CN203999636U - A kind of zooblast adherent culture device - Google Patents
A kind of zooblast adherent culture device Download PDFInfo
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- CN203999636U CN203999636U CN201420408859.1U CN201420408859U CN203999636U CN 203999636 U CN203999636 U CN 203999636U CN 201420408859 U CN201420408859 U CN 201420408859U CN 203999636 U CN203999636 U CN 203999636U
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Abstract
The utility model discloses a kind of zooblast adherent culture device, comprise taper culturing bottle, the bottle mouth position of described culturing bottle is provided with dismountable bottleneck inner sleeve, in Erlenmeyer flask, be provided with and carry bucket peg, the upper end of carrying bucket peg is removably mounted in bottleneck and puts, and the lower end of carrying bucket peg is provided with zero load and carries bucket; In bottleneck, put and be provided with for sealing the upper cover of bottleneck inner sleeve.Autoclaving can Reusability after clean for this device, with low cost; In the preparation of engineering cell antibody and expression process, can supporting CO
2incubator or cell cultures shaking table carry out the cell cultures of 1L-10L scale and amplify antibody preparation, the simple convenient operation of method; For different cell kinds, tie up to when adopting this device to select carrier, also can select other commercialization carriers according to self cell characteristics, greatly expanded the range of application of this device, can provide strong support for Cell Biology Experiment.
Description
Technical field
The utility model relates to cell engineering field, relates generally to a kind of zooblast adherent culture device, for the engineering cell of all kinds of adherent growth, as Chinese hamster ovary celI, HEK293 cell and hybridoma etc.
Background technology
The basis of animal cell culture is histocyte culture technique.Recently due to bio-science with technological sciences interpenetrate, genetics and biochemical mutually combining, the new disciplines such as molecular genetics, molecular biology and cell engineering develop rapidly.The formation of these new subjects is all closely related with tissue culture technique with development, current histocyte culture technique is not only for studying the important technology method of life scientific theory, also day by day becomes means of production and the means of biotechnology, genetic engineering pharmaceutical simultaneously.
Adherent culture (anchorage-dependent culture) is to allow cell be attached to the cultural method of breeding in certain matrix, is mainly applicable to all attached cells, is also applicable to facultative attached cell.The cell category in actual production with adherent growth characteristic is more, as Chinese hamster ovary celI, bhk cell and Vero cell etc.
The major advantage of adherent culture is that cell can be attached at developing medium surfaces externally and internally, cell is by effectively expressing product, easily carry out the replacing of nutrient solution simultaneously, the pattern that easily adopts perfusion to cultivate, culturing process not section is added fresh medium, remove meta-bolites, thereby make unit volume inner cell density higher, compare maintainable culture cycle with suspension culture relatively long.And the weak point of adherent culture is to operate more loaded down with trivial detailsly, need suitable patch material and enough area of attachment, culture condition heterogeneity.Due to the adherent requirement of attached cell, the effective ways that improve cell density make specific surface area in reaction system maximize exactly.Traditional attached cell cultural method comprises that rolling bottle (roller bottle), multi-layer planar cultivate as the cell factory of Nune company, CellCube and Costar company (cell factories), and the stirring carrying agent suspension culture that declines.
Existing attached cell culture technique, there is considerable drawback, as the consumption of work, equipment, consumptive material has increased production cost greatly, as the medical disposable material equipment of ten thousand yuan of the value such as the dish-shaped carrier of the 50mg specification disc of the 5L volume cell factory system HeGE company of Costar company; The rolling bottle of large volume, cell factory are because clean reason is difficult to reuse, and for increasing its adherent area, volume or quantity is corresponding increase also, operation, pollution are taken precautions against and become more difficult; Due to cell attachment growth, after cultivation finishes, adherent dielectric surface can adhere to a large amount of Fibronectins and cell debris, makes the clean and quality control of system become more difficult, and the control that external source is polluted has increased again extra R&D costs.So these factors have greatly limited attached cell and have cultivated the application in antibody R&D process.
Summary of the invention
The purpose of this utility model is, a kind of device for zooblast adherent culture is provided, can be for the zooblast of the adherent growth such as Chinese hamster ovary celI, HEK293 cell and hybridoma, carry out the cell cultures of pilot scale, carry out the expression of the target protein antibody of hundred milligrams of levels, improve antibody drug development efficiency in early stage, reduce R&D costs, simultaneously for the Related Experimental Study such as tumour or the external a large amount of amplifications of histocyte and DNA vector in-vitro transfection provide strong support.
The utility model is by the following technical solutions:
A kind of zooblast adherent culture device, comprise taper culturing bottle, the bottle mouth position of described culturing bottle is provided with dismountable bottleneck inner sleeve, is provided with and carries bucket peg in Erlenmeyer flask, the upper end of carrying bucket peg is removably mounted in bottleneck and puts, and the lower end of carrying bucket peg is provided with zero load and carries bucket; In bottleneck, put and be provided with for sealing the upper cover of bottleneck inner sleeve.
Further, the described bucket peg of carrying comprises knee, and knee is by the epimere bar being parallel to each other and hypomere bar and be connected epimere bar and the union lever of hypomere bar forms, and is integral type structure; The length of epimere bar is greater than the length of hypomere bar, and the top of epimere bar is consolidated with the card that width is greater than epimere shank diameter, is processed with the outside screw of carrying bucket for zero load is installed on hypomere bar.
Further, carry bucket peg and be arranged on while putting in bottleneck, propose bucket peg and zero load and carry the lower end of bucket and all do not contact with culturing bottle bottom surface.
Further, described zero load is carried bucket for hollow cuboid structure, in zero load, carry on the sidewall of bucket and be evenly distributed with a plurality of circular holes that penetrate sidewall, zero load is carried the lower fitting nut that tighten nut and bottom of bucket by its top and is arranged on the hypomere bar of carrying bucket peg.
Further, described bottleneck inner sleeve comprises annular cover and the sleeve fixed with annular cover coaxial inner conductor, and the internal diameter of annular cover is identical with the internal diameter of sleeve, and the external diameter of sleeve is less than the internal diameter of culturing bottle bottleneck, and the external diameter of annular cover is greater than the external diameter of culturing bottle bottleneck.
Further, on the inwall of annular cover and sleeve, be processed with vertically the draw-in groove of carrying bucket peg for installing, draw-in groove length is less than the axial length of bottleneck inner sleeve.
Further, described zero load is carried in bucket carrier is housed, and the position, middle and lower part that bucket is positioned at culturing bottle is carried in zero load.
Further, described zero load is carried bucket and in culturing bottle, is arranged 1～4.
Technological merit of the present utility model:
1) this device can be applicable to zooblast adherent culture, in cytobiology and cell engineering field, has a wide range of applications, and is applicable to cell proliferation cultivation or engineering cell and cultivates antibody and the Related Experimental Study such as prepare;
2) this device can be to vibrator speed of rotation, put forward bucket peg type, adjustment is selected, selected to adherent carrier classification, to optimize the cell of culture systems, attaches ability, increases cell culture period, improves cell density and product output;
3) this device is easy and simple to handle, with low cost, carries bucket and culturing bottle and can after alkali lye processing is clean, after high pressure, reuse.
Accompanying drawing explanation
Fig. 1 is the one-piece construction schematic diagram of this device;
Fig. 2 is the front view of putting forward bucket peg part;
Fig. 3 is the side-view of putting forward bucket peg part;
Fig. 4 is the axial sectional view of bottleneck inner sleeve;
Fig. 5 is the vertical view of bottleneck inner sleeve;
Fig. 6 is three kinds of cell square vases and the contrast of this device adherent culture density;
Fig. 7 is the microscopic examination of three kinds of cell borate glass fiber adherent culture;
Fig. 8 is adherent transfection eGFP-N1 plasmid egfp expression in 293T cell Erlenmeyer flask;
Fig. 9 is that comparison diagram is prepared in the H18 chimeric antibody expression of comparison example 3;
Figure 10 is that the CypA antibody expression of comparison example 4 is prepared comparison diagram;
Figure 11 is that comparison diagram is prepared in the H18 chimeric antibody expression of comparison example 5;
Number in the figure representative: 1-upper cover, 2-sleeve, 3-culturing bottle, 4-epimere bar, 5-union lever, 6-tighten nut, 7-lid, bucket is carried in 8-zero load, 9-hypomere bar, 10-lower fitting nut, 11-annular cover, 12-draw-in groove, 13-card;
The present invention is described in more detail for the embodiment providing below in conjunction with accompanying drawing and contriver.
Embodiment
Defer to technique scheme, as shown in Figure 1 to Figure 3, a kind of zooblast adherent culture device, comprise taper culturing bottle 3, the bottle mouth position of described culturing bottle 3 is provided with dismountable bottleneck inner sleeve, in Erlenmeyer flask, be provided with and carry bucket peg, the upper end of carrying bucket peg is removably mounted in bottleneck and puts, and the lower end of carrying bucket peg is provided with zero load and carries bucket 8; In bottleneck, put and be provided with for sealing the upper cover 1 of bottleneck inner sleeve.
The agent structure of this device is transparent taper culturing bottle 3, is provided with bottleneck inner sleeve on the bottleneck of culturing bottle 3.As shown in Figure 4, bottleneck inner sleeve comprises annular cover 11 and overlaps the fixed sleeve of 11 coaxial inner conductors 2 with annular, the internal diameter of annular cover 11 is identical with the internal diameter of sleeve 2, the external diameter of sleeve 2 is less than the internal diameter of culturing bottle 3 bottlenecks, the external diameter of annular cover 11 is greater than the external diameter of culturing bottle 3 bottlenecks, during use by bottleneck inner sleeve plug on the bottleneck of culturing bottle 3, be used for installing and carry bucket peg.In order to guarantee the stopping property of culturing bottle 3, the external diameter of sleeve 2 should be slightly less than the internal diameter of bottleneck.
In bottleneck, put and be provided with upper cover 1, the longitudinal cross-section of upper cover 1 is inverted trapezoidal, while being sleeved on the bottleneck of culturing bottle 3 in bottleneck, by upper cover 1 in order to seal bottleneck inner sleeve.
Carry bucket peg and comprise knee, knee is by the epimere bar 4 being parallel to each other and hypomere bar 9 and be connected epimere bar 4 and the union lever 5 of hypomere bar 9 forms, and is integral type structure; The length of epimere bar 4 is greater than the length of hypomere bar 9, and the top of epimere bar 4 is consolidated with the card 13 that width is greater than epimere bar 4 diameters, and the effect of card 13 is in the draw-in groove 12 being stuck in bottleneck inner sleeve; On hypomere bar 9, be processed with the outside screw of carrying bucket 8 for zero load is installed.Carry bucket peg and be arranged on while putting in bottleneck, carry bucket peg and unloaded 8 the lower end of struggling against of carrying does not all contact with culturing bottle 3 bottom surfaces, maintain a certain distance.
Zero load is carried bucket 8 for hollow cuboid structure, lid 7 is arranged at its top, in zero load, carry on bucket 8 sidewall and be evenly distributed with a plurality of circular holes that penetrate sidewall, zero load is carried bucket 8 top and bottom and is respectively arranged with and tightens nut 6 and lower fitting nut 10, by two nuts, by its fastening being arranged on the hypomere bar 9 of carrying bucket peg, and can dismantle.
On the inwall of bottleneck inner sleeve,, on the inwall of annular cover 11 and sleeve 2, along it, be axially processed with the draw-in groove 12 of carrying bucket peg for installing, as shown in Figure 5; Draw-in groove 12 length are less than the axial length of bottleneck inner sleeve, draw-in groove 12 with respect to bottleneck inner sleeve be axially not penetrating, the card 13 of carrying bucket peg is placed in draw-in groove 12 along the bearing of trend of draw-in groove 12, can be used for carry zero load and carry bucket 8; And will carry bucket peg, from draw-in groove 12, proposes, can realize and carry the separated of struggle against peg and bottleneck inner sleeve.Zero load is carried bucket 8 and preferably in culturing bottle 3, is arranged 1～4.
Bottleneck inner sleeve, carry bucket peg, zero load carry bucket 8 and tighten nut 6, lower fitting nut 10 all adopts 316L stainless steel to make, bottleneck inner sleeve, carry bucket peg and carry with zero load 8 surfaces externally and internallies that struggle against and all pass through polished finish.
This device adopts suspension design that stainless steel zero load is carried to the middle and lower part that bucket 8 hangs on culturing bottle 3, can change the zero load of peg type adjustment by mistake and put forward bucket 8 distances apart from Erlenmeyer flask center, thereby adjust liquid stream less turbulence.
The carrier of carrying in bucket 8 in zero load cleans and fills before autoclaving, carrier is borate glass fiber yarn, use poly-lysine to carry out surface treatment, increase cell and attach dynamics, this device can select to load the commercialization carrier that huge carrier, dish-shaped carrier equal diameter are greater than 2mm simultaneously.
Clean and the installation of this device
1) using 5%Decon90 scavenging solution to soak culturing bottle 3, carrier and all parts spends the night;
2) use respectively tap water and pure water fully to rinse bottle, carrier and all parts;
3) take same day pure water pH is standard, uses the residual water pH of pH each parts of detection paper value, until equate with pure water pH;
4) take appropriate carrier (borate glass fiber or commercialization carrier), open the lid 7 that bucket 8 is carried in zero load, pack carrier into zero load and carry in bucket 8; Zero load is carried to bucket 8 lid 7 and by lower end, penetrated and carried in bucket peg, connect and carry bucket peg and lower fitting nut 10, bucket 8 is carried in sealing zero load, screws to carry to tighten nut 6 above bucket peg;
5) zero load of installation is carried to bucket 8 and slowly vertically put into successively taper culturing bottle 3;
6) bottleneck inner sleeve cover is packed in taper culturing bottle 3, will carry bucket peg upper end bayonet socket and be installed
Enter in the draw-in groove 12 of bottleneck inner sleeve, cover taper culturing bottle 3 upper covers 1;
7) put into autoclave, taper culturing bottle 3 upper covers 1 are outwarded winding to 1/3, start autoclaving, 121 ℃ of high pressure 30min;
8) after autoclaving completes, the upper cover 1 of sealing taper culturing bottle 3, stand-by.
Borate glass fibrous carrier is processed
By poly-lysine, process the cell attaching ability that can improve borate glass fiber, experiment finds that tumour cell, Chinese hamster ovary celI, HEK293 cell and BHK21 cell have good attaching ability to borate glass fiber except hybridoma and Vero extracellular.So, can select whether to carry out poly-lysine processing according to concrete cultivation situation.
1) use PBS to prepare 100 μ g/ml Poly-L-Lysine Solutions, 0.22 μ m filter filtration sterilization;
2) aseptic PBS is diluted to 25 μ g/ml, adds in the aseptic taper culturing bottle 3 of this device, and liquid answers submergence zero load to carry the top of bucket 8;
3) ambient temperature overnight, PBS rinses after 3 times, stand-by;
Cell cultures growth example 1
1. cell inoculation
By approximately 1 * 10
7the CHO-k1 of cells quantity, HEK293T, H18 hybridoma are seeded in the taper culturing bottle 3 of the difference equipped with glass fiber JiGE commercialization disc of company dish carrier, add 1640 interpolation 10% foetal calf serum substratum to 500ml, be positioned in cell shaking table;
1) cultivate and observe
Cell suspension situation in microscopic examination every day supernatant liquor;
2) cell counting
Cultivate after 5 days, abandoning supernatant, cell in bucket is respectively carried in 50ml trysinization, and hematimeter carries out cell counting;
3) experimental result
After inoculation 24h, cell is all attached in carrier, and substantially acellular suspension is observed in supernatant sampling, can be observed supernatant liquor flavescence gradually between 48-72h, and after 72h, in supernatant liquor, cast-off cells and fragment increase gradually; Reclaim digestion count results and see Fig. 6, this device of result display application can be realized cell attachment and cultivate a large amount of amplifications, and the adherent carrier culture effect of commercialization carrier disc is slightly better than borate glass fibrous carrier, and fiberglass carrier displaing micro picture is shown in Fig. 7.
Plasmid DNA transfection example 2
1) cell inoculation
By approximately 1 * 10
7cells quantity HEK293T is seeded to and installs additional in borate glass fiber taper culturing bottle 3, adds 1640 interpolation 10% foetal calf serum substratum to 450ml, is positioned over incubated overnight in cell shaking table;
2) plasmid transfection
I. according to PEI (sigma) working instructions, with PEI/DNA1:2 ratio, mix eGFP-N1 plasmid DNA and PEI solution, plasmid DNA assay concentration 2ug/ml, room temperature is placed 10min;
Ii. discard the interior blood serum medium of culturing bottle 3, use aseptic PBS solution to rinse 1-2 time gently, add 450ml1640 substratum;
Iii. plasmid DNA and PEI copolymer solution are added in culturing bottle 3, put into cell shaking table and cultivate after 4h, add 10% foetal calf serum, continue to cultivate;
3) fluorescent protein expression is observed
After 24h, take out fiberglass carrier, fluorescence microscopy Microscopic observation egfp expression situation;
4) experimental result
As Fig. 8 shows, after 24h, 293T cell attachment is in good condition, after transfection eGFP-N1 plasmid, and green fluorescent protein normal expression, and there is higher fluorescence intensity.
H18 antibody expression is prepared comparison example 3
1) cell inoculation
I. by approximately 1 * 10
7the H18 hybridoma of cells quantity is seeded in culturing bottle 3, in bottle, loads respectively the disc of GE company carrier and fiberglass carrier, adds respectively 1640 interpolation 10% foetal calf serum substratum to 500ml;
Ii. culturing bottle 3 is positioned over to CO
2cultivate in shaking table, rotational oscillation, arranges rotating speed 50rpm/min, observes and cultivates;
After iii.48h, cell factory is added 1L1640 and is added 10% foetal calf serum substratum, and culturing bottle 3 progressively regulates vibrator rotating speed to 80rpm/min;
After iv.5 days, reclaim supernatant liquor, AKTA affinity chromatography system purification of target antibody
2) preparation detects
Through cultivation in 5 days, cell factory reclaimed upper honest and upright and thrifty 1.9L, obtains antibody 85.8mg, and yield is 45.16mg/L; This device reclaims supernatant 0.85L, obtains antibody 38.6mg, and yield is 45.4mg/L, and output is without significant difference; Through SDS-PAGE, detect, antibody purity and molecular weight no significant difference, as shown in Figure 9.
CypA antibody expression is prepared comparison example 4
1) cell inoculation
I. by approximately 1 * 10
7the H18 hybridoma of cells quantity is seeded in culturing bottle 3, in bottle, loads respectively the disc of GE company carrier and fiberglass carrier, adds respectively 1640 interpolation 10% foetal calf serum substratum to 500ml;
Ii. culturing bottle 3 is positioned over to CO
2cultivate in shaking table, rotational oscillation, arranges rotating speed 60rpm/min, observes and cultivates;
After iii.48h, culturing bottle 3 progressively regulates vibrator rotating speed to 100rpm/min;
After iv.5 days, reclaim supernatant liquor, AKTA affinity chromatography system purification of target antibody.
2) preparation detects
Through cultivation in 5 days, the disc of Ge company carrier recovery supernatant 0.45L, obtained antibody 5.15mg, and yield is 11.4mg/L; Fiberglass carrier reclaims supernatant 0.45L, obtains antibody 4.28mg, and yield is 9.5mg/L, and output is without significant difference; Through SDS-PAGE, detect, antibody purity and molecular weight no significant difference, as shown in figure 10.
H18 chimeric antibody is expressed preparation comparison example 5
1) cell inoculation
I. by approximately 1 * 10
7the H18 hybridoma of cells quantity is seeded in culturing bottle 3, in bottle, loads respectively the disc of GE company carrier and fiberglass carrier, adds respectively 1640 interpolation 10% foetal calf serum substratum to 500ml;
Ii. culturing bottle 3 is positioned over to CO
2cultivate in shaking table, rotational oscillation, arranges rotating speed 60rpm/min, observes and cultivates;
After iii.48h, culturing bottle 3 progressively regulates vibrator rotating speed to 100rpm/min;
After iv.5 days, reclaim supernatant liquor, AKTA affinity chromatography system purification of target antibody
2) preparation detects
Through cultivation in 5 days, the disc of Ge company carrier recovery supernatant 0.45L, obtained antibody 37.26mg, and yield is 82.8mg/L; Fiberglass carrier reclaims supernatant 0.45L, obtains antibody 39.42mg, and yield is 87.6mg/L, and output is without significant difference; Through SDS-PAGE, detect, antibody purity and molecular weight no significant difference, as shown in figure 11.
Claims (8)
1. a zooblast adherent culture device, comprise taper culturing bottle (3), it is characterized in that, the bottle mouth position of described culturing bottle (3) is provided with dismountable bottleneck inner sleeve, in Erlenmeyer flask, be provided with and carry bucket peg, the upper end of carrying bucket peg is removably mounted in bottleneck and puts, and the lower end of carrying bucket peg is provided with zero load and carries bucket (8); In bottleneck, put the upper cover (1) being provided with for sealing bottleneck inner sleeve.
2. zooblast adherent culture device as claimed in claim 1, it is characterized in that, the described bucket peg of carrying comprises knee, knee is by the epimere bar (4) being parallel to each other and hypomere bar (9) and be connected epimere bar (4) and the union lever (5) of hypomere bar (9) forms, and is integral type structure; The length of epimere bar (4) is greater than the length of hypomere bar (9), the top of epimere bar (4) is consolidated with the card (13) that width is greater than epimere bar (4) diameter, on hypomere bar (9), is processed with the outside screw of carrying bucket (8) for zero load is installed.
3. zooblast adherent culture device as claimed in claim 1, is characterized in that, carries bucket peg and is arranged on while putting in bottleneck, carries the lower end that bucket peg carries bucket (8) with zero load and does not all contact with culturing bottle (3) bottom surface.
4. zooblast adherent culture device as claimed in claim 2, it is characterized in that, described zero load is carried bucket (8) for hollow cuboid structure, in zero load, carry on the sidewall of bucket (8) and be evenly distributed with a plurality of circular holes that penetrate sidewall, zero load is carried the lower fitting nut (10) that tighten nut (6) and bottom of bucket (8) by its top and is arranged on the hypomere bar (9) of carrying bucket peg.
5. zooblast adherent culture device as claimed in claim 1, it is characterized in that, described bottleneck inner sleeve comprises annular cover (11) and the sleeve (2) fixed with annular cover (11) coaxial inner conductor, the internal diameter of annular cover (11) is identical with the internal diameter of sleeve (2), the external diameter of sleeve (2) is less than the internal diameter of culturing bottle (3) bottleneck, and the external diameter of annular cover (11) is greater than the external diameter of culturing bottle (3) bottleneck.
6. zooblast adherent culture device as claimed in claim 5, it is characterized in that, on the inwall of annular cover (11) and sleeve (2), be processed with vertically the draw-in groove (12) of carrying bucket peg for installing, draw-in groove (12) length is less than the axial length of bottleneck inner sleeve.
7. zooblast adherent culture device as claimed in claim 1, is characterized in that, described zero load is carried in bucket (8) carrier is housed, and the position, middle and lower part that bucket (8) is positioned at culturing bottle (3) is carried in zero load.
8. zooblast adherent culture device as claimed in claim 1, is characterized in that, described zero load is carried bucket (8) and arrange 1～4 in culturing bottle (3).
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| CN201420408859.1U CN203999636U (en) | 2014-07-23 | 2014-07-23 | A kind of zooblast adherent culture device |
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| CN201420408859.1U CN203999636U (en) | 2014-07-23 | 2014-07-23 | A kind of zooblast adherent culture device |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754369A (en) * | 2016-12-22 | 2017-05-31 | 青岛农业大学 | Blake bottle |
| CN107881107A (en) * | 2017-11-15 | 2018-04-06 | 中国航天员科研训练中心 | The image capturing system of automated cell culture device |
-
2014
- 2014-07-23 CN CN201420408859.1U patent/CN203999636U/en active Active
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754369A (en) * | 2016-12-22 | 2017-05-31 | 青岛农业大学 | Blake bottle |
| CN106754369B (en) * | 2016-12-22 | 2023-06-09 | 青岛农业大学 | Culture bottle |
| CN107881107A (en) * | 2017-11-15 | 2018-04-06 | 中国航天员科研训练中心 | The image capturing system of automated cell culture device |
| CN107881107B (en) * | 2017-11-15 | 2023-06-06 | 中国航天员科研训练中心 | Image acquisition system of automatic cell culture equipment |
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