system of that increase reactor exchange of substance efficiency, that pipeline structure is succinct large scale culturing zooblast,
For reaching above technical purpose, the technical solution used in the present invention is as follows:
A kind of system of large scale culturing zooblast, it comprises for zooblast providing the cell factory bio-reactor in growth place, providing the oxygen supply of oxygen for described cell factory bio-reactor, storing the liquid storage bottle of substratum, the peristaltic pump for driving described substratum to flow in this system, and by the circulation line of described cell factory bio-reactor, oxygen supply, liquid storage bottle, peristaltic pump conducting connection; Described circulation line comprises: the first branch pipeline and the second branch pipeline, and it is respectively equipped with two pinch valves and the first interface that is opened between these two pinch valves and the second interface; The left end of described two branch pipeline is interconnected to form the 3rd interface; The right-hand member of described two branch pipeline is interconnected to form the 4th interface; 3rd branch pipeline, its left end and right-hand member are connected with described first interface and the second interface respectively, and described oxygen supply, liquid storage bottle and peristaltic pump are connected in series by described 3rd branch pipeline; Connect the 4th branch pipeline of described first interface and cell factory bio-reactor; And connect the 5th branch pipeline of described second interface and cell factory bio-reactor.
Accordingly, described cell factory bio-reactor is provided with the first port and the second port that are connected with described 4th branch pipeline and the 5th branch pipeline respectively.
Preferably, described cell factory bio-reactor is microcarrier bio-reactor.
Further, this system is also included in thread detector; Described circulation line also comprises and is connected to the 6th branch pipeline that is in described 3rd branch pipeline and that be connected in series with described on-line monitoring device with T-valve.
Further, this system also comprises the heating plate be serially connected between described peristaltic pump and liquid storage bottle by described 3rd branch pipeline.
Preferably, described oxygen supply is membrane oxygenator.
For realizing automatization, this system also comprises the master controller regulating and affect the controling parameters of each integral part of cell culture condition in this system.
Compared with prior art, the present invention has following advantage:
(1) system of large scale culturing zooblast of the present invention, circulation power is provided by peristaltic pump, by appropriate design circulation line and utilize two pairs of pinch valve alternating open and close control the direction that liquid (substratum) enters reactor tank body, the periodicity two-way filling of substratum can prevent reactor tank cellular metabolic product from piling up at a place, and avoid being fed into the mouth cells oxygen supply well situation of tank outlet cell hypoxia and nutritive substance, effectively eliminate and cultivate dead space formation;
(2) system of large scale culturing zooblast of the present invention, widely used membrane oxygenator in surgical operation is adopted to be used as oxygen supply, effectively improve the gaseous interchange situation in animal cell culture process, contribute to the quality improving animal cell culture;
(3) system of large scale culturing zooblast of the present invention, be provided with on-line checkingi device, for detecting pH and the dissolved oxygen content of substratum, the microenvironment in bio-reactor can be understood in real time, can adjustment be made according to the numerical value of monitoring to reactor parameter in time and improve microenvironment in reactor tank body, also can avoid because sampling detects and pollute;
(4) system of large scale culturing zooblast of the present invention, adopts Microcarrier Culture Techniques to zooblast, suspension culture and adherent culture is merged, has both advantages concurrently.
Accompanying drawing explanation
Fig. 1 is the structural representation of the system of large scale culturing zooblast of the present invention, illustrate when the first pinch valve and the 4th pinch valve open, the second pinch valve and the 3rd pinch valve close time this system in the flow direction of substratum.
Fig. 2 is the system architecture schematic diagram of large scale culturing zooblast of the present invention, and the flow direction of the substratum in this system when the first pinch valve and the closedown of the 4th pinch valve, the second pinch valve and the 3rd pinch valve are opened is shown.
The growing state of the human liver cell that the system that Fig. 3 is employing large scale culturing zooblast of the present invention is cultivated.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.
With reference to figure 1, the system of large scale culturing zooblast of the present invention comprises cell factory bio-reactor 1, liquid storage bottle 2, heating plate 3, peristaltic pump 4, oxygen supply 5, on-line checkingi device 6 and the circulation line 7 by orderly for above-mentioned all parts conducting connection.In the present embodiment, the order of connection of above-mentioned each parts is followed successively by cell factory bio-reactor 1---liquid storage bottle 2---heating plate 3---peristaltic pump 4---oxygen supply 5---cell factory bio-reactor 1, the above-mentioned major cycle pipeline being connected to form native system; Further, described on-line checkingi device 6 is connected in parallel between described cell factory bio-reactor 1 and liquid storage bottle 2.
Particularly, described cell factory bio-reactor 1 is the growth place of zooblast, can gather in the crops the target metabolite of zooblast or zooblast after culturing process completes from this cell factory bio-reactor 1.For the growth characteristic of zooblast, described cell factory bio-reactor 1 preferably adopts microcarrier bio-reactor; Further, for the cultivation of human liver cell C3A, Cytodex3 microcarrier can be adopted.For the cultivation microenvironment of optimizing animal cell, the both sides up and down of the tank body of described cell factory bio-reactor 1 offer the first port 11 and the second port one 2 for perfusion culture base respectively.
Described liquid storage bottle 2 is for storing fresh liquid nutrient medium, and the bottleneck of this liquid storage bottle 2 is provided with diplopore bottle stopper, and the holes of described bottle stopper connects respectively for flowing into or the pipeline of flowing liquid substratum.
Described heating plate 3 is electroluminescent heater members, and this heating plate 3 can heat the liquid nutrient medium flowing through this heating plate 3, with meet zooblast increase in vitro in requirement to temperature.
Described peristaltic pump 4 in the system of described cultivation zooblast flowing fluid substratum provide the motivating force of flowing, the parameter that corresponding control device can be utilized to regulate the flow velocity etc. of this peristaltic pump 4 affect liquid nutrient medium circulate.
Described oxygen supply 5 for described cell factory cell reactor 1 provide animal cells in vitro increase needed for oxygen, described " oxygen " is not pure oxygen, but the gas mixture of the carbonic acid gas of 5% and 95% oxygen, accordingly, widely used membrane oxygenator in surgical operation is adopted to be used as described oxygen supply 5, the gas exchange process of liquid nutrient medium can be optimized, ensure that the oxygen level of liquid nutrient medium meets the requirement of animal cells in vitro amplification.
Described on-line checkingi device 6 is connected in parallel by the major cycle pipeline of a T-valve 761 with this system, for the pH value of on-line checkingi substratum and the content of dissolved oxygen as required, the real time data detecting gained is used to guide the adjustment of the parameter of other devices of the system of this cultivation zooblast.
Described circulation line 7 comprises the first branch pipeline 71, second branch pipeline 72, the 3rd branch pipeline 73, the 4th branch pipeline 74, the 5th branch pipeline 75 and the 6th branch pipeline 76.Wherein, described first branch pipeline 71 is provided with the first pinch valve 712 and the second pinch valve 713, and pipeline between described two pinch valves offers first interface 711; Described second branch pipeline 72 is provided with the 3rd pinch valve 722 and the 4th pinch valve 723, and pipeline between described two pinch valves offers the second interface 721; Further, the left end of described first branch pipeline 71 and the left end of the second branch pipeline 72 are interconnected to form the 3rd interface 731, and the right-hand member of described first branch pipeline 71 and the right-hand member of the second branch pipeline 72 are interconnected to form the 4th interface 732; The left end of described 3rd branch pipeline 73 is connected with described 3rd interface 731, and the right-hand member of the 3rd branch pipeline 73 is connected with described 4th interface 732, and described liquid storage bottle 2, heating plate 3, peristaltic pump 4 and oxygenator 5 are connected in series by the 3rd branch pipeline 73; Described 4th branch pipeline 74 one end is connected with described first interface 711, and the other end is connected with described cell factory bio-reactor 1 by described first port 11; One end of described 5th branch pipeline 75 is connected with described second interface 721, and the other end is connected with described cell factory bio-reactor 1 by described second port one 2; One end of described 6th branch pipeline 76 is connected with described 3rd branch pipeline 73 by T-valve 761, and the other end connects described on-line checkingi device 6.
Further, the open and close state of described first pinch valve 712, second pinch valve 713, the 3rd pinch valve 722 and the 4th pinch valve 723 have impact on the flow direction of liquid nutrient medium in the system of this cultivation zooblast, and particular case is as follows:
(1) as shown in Figure 1, suppose that described peristaltic pump 4 drives liquid nutrient medium to flow left, described first pinch valve 712 and the 4th pinch valve 723 are in the state opened, and described second pinch valve 713 and the 3rd pinch valve 722 are in the state of closedown; Liquid nutrient medium in this system flows out from described liquid storage bottle 2 from left side, along described 3rd branch pipeline 73 successively through described heating plate 3, peristaltic pump 4, oxygen supply 5, described liquid nutrient medium enters described first branch pipeline 71 after described 3rd interface 731, then enters described 4th branch pipeline 74 through described first interface 711 and pours into described cell factory bio-reactor 1 by described first port 11; The liquid nutrient medium of spill-over in described cell factory bio-reactor 1 flows out from described second port one 2 and enters described 5th branch pipeline 75, described second branch pipeline 72 is entered through described second port 721, described 3rd branch pipeline 73 is again entered again through described 4th interface 732, until be back to described liquid storage bottle 2, complete one with this and take turns circulation (circulation route with reference to dotted arrow instruction).
(2) as shown in Figure 2, suppose that described peristaltic pump 4 drives liquid nutrient medium to flow left, described first pinch valve 712 and the 4th pinch valve 723 are in the state of closedown, and described second pinch valve 713 and the 3rd pinch valve 722 are in the state opened; Liquid nutrient medium in this system flows out from described liquid storage bottle 2 from left side, along described 3rd branch pipeline 73 successively through described heating plate 3, peristaltic pump 4, oxygen supply 5, described liquid nutrient medium enters described second branch pipeline 72 after described 3rd interface 731, then enters described 5th branch pipeline 75 through described second interface 721 and pours into described cell factory bio-reactor 1 by described second port one 2; The liquid nutrient medium of spill-over in described cell factory bio-reactor 1 flows out from described first port 11 and enters described 4th branch pipeline 74, described first branch pipeline 71 is entered through described first port 711, described 3rd branch pipeline 73 is again entered again through described 4th interface 732, until be back to described liquid storage bottle 2, complete one with this and take turns circulation (circulation route with reference to dotted arrow instruction).
As can be seen here, the open and close state of described first pinch valve 712, second pinch valve 713, the 3rd pinch valve 722 and the 4th pinch valve 723 have impact on the direction that liquid nutrient medium pours into described cell factory bio-reactor 1.
For realizing the automatization of animal cell culture further, this system can also arrange the master controller regulating and affect the controling parameters of each integral part of cell culture condition in this system.This master controller at least should comprise and regulates the heat control unit of the heating temp of described heating plate 3, regulates the flow rate control unit of the flow velocity of described peristaltic pump 4, monitors the oxygen supply control unit of the gaseous interchange degree of described oxygen supply 5, the impeller control unit controlling the speed of rotation of the paddle wheel in described cell factory bio-reactor 1 and control and show the detecting unit of the detection data of described on-line checkingi device 6.
Further, adopt the concrete operations flow process of systematic cultivation human liver cell C3A of the present invention as follows:
1, the preparation of Cytodex3 microcarrier: beyond culturing bottle, 2% dichlorodimethylsilane silication all used by all glasswares such as suction pipe, culture dish contacted with microcarrier.Take 2.5gCytodex3 microcarrier and add 100ml in siliconized glass bottles, add 100ml fresh PBS damping fluid aquation at least 4 hours or spend the night, change PBS liquid and wash 3 times, renew fresh PBS damping fluid 100ml soaked overnight again, 120 DEG C of 20min sterilizings, the fibronectin liquid 0.5ML added containing 10% foetal calf serum (FBS) perfect medium and 1mg/ml soaks, and is placed in 4 DEG C of refrigerators and saves backup.
2, the preparation of cell factory bio-reactor:
Need before the pipeline use of 2.1 reactors with first rinsing (20ML syringe water filling washing pipe) 1-2 time with clean-out system, then distilled water flushing (20ML syringe water filling washing pipe) 1-2 time is used, spend the night with distilled water immersion, drain the water, put baking box into and dry.
2.2 reactor tank bodies first rinse 2-3 time with clear water before using, then use clear water soaked overnight, more first rinse 2-3 time with distilled water, drain the water, put baking box 60 degree into and dry.
2.3 reactor pipelines and tank body carry out Low-temperature Plasma Sterilization or oxirane disinfection for subsequent use.
3, the preparation of liquid storage bottle/wide-necked bottle: first rinse 2-3 time with clear water, use clear water soaked overnight again, be put into after draining the water in vitriol oil pond and soak 1-2 days, rinse well with clear water after taking-up, then spend the night with distilled water immersion, more first rinse 2-3 time with distilled water, be put into 60 DEG C of baking boxs to dry, package, with disinfection with high pressure steam (25 minutes, 120 DEG C).
4, the pipeline of cell factory bio-reactor is installed: wear sterile gown, wear sterile gloves, pipeline is arranged in Bechtop and carries out.
5, the C3A cell required for the inoculation of cell factory bio-reactor is prepared: recovery with square vase amplification C3A cell, the inoculum size required for reactor is 2 × 10 8.Under phase microscope, confirm that cell has covered with about 80%, in good condition and pollution-free.Collect the cell of square vase amplification in Bechtop digestion, blood cell counting plate counting cells total amount is about 2 × 10 8, Trypan Blue Cell viability is greater than 95%.
6, the inoculation of cell factory bio-reactor: the cell gathered is added reactor tank body together with 2.5gCytodex3 microcarrier, the perfect medium containing 10% foetal calf serum is filled it up with in wide-necked bottle, peristaltic pump is utilized slowly substratum to be pumped into 1/3 (be about 150ml) of reactor tank body to reactor cumulative volume, reactor tank body is put into constant temperature (37 DEG C) shaking table low speed (40rpm) and rock 6-8 hour, namely the time required for C3A cell attachment, C3A cell is impelled can better to be attached on Cytodex3 microcarrier.In Bechtop, situation should be attached with asepsis injector by the thief hole sampling observation of cell of reactor tank body after having attached.
7, preliminary filling reactor pipeline and tank body be installed to controller: utilize peristaltic pump slowly perfect medium to be full of reactor pipeline and the remaining space of tank body, arrange remaining air in system.Reactor is installed to the controller of cell factory, membrane oxygenator is connected the gas mixture containing 5% carbonic acid gas 95% oxygen, with the flow velocity of 3L/min to reactor oxygen supply.
8, the parameter setting of cell factory controller: when starting to rotate microgravity cultivation, the speed of rotation 10rpm of reactor tank body, peristaltic pump flow velocity is 60ml/min, and the commutation cycle is 90s.Reactor tank body speed of rotation can be strengthened gradually from second day, suspend in reactor tank body so that cell and microcarrier just can be made and be advisable.Usually, speed of rotation was 15rpm at second day, the 3rd day 20rpm, and the 4th to the 7th day is 30rpm.Flow velocity starts to be arranged on 60ml/min can be maintained until the 3rd day, within the 4th to the 7th day, can be set to 80ml/min.
9, cell factory bio-reactor change liquid and sampling:
9.1 on-line checkingi pH and dissolved oxygens: when the firm outflow reactor tank body of Cyclic culture base, by the T-valve of line connection by substratum drainage to online pH and dissolved oxygen probe, directly on controller screen, read the pH of Cyclic culture base and the numerical value of dissolved oxygen.
9.2 samplings: close peristaltic pump on the controller and take off pump line, stopped reaction device tank body rotates, and opens bilateral pinch valve and takes off pipeline, move reactor to super clean bench together with pipeline.With 75% cotton ball soaked in alcohol wiping thief hole, turn on calparine cap on liquid pumping hole, syringe is inserted into liquid pumping hole, drawing liquid is carried out in preparation, first jiggle reactor tank body, in question response device, carrier mixes laggard line sampling, extracts 1ML cell microcarrier sample, take off syringe, after cell microcarrier is sunk, extract 5ml cells and supernatant sample.Finally utilize the gas in the emptying reactor tank body of syringe, with 75% cotton ball soaked in alcohol wiping thief hole, then screw on calparine cap.
9.2 change liquid: in Bechtop, change 500ml liquid storage bottle after sampling, have 500ml to contain 10% foetal calf serum perfect medium in new liquid storage bottle.
10, sample preparation:
10.1 cell countings: the cell microcarrier sample of extraction is got 0.5ml and is placed in 1.5ml in EP pipe, leave standstill 5min, after the microcarrier of adherent cell sinks, suck nutrient solution as far as possible, add 0.1M Trisodium Citrate and 0.1% Viola crystallina to 1ml, 37 DEG C of overnight incubation, count fine karyon on blood cell counting plate, draws growth curve.
10.2 cell viabilities are observed: 50mgMTT is dissolved in 10mlPBS, 0.22um filtration sterilization, keeps in Dark Place at 4 DEG C.60u1MTT solution is added in the flat board being equipped with 0.5ml cell microcarrier suspension during detection.Then sample is hatched four hours at 37 DEG C, under inverted phase contrast microscope, observe the motility rate situation of culture hepatocyte.
10.3 cell-specific Function detection: after centrifugal for the supernatant liquor of collection, automatic clinical chemistry analyzer detects its ALT, AST, urea content.
Further, with reference to figure 3, adopt the human liver cell C3A of systematic cultivation of the present invention to be attached to fully on Cytodex3 microcarrier, and maintain the specific function of this cell.
In sum, the system pipeline of large scale culturing zooblast of the present invention is simple for structure, effectively increases reactor exchange of substance efficiency, improves animal cell culture quality.
Above-described embodiment is the present invention's preferably embodiment; but be not merely restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be all included within protection scope of the present invention.

Claims (7)

1. the system of a large scale culturing zooblast, it is characterized in that, it comprises for zooblast providing the cell factory bio-reactor in growth place, providing the oxygen supply of oxygen for described cell factory bio-reactor, storing the liquid storage bottle of substratum, the peristaltic pump for driving described substratum to flow in this system, and by the circulation line of described cell factory bio-reactor, oxygen supply, liquid storage bottle, peristaltic pump conducting connection; Described circulation line comprises:
First branch pipeline and the second branch pipeline, it is respectively equipped with two pinch valves and the first interface that is opened between these two pinch valves and the second interface; The left end of described two branch pipeline is interconnected to form the 3rd interface; The right-hand member of described two branch pipeline is interconnected to form the 4th interface;
3rd branch pipeline, its left end and right-hand member are connected with described first interface and the second interface respectively, and described oxygen supply, liquid storage bottle and peristaltic pump are connected in series by described 3rd branch pipeline;
Connect the 4th branch pipeline of described first interface and cell factory bio-reactor; And
Connect the 5th branch pipeline of described second interface and cell factory bio-reactor.
2. the system of large scale culturing zooblast as claimed in claim 1, is characterized in that: described cell factory bio-reactor is provided with the first port and the second port that are connected with described 4th branch pipeline and the 5th branch pipeline respectively.
3. the system of large scale culturing zooblast as claimed in claim 2, is characterized in that: described cell factory bio-reactor is microcarrier bio-reactor.
4. the system of large scale culturing zooblast as claimed in claim 1, is characterized in that: this system is also included in thread detector; Described circulation line also comprises and is connected to the 6th branch pipeline that is in described 3rd branch pipeline and that be connected in series with described on-line monitoring device with T-valve.
5. the system of large scale culturing zooblast as claimed in claim 1, is characterized in that: this system also comprises the heating plate be serially connected between described peristaltic pump and liquid storage bottle by described 3rd branch pipeline.
6. the system of large scale culturing zooblast as claimed in claim 1, is characterized in that: described oxygen supply is membrane oxygenator.
7. the system of the large scale culturing zooblast as described in claim 1 ~ 6 any one, is characterized in that: this system also comprises the master controller regulating and affect the controling parameters of each integral part of cell culture condition in this system.
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