CN102304476B - Three-dimensional cell dynamic culture reactor - Google Patents

Three-dimensional cell dynamic culture reactor Download PDF

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Publication number
CN102304476B
CN102304476B CN 201110242276 CN201110242276A CN102304476B CN 102304476 B CN102304476 B CN 102304476B CN 201110242276 CN201110242276 CN 201110242276 CN 201110242276 A CN201110242276 A CN 201110242276A CN 102304476 B CN102304476 B CN 102304476B
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reactor
cell
housing
bottle
valve
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Expired - Fee Related
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CN 201110242276
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CN102304476A (en
Inventor
张志琪
牟朝丽
祁小妮
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Shaanxi Normal University
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Shaanxi Normal University
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    • CCHEMISTRY; METALLURGY
    • 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
    • C12M23/00Constructional details, e.g. recesses, hinges
    • C12M23/38Caps; Covers; Plugs; Pouring means
    • CCHEMISTRY; METALLURGY
    • 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
    • C12M29/20Degassing; Venting; Bubble traps

Abstract

The invention discloses a three-dimensional cell dynamic culture reactor. An internal shell of the reactor is arranged in the external shell of the reactor with a clearance in between; the lower part of the left side of the external shell of the reactor extends into an air inlet pipe in the lower internal part of the internal shell of the reactor; the upper end of the internal shell of the reactor is provided with a sealing upper cover with an exhaust one-way valve; the side wall of the internal shell of the reactor is provided with a liquid outlet pipe; the lower internal part of the internal shell of the reactor is provided with a supporting plate in which a vent hole is formed; the supporting plate is provided with a lower waterproof breathable film of which the upper part is provided with a cell culture bracket; the upper surface of the cell culture bracket is provided with a waterproof breathable film; the lower end of the liquid inlet pipe is inserted onto the upper surface of the lower water; the liquid inlet pipe is communicated with the outlet of a peristaltic pump; the inlet of the peristaltic pump is communicated with a cell suspension bottle, a cell culture solution bottle and a buffer solution bottle; and the liquid outlet pipe is communicated with a cell suspension bottle, a cell culture solution bottle and a waste liquid bottle. When the reactor is used, cell dynamic inoculation and cell dynamic culture are realized, equipment is reduced, and the cell culture time is reduced. The reactor can be used for culturing adherent cells.

Description

Three-dimensional cell is dynamically cultivated reactor
Technical field
The invention belongs to cell cultivation equipment or device technique field, be specifically related in carbon dioxide environment, use the culture device of the cell culture fluid that flows.
Background technology
Traditional cell culture processes, namely two-dimentional cell cultures can cause cell to lose its function relevant with tissue, can't reflect cell growth in vivo situation exactly.Though animal model and in vivo test can reflect the true interior environment of cell growth, cost is too high, process is complicated, and is subjected to the restriction of ethics aspect.Under the environment, all cells all are in a kind of three-dimensional space in vivo.Three-dimensional cell is cultivated the interaction that can reappear between cell-cell and the cell-matrix, compares with two-dimentional cell cultures, more can reflect the interior environment of cell growth truly.Therefore, three-dimensional cell is cultivated the bridge that can erect between two-dimentional cell cultures and the animal experiment.Three-dimensional cell is cultivated and has been obtained application in the research aspect cytobiology.
In the three-dimensional cell inoculation and cultured method of routine, the main method that adopts static inoculation and static cultivation.This method has shortcomings such as rate of vaccination is low, inoculation is inhomogeneous, and growing state is inhomogeneous.Dynamically the cultivation of inoculation and perfusion is compared with static cultivation in hepatocellular high-density culture and demonstrated its special advantages, and is good as the adhesivity of cell on support, dispersion is more even, cell survival rate is more high.In order to realize dynamically inoculation and perfusion culture method, some cell reactors have been developed at present both at home and abroad.
At present, the bio-reactor of clinical use roughly is divided into four types: hollow fiber type, single flat template, perfusion bed type, bag are by suspension type.The function singleness of these bio-reactors, there are some instruments to be used for separation or the collection of cell, some instruments are used for the preparation of cell suspension, some instruments are used for mixing of cell and material, some instruments are used for the cultivation of cell and tissue, and the main drawback of these bio-reactors is Operating Complexities, is easy to pollute.
The patent No. is ZL 031106701.2, denomination of invention is the patent of invention of " cell reactor ", number of patent application is 200910229109.1, denomination of invention is the patent of invention of " medicament-induced hepatotoxicity evaluation polysaccharide composite stent perfusion type liver cell reactor system ", number of patent application is 200910242907.8, denomination of invention is the patent of invention of " a kind of cell reactor and comprise the AISS of this reactor ", these three kinds of cell reactors all can not provide the cell growth required temperature, humidity and gas condition, cell reactor must be placed CO2gas incubator just can use, and cell reactor and a kind of cell reactor and the AISS that comprises this reactor can not realize that cell dynamically inoculates, dynamically cultivate.
Summary of the invention
Technical problem to be solved by this invention is to overcome the shortcoming of above-mentioned bio-reactor, provides that a kind of cell is dynamically inoculated, perfusion cultivates, do not need to place the three-dimensional cell of CO2gas incubator dynamically to cultivate reactor cell reactor.
Solving the problems of the technologies described above the technical scheme that adopts is: be provided with the reactor inner housing in the shell of reactor body, shell of reactor body and reactor inner housing maintain the gap, form water stream channel, the bottom, left side of shell of reactor body arranges the inlet pipe that extend into bottom in the reactor inner housing, the upper end of reactor inner housing arranges the sealing loam cake that the outgassing nonreturn valve door is installed, the sidewall of reactor inner housing is provided with drain pipe, bottom in the reactor inner housing arranges the back up pad that is processed with ventilating pit, form the air chamber that links with inlet pipe between back up pad and the reactor inner housing inner bottom part, back up pad is provided with down waterproof ventilated membrane, following waterproof ventilated membrane top is provided with cell culturing bracket, the upper surface of cell culturing bracket is provided with waterproof ventilated membrane, the lower end of liquid-inlet pipe is inserted into and passes the cell culturing bracket upper surface of waterproof ventilated membrane extremely down in the reactor inner housing, liquid-inlet pipe links by conduit and wriggling pump outlet, the import of peristaltic pump links by being installed in supravasal cell suspension fluid valve and cell suspension bottle, link by being installed in supravasal cell culture fluid fluid valve and cell culture fluid bottle, link by being installed in supravasal damping fluid valve and damping fluid bottle, drain pipe links by being installed in supravasal cell suspension liquid flowing valve and cell suspension bottle, link by being installed in supravasal cell culture fluid liquid flowing valve and cell culture fluid bottle, link by being installed in supravasal waste liquid valve and waste liquid bottle.
Cell culturing bracket of the present invention is that porous cell is cultivated support.
Porous cell cultivation support of the present invention is any one in silk fibroin/hydroxyapatite/collagen albumen complex stephanoporate bracket, chitosan/hydroxyapatite three-dimensional porous rack, the polylactic acid-polyglycolic acid multipolymer/hydroxyapatite/collagen albumen complex stephanoporate bracket.
The present invention adopts silk fibroin/hydroxyapatite/collagen albumen complex stephanoporate bracket to be prepared into cell culturing bracket, in carbon dioxide environment, cell adhesion is on cytoskeleton, cell culture fluid flows in the cell culturing bracket cocycle, the cell culturing bracket that does not need to be stained with cell is placed in the CO2gas incubator to be cultivated, realize that cell is dynamically inoculated, cell is dynamically cultivated, simplified technology, reduced equipment, shortened the cell cultures time, reduced the cell cultures expense, the present invention can be used for the cultivation of attached cell.
Description of drawings
Fig. 1 is the structural representation of one embodiment of the invention.
Fig. 2 is that human osteosarcoma cell MG-63 amplifies 1500 times environmental scanning electron microscope figure after dynamically cultivating 48 hours on the support.
Fig. 3 is that human osteosarcoma cell MG-63 amplifies 1000 times environmental scanning electron microscope figure after dynamically cultivating 48 hours on the support.
Fig. 4 is that human colon cancer cell Lovo amplifies 4000 times environmental scanning electron microscope figure after dynamically cultivating 48 hours on the support.
Fig. 5 is that human colon cancer cell Lovo amplifies 1000 times environmental scanning electron microscope figure after dynamically cultivating 48 hours on the support.
Embodiment
The present invention is described in more detail below in conjunction with drawings and Examples, but the invention is not restricted to these embodiment.
Embodiment 1
In Fig. 1, it is by shell of reactor body 1 that the three-dimensional cell of present embodiment is dynamically cultivated reactor, reactor inner housing 2, liquid-inlet pipe 3, last waterproof ventilated membrane 4, drain pipe 5, peristaltic pump 6, damping fluid valve 7, cell culture fluid fluid valve 8, cell suspension fluid valve 9, cell suspension liquid flowing valve 10, cell suspension bottle 11, cell culture fluid liquid flowing valve 12, cell culture fluid bottle 13, damping fluid bottle 14, waste liquid valve 15, waste liquid bottle 16, cell culturing bracket 17, following waterproof ventilated membrane 18, back up pad 19, inlet pipe 20, sealing loam cake 21, outgassing nonreturn valve door 22 connects formation.
Reactor inner housing 2 is installed in shell of reactor body 1, being shaped as of shell of reactor body 1 and reactor inner housing 2 is cylindrical, shell of reactor body 1 and reactor inner housing 2 adopt synthetic glass to make, shell of reactor body 1 maintains the gap with reactor inner housing 2, form water stream channel, left end forms water-in a between shell of reactor body 1 and the reactor inner housing 2, right-hand member forms mouth of a river b, 37 ℃ recirculated water enters from water-in a, flow through water stream channel between shell of reactor body 1 and the reactor inner housing 2 flows out from water outlet b.The bottom, left side of shell of reactor body 1 is equipped with inlet pipe 20, and inlet pipe 20 is inserted in the shell of reactor body 1, and the inner of inlet pipe 20 extend into the bottom in the reactor inner housing 2.
Upper end at reactor inner housing 2 is equipped with sealing loam cake 21 by thread connection, by thread connection outgassing nonreturn valve door 22 is installed on the sealing loam cake 21.There is drain pipe 5 on the right side of reactor inner housing 2, and the outer end of drain pipe 5 is connected as a single entity with reactor inner housing 2 and links for the porose sealed tube of processing, drain pipe 5.Bottom in the reactor inner housing 2 is equipped with back up pad 19, be processed with ventilating pit on the back up pad 19, maintain distance between back up pad 19 and reactor inner housing 2 inner bottom parts, form air chamber, air chamber and inlet pipe 20 link, the ultrapure water that it is 18.2M Ω-cm that the air chamber inner bottom part is equipped with 25 ℃ of resistivity, ultrapure water is used for providing humidity, aseptic carbonic acid gas and Air mixing gas can enter in the air chamber, outgassing nonreturn valve door 22 from sealing loam cake 21 flows out, in carbonic acid gas and the Air mixing gas, the volume ratio of carbonic acid gas and air is 5: 95.Be placed with down waterproof ventilated membrane 18 on the back up pad 19, the commodity of following waterproof ventilated membrane 18 for selling on the market, sold by U.S. Sigma Technonologies Intl.LLC company, following waterproof ventilated membrane 18 is used for ventilation and prevents down under the current on the waterproof ventilated membrane 18.Cell culturing bracket 17 is equipped with on 2 times waterproof ventilated membrane 18 tops of reactor inner housing, the cell culturing bracket 17 of present embodiment adopts porous support, concrete silk fibroin/hydroxyapatite/collagen albumen the complex stephanoporate bracket that adopts, cell culturing bracket 17 is used for adherent cell and holds cell culture fluid, the mixing solutions of the high sugared nutrient solution of the DMEM that cell culture fluid adopts Gbico company to produce and foetal calf serum, in mixing solutions, the volume ratio of DMEM cell culture fluid and bovine serum solution is 9: 1.The upper surface of cell culturing bracket 17 is placed with waterproof ventilated membrane 4, and last waterproof ventilated membrane 4 is identical commodity with following waterproof ventilated membrane 18, and last waterproof ventilated membrane 4 is used for preserving moisture ventilation.The lower end of liquid-inlet pipe 3 is inserted into passes cell culturing bracket 17 to the upper surface of following waterproof ventilated membrane 18 in the reactor inner housing 2, liquid-inlet pipe 3 links by the outlet of conduit and peristaltic pump 6, the import of peristaltic pump 6 links with cell suspension bottle 11 by being installed in supravasal cell suspension fluid valve 9, cell suspension bottle 11 links with drain pipe 5 by being installed in supravasal cell suspension liquid flowing valve 10, and peristaltic pump 6 can be injected into cell suspension on the cell culturing bracket 17 and circulate; The import of peristaltic pump 6 links with cell culture fluid bottle 13 by being installed in supravasal cell culture fluid fluid valve 8, cell culture fluid bottle 13 links with drain pipe 5 by being installed in supravasal cell culture fluid liquid flowing valve 12, and peristaltic pump 6 can be injected into cell culture fluid on the cell culturing bracket 17 and circulate; The import of peristaltic pump 6 links with damping fluid bottle 14 by being installed in supravasal damping fluid valve 7, and peristaltic pump 6 can be injected into phosphate buffer solution on the cell culturing bracket 17, and phosphate buffer solution is used for rinsing and sticks to cell on the cell culturing bracket 17; Waste liquid bottle 16 links with drain pipe 5 by being installed in supravasal waste liquid valve 15, and the phosphate buffer solution after the rinsing flow in the waste liquid bottle 16 through being installed in supravasal waste liquid valve 15.
The three-dimensional cell of this structure is dynamically cultivated reactor, the cell culturing bracket 17 that does not need to be stained with cell is placed in the CO2gas incubator to be cultivated, realize that cell is dynamically inoculated, cell is dynamically cultivated, simplified technology, reduced equipment, shorten the cell cultures time, reduced the cell cultures expense.
The silk fibroin of present embodiment/hydroxyapatite/collagen albumen complex stephanoporate bracket is prepared by the contriver, and its preparation method is as follows:
1, preparation silk fibroin protein solution
Remove the silk cocoon 20g of pupa, add 0.02mol/L Na 2CO 3Aqueous solution 2000g boiled 30 minutes, and taking-up is wrung out, and is clean with distilled water flushing; Repeat aforesaid operations again 2 times, 50 ℃ of vacuum-dryings obtain boiled silk; The 5g boiled silk is placed 50g 9.3mo l/L LiBr solution, 50 ℃ are incubated to boiled silk and dissolve fully, the molecular weight that dams of packing into is in the 3500 daltonian dialysis tubings, dialysis is 5 days in the distilled water of 1ml/min flow velocity, centrifugation, gained supernatant liquor are silk fibroin protein solution, measure the mass concentration of silk fibroin protein solution with the Xylene Brilliant Cyanine G method, being 6%, 5 ℃ with distilled water diluting to the mass concentration of silk fibroin protein solution stores for future use.
2, preparation support
Be the NaCl of 180~250 μ m with the 4g particle diameter, 0.16g hydroxyapatite mixes, the mass ratio of NaCl and hydroxyapatite is 1: 0.04, be packed in the syringe that volume is 10mL, the volume ratio that adds the 4mL massfraction and be 6% collagen solution and massfraction and be 6% silk fibroin protein solution is 1: 3 mixed solution, the piston handle of pushing syringe, make mixed solution by the mixture of NaCl and hydroxyapatite, room temperature was placed 24 hours, go out coherent mass from syringe, be 70% alcohol solution dipping 30 minutes with volume fraction, thoroughly clean with ultrapure water, be prepared into silk fibroin/hydroxyapatite/collagen albumen complex stephanoporate bracket.
Embodiment 2
In the present embodiment, be placed with down waterproof ventilated membrane 18 on the back up pad 19 in the reactor inner housing 2, cell culturing bracket 17 is equipped with on 2 times waterproof ventilated membrane 18 tops of reactor inner housing, the cell culturing bracket 17 of present embodiment adopts chitosan/hydroxyapatite three-dimensional porous rack, and chitosan/hydroxyapatite three-dimensional porous rack is prepared by the contriver.The connecting relation of other component and component is identical with embodiment 1.
The preparation method of chitosan/hydroxyapatite three-dimensional porous rack is as follows:
1, dissolving chitosan and hydroxyapatite
1g chitosan (deacetylation is 80%~95%), 0.1g hydroxyapatite are dissolved in the acetic acid aqueous solution of 100mL 20g/L, stirred 6 hours, the centrifugal bubble that degass obtains the mixed solution of chitosan and hydroxyapatite.
2, preparation chitosan/hydroxyapatite three-dimensional stephanoporate compound stent
With SiO 2Microballoon is pore-creating agent, is the SiO of 100~150 μ m with particle diameter 2Microballoon adds in the syringe of 20mL band syringe needle, with pestle extruding, compacting SiO 2Microballoon, till its position in syringe does not change, SiO 2The add-on of microballoon is 0.25 of syringe volume, adds the mixed solution of chitosan and hydroxyapatite again in the syringe, the add-on of mixed solution and SiO 2The volume of microballoon is identical, and the piston handle of pushing syringe makes mixed solution flow through compact arranged SiO 2Microballoon, syringe was placed 60 ℃ of dryings of vacuum drying oven 24 hours, from syringe, take out coherent mass, cut the upper and lower upper layer of coherent mass with cutter, place the NaOH aqueous solution of 0.05g/mL to boil 3 hours the coherent mass that cuts upper and lower upper layer, the water flushing is to neutral,-20 ℃ of pre-freezes are 12~24 hours in refrigerator, with liquid nitrogen pre-freeze 30 minutes ,-50 ℃ of lyophilizes 24 hours were prepared into chitosan/hydroxyapatite three-dimensional stephanoporate compound stent material.
Embodiment 3
In the present embodiment, be placed with down waterproof ventilated membrane 18 on the back up pad 19 in the reactor inner housing 2, cell culturing bracket 17 is equipped with on 2 times waterproof ventilated membrane 18 tops of reactor inner housing, the cell culturing bracket 17 of present embodiment adopts polylactic acid-polyglycolic acid multipolymer/hydroxyapatite/collagen albumen complex stephanoporate bracket, and polylactic acid-polyglycolic acid multipolymer/hydroxyapatite/collagen albumen complex stephanoporate bracket is prepared by the contriver.The connecting relation of other component and component is identical with embodiment 1.
Multipolymer/hydroxyapatite/collagen albumen complex stephanoporate bracket 100g is example with the preparation polylactic acid-polyglycolic acid, and used raw material and proportioning thereof are:
Polylactic acid-polyglycolic acid multipolymer 95g
Hydroxyapatite 2.5g
Collagen protein 2.5g
Its preparation method is as follows:
Get polylactic acid-polyglycolic acid multipolymer 95g, hydroxyapatite 2.5g, collagen protein 2.5g, mix, being cold-pressed into diameter with tabletting machine is that 3cm, thickness are the disk of 1.5cm, and the concrete size of disk can be that 2~5cm, thickness are that the size according to reactor is determined in the scope of 1~2cm at diameter.Compressing tablet is put into mould, and mould places supercritical CO 2In the reactor, close CO 2Purging valve, setting reaction pressure are that 18MPa, temperature of reaction are 45 ℃, CO 2Flow velocity is 10mL/ minute, opens CO 2Steel cylinder valve, high-pressure pump and temperature control valve make pressure and temperature reach set(ting)value, react 45 minutes, open CO 2Purging valve cuts out CO 2The switch of steel cylinder valve, high-pressure pump and reactor, pressure take out mould after being down to normal pressure from reactor, be prepared into composite porous support material.
Using method of the present invention and operation steps are as follows:
1, dynamically inoculation
Earlier three-dimensional cell is dynamically cultivated reactor (except peristaltic pump) sterilising treatment, inlet pipe 20 is by pipeline and dioxide bottle UNICOM, the aseptic gas mixture of carbon dioxide and air is input in the reactor inner housing 2, with peristaltic pump 6 cell suspension being entered cell culturing bracket 17 with the flow velocity of 5ml/min in liquid-inlet pipe 3 pumps into reactor inner housing 2 circulates, dynamically inoculated in lasting 1 hour, after dynamically inoculation is finished, close peristaltic pump 6, left standstill 3 hours, make cell adhesion on cell culturing bracket 17, with peristaltic pump 6 the phosphoric acid buffer pouring is washed off the cell that does not adhere to and adhered to unstable cell with the flow velocity of 1ml/min on cell culturing bracket 17, acellular outflow ends in reactor inner housing 2 effluent liquid, cell is sticked on the cell culturing bracket 17 fully, and the phosphoric acid buffer after the flushing flows into waste liquid bottle 16.
2, dynamically cultivate
With peristaltic pump 6 cell culture fluid is entered cell culturing bracket 17 with the flow velocity of 1ml/min in liquid-inlet pipe 3 pumps into reactor inner housing 2 and circulate, continue 48 hours, finish the dynamic cultivation of cell.
In order to verify beneficial effect of the present invention, the contriver adopts the three-dimensional cell of the embodiment of the invention 1 preparation dynamically to cultivate reactor human osteosarcoma cell MG-63, human colon cancer cell Lovo is dynamically cultivated, and various experiment situations are as follows:
Cultural method is identical with above-mentioned dynamic cultural method.Dynamically cultivate after 48 hours, observe with the Quanta200 environmental scanning electron microscope, the results are shown in Figure 2~Fig. 5.
Shown that by Fig. 2~Fig. 5 cell is evenly distributed in the support, in the hole of support and the bore edges cell that all distributing uniformly.The pseudopodium of cell surface and microvillus showed cell growth conditions are good.Therefore, utilizing this cell dynamically to cultivate reactor can make cell be grown in more equably on the cell culturing bracket 17.

Claims (2)

1. a three-dimensional cell is dynamically cultivated reactor, it is characterized in that: in shell of reactor body (1), be provided with reactor inner housing (2), shell of reactor body (1) maintains the gap with reactor inner housing (2), form water stream channel, the bottom, left side of shell of reactor body (1) arranges the inlet pipe (20) that extend into the interior bottom of reactor inner housing (2), the upper end of reactor inner housing (2) arranges the sealing loam cake (21) that outgassing nonreturn valve door (22) is installed, the sidewall of reactor inner housing (2) is provided with drain pipe (5), bottom in the reactor inner housing (2) arranges the back up pad (19) that is processed with ventilating pit, form the air chamber that links with inlet pipe (20) between back up pad (19) and reactor inner housing (2) inner bottom part, back up pad (19) is provided with down waterproof ventilated membrane (18), following waterproof ventilated membrane (18) top is provided with cell culturing bracket (17), the upper surface of cell culturing bracket (17) is provided with waterproof ventilated membrane (4), the lower end of liquid-inlet pipe (3) is inserted into and passes cell culturing bracket (17) upper surface of waterproof ventilated membrane (18) extremely down in the reactor inner housing (2), liquid-inlet pipe (3) links by the outlet of conduit and peristaltic pump (6), the import of peristaltic pump (6) links with cell suspension bottle (11) by being installed in supravasal cell suspension fluid valve (9), link with cell culture fluid bottle (13) by being installed in supravasal cell culture fluid fluid valve (8), link with damping fluid bottle (14) by being installed in supravasal damping fluid valve (7), drain pipe (5) links with cell suspension bottle (11) by being installed in supravasal cell suspension liquid flowing valve (10), link with cell culture fluid bottle (13) by being installed in supravasal cell culture fluid liquid flowing valve (12), link with waste liquid bottle (16) by being installed in supravasal waste liquid valve (15);
Above-mentioned cell culturing bracket (17) is cultivated support for porous cell.
2. dynamically cultivate reactor according to the described three-dimensional cell of claim 1, it is characterized in that: described porous cell cultivation support is any one in silk fibroin/hydroxyapatite/collagen albumen complex stephanoporate bracket, chitosan/hydroxyapatite three-dimensional porous rack, the polylactic acid-polyglycolic acid multipolymer/hydroxyapatite/collagen albumen complex stephanoporate bracket.
CN 201110242276 2011-08-22 2011-08-22 Three-dimensional cell dynamic culture reactor Expired - Fee Related CN102304476B (en)

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CN106754356A (en) * 2016-11-30 2017-05-31 广州迈普再生医学科技有限公司 Three-dimensional perfused culture system and the histoorgan of 3D printing
CN110892060A (en) * 2017-04-07 2020-03-17 埃皮博恩股份有限公司 System and method for seeding and culturing

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CN104623733B (en) * 2015-01-12 2017-01-18 中国人民解放军第三军医大学野战外科研究所 Three-dimensional dynamic composite culture system and method for urethra of functional tissue engineering
TW202140769A (en) * 2020-04-29 2021-11-01 財團法人醫藥工業技術發展中心 Cell culture device

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CN106754356A (en) * 2016-11-30 2017-05-31 广州迈普再生医学科技有限公司 Three-dimensional perfused culture system and the histoorgan of 3D printing
CN106754356B (en) * 2016-11-30 2019-07-23 广州迈普再生医学科技股份有限公司 Three-dimensional perfused culture system and the histoorgan of 3D printing
CN110892060A (en) * 2017-04-07 2020-03-17 埃皮博恩股份有限公司 System and method for seeding and culturing
IL269839A (en) * 2017-04-07 2022-02-01 Epibone Inc System and method for seeding and culturing

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