CN102140445B - Carrier cell immobilization method - Google Patents
Carrier cell immobilization method Download PDFInfo
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- CN102140445B CN102140445B CN201010618505.6A CN201010618505A CN102140445B CN 102140445 B CN102140445 B CN 102140445B CN 201010618505 A CN201010618505 A CN 201010618505A CN 102140445 B CN102140445 B CN 102140445B
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- 238000000034 method Methods 0.000 title claims abstract description 36
- 238000007444 cell Immobilization Methods 0.000 title abstract 3
- 238000011081 inoculation Methods 0.000 claims abstract description 83
- 210000004027 cell Anatomy 0.000 claims abstract description 54
- 210000001822 immobilized cell Anatomy 0.000 claims abstract description 37
- 229910001220 stainless steel Inorganic materials 0.000 claims abstract description 29
- 239000010935 stainless steel Substances 0.000 claims abstract description 29
- 239000007788 liquid Substances 0.000 claims abstract description 20
- 235000015097 nutrients Nutrition 0.000 claims description 32
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- 238000002360 preparation method Methods 0.000 claims description 11
- 230000002411 adverse Effects 0.000 claims description 9
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- NJPPVKZQTLUDBO-UHFFFAOYSA-N novaluron Chemical compound C1=C(Cl)C(OC(F)(F)C(OC(F)(F)F)F)=CC=C1NC(=O)NC(=O)C1=C(F)C=CC=C1F NJPPVKZQTLUDBO-UHFFFAOYSA-N 0.000 claims description 4
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- 101710159104 Flavor peptide Proteins 0.000 description 2
- CKLJMWTZIZZHCS-UWTATZPHSA-N L-Aspartic acid Natural products OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 2
- 102000004882 Lipase Human genes 0.000 description 2
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Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention relates to a method for making immobilized cells in a carrier uniformly distributed, in particular to a stainless steel inoculation barrel which is provided with a plug and of which the wall is provided with small holes densely, the carrier is sleeved on the barrel, seed liquid is transferred to the carrier from the barrel through pressurization, and the immobilized cells are uniformly distributed in the carrier by matching a special recoiling device. Compared with the conventional immobilization method, the method for making the immobilized cells in the carrier uniformly distributed has the advantages that: the method is easy to operate, has low influence on the activity of the cells, ensures high density of immobilized cells, and is suitable for large-scale industrial production; the stainless steel inoculation barrel is designed, and can be used for immobilization of the cells and production of the immobilized cells after being properly amplified; and the carrier is integrally used for the immobilization of the cells, and is divided into carrier particles of proper size according to requirements, and defects of nonuniform cell immobilization and low carrier utilization rate caused by direct application of a small-particle carrier to the cell immobilization are effectively avoided.
Description
Technical field
The present invention relates to a kind of carrier immobilized method of cell.
Background technology
Immobilization technology refers to and utilizes various physico-chemical methods that free cell or enzyme are strapped in specific area of space, improves the concentration of cell or enzyme, and a kind of technology that keeps biological activity and recycle.Utilize cell or the enzyme that this technology makes to be called immobilized cell or immobilized enzyme.
The hydrolysis that zymotechnic is often applied to biomacromolecule has the various small-molecule substances of physiologically active with preparation.Yet resolvase, after reaction finishes, is mixed in the difficulty that has increased product separation in product, and resolvase is directly exposed in environment, and its catalytic activity is subject to the impact of various chemical factors larger.First Nelson in 1916 and Griffin prepare immobilized enzyme, and until the talents such as nineteen fifty-three Grubhofer really start effective research.Compare with resolvase, immobilized enzyme has the following advantages: the catalytic efficiency of enzyme is high; Can reuse, productive expense is low; Do not have enzyme to depart from product, additional processing is few; It is little that enzyme is subject to external influence, and the stability of enzyme is high; Improved the behavior of enzyme; Can realize serialization and produce, controllability is strong, and quality product is guaranteed; Be conducive to the utilization of multienzyme system.
Yet enzyme immobilization technology still needs first lipase to be extracted from cell, the common more complicated of extraction process, and enzyme easy inactivation in extraction and immobilization process.Nineteen fifty-nine, Hattori and Furusaka are adsorbed on intestinal bacteria E.coli on resin first, have realized cell fixation.1973, the microorganism that contains L-Aspartic acid enzyme was immobilized, and for the production of L-Aspartic acid.Late 1970s, animal, plant immobilized cell technology have also obtained development fast.At present along with the further investigation to immobilized cell technique, all microorganism fermentation process have almost been contained in immobilized growing cell or the research work that is called fixed hyperplasia cell, existing a large amount of Research Literature is delivered, and part Study achievement has been applied to production practice, such as beer production, amino acid, organic acid fermentation, production of antibiotics and sewage disposal etc.
Compare with immobilized enzyme, immobilized cell mainly has following advantage:
One, do not need enzyme to extract from microorganism cells and purifying in addition, enzyme activity loss is little, cost is low.
Two, Growth of Cells is short dead time, and cell is many, reaction is fast, and contamination resistance is strong, can continuously ferment, and Reusability, application cost is low.
Three, in the environment of enzyme in n cell, stability is high.
Four, use immobilized cell reactor, can add nutrient solution in limit, limit is cultivated and is discharged fermented liquid, can effectively avoid feedback inhibition and product consumption.
Five, be suitable for carrying out multienzyme order successive reaction.
Six, be easy to carry out the regeneration of cofactor, thereby be more suitable in the reaction that needs cofactor, as redox reaction, building-up reactions etc.Therefore, the application of this technology has surpassed the application of immobilized enzyme.
Although immobilized cell technology has broad application prospects, be to study in laboratory scale mostly, real practical or industrial application, also have following problem to need to solve:
One, the stability problem of immobilized cell, because immobilized material all has impact to make part cell inactivation on cell on the heat release in the toxicity of microorganism, fixing condition, immobilization process etc.
Two, the exploitation of immobilized cell batch production device, this is that immobilized cell technology moves towards the essential step of practical application from laboratory.
Three, the fixation support of cheap and durable and the research and development of compound immobilization technology.
Four, the exploitation of efficient immobilization reactor.What immobilized cell technology mainly relied on is the cell in set state in carrier, how to make the enough cells of fixing foot in carrier have an important significance undoubtedly for the High-efficient Production that realizes immobilized cell reactor.
The fixing means of cell mainly can be divided into four kinds according to fixing mechanism and supporting dielectric: surface adsorption, embedding, film are fixed, self aggregation.Surface adsorption is simple, but has the shortcoming that adsorption rate is low, cell is easily received environmental influence; The fixing method of film is suitable for producing the macromolecular substance of cell exocrine, but product and nutrient solution composition are easily at film surface deposition, stop up fenestra; The impact that is subject to nutrient solution composition and culture condition of self aggregation, but also very unclear so far; Entrapping method is the method for applying maximum immobilized cells in current industrial, its principle is to make cellular invasion enter porous carrier inside or cell is wrapped in gel network structure or in semipermeability film, small molecules substrate and product can free diffusings, and cell can not be diffused in surrounding medium and go.This method is simple to operate, little on cytoactive impact, although embedded material can to a certain degree hinder substrate and oxygen diffusion, and inapplicable to macromolecule substrate, but still be the process for fixation the most widely of research at present.There is the shortcoming that physical strength is low, resistance to mass transfer is larger in the process for fixation that the gel of take is embedding medium, and while adopting porous support embedding cell, the distribution of the carrying capacity of carrier and set state cell is wayward.
Summary of the invention
The method the present invention relates to is that the shortcoming that exists when adopting porous support embedding cell is improved and formed, and object is make the abundant cell of set in carrier and realize cell being uniformly distributed in carrier.
Object of the present invention is achieved through the following technical solutions:
A carrier immobilized method for cell, is characterized in that comprising the following steps:
1) seed liquor that preparation comprises the cell that is fixed;
2) preparation is applicable to the nutrient solution of Growth of Cells;
3) in being placed in the inoculation cylinder of reaction vessel, inject nutrient solution, until nutrient solution volume accounts for 70% ~ 80% of reaction vessel volume, wherein, and this inoculation cylinder upper and lower opening sealing, barrel is uniformly distributed through hole, and the outer socket of cylinder one monoblock is the carrier of hollow cylindrical;
4) seed liquor is caused in inoculation cylinder, seed liquor add-on is 1% ~ 10% of nutrient solution volume;
5) continue to inject nutrient solution in inoculation cylinder, open discharge nozzle simultaneously, make the total liquid volume in reaction vessel remain at below 90% of reaction vessel volume;
6) by turbidometer, monitor the cell concn in the liquid that penetrates carrier, judgement immobilization terminal.
Optimally, the step 6) of above-mentioned process for fixation replaces with: by turbidometer, monitor the cell concn in the liquid that penetrates carrier, while reaching default adverse current value, start the adverse current program of 30 minutes: the liquid reverse osmosis carrier outside carrier is extremely inoculated in cylinder, after adverse current EP (end of program), be immobilization terminal.
Optimally, in described reaction vessel, be arranged with at least two inoculation cylinders.
Optimally, the through-hole diameter of described inoculation cylinder is 0.2mm ~ 1mm.
What the present invention was more detailed is described below:
A kind of equally distributed method of immobilized cell that makes in carrier realizes by designing a kind of stainless steel inoculation cylinder.This cylinder 1-2 bottom is open, and cylinder outward flange has a circle screw thread, can engage with a stainless steel chassis, thereby make closed bottom, and seed liquor enters in inoculation cylinder from stream 4-14,4-15,4-16.The top tool plug of inoculation cylinder, there is agitator access port in stopper central authorities, the barrel aperture that gathers.
During use, inoculation cylinder is installed on stainless steel chassis, chassis central authorities have a pedestal, can place stirrer shaft, and inoculation cylinder top stopper will be inoculated cylinder sealing through stir shaft, and porous support is designed to open circles tubular, inner diameter d
1with inoculation cylinder outside diameter d
2meet d
1=d
2, thereby carrier can be enclosed within on inoculation cylinder.By inoculation cylinder, to adding fermentation culture to final volume in classification inoculation apparatus, be 70% ~ 80% of reaction vessel volume again.To being added with the reaction vessel of fermentation culture, carry out after autoclaving, the inoculum size according to 1% ~ 10% is added seed liquor in inoculation cylinder.Finally, use fermentation culture fresh, sterilizing instead and pass into from stream 4-15,4-16, original seed liquor in displacement cylinder, the seed liquor aperture on barrel flows into carrier and is finally fixed in carrier.Or above-mentioned nutrient solution is all prior sterilizing.
As a preferred version of the present invention, the size of aforesaid stainless steel inoculation cylinder can determine according to the size of immobilized cell bioreactor and required carrier, can design one to several hickeys on chassis, to be connected to several inoculations tin with one.When connecting two above inoculation cylinders, cancel the built-in design of stir shaft, be about to stir shaft and be placed in outside inoculation cylinder, meanwhile, inoculation pipeline is no longer from inoculation cylinder bottom access inoculation cylinder but enter inoculation cylinder by original stir shaft present position.
Aforesaidly make the equally distributed method of immobilized cell in carrier, its stainless steel inoculation cylinder can also be designed to as shown in Figure 3.Inoculation cylinder closed bottom, stir shaft is external, and on stainless steel chassis, design can be laid the groove of stainless steel inoculation cylinder.This design is applicable to install the situation of a plurality of inoculation cylinders simultaneously.
Aforesaidly make the equally distributed method of immobilized cell in carrier, the hole diameter distributing on the barrel of its stainless steel inoculation cylinder is 0.2mm ~ 1mm, in actual use, according to the characteristics design of the classification of bacterial classification and seed liquor, becomes different sizes.
Aforesaidly make the equally distributed method of immobilized cell in carrier, it treats, in the external environment of immobilization carrier, a turbidometer is installed, the concentration of the free cell in the nutrient solution can Real-Time Monitoring flowing out through carrier, thus judge the terminal of cell fixation.
Aforesaidly make the equally distributed method of immobilized cell in carrier, its nutrient solution flowing out through carrier first enters container 4-4 through stream 4-17, then forms a circulation loop by stream 4-17 first half section pipeline, stream 4-18, stream 4-15, stream 4-16.So repeatedly circulation, until reach best carrying capacity in carrier.
Aforesaidly make the equally distributed method of immobilized cell in carrier, its carrier of inoculating can be directly used in as required immobilization fermentation reaction or under aseptic condition, be divided into the carrier granule of suitable size.
Aforesaidly make the equally distributed method of immobilized cell in carrier, its stream can counter movement, it is adverse current program, be that the nutrient solution that flows out from carrier in container 4-4 is under the effect of pump 4-6, through stream, 4-17 flows back to reactor, and meanwhile, the nutrient solution in inoculation cylinder, under the effect of pump 4-5, flows back in container 4-2 through stream 4-16,4-15.So repeatedly circulation, makes the cell density in outside in hollow cylindrical carrier consistent, realizes being uniformly distributed of immobilized cell, adverse current program preferably 30 minutes.
Aforesaidly make the equally distributed method of immobilized cell in carrier, stainless steel inoculation cylinder wherein can be in a kind of novel immobilized cell bioreactor (Fig. 5), for the continuous enzymolysis enrichment of the marine active substances such as DHA/EPA, flavor peptides and repeatedly enzymolysis preparation.After cell is fixed in carrier, with stock liquid, replace initial incubation liquid, continue to utilize recycle pump 4-5 that stock liquid is pumped into stainless steel inoculation cylinder through stream 4-15, stream 4-16, simultaneously, ON cycle pump 4-6, extracts the reaction solution being seeped into outside carrier out, and through stream, 4-17 enters in receiving flask.So continuous enzymolysis enrichment of realize target product.
Aforesaidly make the equally distributed method of immobilized cell in carrier, stainless steel inoculation cylinder wherein can be in a kind of novel immobilized cell bioreactor, for the continuous enzymolysis enrichment of the marine active substances such as DHA/EPA, flavor peptides and repeatedly enzymolysis preparation, wherein said repeatedly enzymolysis preparation is to realize by stream as shown in Figure 4.After stock liquid adds in stainless steel inoculation cylinder, valve-off 4-9, valve 4-11, utilize recycle pump that the reaction solution being seeped into outside carrier is pumped in stainless steel inoculation cylinder again through stream 4-17, stream 4-18, stream 4-15, stream 4-16, carry out secondary enzymolysis reaction.By timing sampling, detect, determine the number of times that repeats enzymolysis.The so repeatedly enzymolysis of realize target product preparation.
Method for immobilizing cell of the present invention is applicable to common micro-organisms cell and enzyme etc., is particularly useful for candida cell.
Compare with existing process for fixation, the equally distributed method of immobilized cell in carrier that makes the present invention relates to has following advantage:
(1) simple to operate, little on cytoactive impact, set state cell density is large, be applicable to large-scale industrialization and produce;
(2) design a kind of stainless steel inoculation cylinder, after suitably amplifying, can be used for the immobilization of cell, can be used for again immobilized cell and produce;
(3) carrier is first whole for cell fixation, then is divided into as required the carrier granule of appropriate size, has effectively avoided small-particle carrier to be directly used in the shortcoming that cell set is inhomogeneous and carrier utilization ratio is not high that cell fixation produces.
Accompanying drawing explanation
The monotubular of Fig. 1 the present invention design, there are each parts of hickey classification inoculation apparatus and assembling schematic diagram thereof.
Many of Fig. 2 the present invention design, have each parts of hickey classification inoculation apparatus and an assembling schematic diagram thereof.
Many of Fig. 3 the present invention design, without each parts of hickey classification inoculation apparatus and assembling schematic diagram thereof.
Each stream schematic diagram of immobilization process of Fig. 4 the present invention design.
The bio-reactor schematic diagram of being fixed of the inoculation cylinder cells produce of Fig. 5 application the present invention design.
In figure, each Reference numeral implication sees the following form:
Embodiment
Below in conjunction with accompanying drawing, object of the present invention, advantage and disadvantage are further detailed.That given is preferential embodiment, and diagram and explanation do not form a kind of restriction to the claims in the present invention.
Embodiment mono-: as shown in Figure 1 and Figure 2, the present invention realizes immobilized cell being uniformly distributed in carrier by designing a kind of cylinder of inoculating.This classification inoculation apparatus can be comprised of single inoculation cylinder, and matching component comprises agitator, chassis and various pipeline.Classification inoculation apparatus diameter d
3=240mm, height H=360mm, chassis diameter d
4=200mm, d
3/ d
4=1.2:1, height h
1=10mm, is fixed on reactor bottom central authorities by welding or screw.Chassis central authorities leave a cylindrical space, diameter d
5=50mm, d
5/ d
3be 0.25, degree of depth h
2=10mm, h
2/ h
1=1.0, stir shaft pedestal is arranged on space central authorities, its height h
3=8mm, h
3/ h
2=0.8, ventpipe circular line is around pedestal, and the lateral duct of feed-pipe and ventpipe is built in chassis, and runs through chassis.Top, space corresponding part is fixed with a hickey, its inner diameter d
6=50mm, height h
4=10mm, ring inner surface is carved with screw thread, coincide, thereby stainless steel inoculation cylinder is fixed on chassis with stainless steel inoculation cylinder threaded area, lower end.Stainless steel inoculation cylinder threaded area outside diameter d
2=50mm, inner diameter d
7=50mm, thickness h
5=0.5mm, height h
6=300mm, h
6/ H=0.833.Cylinder upper end tool plug, stopper central authorities are provided with a sealing-ring, allow stir shaft through stopper, and screw thread is carved with in lower end, threaded area length h
7=10mm.Cylindrical wall, except position and threaded area, lower end that upper end contacts with stopper, rest part is uniformly distributed some apertures, aperture d
8=1mm.Stir shaft is built in stainless steel inoculation cylinder, stirring rake is installed, diameter of stirring paddle d on axle
9=40mm.Carrier adopts the many empty carriers of a monoblock to make, and is shaped as hollow cylindrical, inner diameter d
1=52mm, outside diameter d
10=160mm, height h
8=270mm, h
8/ h
6=0.9.During use, directly carrier is enclosed within on stainless steel inoculation cylinder, first utilize recycle pump that fermentation culture is pumped in stainless steel inoculation cylinder through stream 4-15, after it is full of inoculation cylinder, continue to add, nutrient solution flows out through stainless steel inoculation cylinder, carrier successively, to nutrient solution final volume be classification inoculation apparatus volume 70% time stop adding nutrient solution, in this timer, nutrient solution volume is about 10.5L.Take again flame inoculation method in inoculation bottle, to inject 0.525L seed liquor by 5% inoculum size, utilize the pressurized air of filtration sterilization that seed liquor is pressed in stainless steel inoculation cylinder from stream 4-14, stream 4-15, now, throttling valve 4-8 is in closing condition, and throttling valve 4-7, throttling valve 4-9 are in opened condition.Valve-off 4-7, Open valve 4-8, utilize recycle pump 4-5 that fermentation culture is pumped in inoculation cylinder through stream 4-15, stream 4-16, the seed liquor in former inoculation cylinder will be rushed in carrier, the nutrient solution flowing out from carrier enters receiving flask through stream 4-17, now, valve 4-11 is in opened condition, and valve 4-10 is in closing condition.When turbidometer outside carrier shows in carrier the enough cells of fixing foot, back flush again, even if the nutrient solution in container 4-4 is flowed back in classification inoculation apparatus by stream 4-17, nutrient solution in inoculation cylinder is flowed out by stream 4-16, stream 4-15 simultaneously, make a kind of pressure difference of the inside and outside generation of inoculation cylinder, the nutrient solution outside carrier flows to inoculation cylinder by carrier.In this process, liquid flows regressive erosion carrier, makes the distribution trend of immobilized cell wherein evenly, and this adverse current program is 30 minutes.
Embodiment bis-: the stainless steel inoculation cylinder of the present invention's design can be assembled into a fixedly immobilized cell reactor, for the enzymolysis enrichment of crude fish oil glyceryl ester type DHA, EPA, as shown in Figure 5.After cell is fixed in carrier, former inoculation cylinder can be for holding stock liquid, concrete operations are: ON cycle pump 4-5 and throttling valve 4-8, close throttling valve 4-7, the liquid medium that contains crude fish oil is pumped into stainless steel inoculation cylinder, ON cycle pump 4-6, throttling valve 4-11, keep throttling valve 4-10 in closing condition, by the nutrient solution extraction device outside carrier simultaneously.During this period, being aided with stirrer stirs.Stock liquid is under the dual function of recycle pump and agitator, successively through stainless steel inoculation cylinder, carrier.Stock liquid enters after carrier, under the effect of the lipase of secreting at set state microbes producing cellulase, decomposes and generates target product, finally flows into carrier peripheral space, and enters receiving flask through stream 4-17.
Claims (2)
1. a carrier immobilized method for cell, is characterized in that comprising the following steps:
1) seed liquor that preparation comprises the cell that is fixed;
2) preparation is applicable to the nutrient solution of Growth of Cells;
3) in being placed in the inoculation cylinder of reaction vessel, inject nutrient solution, until nutrient solution volume accounts for 70% ~ 80% of reaction vessel volume, wherein, and this inoculation cylinder upper and lower opening sealing, barrel is uniformly distributed through hole, and the outer socket of cylinder one monoblock is the porous support of hollow cylindrical;
4) seed liquor is caused in inoculation cylinder, seed liquor add-on is 1% ~ 10% of nutrient solution volume;
5) continue to inject nutrient solution in inoculation cylinder, open discharge nozzle simultaneously, make the total liquid volume in reaction vessel remain at below 90% of reaction vessel volume;
6) by turbidometer, monitor the cell concn in the liquid that penetrates porous support, judgement immobilization terminal;
Wherein, described reaction vessel is immobilized cell bioreactor, and this inoculation cylinder bottom is open, and cylinder outward flange has a circle screw thread, can engage with the stainless steel chassis of immobilized cell bioreactor, thereby make closed bottom, by seed liquor access inoculation cylinder; The top tool plug of inoculation cylinder, there is agitator access port in stopper central authorities, the barrel aperture that gathers, through-hole diameter is 0.2mm ~ 1mm; Stir shaft is built in inoculation cylinder, stirring rake is installed, diameter of stirring paddle 40mm on axle; During use, inoculation cylinder is installed on stainless steel chassis, and chassis central authorities have one for placing the pedestal of stirrer shaft, and inoculation cylinder top stopper will be inoculated cylinder sealing through stir shaft, porous support is designed to open circles tubular, thereby porous support can be enclosed within on inoculation cylinder.
2. the carrier immobilized method of cell according to claim 1, is characterized in that comprising the following steps:
1) seed liquor that preparation comprises the cell that is fixed;
2) preparation is applicable to the nutrient solution of Growth of Cells;
3) in being placed in the inoculation cylinder of reaction vessel, inject nutrient solution, until nutrient solution volume accounts for 70% ~ 80% of reaction vessel volume, wherein, and this inoculation cylinder upper and lower opening sealing, barrel is uniformly distributed through hole, and the outer socket of cylinder one monoblock is the carrier of hollow cylindrical;
4) seed liquor is caused in inoculation cylinder, seed liquor add-on is 1% ~ 10% of nutrient solution volume;
5) continue to inject nutrient solution in inoculation cylinder, open discharge nozzle simultaneously, make the total liquid volume in reaction vessel remain at below 90% of reaction vessel volume;
6) by turbidometer, monitor the cell concn in the liquid that penetrates carrier, while reaching default adverse current value, start the adverse current program of 30 minutes: make liquid reverse osmosis carrier outside carrier to inoculation cylinder, after adverse current EP (end of program), be immobilization terminal.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010618505.6A CN102140445B (en) | 2010-12-31 | 2010-12-31 | Carrier cell immobilization method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010618505.6A CN102140445B (en) | 2010-12-31 | 2010-12-31 | Carrier cell immobilization method |
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| Publication Number | Publication Date |
|---|---|
| CN102140445A CN102140445A (en) | 2011-08-03 |
| CN102140445B true CN102140445B (en) | 2014-09-03 |
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|---|---|---|---|
| CN201010618505.6A Expired - Fee Related CN102140445B (en) | 2010-12-31 | 2010-12-31 | Carrier cell immobilization method |
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