CN101100641A - Curtain-shape anchoring culture bioreactor and method for culturing plant cell, tissue or organ using the same - Google Patents
Curtain-shape anchoring culture bioreactor and method for culturing plant cell, tissue or organ using the same Download PDFInfo
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- CN101100641A CN101100641A CNA2007100351563A CN200710035156A CN101100641A CN 101100641 A CN101100641 A CN 101100641A CN A2007100351563 A CNA2007100351563 A CN A2007100351563A CN 200710035156 A CN200710035156 A CN 200710035156A CN 101100641 A CN101100641 A CN 101100641A
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Abstract
A curtain-type anchoring biological culturing reactor and its method for culturing vegetable cell, tissue or organs are disclosed. The reactor consists of bed(E), curtain-type netted double-layer anchoring culturing bed(A), supply system(B), hanger(C), composite circulation feeder(F) and disposable sterile sealing film(G). (A) is hung on (C) and has (E), (F) is arranged on top of (E), (E) is sleeved with (G). It's simple, safe and cheap. It has less consumption and can be used for continuous production.
Description
Technical field
The present invention relates to a kind of bio-reactor and utilize this bio-reactor to carry out a large amount of cultured method of vegetable cell, tissue or organ, be applicable to the cultivation of cell, tissue or the organ of microorganism, plant, animal, belong to biotechnology culture plant cell engineering field, or belong to crossing domain---the biological industry production field of biotechnology and Chemical Engineering and Technology according to range of application.
Background technology
In order to realize vegetable cell, tissue and organ are cultivated batch production in a large number and are produced useful secondary metabolite, from Routies in 1956 and Nichell apply for the 1st utilize the patent of the useful secondary metabolites of bubbling column reactor culturing plants cells produce since, mechanical agitation type STR reactor has been developed in the whole world, comprise spiral agitating type STR withspiral impeller, promote stirring-type STR with cell lift impeller, the film ventilating/stirring, common stirring-type STR with combined membrane aerator and agitator[Huang is gorgeous, Zhao Dexiu, Li Zuohu. BULLETIN OF BOTANY Vol., 2001,18 (6): 665-671], duplex band shape stirring rake reactor Doublehelical-ribbon impeller bioreactor[Jolicoeur M, et al.Biotech.Bioeng.1992,39:511-521], stainless (steel) wire gas distributor cell hoisting type stirring rake coupled mode reactor, micropore metal silk screen air-distributor three bladed propeller reactor MRP[clock Kien Giang, Shen Linnan, Wang Sijing. the latest developments of culture plant cell engineering [J]. industrial microorganism, 1995,25 (1): 25-29] etc.; Air lift type (AL) reactor comprises outside circulation gas injection lift-type External boop airlift, interior circulation air lift type Internal boop airlift[Liu Chun court, Wang Yuchun, Ou Yangfan etc. Botany Gazette, 1999,41 (2): 181-183; Sajc L, Obradovic B, VukovicD, et al.Biotechnol Prog, 1995,11:420], internal cross-current type Alicrof, Air lift internalcross flow[ZL96222807], guide air lift type Dranght tube airlift[Huang is gorgeous, Zhao Dexiu, Li Zuohu. BULLETIN OF BOTANY Vol., 2001,18 (6): 665-671], circulation bioreactor in the stainless (steel) wire guide shell, center polyethylene fixed air lift-type, circulation air lift type in the built-in multilayer screen cloth [Liu Chunchao, Wang Yuchun, Ou Yangfan etc. chemical industry metallurgical, 1999,20 (3): 329-336] etc.; Bubbling column reactor comprises that bubbling style Bubble column, inclination bubbling style Slanted bottom bubble column[Huang are gorgeous, Zhao Dexiu, Li Zuohu. BULLETIN OF BOTANY Vol., 2001,18 (6): 665-671] etc.; Revolving drum type bioreactor; The fluidized bed bio reactor comprises bubbling style circulating fluid bed reactor [Dubuis B, Kut O M, Prenosil J E.Plant Cell Tissue Organ Cult, 1995,43:171]; Packed bed bio-reactor [Panda A K, Mishhra S, Bisaria U S, Bhojwani SS.Plant cell reactor, a perspective.Enzyme Microb Technol, 1989,11:386-397]; Membrane bioreactor, comprise hollow-fibre membrane, spirally coiling reactor [Liu Chunchao, Wang Yuchun, Ou Yangfan etc. chemical industry metallurgical, 1999,20 (3): 329-336], composite film reactor, agitated-film reactor [Panda A K, MishhraS, Bisaria U S, Bhojwani S S.Plant cell reactor, a perspective.Enzyme MicrobTechnol, 1989,11:386-397], dual hollow fiber reactor Dual hollow fibrereactor[Kim.D.J., Chang, H.N.Biotech.Bioeng.1990,36:460-466], drip membranous type trickling film reactor [Yokoi, H.et al.J.Ferment.Bioeng.1993,75:48-52] etc.; Micelle fixed reactor [Panda A K, Mishhra S, Bisaria U S, Bhojwani S S.Plant cellreactor, a perspective.Enzyme Microb Technol, 1989,11:386-397]; Continous pouring formula bio-reactor [Lan Wenzhi, surplus Longjiang, Wu Yuanxi. Central China University of Science and Technology's journal (natural science edition), 2002,30 (5): 91-93]; The atomizing reactor comprises built-in screen cloth spraying type, built-in ring type filling spraying type, ultrasonic atomization reactor, sprays spray flow pouring reactor etc.; Artificial hydrophilic fibers guide flow type multilayer column plate bio-reactor; The rectangle bio-reactor; The bell reaction device; Radial flow multilayer column plate bio-reactor; Intermittent immersed reactor; Spraying gun separates band of gas formula culture systems with incubator; Spin filtering type reactor [Liu Chunchao, Wang Yuchun, Ou Yangfan etc. chemical industry metallurgical, 1999,20 (3): 329-336]; Trickle-bed reactor; Gas blowing type reactor [Mckelvey SA, etal.Biotechnol Prog, 1993,9:315-312] [Rama Krishnan D.et al.BiotechnologyTechniques, 1994,8 (9): 639-644]; Spouting of liquid formula loop reactor [Buitelaar RM, etal.Enzy Microb Technol, 1991,13:487-494]; Centrifugal impeller reactor centrifugal impellerbioreactor[Wang S J, Zhong J J.Biotechnol Bioeng, 1995,51:520]; Cell lift-type reactor cell-lift bioreactor[clock Kien Giang, Shen Linnan, Wang Sijing. the latest developments of culture plant cell engineering [J]. industrial microorganism, 1995,25 (1): 25-29]; Upflow bioreactor lift-streambioreactor[Liu Zhiwei, Guo Yong, open morning. the progress of culture plant cell bio-reactor [J]. modern chemical industry, 1999,19 (8): 14-16] etc., scale is raised to 75000 liters and is not waited by several, the plant of having used has ivy, Echinacea purpurea Moench, Asian puccoon, genseng, Herba Artemisiae annuae, the coptis, tens of kinds of Ramulus et folium taxi cuspidatae etc., Japan takes the lead in having realized Asian puccoon, genseng, coptis cell large scale is cultivated the preparation shikonin, the suitability for industrialized production of ginsenoside and berberine [Hara Y.Research on the production of useful compounds by plant cell cultures in Japan.In:Di Cosmo F, Misawa M, eds.Plant Cell Culture Secondary Metabolism TowardIndustrial Applicatong.Boca Ratton; CRC Press, 1996,187-202].But most of technology still rest on laboratory or industrialization scale-up stage.These researchs and technological achievement all have a common feature, promptly adopt and at all events plant reactor, and what it followed substantially all is relevant theory, method and the principle of microbial fermentation, cultivate and all carry out in encloses container, all need step to amplify.Because the culture cycle of plant is more much longer than microorganism, thereby the insoluble problems such as lasting maintenance of the various uncertainties that caused in digging system, cell growth and the conglomeration of the aseptic condition of the consistence control of the long-term lasting maintenance that continues all will face in the culturing process good hereditary property of vegetable cell, tissue and organ and physio-biochemical characteristics, cell growth, culturing process and each physical and chemical factor, gas-liquid, liquid-solid and energy mass transfer characteristic, be difficult to the industrialization amplification more.Too high cost of investment and cause too high operation cost because of too much, long nonproductive process, add uncertainty production brings to large-scale industrialization too high, the unaffordable commercial risks of enterprise in the too many control, seriously hindered the realization of its large-scale commercial applications production.Particularly step amplifies, especially with the amplification at double of cost risk, to the end, as long as once failure just may cause business failure.Vegetable cell, tissue and organ culture required time are long, and parameter is too many in the process, only control aseptic just very difficult.So if the cost undue concentration of disposable input, the trade investment risk of its large scale culturing suitability for industrialized production is just huge unusually, any enterprise all can't bear.
In addition, the vegetable cell of reporting comprises tissue at present, organ is cultivated the useful secondary metabolite technology of producing in a large number, its method of taking no matter, approach, means etc. how, net result all without exception the plant that makes becomes callus by the complexity simple plant that becomes by the explant dedifferentiation that complex construction is arranged, become rudimentary or comparatively original by senior by comparatively evolving to, promptly become low the grade or the primary cell mass by the higher plant that various organ differentiation are arranged, even unicellular, become vegetative propagation by sexual propagation, become heterotrophism by autotrophy, by Lu Sheng to aquatic cell or the cell mass that becomes fluid suspension culture by the living plant in ground.The promptly existing a large amount of whole processes of cultivating of cell, tissue, organ that plant is implemented, actual capabilities are the processes that the counter-evolution domestication is cultivated, people's attempt is tamed plant culturing as microorganism, can produce by the mode of fermentation, the result has but ignored the form that this process has not only changed plant, has more likely changed physio-biochemical characteristics and the metabolism of plant.Therefore, aforementioned vegetable cell is cultivated suitability for industrialized production variety of issue that useful secondary metabolite is met in a large number, should have some relations therewith.
Summary of the invention
Purpose of the present invention is exactly the problems referred to above that exist at the bio-reactor that present culture plant cell engineering field is developed; a kind of curtain-shape anchoring culture bioreactor is provided and utilizes this bio-reactor to carry out vegetable cell; tissue and a large amount of cultured method of organ; it is applicable to vegetable cell; tissue and organ growth and production are cultivated; being particularly useful for long-term continuous large-scale production cultivates; also more suitable to microorganism and animal; have simple in structure; low cost of manufacture; energy consumption is low; nonproductive process is short and simple; be not subjected to the uncertain restriction of culturing process; the commercial operation risk is low; process control is simply easy; being easy to industrialization amplifies; no scale restriction; multi-use; but cultured continuously is gathered in the crops repeatedly; be easy to advantages such as online in good time enforcement regulation and control; can greatly reduce the large-scale production cost of the manufacturing cost and the plant isolated culture of bio-reactor, disperse to dissolve scale and risk.
For achieving the above object, embodiment of the present invention are: a kind of curtain-shape anchoring culture bioreactor, comprise that the netted two-sided anchoring of curtain formula is culture bed, the keeping system, suspension system, culture bed the hanging on the suspension system of the netted two-sided anchoring of curtain formula, the netted two-sided anchoring of described curtain formula is culture bed to have a body, compound streamer feed supplement device and disposable sterilized envelope film, the bed body has the double-deck net of supporting, support that net is installed in the frame, two support to have between net the space to form the bed inner chamber, supporting to be attached with the suede adhesion layer on the net, compound streamer feed supplement device is installed in the top of a body, has streamer feed supplement groove, streamer feed supplement groove is communicated with the bed inner chamber and the liquid-inlet pipe that is arranged on the compound streamer feed supplement device of bed body, and the bed extracorporeal set is the aseptic envelope film of property once, and disposable sterilized envelope film is provided with liquid discharge pipe and air communication pipe; The keeping system has tubing and container for storing liquid, and curtain-shape anchoring culture bioreactor is communicated with container for storing liquid by liquid-inlet pipe and tubing.
Keeping of the present invention system is a circulation keeping system, have container for storing liquid and filter container for storing liquid down, container for storing liquid is communicated with liquid-inlet pipe on the compound streamer feed supplement device by tubing, can the netted two-sided anchoring of the injection curtain formula of the nutrient solution in the container for storing liquid is culture bed by air pressurization pump, filtering container for storing liquid down is communicated with liquid discharge pipe under the disposable sterilized envelope film by liquid returning tube, return line and following the filtration between the container for storing liquid are provided with the instantaneous sterilization device, container for storing liquid and following the filtration between the container for storing liquid have communicating pipe, the backflow nutrient solution that descends to filter in the container for storing liquid can be squeezed in the container for storing liquid by vacuum pump.
Described suspension system has spherical slide rail, is provided with a plurality of spherical sliding hanging of hanging in the spherical slide rail, and the netted two-sided anchoring of a plurality of curtain formulas is culture bed can be suspended on spherical sliding the hanging on of hanging.
Gapped regulation and control plate between two supporting networks of described bed body, frame are provided with the thick frame of limit, and the upper side frame of frame disposes load-bearing outward and connects crossbeam.The suede adhesion layer is that the fiber with strongly hydrophilic is made.
Described compound streamer feed supplement utensil has riveting door bolt and rivet hole, by riveting door bolt and rivet hole compound streamer feed supplement device is installed on the top of a body, and the top of compound streamer feed supplement device is equipped with suspension ring.
The plastic film bag that described disposable sterilized envelope film is made for the high-temperature resistance plastice film, Yi Bian it has the adhesive tape for the treatment of at least, all the other bandings close, and bottom is obliquely installed, and liquid discharge pipe is located at following low edge.
When indoor when carrying out vegetable cell, tissue or organ culture, illumination if desired, the present invention also needs lighting system, described lighting system can be a solar collector transmission lighting system, form by sunshine collector, transmission cable and luminous end, sunshine collector is installed on the roof of culturing room, and it is between any two culture bed that luminous end is installed in the netted two-sided anchoring of curtain formula.
The present invention also comprises press for extracting juice formula sterilization cabinet, press for extracting juice formula sterilization cabinet (H) side of having body cabinet gap frame C, in be provided with the movable plate that presses, frame is equipped with handwheel outward, can move the control activity by the operation handwheel and press plate.
Described press for extracting juice formula sterilization cabinet also is furnished with buffering dividing plate that adapts with bed body length and width and the bed inner chamber infill panel that adapts with bed inner chamber length and width.The buffering dividing plate is the middle double-layer plate that accompanies rubber pad.
The present invention also provides a kind of method of utilizing above-mentioned curtain-shape anchoring culture bioreactor to carry out vegetable cell, tissue and organ culture, may further comprise the steps:
A, inject for the bed body to solidify the substratum horizontal high voltage steam sterilizing sterilization of going forward side by side;
B, will treat that cultivated material is anchored on the suede adhesion layer of a body by solidifying culture medium inoculated;
After c, the inoculation of bed body, under aseptic condition, it is culture bed that aseptic successively respectively separately installation load-bearing connection crossbeam, compound streamer feed supplement device and disposable sterilized envelope film are assembled into the netted two-sided anchoring of curtain formula, then with culture bed being suspended on the suspension system of the netted two-sided anchoring of each curtain formula, push culturing room's preposition, and with the netted two-sided anchoring of each curtain formula liquid-inlet pipe and liquid discharge pipe and the keeping system connectivity on culture bed;
D, adjusting are cultivated room temperature, humidity to required value, behind required cultivation certain hour, start the keeping system, beginning supplements the nutrients in the bed body, reach increment until cultivating bulk-growth with results value, close with the keeping system between be connected valve, the netted two-sided anchoring of aseptic dismounting curtain formula culture bed with the keeping system between be connected;
E, the culture bed immigration of the netted two-sided anchoring of curtain formula that will be to be gathered in the crops aseptic processing room, tear cover disposable sterilized envelope film thereon, along the culture bed frame of the netted two-sided anchoring of curtain formula aseptic flat cut or straight snips under grow the cultivation body of frame, remaining part is retained in the seed of cultivating as next cycle on the bed surface;
F, change a new disposable sterilized envelope film and again culture bed cover is sealed, push back culturing room, a beginning new round is cultivated.
Among the step a, it is described that will to solidify method that substratum injects bed body and sterilization be the bed body that load-bearing connects crossbeam will also not to be installed insert the sterilizing bag that is contained in advance in the press for extracting juice formula sterilization cabinet, insert a buffering dividing plate between any two at the bed body, in the two bed inner chambers of supporting between the net, insert a bed inner chamber infill panel again; Rotate press for extracting juice formula sterilization handwheel cashier's office in a shop and move the movable plate that presses, with each the member jam-packed that inserts in the press for extracting juice formula sterilization cabinet; Be fed into a body with preparing not solidified curing substratum in advance from the upper side frame of bed body and the slit between the suede adhesion layer, substratum is full of between the buffering dividing plate of suede adhesion layer and insertion; After sterilizing bag tightened press for extracting juice formula sterilization cabinet H being pushed the high pressure steam sterilization chamber sterilizes and gets final product.
Among the step a, also can earlier the bed body be carried out high pressure steam sterilization, then between aseptic technique in the perfusion aseptic curing substratum.
Among the step b, after treating that cultivated material is seeded on the curing substratum of a body,, on the bed body, make and treat that cultivated material embeds in the curing substratum deeply up to fully contacting with the suede adhesion layer, so that cultivated material can continue to obtain the required nutriment of its growth by the suede adhesion layer with buffering dividing plate concora crush.
The method of injecting curing substratum, disinfection with high pressure steam sterilization and inoculation for before the step c of the present invention the bed body also can be, earlier the bed body is carried out high pressure steam sterilization, then substratum is planted plate simultaneously together with culturing cell and be anchored on the suede adhesion layer of a body, plant plate thickness so that cell grows up to can fully contact with the suede adhesion layer behind the callus is as the criterion.
It is the theory and the principle of the culture plant cell engineering of reference with the microbial fermentation at present that plant culturing bio-reactor design construction principle provided by the present invention is different from, but higher plant is highly broken up the bionical of constitutional features.Higher plant is under state of nature, organ differentiation is promptly arranged, differentiation in a organized way again, and the division of labor on the function is arranged according to differentiation, as wherein microtubule tissue is to connect each cell, tissue and organ, and the transfusion tissue of material and signal conduction is provided for its growth, differentiation and defence etc.Therefore, the present invention according to higher plant at cell, the level that the height division of labor on tissue and the organ level and mutual organic connections and target are cultivated body anatomical structure of living in former plant materials, so that plant culturing body and organic the combining closely of culture bioreactors energy, be grown to purpose continually and steadily, rather than after plant materials cultivated the biological condition tame near microorganism it is grown in culture bioreactors as microorganism, bio-reactor is only just cultivated the implements of usefulness, the two is not closely linked each integral part in the plant plant, be full of vascular bundle in the bionic plant fruit, aim at embryo provide moisture and nutrition plant placenta design construction the netted two-sided anchoring of curtain formula culture bed, the net suede that is made into the high-hydrophilic fiber replaces the vascular bundle on the plant placenta, circulation provisions system continuously flows to moisture and nutrition by this bionical " microtubule system " adheres on the plant culturing body that is anchored on this net suede by solidifying substratum as plant " embryo ", along with the continuous growth of cultivating body, the two embryo and placenta just as plant are closely linked, form integral body until combination, so only need inoculation once, just but repeated multiple times ground gathers in the crops its biologic, reach the continuous large-scale batch production and produce the purpose of plant culturing body, and make vegetable cell, the a large amount of initial costs of investment of cultivating of tissue and organ are greatly reduced, and disperse to dissolve the investment scale and risk that is caused because of the capital contribution undue concentration.
Description of drawings
Fig. 1 forms synoptic diagram for the system of curtain-shape anchoring culture bioreactor of the present invention;
Fig. 2 is the axle side synoptic diagram after bed body of the present invention, compound streamer feed supplement device and the assembling of disposable sterilized envelope film form the culture bed A of the netted two-sided anchoring of curtain formula;
Fig. 3 is the cut view of Fig. 2 along the I-I direction;
Fig. 4 is the axle side synoptic diagram after bed body of the present invention and the assembling of compound streamer feed supplement device;
Fig. 5 is the axle side synoptic diagram of bed body of the present invention;
Fig. 6 is the layered structure axle side synoptic diagram of bed body of the present invention;
Fig. 7 is the vertical profile structural representation and the positive elevational schematic view of the compound streamer feed supplement of the present invention device;
Fig. 8 is the sectional view of Fig. 7 along the II-II direction;
Fig. 9 is the structural representation of the disposable sterilized envelope film of the present invention;
Figure 10 is the structural representation of suspension system of the present invention;
Figure 11 hangs axle side synoptic diagram behind the installation in position for curtain-shape anchoring culture bioreactor of the present invention;
Figure 12 is packed into the axle side synoptic diagram of press for extracting juice formula sterilization cabinet of a body of the present invention;
Figure 13 is housed to a body for the present invention and the axle side synoptic diagram of the press for extracting juice formula of a body of not packing into sterilization cabinet.
Wherein:
The culture bed B-of the netted two-sided anchoring of A-curtain formula supports G-the aseptic envelope H-film of property of the C-of system suspension system D-solar collector transmission lighting system E-bed body F-compound streamer feed supplement device press for extracting juice formula sterilization cabinet
The 1-load-bearing connects sliding the hanging of the crossbeam 11-streamer feed supplement spherical suspension of groove 21-
2-limits the spherical slide rail of thick frame 12-groove cover 22-
3-frame 13-riveting door bolt 23-container for storing liquid
4-lower frame 14-rivet hole 24-tubing
5-upper side frame 15-liquid-inlet pipe 25-goes up air communicating pipe
6-suede adhesion layer 16-suspension ring 26-air pressurization pump
7-supports net 17-to treat adhesive tape 27-communicating pipe
8-gap regulation and control plate 18-film 28-feed supplement pipe
9-pre-opened hole 19-air communication pipe 29-air-pressure tube
10-bed inner chamber 20-liquid discharge pipe 30-goes up valve tube
The last liquid discharge pipe 47-of 31-screw mandrel 63-vacuum pump main valve
32-filters container for storing liquid 48-handwheel 64-steam inlet valve down
33-filtering net 49-presses vacuum pipe valve under the base plate 65-
34-filter screen operation handle arm 50-frame beam 66-sterilization filter
Air communication pipe 51-buffering dividing plate 67-feed supplement pipe valve under the 35-
Valve tube 52-universal wheel 68-is communicated with pipe valve under the 36-
37-vacuum pump 53-bed inner chamber infill panel 69-air communication pipe valve
Liquid discharge pipe 54-air communication pipe valve 70-sterilization filter under the 38-
39-instantaneous sterilization device 55-sterilization filter 71-is the liquid pipe valve next time
40-liquid returning tube 56-throttling valve 72-goes up the discharge opeing pipe valve
Discharge opeing pipe valve under the 41-sunshine collector 57-arm variable valve 73-
42-transmission cable 58-sterilization filter 74-sterilizer admission valve
The luminous terminal 59-pneumatics pipe valve 75-sterilizer gas outlet valve of 43-
44-is front apron 60-pneumatics manifold valve 76-sterilizer water discharge valve fixedly
45-is rear deflector door 61-sterilization filter 77-steam inlet duct fixedly
The movable plate 62-that presses of 46-goes up vacuum pipe valve 78-backflow manifold valve
Embodiment
As Fig. 1, shown in Figure 11, a kind of curtain-shape anchoring culture bioreactor, comprise the culture bed A of the netted two-sided anchoring of a plurality of curtain formulas, the circulation keeping B of system and suspension system C, the culture bed A of the netted two-sided anchoring of curtain formula is suspended on the suspension system C, and the culture bed A of the netted two-sided anchoring of each curtain formula all is connected with the circulation keeping B of system.
As shown in Figure 1, the circulation keeping B of system is actual to be that system is supported in an independently aseptic feed supplement, by last container for storing liquid 23 and filter container for storing liquid 32 and accessory tubing 24 thereof down, go up air communicating pipe 25, air pressurization pump 26, communicating pipe 27, feed supplement pipe 28, air-pressure tube 29, go up valve tube 30, go up liquid discharge pipe 31, filtering net 33, filtering net operation handle arm 34, down air communication pipe 35, down valve tube 36, vacuum pump 37, liquid discharge pipe 38, high-temperature instantaneous sterilization device 39 and liquid returning tube 40 are formed down.This system all and substratum contact part are stainless steel and make, and can wash in the system, high-temperature steam sterilization, carry out technological operations such as high-temperature instantaneous sterilization and filtration to cultivating tail washings.Described circulation supports the container for storing liquid 23 of the B of system to be connected with the liquid-inlet pipe 15 of each culture bed E by tubing 24, and tubing 24 is at the bottom of the access of container for storing liquid 23 tops and extending jar.Communicating pipe 27 1, end inserted from last container for storing liquid 23 top sidewalls, and the mouth of pipe is stretched at last container for storing liquid 23 near tops, and the other end is from the top access of filtration container for storing liquid 32 down, at the bottom of passing filtering net 33 and directly reaching jar.Filtering container for storing liquid 32 down is connected by the culture bed liquid discharge pipe 20 of disposable aseptic envelope film G below of following liquid returning tube 40 and each, liquid returning tube 40 is through high-temperature instantaneous sterilization device 39, by under filter container for storing liquid 32 tops and insert, the mouth of pipe is stretched above filtering net 33, allows phegma reflux behind disinfection filtering and filters container for storing liquid 32 down.Implement sterile pressurized for last container for storing liquid 23 by air pressurization pump 26 and wherein nutrient solution is sent to each is culture bed, and regulate and control the keeping amount by regulating and control wherein the size of pressure.Make down to filter in the container for storing liquid 32 by vacuum pump 37 to produce negative pressure, into filter container for storing liquid 32 down to impel the unabsorbed nutrient solutions in an inner chamber 10 backs of flowing through to reflux.Make in the container for storing liquid 23 by vacuum pump 37 to produce negative pressure, reflux by communicating pipe 27 and advance to go up container for storing liquid 23 so that filter nutrient solution in the container for storing liquid 32 down.Last container for storing liquid 23 all is equipped with air valve and air filtration Sterilizers with the pipeline 29,30,36 that air pressurization pump 26 is connected with vacuum pump 37 respectively with the following container for storing liquid 32 that filters.Two tank tops were equipped with the last air communicating pipe 25 and the following air communication pipe 35 of band air filtration Sterilizers and pressure lock respectively, made container for storing liquid 23 and the following container for storing liquid 32 that filters can carry out air inerchange outward with jar in case of necessity.Communicating pipe 27, nearly container for storing liquid 23 ends of going up were connected to a feed supplement pipe 28 with threeway, were equipped with to use for last container for storing liquid 23 additional nutrient solutions.The last container for storing liquid 23 and the following end of filtering container for storing liquid 32, are taper shape, and awl bottom be equipped with respectively liquid discharge pipe 31, on discharge opeing pipe valve 72 and following liquid discharge pipe 38, discharge opeing pipe valve 73 down, for cleaning the usefulness of discharging water of condensation behind tank body sewage effluent and the high-temperature steam sterilization.Filter down container for storing liquid 32 internal upper part, be positioned at down the below of liquid returning tube 40 have two respectively solid welding filter on container for storing liquid 32 sidewalls down, to intermediate, inclined, middle movable, as to overturn downwards filtering net 33, with to filtering from the following residual media of liquid returning tube 40, middle a slice filter screen can be movable, operation filtering net operation handle arm 34 can make its downward upset, is convenient to clean be trapped in online resistates.
As Fig. 2-shown in Figure 6, the culture bed A of the netted two-sided anchoring of curtain formula, have a body E, compound streamer feed supplement device F and disposable sterilized envelope film G, bed body E is the window formula curtain shape structure about the about 2cm of an integral thickness, have two-layer support net 7, support that net 7 is attached with suede adhesion layer 6, support net 7 and suede adhesion layer 6 to press embedding to be installed in the frame 3, frame 3 is provided with the thick frame 2 of limit, and two-layer support net 7 is over against accompanying regulation and control plate 8 in gap between the thick frame 2 vertical frames of limit.2 load-bearing that bed body E top is two connect crossbeam 1 usefulness screw mandrel and nut is installed on the body E, be the bearing beam of a body E when hanging as curtain, on pre-opened hole 9 for cover riveting compound streamer feed supplement device F is arranged, just install under aseptic condition behind bed body E perfusion culture base, high pressure steam sterilization, the size of its size, thickness, material, mechanical characteristic and last pre-opened hole thereof and what are all decided because of size, the weight of bed.Limit thick frame 2, suede adhesion layer 6, support net 7 and be clipped in two and support that gap regulation and control plate 8 each layers between net closely are fixed together with screw, nut, also can press and be fixed together, all must and support net 7 enough to tighten when each layer is fixed together, guarantee keeping the certain intervals space between the two support nets 7 under the interval action of gap regulation and control plate 8 suede adhesion layer 6.Gap regulation and control plate 8 thick 1.2mm ~ 2.3mm, wide, long with the vertical frame 3 of the thick frame 2 of limit, being clipped in two supports between the net 7, make two to support the net interbody spacer to form the thin layer feed supplement passage bed inner chamber 10 of one 1.2mm ~ 2.3mm, pass when feed supplement flows down by this inner chamber 10 and support net 7 to infilter suede adhesion layer 6, the cilium suede by suede adhesion layer 6 cultivates body for the vegetable cell that sticks on the culture bed A of the netted two-sided anchoring of curtain formula, tissue, organ etc. nutrient solution water conservancy diversion mass transfer.Respectively have on the two sides of gap regulation and control plate 8 one with the corresponding strip projection of the thick frame 2 vertical frame inner surface recesses of limit, and the two can be chimeric mutually, with embedding press fit suede adhesion layer 6 with support net 7, the prevention nutrient solution oozes out from the both sides of bed.Suede adhesion layer 6 is to utilize to have the made fibrous reticulum suede of extremely strong hydrophilic fiber, it is netted towards the one side in the bed, one side outside the bed thick cilium suede of 5mm of having an appointment, working to adhere to curing substratum and water conservancy diversion nutrient solution supplies with nutrition for cultivation body mass transfer, it is the key member that adheres to substratum, vegetable cell, tissue and organ, its upper limb is mutually concordant with the upper limb of thick frame 2 upper side frames 5 of limit, from its upper limb downwards on the load-bearing tie-beam 1 riveting door bolt pre-opened hole 9 places also correspondingly be equipped with pre-opened hole, with favourable compound streamer feed supplement device F suit.Support net 7 to be pressed solidly a kind of high strength net 8 two of gap regulation and control plates; get up and protect suede adhesion layer 6, prevent the effect that inner chamber 10 blocks; its upper limb is mutually concordant with suede adhesion layer 6, with suede adhesion layer 6 on the corresponding position of pre-opened hole also be equipped with pre-opened hole.
As Fig. 3, Fig. 7, shown in Figure 8, compound streamer feed supplement device F is made up of streamer feed supplement groove 11 and groove cover 12 and accessory riveting door bolt 13, rivet hole 14, liquid-inlet pipe 15 and suspension ring 16, be to aim at the parts that the culture bed A after-teeming of the netted two-sided anchoring of curtain formula distributes nutrition, aseptic being installed in is poured on the bed body E that solidifies behind substratum, high pressure steam sterilization, the aseptic installation load-bearing crossbeam 1.It is that all the other were high strength, thermostability engineering plastic is made stainless steel was made except that suspension ring, and makes after sterilising treatment is that sterile product, sterile packed are standby, but high temperature and high pressure steam is sterilized, used repeatedly.Described streamer feed supplement groove 11 is that groove cover 12 shape of cross sections that are clipped in innermost layer add isosceles triangle by a rectangle and formed the pentagon elongated slot in fact, the pentagonal end, be the groove top up, pentagonal top is bottom land down, groove is equipped with a liquid-inlet pipe 15 on the top, bottom land trilateral wedge tip has 1.0mm～1.8mm narrow slit, extend into a body E two during installation and support in the bed inner chamber 10 of 7 on net that the feed supplement nutrient solution is gone in the bed inner chamber 10 of a body E through the slit beam of feed supplement groove 11 bottoms by liquid-inlet pipe 15.Groove cover 12 has suspension suspension ring 16, and hanging suspension ring has riveting door bolt pre-opened hole 14 for 16 times.Streamer feed supplement groove 11 and bed body E are equipped with an end uniform thickness of load-bearing tie-beam 1, groove overlaps 12 inner chambers and bed body E, and that one end etc. of load-bearing tie-beam 1 is housed is big, during assembling bed body E is equipped with load-bearing tie-beam 1 end insertion groove cover 12, in riveting door bolt pre-opened hole 14, latch riveting door bolt 13 and get final product.When the culture bed A of the netted two-sided anchoring of curtain formula was hanging on the suspension system C, its all wts was by 16 carryings of the suspension suspension ring on the groove cover 12, and culture bed A hangs one of key member of load-bearing so compound streamer feed supplement device F is again the netted two-sided anchoring of curtain formula.
As Fig. 2, shown in Figure 9, disposable sterilized envelope film G is actual to be exactly a plastic film bag of being made by high-temperature resistance plastice film 18, remain adhesive tape 17, air communication pipe 19 and liquid discharge pipe 20 on it, has good light transmittance, airtight, plaing a part has the collarium border to separate, keep inner aseptic with extraneous the culture bed A of the netted two-sided anchoring of described curtain formula.Make after sterilization becomes sterile product standby, be expendable consumed product, promptly when a culture cycle finished, gathers in the crops the cultivation body, disposable sterilized envelope film G just was torn and changes new film into.Air communication pipe 19 hot pressing rivet clasps in film bag front or upper rear portion by edge, be in the bed with the outer passage that carries out air inerchange of bed, strainer is housed on it, can filter airborne harmful bacterium, guarantee that an interior air and an outer air carry out secure exchange.The unabsorbed remaining culture liq of an inner chamber of flowing through refluxes through liquid discharge pipe 20 and advances to circulate the B of keeping system.The below of disposable sterilized envelope film G is obliquely installed, and liquid discharge pipe 20 is fixed on the top ends of below of film bag by hot sealing, and inwardly puts in about 3cm, and seal at the top, and interior Na Yekou opens in the side at pipe top, in case the cultivation body that the falls obstruction mouth of pipe.Before the installation, the side that film is long and the lower tilt edge of film are seal strip, and residue two sides are band to be sealed, belt edge to be sealed be scribble viscose glue treat adhesive tape 17, layer protecting film is arranged on it, tear protective membrane during installation and can implement to bond and seal film.The bed body E that is poured behind substratum, sterilization, inoculation, aseptic installation load-bearing crossbeam 1 and the compound streamer feed supplement device F gets final product suspension positioning after the disposable aseptic envelope film G sealing on the quilt cover again, opens liquid discharge pipe 20 and implements aseptic the butt joint with the circulation provisions B of system.
As Fig. 5, shown in Figure 12, the thin about 0.3cm～0.5cm of outward flange of the vertical frame 3 of the thick frame 2 of limit of bed body E and lower frame 4, the thicker about 0.5cm～0.8cm of preglabellar field is equivalent to the thickness that solidifies substratum that adheres to, and plays the restriction bed surface and pours into curing substratum thickness.It flat one faces an internal layer, one of injustice faces outside the bed, the thicker side frame of frame outside surface is straight and strict in same plane so that its in press for extracting juice formula sterilization cabinet H can and 44 seamless presses for extracting juice of buffering dividing plate tight, make institute pour into the curing substratum and when injection and autoclaving, can not leak.The upper side frame 5 of frame is thinner, and other frames back are thick, and 6 of the suede adhesion layers of upper side frame 5 and internal layer have an about 3mm～5mm slit, just stitches injection in press for extracting juice formula sterilization cabinet H when substratum is solidified in perfusion thus to bed surface.
As shown in figure 10, suspension system C by the sphere of band suspension hook hang sliding hang 21 and spherical slide rail 22 formed, the two is all made with stainless steel or highstrenghtpiston, spherical slide rail 22 is fixed on screw, nut on the steel I-beam on culturing room top, its design strength is according to the size dimension of the culture bed A of the netted two-sided anchoring of curtain formula and overall weight and decide, and is culture bed and can slide and be as the criterion freely to be enough to hang this of any amount.Described spherical the suspension slidingly hangs 21 for having the solid sphere of suspension hook, and the section of spherical slide rail 22 be circular, and spherical the suspension slidingly hung 21 and can be entrenched in just that chute is interior to be free to slide along spherical slide rail 22.Spherical slide rail 22 is fixed on the steel I-beam of indoor smallpox end face of the culture bed A transfer path of curtain-shape anchoring.The spherical suspension slidingly hung 21 and can be done 90 ° of right angles or other is turned at any angle along spherical slide rail 22.Spherical hang slidingly hang 21, the size of spherical slide rail 22 diameters, size and the weight on the culture bed A of the netted two-sided anchoring of curtain formula requires to decide to the strength of materials and mechanics fully.
As shown in Figure 1, solar collector transmission lighting system D is made up of sunshine collector 41, transmission cable 42 and luminous terminal 43, and luminous terminal 43 can stretch into the culture bed A of the netted two-sided anchoring of curtain formula between any two.Solar collector transmission lighting system D is an independent light lighting system, a plurality of huge sunshine collectors 41 are installed on the roof of culturing room, be transferred to by transmission cable 42 outdoor natural light that it is collected and be clipped in luminous terminal 43 between culture bed of installation in position, for the plant culturing body on the bed provides illumination.
As shown in Figure 12 and Figure 13, press for extracting juice formula sterilization cabinet H is an independently auxiliary sterilization equipment of bed body E special use, actual be one by fixing front apron 44, fixedly rear deflector door 45, movable press for extracting juice plate 46, handwheel 48, press for extracting juice base plate 49 and frame beam 50 are formed.The movable press for extracting juice on the plate 46 is equipped with 1～2 crin bar 47, after passing, screw mandrel 47 refills the handwheel 48 of band internal thread from fixing rear deflector door 45, as long as rotate handwheel 48, just can the drive activity press plate 46 and move forward and backward, also do not load onto load-bearing in the frame and connect a body E jam-packed of crossbeam 1 or loosen thereby will be clipped in to press.Fixedly screw mandrel 47 passes the hole at place in the rear deflector door 45, adds beam with borehole jack and reinforces, and utilizes the screw press method will press each member in the frame to press the due mechanical property of associated materials when tight to satisfy.Press base plate four universal wheels 52 are housed for 49 times, being convenient to whole press for extracting juice formula sterilization cabinet H can move freely.The high roomy little of press for extracting juice formula sterilization cabinet H all depended on a body E, and integral body can be sterilized by high temperature and high pressure steam.Its accessory buffering dividing plate 51 and bed inner chamber infill panel 53 only use when sterilization, and the former is inserted in a body E between any two, and the latter inserts in the bed inner chamber 10 of a body E.Described buffering dividing plate 51 is middle upright straight double-deck stainless steels that are lined with a circle rubber pad, during high pressure steam sterilization, enclose forward and backward, an airtight lamella space in below and both sides by its thick frame 2 of limit, suede adhesion layer 6 and bed inner chamber infill panel 53 with bed body E temporarily, for fill solidifying substratum, the effect that rubber pad is therebetween got up and the slit occurred between the thick frame 2 of limit that buffering expands with heat and contract with cold, prevents body E when the autoclave sterilization postcooling shrinks and the stainless steel buffering dividing plate 51, causes the substratum leakage.
A kind of method of utilizing above-mentioned curtain-shape anchoring culture bioreactor to carry out vegetable cell, tissue and organ culture,
Concrete steps are:
1, the B of circulation keeping system sterilization.
Close pneumatics pipe valve 59, vacuum pump main valve 63, open air communication pipe valve 54, throttling valve 56, arm variable valve 57, pneumatics manifold valve 60, last vacuum pipe valve 62, steam inlet valve 64, following vacuum pipe valve 65, feed supplement pipe valve 67, be communicated with pipe valve 68, air communication pipe valve 69, the liquid pipe valve 71 next time, backflow manifold valve 78, last discharge opeing pipe valve 72 and following discharge opeing pipe valve 73, steam enters from steam inlet duct 77, process is valve tube 36 down, last valve tube 30 enters container for storing liquid 23 respectively, under filter behind the container for storing liquid 32 respectively from tubing 24, last air communicating pipe 25, communicating pipe 27, feed supplement pipe 28, air-pressure tube 29, pneumatics manifold valve 60, last liquid discharge pipe 31, following air communication pipe 35, following liquid discharge pipe 38, following liquid returning tube 40 is discharged, the disinfecting steps of the circulation keeping B of system is finished in the back by working specification, the steam off intake valve 64 then, steam condensate closes after last liquid discharge pipe 31 and 38 discharges of following liquid discharge pipe closes liquid discharge pipe valve 72 and following discharge opeing pipe valve 73, seal tubing 24 and following liquid returning tube 40 and the aseptic associated adapter of docking of curtain-shape anchoring culture bioreactor, the back injects the liquid level of nutrient solution to regulation for last container for storing liquid 23 by feed supplement pipe 28.
2, substratum is solidified in bed body E sterilization, perfusion.
The bed body E that load-bearing connection crossbeam 1 also is not installed is inserted in the high temperature high voltage resistant steam sterilizing bag that is contained in advance in the press for extracting juice formula sterilization cabinet H press for extracting juice frame, insert a buffering dividing plate 51 between any two at bed simultaneously, support to insert a piece size again between the net 7 at two of bed, the bed inner chamber infill panel 53 that thickness is suitable with this gap, the movable handwheel 48 that presses on the plate 46 on the fixing screw mandrel 47 of press for extracting juice formula sterilization cabinet H is rotated in the back, behind each the member jam-packed that inserts among the press for extracting juice formula sterilization cabinet H, not solidified curing substratum is fed into the suede adhesion layer 6 of a body E and the stainless steel of insertion cushions between the dividing plate 51 with preparing in advance, make inject the lower edge that a little higher than load-bearing that will install of substratum connects crossbeam 1, after high pressure resistant sterilizing bag is tightened, push the high pressure steam sterilization chamber and sterilize.
3, press embedding or plant plate inoculation.
The bed body E of the bacterium of having gone out is pushed between aseptic technique, under aseptic condition, bed body E taking-up is lain on the aseptic technique platform after waiting to solidify the abundant cooled and solidified of substratum, it fills up with the buffering dividing plate 51 that takes out simultaneously down, after will treat that cultivated material is seeded on the curing substratum of a body E with proper density, then in bed with buffering dividing plate 51 concora crush, make and treat that cultivated material embeds in the curing substratum deeply up to fully contacting with suede adhesion layer 6, so that cultivated material can continue to obtain the required nutriment of its growth by suede adhesion layer 6, also connect kind in kind for another side.Aseptically filling curing substratum in also can be earlier carrying out behind the high pressure steam sterilization bed body E again between aseptic technique with press for extracting juice formula sterilization cabinet H or additive method, or with aseptic low melting point nutrient agar the higher density suspended culture cell is planted plate and be fixed in the suede adhesion layer 6 upper transplating board thickness of a body E in 3mm, can fully contact with suede adhesion layer 3 after making cell grow up to callus, to obtain to continue the needed nutrition of growth.
4, assembling one envelope one suspension one butt joint one cultivation one results are cultivated again and again
After the inoculation, equally under aseptic condition, aseptic successively installation load-bearing connects crossbeam 1, compound streamer feed supplement device F and disposable sterilized envelope film G, the sphere that suspension ring 16 on the compound streamer feed supplement device F of the culture bed A of the netted two-sided anchoring of curtain formula that then aseptic assembling is finished, connect kind hang suspension system C hangs sliding 21 the suspension hook that hangs, the spherical slide rail 22 of edge of bed is pushed culturing room's preposition, liquid-inlet pipe on it 15 and liquid discharge pipe 20 are implemented aseptic the butt joint with tubing 24, the liquid returning tube 40 of the circulation keeping B of system respectively.Then room temperature is transferred to 25 ℃ ± 5 ℃, humidity transfers to 80% ± 5%, treat that its cultivation is after about 20 days, valve tube valve 62 is closed in the pass, air communication pipe valve 54, feed supplement pipe valve 67, be communicated with pipe valve 68 and pneumatics manifold valve 60, open throttling valve 56, arm variable valve 57 and pneumatics pipe valve 59, start air pressurization pump 26, beginning supplements the nutrients in culture bed, regulate air communication pipe valve 54 and arm variable valve 57, the nutritious drop in bottom that makes pressure in the container for storing liquid can keep the bed inner chamber 10 of culture bed E intermittently flows out, the beginning feed supplement is cultivated, grow bed surface until cultivating body, volume increases more than 3.5 times, it is a culture cycle (with southern taxus callus is example, and this cycle is about 70 ~ 80 days) that growth slows down when being close to stagnation gradually.Close arm variable valve 57 and backflow manifold valve 78 then, the aseptic liquid-inlet pipe 15 that disassembles, the tubing 24 of liquid discharge pipe 20 and the circulation keeping B of system, the connection that liquid returning tube is 40, culture bed A shifts out in culturing room along spherical slide rail 22 with the netted two-sided anchoring of curtain formula, move into the aseptic processing room, tear cover disposable sterilized envelope film G thereon, with a sharp aseptic rimmer knife, the two vertical frame 3 flat cultivation bodies that grow frame of cutting down near the culture bed A of the netted two-sided anchoring of curtain formula, remaining part still is retained on the bed surface, after change a new disposable sterilized envelope film G and again culture bed cover sealed, push back culturing room along spherical slide rail 22 again, a beginning new round is cultivated.
5, nutrient solution circulation
Intermittently drip effusive residue nutritive medium in the culturing process from bed inner chamber 10 bottoms and reflux after the sterilization again through high-temperature instantaneous sterilization device 39 and filter down on the filtering net 33 of container for storing liquid 32, standby in being stored in after filtering jar.If it is higher to filter the position of container for storing liquid 32 down, phegma can not rely on the self gravitation effect to flow to down and filter container for storing liquid (32), then can close air communication pipe valve 69, be communicated with pipe valve 68, go up vacuum pipe valve 62, steam inlet valve 64, open down vacuum pipe valve 65, vacuum pump main valve 63, start vacuum pump 37, make down to filter in the container for storing liquid 32 to produce negative pressure, into filter container for storing liquid 32 down to impel phegma to reflux.
Instantly the phegma that filters in the container for storing liquid 32 runs up to a certain amount of, liquid level is during soon near the height of filtering net, close throttling valve 56, air pressurization pump 26 shuts down, open air communication pipe valve 54, make container for storing liquid 23 be depressurized to air balance after close air communication pipe valve 54 again, pneumatics pipe valve 59, following vacuum pipe valve 65, steam inlet valve 64, pneumatics manifold valve 60 and feed supplement pipe valve 67, open vacuum pump main valve 63, last vacuum pipe valve 62, air communication pipe valve 69, the liquid pipe valve 71 next time, be communicated with pipe valve 68, start vacuum pump 37, make in the container for storing liquid 23 and produce negative pressure, the backflow nutrient solution that filters down in the container for storing liquid 32 just can be by refluxing upward container for storing liquid 23 communicating pipe 27, treating down to filter backflow nutrient solution in the container for storing liquid 32 shifts and just makes full cycle support the B of system to come back to the keeping state after finishing, circulation so repeatedly, cultivation is continued to carry out again and again.If accumulated harmful metabolite of too much cultivation body under refluxing in the phegma of filtration container for storing liquid 32, then phegma can be discharged to harmful metabolite of removing or extract in the relevant treatment system wherein from following liquid discharge pipe 38, the backflow nutrient solution through having purified can enter the circulation keeping B of system again and utilize.
The present invention has the following advantages and positively effect:
1. nonproductive process is short.With the callus culture is example, by the triangular flask kind, or one step of culture dish kind, plastics bag kind arrive and produce inoculation, removed loaded down with trivial details step from and amplified inoculation.
2. can once inoculate multiple batches of cultivation results.Owing to adopted adhesion anchoring, circulation feed supplement to cultivate and partly kept the results technology, thus inoculation once as long as the back is not contaminated, just cultivation that can be multiple batches of, multiple batches of results do not need every batch of inoculation, make production process be simplified and shorten greatly.
3. device fabrication is simple, and cost is low.The effective culture area of one single face 5M2 culture bed, manufacturing cost valency can not surpass 1000 yuan, but its culture space that has is equivalent to 300L, and the bio-reactor of an existing 300L any type, its cost is all more than 100,000 yuan.
4. operation control is simple, easily realizes intelligent full automatic control.Because of culture bed among the present invention is a relatively independent sterile system, and the process of its incubation growth is a complete bionical process, therefore do not need the biosensor Information Monitoring of various complexity, implement regulation and control, and only need temperature, the humidity of culturing room are carried out appropriate regulation.The entire operation technical process have only sterilization, inoculation, assembling, in place, cultivate, results, cultivate 7 nodes again, and do not need repeatedly subculture, multistage spreading cultivation, so more easily realize stdn, intelligent full automatic control.
5. the production risk cost is low.Have only two cultivation faces because the institute's curtain formula that adopts suspension is culture bed among the present invention, and per two cultivation faces constitute a relatively independent culture system, so just in case go wrong in certain culturing process, it can cause single failure, and can not influence other, the loss that is caused is minimum, whole relatively large scale culturing, and it almost can be ignored.
6. energy consumption is low, and operation cost is low.Because nutrient solution has adopted the recycle mode in the whole culturing process, circulation power has adopted air pressure type, needs the power consumption so whole culturing process is safeguarded except that temperature, the humidity of culturing room, and the others power consumption is extremely low.Cultivate required illumination,, then can adopt the natural lighting of glass canopy to cultivate if consider the cost of solar collector transmission lighting system.
7. the industrialization that is easy to random scale is amplified.Because curtain-shape anchoring culture bioreactor of the present invention is to hang the folded system that the relatively independent cultivation unit of curtain sample is formed in culturing room of piling up neatly one by one, the curtain that culturing room every 10cm crosscut space just can hold among the present invention at least is culture bed, and the expansion scale is as long as increase the curtain number.So the scale of culturing room has much, cultivating the scale of producing just can have much.
But 8. multi-use.Because the cultivation unit of curtain-shape anchoring culture bioreactor of the present invention is the curtain shape bed of a hollow, so its pair cell, tissue, organ, plant, animal and microorganism should be all more suitable.
9. be easy to the on-line implement regulation and control.Because what the present invention carried out is feed supplement cultured continuously method, implement regulation and control pointedly so can add special additive in any stage of cultivating.
10. comparatively unique bionics principle.The culture bed A of the netted two-sided anchoring of curtain formula in the curtain-shape anchoring culture bioreactor of the present invention, be based on bionical to placenta in the plant ovary, the cultivation body adheres on the suede adhesion layer 6 that is anchored at the culture bed A of the netted two-sided anchoring of curtain formula to be cultivated, also being based in the fruit seed and is born in the bionical of placenta, to cultivate body and culture bioreactors and be considered as integral body and consider, be the most valuable place of the present invention.
Now with the present invention carried out reaching 1 year 6 totally batches southerm yew Taxus chinensis varmariei callus entity cultivate, callus output can reach 392g dry weight/day M
2, content of taxol reaches as high as 0.0465% of callus dry weight, is 40 times of content in the natural plant.
Claims (13)
1, a kind of curtain-shape anchoring culture bioreactor, comprise the netted two-sided anchoring of curtain formula culture bed (A), keeping system (B), suspension system (c), the netted two-sided anchoring of curtain formula culture bed (A) hangs on the suspension system (c), the netted two-sided anchoring of described curtain formula culture bed (A) has a body (E), compound streamer feed supplement device (F) and disposable sterilized envelope film (G), bed body (E) has the double-deck net (7) of supporting, support net (7) to be installed in the frame (3), two support to have between net (7) space to form bed inner chamber (10), supporting to be attached with suede adhesion layer (6) on the net, compound streamer feed supplement device (F) is installed in the top of a body (E), has streamer feed supplement groove (11), streamer feed supplement groove (11) is communicated with the bed inner chamber (10) of bed body (E) and is arranged on liquid-inlet pipe on the compound streamer feed supplement device (F), bed body (E) is outer to be set with disposable sterilized envelope film (G), and disposable sterilized envelope film (G) is provided with liquid discharge pipe and air communication pipe; Keeping system (B) has tubing (24) and container for storing liquid (23), and curtain-shape anchoring culture bioreactor is communicated with container for storing liquid (23) by liquid-inlet pipe (15) and tubing (24).
2, curtain-shape anchoring culture bioreactor according to claim 1, it is characterized in that, described keeping system (B) is a circulation keeping system, have container for storing liquid (23) and filter container for storing liquid (32) down, container for storing liquid (23) is communicated with liquid-inlet pipe (15) on the compound streamer feed supplement device (F) by tubing (24), nutrient solution in the container for storing liquid (23) can be injected the netted two-sided anchoring of curtain formula culture bed (A) by air pressurization pump (26), filtering container for storing liquid (32) down is communicated with liquid discharge pipe (20) under the disposable sterilized envelope film (G) by liquid returning tube (40), return line (40) and following the filtration between the container for storing liquid (32) are provided with the instantaneous sterilization device, container for storing liquid (23) and following the filtration between the container for storing liquid (32) have communicating pipe (27), the backflow nutrient solution that descends to filter in the container for storing liquid (32) can be squeezed in the container for storing liquid (23) by vacuum pump (37).
3, curtain-shape anchoring culture bioreactor according to claim 1, it is characterized in that, described suspension system (C) has spherical slide rail (22), be provided with a plurality of spherical hang sliding hang (21) in the spherical slide rail (22), the netted two-sided anchoring of a plurality of curtain formulas culture bed (A) can be suspended on spherical sliding the hanging on (21) of hanging.
4, curtain-shape anchoring culture bioreactor according to claim 1, it is characterized in that, gapped regulation and control plate (8) between two supporting networks (7) of described bed body (E), frame (3) is provided with limit thick frame (2), the outer load-bearing connection crossbeam (1) that disposes of the upper side frame (5) of frame (3), suede adhesion layer (6) is made for the fiber with strongly hydrophilic.
5, curtain-shape anchoring culture bioreactor according to claim 1, it is characterized in that, described compound streamer feed supplement device (F) has riveting door bolt (13) and rivet hole (14), fasten (13) and rivet hole (14) with a bolt or latch by riveting is installed on the top of a body (E) with compound streamer feed supplement device (F), and the top of compound streamer feed supplement device (F) is equipped with suspension ring (16).
6, curtain-shape anchoring culture bioreactor according to claim 1, it is characterized in that, the plastic film bag that described disposable sterilized envelope film (G) is made for high-temperature resistance plastice film (18), it has the adhesive tape for the treatment of (17) at least on one side, all the other bandings close, bottom is obliquely installed, and liquid discharge pipe (20) is located at following low edge.
7, curtain-shape anchoring culture bioreactor according to claim 1, it is characterized in that, it also has lighting system, described lighting system is a solar collector transmission lighting system (D), form by sunshine collector (41), transmission cable (42) and luminous end (43), sunshine collector (41) is installed on the roof of culturing room, and luminous end (43) is installed in the netted two-sided anchoring of curtain formula culture bed (A) between any two.
8, curtain-shape anchoring culture bioreactor according to claim 1, it is characterized in that, it also has press for extracting juice formula sterilization cabinet (H), press for extracting juice formula sterilization cabinet (H) side of having body cabinet gap frame C, in be provided with the movable plate (46) that presses, frame is equipped with handwheel (48) outward, can move the control activity by operation handwheel (48) and press plate (46).
9, curtain-shape anchoring culture bioreactor according to claim 8, it is characterized in that, described press for extracting juice formula sterilization cabinet (H) is furnished with buffering dividing plate (51) that adapts with bed body (E) length and width and the bed inner chamber infill panel (53) that adapts with bed inner chamber (10) length and width, and buffering dividing plate (51) accompanies the double-layer plate of rubber pad in the middle of being.
10, a kind of method of utilizing the described curtain-shape anchoring culture bioreactor of claim 1 to carry out vegetable cell, tissue and organ culture is characterized in that, may further comprise the steps:
A, inject for a bed body (E) to solidify the substratum horizontal high voltage steam sterilizing sterilization of going forward side by side;
B, will treat that cultivated material is anchored on the suede adhesion layer (6) of a body (E) by solidifying culture medium inoculated;
After c, bed body (E) inoculation, under aseptic condition, respectively aseptic successively separately installation load-bearing connects crossbeam (1), compound streamer feed supplement device (F) and disposable sterilized envelope film (G) and is assembled into the netted two-sided anchoring of curtain formula culture bed (A), then the netted two-sided anchoring of each curtain formula culture bed (A) is suspended on the suspension system (C), push culturing room's preposition, and liquid-inlet pipe (15) in the netted two-sided anchoring of each curtain formula culture bed (A) and liquid discharge pipe (20) are communicated with keeping system (B);
D, adjusting are cultivated room temperature, humidity to required value, behind required cultivation certain hour, start keeping system (B), beginning supplements the nutrients in bed body (E), reach increment until cultivating bulk-growth with results value, close with keeping system (B) between be connected valve, being connected between the netted two-sided anchoring of aseptic dismounting curtain formula culture bed (A) and the keeping system (B);
E, the netted two-sided anchoring of curtain formula that will be to be gathered in the crops culture bed (A) move into the aseptic processing room, tear cover disposable sterilized envelope film (G) thereon, along the frame (3) of the netted two-sided anchoring of curtain formula culture bed (A) aseptic flat cut or straight snips under grow the cultivation body of frame, remaining part is retained in the seed of cultivating as next cycle on the bed surface;
F, change a new disposable sterilized envelope film (G) and again culture bed cover is sealed, push back culturing room, a beginning new round is cultivated.
11, method of carrying out vegetable cell, tissue and organ culture according to claim 10, it is characterized in that, among the step a, it is described that will to solidify method that substratum injects bed body (E) and sterilization be the bed body (E) that load-bearing connects crossbeam (1) will also not to be installed insert the sterilizing bag that is contained in advance in the press for extracting juice formula sterilization cabinet (H), insert a buffering dividing plate (51) between any two at bed body (E), in the two bed inner chambers of supporting between the net (7) (10), insert a bed inner chamber infill panel (53) again; The handwheel (48) that rotates on the press for extracting juice formula sterilization cabinet (H) moves the movable plate (46) that presses, and will insert each the member jam-packed in the press for extracting juice formula sterilization cabinet (H); To prepare not solidified curing substratum in advance and be fed into a body (E), substratum is full of between the buffering dividing plate (51) of suede adhesion layer (6) and insertion from the upper side frame (5) of bed body (E) and the slit between the suede adhesion layer (6); After sterilizing bag tightened press for extracting juice formula sterilization cabinet (H) being pushed the high pressure steam sterilization chamber sterilizes and gets final product.
12, method of carrying out vegetable cell, tissue and organ culture according to claim 10, it is characterized in that, among the step b, after will treating that cultivated material is seeded on the curing substratum of a body (E), with cushioning dividing plate (51) concora crush on bed body (E), make and treat that cultivated material embeds in the curing substratum deeply up to fully contacting with suede adhesion layer (6), so that cultivated material can continue to obtain the required nutriment of its growth by suede adhesion layer (6).
13, method of carrying out vegetable cell, tissue and organ culture according to claim 10, it is characterized in that, the method of injecting curing substratum, disinfection with high pressure steam sterilization and inoculation for before the step c bed body (E) is, earlier bed body (E) is carried out high pressure steam sterilization, then substratum is planted plate simultaneously together with culturing cell and be anchored on the suede adhesion layer (6) of a body (E), plant plate thickness so that cell grows up to can fully contact with suede adhesion layer (3) behind the callus is as the criterion.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2007100351563A CN101100641B (en) | 2007-06-13 | 2007-06-13 | Curtain-shape anchoring culture bioreactor and method for culturing plant cell, tissue or organ using the same |
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| CN2007100351563A CN101100641B (en) | 2007-06-13 | 2007-06-13 | Curtain-shape anchoring culture bioreactor and method for culturing plant cell, tissue or organ using the same |
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| CN101100641A true CN101100641A (en) | 2008-01-09 |
| CN101100641B CN101100641B (en) | 2010-12-29 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102304470A (en) * | 2011-08-13 | 2012-01-04 | 王敬华 | Multichannel automatic bacteria rapid inoculator and using method thereof |
| CN105647826A (en) * | 2015-11-11 | 2016-06-08 | 深圳市绿得宝保健食品有限公司 | Culture method and device of photosynthetic microorganism |
| CN110999789A (en) * | 2019-12-18 | 2020-04-14 | 南京师范大学 | Sterile nutrient supplement device and method for plant culture bioreactor system |
| WO2021129472A1 (en) * | 2019-12-25 | 2021-07-01 | Cesco Bioengineering Co., Ltd. | A carrier for cell biomass production and cell culture device comprising the same |
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Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4416993A (en) * | 1981-06-11 | 1983-11-22 | Mcleod & Miller (Engineers) Limited | Apparatus with semi-permeable membrane, and method for cultivating micro-organisms |
| CN1051575C (en) * | 1996-05-15 | 2000-04-19 | 中国科学院化工冶金研究所 | Reactor for nutrient liquid atomizing culture of plant |
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2007
- 2007-06-13 CN CN2007100351563A patent/CN101100641B/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102304470A (en) * | 2011-08-13 | 2012-01-04 | 王敬华 | Multichannel automatic bacteria rapid inoculator and using method thereof |
| CN102304470B (en) * | 2011-08-13 | 2014-02-19 | 王敬华 | Multichannel automatic bacteria rapid inoculator and using method thereof |
| CN105647826A (en) * | 2015-11-11 | 2016-06-08 | 深圳市绿得宝保健食品有限公司 | Culture method and device of photosynthetic microorganism |
| CN105647826B (en) * | 2015-11-11 | 2019-05-10 | 深圳市绿得宝保健食品有限公司 | A kind of cultural method and device of photosynthetic microorganism |
| CN110999789A (en) * | 2019-12-18 | 2020-04-14 | 南京师范大学 | Sterile nutrient supplement device and method for plant culture bioreactor system |
| WO2021129472A1 (en) * | 2019-12-25 | 2021-07-01 | Cesco Bioengineering Co., Ltd. | A carrier for cell biomass production and cell culture device comprising the same |
| GB2606928A (en) * | 2019-12-25 | 2022-11-23 | Cesco Bioengineering Co Ltd | A carrier for cell biomass production and cell culture device comprising the same |
| GB2606928B (en) * | 2019-12-25 | 2023-11-01 | Cesco Bioengineering Co Ltd | A carrier for cell biomass production and cell culture device comprising the same |
| US11299700B1 (en) | 2021-02-19 | 2022-04-12 | Acequia Biotechnology, Llc | Bioreactor containers and methods of growing hairy roots using the same |
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