CN103146578A - Transgenic cell culture device - Google Patents
Transgenic cell culture device Download PDFInfo
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- CN103146578A CN103146578A CN2013101063520A CN201310106352A CN103146578A CN 103146578 A CN103146578 A CN 103146578A CN 2013101063520 A CN2013101063520 A CN 2013101063520A CN 201310106352 A CN201310106352 A CN 201310106352A CN 103146578 A CN103146578 A CN 103146578A
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Abstract
The invention discloses a transgenic cell culture device and belongs to technical field of medical treatment and laboratory equipment. The transgenic cell culture device comprises a bottle cover, a bottle opening, a bottle head, a bottle neck, an upper bottle, a lower bottle, a bottle body, a bottle cavity, a support column, a growth plate, a liquid permeating plate, an upper liquid box cavity, growth plate insertion holes, a liquid box, a bottle bottom, a lateral bottle side, a liquid permeating plate body, supports among plates, a support shaft, growth plate clamp grooves, a support layer vertical wall, a support layer, a liquid absorbing layer, a growth liquid permeation adherent layer, liquid permeating holes, support shaft holes and a selection device. The device is characterized in that the growth plate comprises the vertical wall support layer, the growth liquid permeation adherent layer, the liquid absorbing layer, the support layer, the liquid permeating holes and the support shaft holes; the growth liquid permeation adherent layer is a fiber membrane, the liquid permeating holes are arranged in the fiber membrane, so that cells inoculated onto the outer surface of the membrane can favorably grow; the liquid absorbing layer can absorb the culture liquid; and the plastic front edge and the two plastic lateral edges of the support layer can be folded up to form the support layer vertical wall so as to prevent the culture liquid from flowing to the outside. The transgenic cell culture device has beneficial effects of simpleness in manufacture, multiple functions, strong operability, low cost and obvious effects.
Description
Technical field
The present invention relates to a kind of transgenic cell culture apparatus, belong to medical treatment and laboratory equipment technical field.
Background technology
Transgenic technology is manually to separate and the gene of modified imports in the organism genome, due to the expression of quiding gene, causes heritable modification of the proterties of organism, and this technology is referred to as transgenic technology Transgene technology." genetic engineering " that it has often been said, " genetically engineered ", " genetic transformation " are genetically modified synonym.It is exactly the animal that contains foreign gene in genome such as Animal Transgenic, these animals are the designs according in advance, by cytogamy, cell restructuring, transfer of genetic material, chromosome engineering and genetic engineering technique, foreign gene is imported sperm, ovum or zygote, with the reproduction engineering technology, might breed transgenic animal again.Animal Transgenic Technology is obtained certain achievement in the domestic animals such as ox, sheep, pig, chicken, fish, such as passing through the transgenosis such as growth hormone gene, voluminous gene, short ovum plain gene, high lactation amount gene, lean meat species gene, keratin gene, parasiticide gene, antiviral gene, growth cycle can be bred short, farrowing, raw egg is many and lactation amount is high, meat, fur quality and good processability, and have the animal varieties of disease resistance.In the transgenic technology research process, cell cultures is the most basic experimental implementation, there is no cell, and all are not all known where to begin.At present, in cell cultivation process, in order to reach specific purpose, transgenic cell such as bovine fibroblasts etc. need to be cultivated quickly and easily, the material consumption that whole process need is larger, sterilization process particularly, the utensil that uses is more, needs the article that consume more, experimental cost also increases thereupon, so the experiment of cells involved all needs a large amount of financial supports, otherwise just can't complete.Through long-term practical observation, we find that the usage quantity of cell culture apparatus in the transgenic experiments process is maximum, are also links the most rambunctious, and little error sometimes just may cause large-area pollution, brings larger impact to experiment.therefore the chance that reduces to pollute just becomes extremely important, how to design a kind of can reduce the cell culture apparatus usage quantity and make the transfectional cell of cultivation can be in an airtight environment can be shifted and then reduce the contaminated chance of transfectional cell of cultivating by picking become and be badly in need of a great problem that solves, thereby can the manual method of picking device so utilize the adherent area that increases Growth of Cells in cell culture apparatus to reduce the cell culture apparatus usage quantity and add one, invent that a kind of to have multilayered structure and pick device and can be necessary at the outer transgenic cell culture apparatus that pick by hand the purpose cell of device for cell attachment growth.
Summary of the invention
to use too much because of cell culture apparatus in the Cell Biology Experiment process and repeatedly often increase the difficult problem of various risks to experiment in the transitional cell process in the culturing process that goes down to posterity in order to overcome, the invention provides a kind of transgenic cell culture apparatus, this transgenic cell culture apparatus utilizes increases the growth plate that multilayer can supply cell attachment to grow in cell culture apparatus, the liquid box is set guarantees that every growth plate all can make the cell on it obtain sufficient nutrient solution, arranging simultaneously can be by the manually operated device of picking of operator outside device, thereby reach the purpose of better completing cell cultures.
The technical solution adopted for the present invention to solve the technical problems is:
transgenic cell culture apparatus of the present invention is by bottle cap 1, bottleneck 2, bottle 3, bottleneck 4, bottle top 5, bottle following 6, bottle 7, bottle chamber 8, pillar stiffener 9, growth plate 10, liquid permeable plate 11, liquid box epicoele 12, growth plate jack 13, liquid box 14, the bottle end 15, bottle sidewall 16, liquid permeable plate body 17, support 18 between plate, back shaft 19, growth plate draw-in groove 20, holder layer wall 21, holder layer 22, liquid-adsorption layer 23, the adherent layer 24 of the liquid of growing, liquid drain hole 25, supporting axis hole 26 forms, it is characterized in that: growth plate 10 is by holder layer wall 21, the adherent layer 24 of the liquid of growing, liquid-adsorption layer 23, holder layer 22, liquid drain hole 25, supporting axis hole 26 forms, the long 4-14 of growth plate 10 centimetre, wide 2.5-7 centimetre, thick 1-3 millimeter, comprise three-decker, be followed successively by from top to bottom the adherent layer 24 of the saturating liquid of growth, liquid-adsorption layer 23, holder layer 22.The adherent layer 24 of the liquid of growing is one deck tunica fibrosas, thick 0.01-0.1 millimeter, the above has a lot of liquid drain holes 25, the nutrient solution that adsorbs from liquid box 14 in liquid-adsorption layer 23 can successfully be penetrated into the outside surface of the adherent layer of the saturating liquid of growth 24 film by liquid drain hole 25, in order to the growth that is seeded in cell on film outer surface; Liquid-adsorption layer 23 is one deck absorbent cotton, and thickness is the 0.69-1.9 millimeter, can adsorb nutrient solution; Holder layer 22 plastics quality or glass quality, the preferred glass quality, thick 0.3-1 millimeter, leading edge and both side edges are upwards turned up, and form holder layer wall 21, in order to avoid nutrient solution outwards oozes out.Support axis hole 26 and be on growth plate 10 strip hole with back shaft 19 corresponding sections, length is that 3-13 centimetre, width are the 2.1-3.1 millimeter, after being used for the interspersed growth plate 10 of back shaft 19, together with supporting 18 between plate, growth plate 10 is fixed on specific position, and growth plate 10 can be to support axis hole 26 edges and back shaft 19 formation slideways, advance between slave plate and plate or pull out, so that the growing state of cell on the observation growth plate perhaps cleans dead cell at growth plate 10, picks the operations such as monoclonal cell.
Described bottle cap 1 profile plastics quality, profile and structure be with known bottle cap, and lid mouthful internal surface has the silk that revolves of protrusion, can with the revolving together with silk is screwed in mutually of bottle 3 an outside surface evagination so that bottle cap 1 seals bottleneck 2, prevent that in bottle chamber 8, liquid flows out.Bottleneck 2 is openings of bottle 3 free end, namely adds liquid and injects the opening that the liquid in bottle chamber 8 or bottle chamber 8 flow out.Bottle 3 is the outwardly directed one section cirque structure of bottleneck 4 ends, and internal surface is smooth, and outside surface has the silk that revolves of evagination.Bottleneck 4 is the parts that are connected between bottle 3 and bottle 7, lower face length, above short, make bottle 3 be arranged in the top of bottle 7 axial planes, in order to avoid after cell culture apparatus was placed on horizontal plane, liquid flowed out from bottleneck 2, bottleneck 4 inner chambers and bottle 3 and a bottle chamber 8 are connected.
Described bottle 7 cuboids, long 5-15 centimetre, wide 3-8 centimetre, high 2-5 centimetre, its wall thickness 1-3 millimeter, quality is plastics quality or glass quality, preferred glass quality, front do not have a bottle wall, are openings, after being placed on horizontal plane, the above is above bottle 5, and the below be below bottle 6, and both sides are a bottle sidewall 16, the back is the bottle end 15, and the cavity that is surrounded by each face in it is bottle chamber 8.
Described pillar stiffener 9 be connected in above bottle 5 and bottle below 6 center column constructions, plastics quality or glass quality, the preferred glass quality, comprise support 18 between plate, back shaft 19, growth plate draw-in groove 20.Support 18 cylindrical ring sets between plate, internal diameter is the 2.2-3.2 millimeter, and external diameter is the 3-4 millimeter, and length is the 2-3 millimeter; Back shaft 19 is the axle of pillar stiffener 9 central authorities, and is cylindrical, and diameter is the 2-3 millimeter; The gap of supporting between 18 between the plate of two of up and down forms growth plate draw-in groove 20, is used for blocking growth plate 10, makes between adjacent two growth plates 10 at a distance of the 2-3 millimeter.
Described liquid permeable plate 11 is the cuboid plates that connect bottle 7 two sides and lower wall, plastics quality or glass quality, preferred glass quality, comprise growth plate jack 13 and liquid permeable plate body 17 two portions, long 2.8-6.4 centimetre, wide 1.6-3.8 centimetre, thickness is the 1-3 millimeter.Growth plate jack (13) is comprised of cell cultures growth plate jack (49) and mono-clonal screening growth plate jack (51), it is the duct that is bathed in after the plug that stretches out later of growth plate (10) inserts in the interior nutrient solution of liquid box (14), length is 0.5-1 centimetre, width is 0.1-0.3 centimetre, be positioned at the both sides of liquid permeable plate body 17, simultaneously also can and pillar stiffener (9) form the supporting structure of growth plate (10), between adjacent two growth plate jacks (13) up and down at a distance of the 2-3 millimeter.
Described liquid box 14 is box like structure of liquid permeable plate 11 and the formation of bottle at the end 15, hatch liquid bath 46, transfection reagent by trypsin solution groove 38, D-hanks liquid tank 39, screening and culturing liquid bath 40, waste liquid tank 41, buffering liquid groove 42, grown cultures liquid bath 43, transfection reagent groove 44, plasmid groove 45, plasmid and hatch liquid bath 47, syringe needle jack 48, cell cultures growth plate jack 49, waste liquid outlet orifice 50, mono-clonal screening growth plate jack 51 and form, be used for containing nutrient solution; Liquid box epicoele 12 is the space between liquid permeable plate 11 and bottle top 5, is the opening of nutrient solution liquid in-out box 14.
The described device 37 of picking is turned ball 28, flexible tube sealing 29, Glass tubing 30, breather cheek valve 31, unidirectional control valve 32, glass tube neck 33, chooses cell pipe 34, advances cytostome 35, is gone out cytostome 36 and form by suction glue head 27, bottle cap; Suction glue head 27 is glue heads that commercially available glue head dropper is used, and diameter is 0.5-1 centimetre, and length is 1-2 centimetre, and glue head wall thickness is the 1-2 millimeter, glue draught animals place round shape, and diameter is 0.5 centimetre, length is 0.5-1 centimetre; It is the spheroidal sealing member of 1-1.5 centimetre for diameter that bottle cap turns ball 28, formed by shell and endosphere, the shell disc, totally 2, the diameter of every is 1-1.5 centimetre, thickness is 0.5-0.75 centimetre, available bolt turns ball 28 with bottle cap and is fixed on bottle cap 1 bottom surface, two shells are by stacking the right cylinder of formation simultaneously, in this right cylinder central authorities, a diameter being arranged is the spheroidal space of 0.75-1.25 centimetre, is fixed in this space and can rotates therein so that fixed diameter is the endosphere of 0.75-1.25 centimetre; Crossing endosphere central authorities, a diameter is arranged is the duct of 0.5 centimetre, it is the space that Glass tubing 30 inserts, after Glass tubing 30 inserts the endosphere duct, the operator is enclosed within Glass tubing 30 1 ends with suction glue head 27, because endosphere can freely rotate and good seal performance, the operator just can control bottle inside of picking device and be divided into line operate; Flexible tube sealing 29 is the telescopic long tube of plastics matter, and back cut diameter is 0.75-1.25 centimetre, and lower port diameter is 0.5 centimetre, and structure can be stretched with the crook of known milk pipet, and whole flexible tube sealing 29 is enclosed within the outside of Glass tubing 30; Glass tubing 30 is that diameter is the long tube of 0.5 centimetre of glass quality, and total length is whole device inner length, wall thickness 1-2 millimeter, and the outer end turns ball 28 from bottle cap and passes bottle cap 1, and opening part has millipore filtration to seal; The inner is connected to glass tube neck 33 central authorities, and inner chamber communicates with the inner chamber of glass tube neck 33; Breather cheek valve 31 is positioned on Glass tubing 30 the flexible inner inboard 0.5-1 centimeters of tube sealing 29, and structure is with known breather cheek valve, can allow gas unidirectional in device enter Glass tubing 30 chambeies; Unidirectional control valve 32 structures are with known unidirectional control valve, be positioned at Glass tubing 30 inner ends, when suction glue head 27 is pinched or is unclamped, can and choose cell pipe 34 at glass tube neck 33 and form one suction, allow the cell picked enter and choose cell pipe 34 from advancing cytostome 35, then by going out cytostome 36 out, and then be placed on specific position; Glass tube neck 33 is parts that Glass tubing 30 ends extend, and then base structure is tapered with Glass tubing 30, with the diameter of choosing cell pipe 34 connecting places be 0.2-0.3 centimetre, the length of whole glass tube neck 33 is 1-2 centimetre; Choose cell pipe 34 arcs, radian is 10-30 °, and length is 1-2 centimetre, advances cytostome 35 front, and bore is the 1-2 millimeter, and opening direction is consistent with glass tube neck 33 front opening directions; Go out cytostome 36 rear, bore is the 2-3 millimeter, opening direction and glass tube neck 33 front opening opposite directions.
During described manufacturing transgenic cell culture apparatus, first produce two portions up and down of cell culture apparatus, then add in the inside of bottom that liquid permeable plate 11 forms liquid box 14, insert growth plate 10, place up and down at the support axis hole 26 of growth plate 10 and support 18 between plate, insert back shaft 19, back shaft 19 lower ends are connected to below bottle on 6 internal surface, then two portions up and down of cell culture apparatus are heat sealing at together.
Beneficial effect of the present invention is, the transgenic cell culture apparatus utilizes increases the growth plate that multilayer can supply cell attachment to grow in cell culture apparatus, the liquid box is set guarantees that every growth plate all can make the cell on it obtain sufficient nutrient solution, arranging simultaneously can be by the manually operated device of picking of operator outside device, thereby reaches the purpose of better completing cell cultures.The transgenic cell culture apparatus is made simple, and diverse in function is workable, with low cost, successful.
Description of drawings
The present invention is further described below in conjunction with accompanying drawing.
Fig. 1 is the one-piece construction schematic diagram of transgenic cell culture apparatus of the present invention.
Fig. 2 is the structural representation of the liquid permeable plate of transgenic cell culture apparatus of the present invention.
Fig. 3 is the structural representation of the pillar stiffener of transgenic cell culture apparatus of the present invention.
Fig. 4 is the structural representation of the growth plate of transgenic cell culture apparatus of the present invention.
Fig. 5 is the structural representation of picking device of transgenic cell culture apparatus of the present invention.
Fig. 6 is the structural representation of the liquid box of transgenic cell culture apparatus of the present invention.
1. bottle caps in figure, 2. bottleneck, the bottle head, 4. bottleneck, 5. above the bottle, 6. below the bottle, 7. bottle, the bottle chamber, 9. pillar stiffener, 10. growth plate, 11. liquid permeable plate, 12. liquid box epicoele, 13. growth plate jack, 14. liquid box, 15. the bottle end, 16. bottle sidewall, 17. liquid permeable plate body, 18. support between plate, 19. back shaft, 20. growth plate draw-in groove, 21. holder layer wall, 22. holder layer, 23. liquid-adsorption layer, the adherent layer of liquid 24. grow, 25. liquid drain hole, 26. support axis hole, 27. suction glue head, 28. bottle cap turns ball, 29. flexible tube sealing, 30. Glass tubing, 31. breather cheek valve, 32. unidirectional control valve, 33. glass tube neck, 34. choose the cell pipe, 35. advance cytostome, 36. go out cytostome, 37. pick device, 38. trypsin solution groove, 39. D-hanks liquid tank, 40. screening and culturing liquid bath, 41. waste liquid tank, 42. buffering liquid groove, 43. grown cultures liquid bath, 44. transfection reagent groove, 45. plasmid groove, 46. plasmid is hatched liquid bath, 47. transfection reagent is hatched liquid bath, 48. syringe needle jack, 49. cell cultures growth plate jack, 50. waste liquid outlet orifice, 51. mono-clonal screening growth plate jack, 52. growth plate push-and-pull ring.
Embodiment
Embodiment one:
as shown in the figure, transgenic cell culture apparatus of the present invention is specially: by bottle cap 1, bottleneck 2, bottle 3, bottleneck 4, bottle top 5, bottle following 6, bottle 7, bottle chamber 8, pillar stiffener 9, growth plate 10, liquid permeable plate 11, liquid box epicoele 12, growth plate jack 13, liquid box 14, the bottle end 15, bottle sidewall 16, liquid permeable plate body 17, support 18 between plate, back shaft 19, growth plate draw-in groove 20, holder layer wall 21, holder layer 22, liquid-adsorption layer 23, the adherent layer 24 of the liquid of growing, liquid drain hole 25, support axis hole 26, suction glue head 27, bottle cap turns ball 28, flexible tube sealing 29, Glass tubing 30, breather cheek valve 31, unidirectional control valve 32, glass tube neck 33, choose cell pipe 34, advance cytostome 35, go out cytostome 36, pick device 37, trypsin solution groove 38, D-hanks liquid tank 39, screening and culturing liquid bath 40, waste liquid tank 41, buffering liquid groove 42, grown cultures liquid bath 43, transfection reagent groove 44, plasmid groove 45, plasmid is hatched liquid bath 46, transfection reagent is hatched liquid bath 47, syringe needle jack 48, cell cultures growth plate jack 49, waste liquid outlet orifice 50, mono-clonal screening growth plate jack 51, growth plate push-and-pull ring 52 forms.Bottle cap 1 profile plastics quality, profile and structure be with known bottle cap, and lid mouthful internal surface has the silk that revolves of protrusion, can with the revolving together with silk is screwed in mutually of bottle 3 an outside surface evagination so that bottle cap 1 seals bottleneck 2, prevent that in bottle chamber 8, liquid flows out.Bottleneck 2 is openings of bottle 3 free end, namely adds liquid and injects the opening that the liquid in bottle chamber 8 or bottle chamber 8 flow out.Bottle 3 is the outwardly directed one section cirque structure of bottleneck 4 ends, and internal surface is smooth, and outside surface has the silk that revolves of evagination.Bottleneck 4 is the parts that are connected between bottle 3 and bottle 7, lower face length, above short, make bottle 3 be arranged in the top of bottle 7 axial planes, in order to avoid after cell culture apparatus was placed on horizontal plane, liquid flowed out from bottleneck 2, bottleneck 4 inner chambers and bottle 3 and a bottle chamber 8 are connected.Bottle 7 cuboids, long 5-15 centimetre, wide 3-8 centimetre, high 2-5 centimetre, its wall thickness 1-3 millimeter, quality is plastics quality or glass quality, preferred glass quality, front do not have a bottle wall, are openings, after being placed on horizontal plane, the above is above bottle 5, and the below be below bottle 6, and both sides are a bottle sidewall 16, the back is the bottle end 15, and the cavity that is surrounded by each face in it is bottle chamber 8.Pillar stiffener 9 be connected in above bottle 5 and bottle below 6 center column constructions, plastics quality or glass quality, the preferred glass quality, comprise support 18 between plate, back shaft 19, growth plate draw-in groove 20.Support 18 cylindrical ring sets between plate, internal diameter is the 2.2-3.2 millimeter, and external diameter is the 3-4 millimeter, and length is the 2-3 millimeter; Back shaft 19 is the axle of pillar stiffener 9 central authorities, and is cylindrical, and diameter is the 2-3 millimeter; The gap of supporting between 18 between the plate of two of up and down forms growth plate draw-in groove 20, is used for blocking growth plate 10, makes between adjacent two growth plates 10 at a distance of the 2-3 millimeter.Liquid permeable plate 11 is the cuboid plates that connect bottle 7 two sides and lower wall, plastics quality or glass quality, and the preferred glass quality comprises growth plate jack 13 and liquid permeable plate body 17 two portions, long 2.8-6.4 centimetre, wide 1.6-3.8 centimetre, thickness is the 1-3 millimeter.Growth plate jack (13) is comprised of cell cultures growth plate jack (49) and mono-clonal screening growth plate jack (51), it is the duct that is bathed in after the plug that stretches out later of growth plate (10) inserts in the interior nutrient solution of liquid box (14), length is 0.5-1 centimetre, width is 0.1-0.3 centimetre, be positioned at the both sides of liquid permeable plate body 17, simultaneously also can and pillar stiffener (9) form the supporting structure of growth plate (10), between adjacent two growth plate jacks (13) up and down at a distance of the 2-3 millimeter.Liquid box 14 is box like structure of liquid permeable plate 11 and the formation of bottle at the end 15, is used for containing nutrient solution.Liquid box epicoele 12 is the space between liquid permeable plate 11 and bottle top 5, is the opening of nutrient solution liquid in-out box 14.liquid box 14 is by trypsin solution groove 38, D-hanks liquid tank 39, screening and culturing liquid bath 40, waste liquid tank 41, buffering liquid groove 42, grown cultures liquid bath 43, transfection reagent groove 44, plasmid groove 45, plasmid is hatched liquid bath 46, transfection reagent is hatched liquid bath 47, syringe needle jack 48, cell cultures growth plate jack 49, waste liquid outlet orifice 50, mono-clonal screening growth plate jack 51 forms, trypsin solution groove 38 is positioned at liquid box 14 top one ends, volume is the 5-10 milliliter, in fill trypsinase, it is fixed that tryptic amount is come according to the amount of the required trypsin solution of passage, ratio used is 0.25%.Namely press mass percent and add 0.25 gram trypsinase in 100 milliliters of D-hanks liquid.D-hanks liquid tank 39 volumes are the 5-10 milliliter, and interior Sheng is without the balanced salt damping fluid of calcium ions and magnesium ions, and pH 8.0.Screening and culturing liquid bath 40 volumes are the 15-40 milliliter, are used for the needed nutrient solution of splendid attire mono-clonal screening growth plate, and it is the 400-500 ug/ml that aminoglycoside antibiotics G418, concentration are used in the eukaryotic cell screening; Trypsin solution groove 38 and D-hanks liquid tank 39 are arranged at its top, front side wall is the growth plate jack 13 on liquid permeable plate body 17, there be 5-7, claim again mono-clonal screening growth plate jack 51, each mono-clonal screening growth plate jack 51 length is 0.5-1 centimetre, width is 0.1-0.3 centimetre, is to cultivate growth plate 10 insertions of monoclonal cell and the place that nutrient solution enters growth plate 10.Waste liquid tank 41 volumes are the 20-50 milliliter, be the space of collecting the various waste liquids that produce in the transgenic cell culturing process, there is waste liquid outlet orifice 50 its underpart, so that the waste liquid amount of preserving waste liquid tank 41 volume one halfs, on Bechtop, waste liquid is extracted out with asepsis injector.Waste liquid outlet orifice 50 is positioned at waste liquid tank 41 bottoms, that a diameter is that 1-2 millimeter, length are the Glass tubing of 3-5 millimeter, wall thickness 0.1-0.5 millimeter, waste liquid outlet orifice 50 the inners are opened on waste liquid tank 41 chambeies, the rubber sleave that external port is the 1-2 millimeter by a diameter entangles, in order to avoid in cell cultivation process, extraneous bacterium enters with waste liquid and overflows.Buffering liquid groove 42 volumes are the 20-50 milliliter, interior Sheng PBS phosphate buffered saline buffer.Because in the transgenic cell culturing process, often carry out single job, just must clean picking device, needed damping fluid is more, but due to the limited space of transgenic cell culture apparatus, in order to solve this difficult problem, be provided with syringe needle jack 48 on buffering liquid groove 42 tops, so that syringe inserts, the liquid stream that penetrates of syringe also can rinse preferably and pick device etc. and need the parts that clean simultaneously.The structure of syringe needle jack 48 is with waste liquid outlet orifice 50, also that 1 diameter is that 1-2 millimeter, length are the Glass tubing of 3-5 millimeter, wall thickness 0.1-0.5 millimeter, the inner is opened on buffering liquid groove 42, the rubber sleave that external port is the 1-2 millimeter by a diameter entangles, in order to avoid in cell cultivation process, extraneous bacterium enters and cushions hydrorrhea and goes out.grown cultures liquid bath 43, volume is the 15-40 milliliter, be used for the needed nutrient solution of splendid attire cell cultures growth plate, for example the required nutrient solution of bovine fibroblasts growth by volume percentage ratio add 10 milliliters of standard foetal calf serums in 90 milliliters of DMEM substratum, transfection reagent groove 44 is arranged at its top, plasmid groove 45, plasmid hatches liquid bath 46 and transfection reagent is hatched liquid bath 47, front side wall is that the growth plate jack 13 on liquid permeable plate body 17 only has 1, claim again cell cultures growth plate jack 49, each cell cultures growth plate jack 49 length is 0.5-1 centimetre, width is 0.1-0.3 centimetre, to cultivate growth plate 10 insertions of transgenic cell and the place that nutrient solution enters growth plate 10.Transfection reagent groove 44 volumes are the 3-5 milliliter, in fill PEI polymine transfection reagent, this reagent has been got rid of the impact of use serum on transfection efficiency in the cell cultures, and this reagent transfection efficiency is high, cost is low, good reproducibility, can be good at being applicable to the transfection research of bovine fibroblasts etc.plasmid groove 45 volumes are the 3-5 milliliter, in fill and contain foreign gene and wish the plasmid vector that it is expressed in host cell according to what genetically modified purpose was selected, plasmid expression vector such as the IRES-EGFP plasmid green fluorescence protein gene after linearization process, plasmid concentration is 1 microgram/microlitre, adopt this restriction enzyme in advance the expression vector IRES-EGFP of green fluorescent protein to be carried out enzyme and cut processing, make it become linearizing carrier segments, thereby can be by transfection, be incorporated into better in the genome of bovine fibroblasts, improve the efficient of green fluorescent protein stably express.It is the 3-5 milliliter that plasmid is hatched liquid bath 46 volumes, in fill DMEM substratum (Dulbecco improvement Eagle substratum), be the solution of hatching of plasmid.It is the 3-5 milliliter that transfection reagent is hatched liquid bath 47 volumes, in fill DMEM substratum (Dulbecco improvement Eagle substratum), be the solution of hatching of transfection reagent.Growth plate push-and-pull ring 52 is that diameter is that the 1-5 millimeter is connected to the glass ring of the front of growth plate 10 by handle, every growth plate front arranges 3 growth plate push-and-pull rings 52, lay respectively at the centre between growth plate 10 centres, fronts and centre and edge, when needs are observed on a certain growth plate 10 the transgenic cell growing state, can drop growth plate 10 with picking device, allow growth plate 10 move along supporting axis hole 26 on back shaft 19, and be pulled to the position that device can be observed with inverted microscope.Growth plate 10 is comprised of holder layer wall 21, the adherent layer 24 of the saturating liquid of growth, liquid-adsorption layer 23, holder layer 22, liquid drain hole 25, support axis hole 26, long 4-14 centimetre, wide 2.5-7 centimetre, thick 1-3 millimeter is followed successively by the adherent layer 24 of the saturating liquid of growth, liquid-adsorption layer 23, holder layer 22 from top to bottom.The adherent layer 24 of the liquid of growing is one deck tunica fibrosas, thick 0.01-0.1 millimeter, the above has a lot of liquid drain holes 25, cell only has several microns sizes on the one hand, 0.01-0.1 the thickness of millimeter is enough to provide for Growth of Cells the nutrient solution of the degree of depth that can the submergence cell, on the other hand, the nutrient solution that adsorbs from liquid box 14 in liquid-adsorption layer 23 is can be by liquid drain hole 25 very smooth and be penetrated into continually the outside surface of film, in order to the growth that is seeded in cell on film outer surface; Liquid-adsorption layer 23 is one deck absorbent cotton, and thickness is the 0.69-1.9 millimeter, can adsorb nutrient solution in conjunction with the pressure of liquid box 14 interior liquid, forms the bed course of growth plate 10; Holder layer 22 plastics quality or glass quality, the preferred glass quality, thick 0.3-1 millimeter, leading edge and both side edges are upwards turned up, and form holder layer wall 21, in order to avoid nutrient solution outwards oozes out.Support axis hole 26 and be on growth plate 10 strip hole with back shaft 19 corresponding sections, length is that 3-13 centimetre, width are the 2.1-3.1 millimeter, after being used for the interspersed growth plate 10 of back shaft 19, together with supporting 18 between plate, growth plate 10 is fixed on specific position, and growth plate 10 can be to support axis hole 26 edges and back shaft 19 formation slideways, advance between slave plate and plate or pull out, in order to observe the growing state of cell on growth plate with inverted microscope, perhaps clean dead cell at growth plate 10, pick the operation such as monoclonal cell.Picking device 37 is turned ball 28, flexible tube sealing 29, Glass tubing 30, breather cheek valve 31, unidirectional control valve 32, glass tube neck 33, chooses cell pipe 34, advances cytostome 35, is gone out cytostome 36 and form by suction glue head 27, bottle cap.Suction glue head 27 is glue heads that commercially available glue head dropper is used, diameter is 0.5-1 centimetre, length is 1-2 centimetre, and glue head wall thickness is the 1-2 millimeter, glue draught animals place round shape, diameter is 0.5 centimetre, length is 0.5-1 centimetre, so that suction glue head 27 is enclosed within the Glass tubing outer end when picking transgenic cell, by pinching loose glue head, power and suction are blown in generation, and then mobile needed positive colony cell strain is to the growth plate 10 of appointment.Pick complete after, suction glue head 27 is taken off, paste one deck millipore filtrations in Glass tubing 30 external port flame is other, in order to allow the oxygen in outside air enter in culture apparatus, for the Growth of Cells utilization.It is the spheroidal sealing member of 1-1.5 centimetre for diameter that bottle cap turns ball 28, formed by shell and endosphere, the shell disc, totally 2, the diameter of every is 1-1.5 centimetre, thickness is 0.5-0.75 centimetre, available bolt turns ball 28 with bottle cap and is fixed on bottle cap 1 bottom surface, two shells are by stacking the right cylinder of formation simultaneously, in this right cylinder central authorities, a diameter being arranged is the spheroidal space of 0.75-1.25 centimetre, is fixed in this space and can rotates therein so that fixed diameter is the endosphere of 0.75-1.25 centimetre.Crossing endosphere central authorities, a diameter is arranged is the duct of 0.5 centimetre, it is the space that Glass tubing 30 inserts, after Glass tubing 30 inserts the endosphere duct, the operator is enclosed within Glass tubing 30 1 ends with suction glue head 27, because endosphere can freely rotate and good seal performance, the operator just can control bottle inside of picking device and be divided into line operate.Flexible tube sealing 29 is the telescopic long tube of plastics matter, back cut diameter is 0.75-1.25 centimetre, lower port diameter is 0.5 centimetre, structure is with the crook of known milk pipet, can stretch, whole flexible tube sealing 29 is enclosed within the outside of Glass tubing 30, but one side fixing glass pipe 30, can further seal bottleneck 2 on the other hand, prevent that bacterium is in bottle cap turns gap access to plant ball 28, namely extract out or inject at Glass tubing 30 and set up barrier one when bottle cap turns ball 28, to reduce the contaminated probability of cell.Glass tubing 30 is that diameter is the long tube of 0.5 centimetre of glass quality, total length is whole device inner length, wall thickness 1-2 millimeter, has minimum on wall and is the scale of 1 millimeter, in order to pick and accurately draw quantitative liquid in process, the outer end turns ball 28 from bottle cap and passes bottle cap 1, and opening part has millipore filtration to seal, with needed oxygen in the supply cell growth process; The inner is connected to glass tube neck 33 central authorities, and inner chamber communicates with the inner chamber of glass tube neck 33.Breather cheek valve 31 is positioned on Glass tubing 30 the flexible inner inboard 0.5-1 centimeters of tube sealing 29, and structure is with known breather cheek valve, can allow gas in device enter Glass tubing 30 chambeies.Unidirectional control valve 32 structures are with known unidirectional control valve, be positioned at Glass tubing 30 inner ends, when suction glue head 27 is pinched or is unclamped, can and choose cell pipe 34 at glass tube neck 33 and form one suction, allow the cell picked enter and choose cell pipe 34 from advancing cytostome 35, then by going out cytostome 36 out, and then be placed on specific position.Glass tube neck 33 is parts that Glass tubing 30 ends extend, and then base structure is tapered with Glass tubing 30, with the diameter of choosing cell pipe 34 connecting places be 0.2-0.3 centimetre, the length of whole glass tube neck 33 is 1-2 centimetre.Choose cell pipe 34 arcs, radian is 10-30 °, and length is 1-2 centimetre, advances cytostome 35 front, and bore is the 1-2 millimeter, and opening direction is consistent with glass tube neck 33 front opening directions; Go out cytostome 36 rear, bore is the 2-3 millimeter, opening direction and glass tube neck 33 front opening opposite directions.When making the transgenic cell culture apparatus, first produce two portions up and down of cell culture apparatus, then add in the inside of bottom that liquid permeable plate 11 forms liquid box 14, insert growth plate 10, place up and down at the support axis hole 26 of growth plate 10 and support 18 between plate, insert back shaft 19, back shaft 19 lower ends are connected to below bottle on 6 internal surface, then two portions up and down of cell culture apparatus are heat sealing at together.
For verifying practicality of the present invention, namely the transgenic cell culture apparatus can be carried out cell cultures, plasmid transfection and screening function, and in transgenic cell culture apparatus application process, take bovine fibroblasts as example, through many experiments, preferred concrete operations are as follows:
1. transfection is front 1 day, the bovine fibroblasts of taking the logarithm vegetative period is inoculated on the growth plate 10 of transgenic cell culture apparatus, PBS phosphate buffered saline buffer, 0.25% trypsinase and the required nutrient solution of bovine fibroblasts growth in liquid box 14 different slots adopted respectively in the cleaning of cell afterwards, digestion and cultivation, the growing state of observation of cell after 12 hours, as best Growth of Cells and transfection density, select the operation of carrying out next step transfection on the underlaid growth plate 10 of the best cell of growth conditions when occupying the 70-80% of whole growth plate 10 take the cell attachment area;
2. in liquid box 14 respective grooves of transgenic cell culture apparatus, fill respectively 100 microlitre DMEM substratum, wherein fill the PEI transfection reagent of 8 microlitres in 1 groove, adding 4 microlitre concentration in other 1 groove is IRES-EGFP plasmid after 1 microgram/microlitre linearization process, is hatching respectively 5 minutes in corresponding incubation slot.Reagent in 2 grooves is carried out sufficient mixing, and hatch 15 minutes, thereby obtained the mixture of PEI transfection reagent and IRES-EGFP plasmid;
3. when 2. step carries out, get the cell for the treatment of transfection that step is chosen in 1., discard the covering required nutrient solution of bovine fibroblasts growth thereon, adopt the PBS phosphate buffered saline buffer of 2 milliliters to cover cell, and shake gently the transgenic cell culture apparatus to clean cell, then discard the PBS phosphate buffered saline buffer, and repeat this cleaning step 1 time, introduce afterwards the required nutrient solution of bovine fibroblasts growth of capacity on the growth plate 10 for the treatment of transfection from screening and culturing liquid bath 40;
4. adopt step 2. with picking device 37, the PEI transfection reagent that obtains and the mixture of IRES-EGFP plasmid dropwise to be added drop-wise to the cell surface for the treatment of transfection, gently shake the growth plate 10 of transgenic cell culture apparatus while dripping.Then be placed in the CO of 37 ℃
2After hatching 4 hours in cell culture incubator, discard the mixing solutions of growth plate 10 in the transgenic cell culture apparatus, replace it with the required nutrient solution of the growth of the bovine fibroblasts in the reagent bottle of 2 milliliters.And in the CO of 37 ℃
2Continue in cell culture incubator to cultivate;
5. continue to cultivate and discard the covering required nutrient solution of bovine fibroblasts growth thereon after 48 hours, adopt the PBS phosphate buffered saline buffer of 2 milliliters to cover cell, and shake gently the transgenic cell culture apparatus to clean cell, then discard the PBS phosphate buffered saline buffer, and repeat this cleaning step 1 time, add 0.5 milliliter of 0.25% trypsinase cell dissociation buffer in the surface of cell, adopt 2 milliliters of required nutrient solutions of bovine fibroblasts growth to stop digestion reaction after 1-2 minute, and cell is blown and beaten mixing, then inoculate in the growth plate 10 that is passaged in the transgenic cell culture apparatus, it is 20 milliliters that the required nutrient solution of continuation interpolation bovine fibroblasts growth makes the final liquor capacity in this growth plate 10.And in the CO of 37 ℃
2Continue in cell culture incubator to cultivate;
6. after continuing to cultivate 24 hours, when in the growth plate 10 in the transgenic cell culture apparatus, cell presents the state of adherent growth, discard the solution that is covered on cell, concentration in 20 milliliters of reagent bottles replaces it with aminoglycoside antibiotics G418 solution as the eukaryotic cell screening of 400-500 ug/ml, to carry out drug screening.Changed 1 G418 solution every 2 days, can observe some positive colony cell colonies that send green fluorescence that formed by individual cells in about 7 days under inverted fluorescence microscope.Be the gfp transgene bovine fibroblasts strain of this test kit preparation.
Above demonstration and described ultimate principle of the present invention and principal character and advantage of the present invention.The technician of the industry should be appreciated that; the present invention is not restricted to the described embodiments; that describes in above-described embodiment and specification sheets just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications; these changes and improvements all fall in the claimed scope of the invention, and the claimed scope of the present invention is defined by its equivalent of appending claims.
Claims (8)
1. the transgenic cell culture apparatus is by bottle cap (1), bottleneck (2), bottle head (3), bottleneck (4), above bottle (5), below bottle (6), bottle (7), bottle chamber (8), pillar stiffener (9), growth plate (10), liquid permeable plate (11), liquid box epicoele (12), growth plate jack (13), liquid box (14), the bottle end (15), bottle sidewall (16), liquid permeable plate body (17), support between plate (18), back shaft (19), growth plate draw-in groove (20), holder layer wall (21), holder layer (22), liquid-adsorption layer (23), the adherent layer of the liquid of growing (24), liquid drain hole (25), support axis hole (26), suction glue head (27), bottle cap turns ball (28), flexible tube sealing (29), Glass tubing (30), breather cheek valve (31), unidirectional control valve (32), glass tube neck (33), choose cell pipe (34), advance cytostome (35), go out cytostome (36), pick device (37), trypsin solution groove (38), D-hanks liquid tank (39), screening and culturing liquid bath (40), waste liquid tank (41), buffering liquid groove (42), grown cultures liquid bath (43), transfection reagent groove (44), plasmid groove (45), plasmid is hatched liquid bath (46), transfection reagent is hatched liquid bath (47), syringe needle jack (48), cell cultures growth plate jack (49), waste liquid outlet orifice (50), mono-clonal screening growth plate jack (51), growth plate push-and-pull ring (52) forms, and it is characterized in that: growth plate (10) is by holder layer wall (21), the adherent layer of the liquid of growing (24), liquid-adsorption layer (23), holder layer (22), liquid drain hole (25), support axis hole (26) and form, long 4-14 centimetre of growth plate (10), wide 2.5-7 centimetre, thick 1-3 millimeter comprises three-decker, is followed successively by from top to bottom the growth adherent layer of saturating liquid (24), liquid-adsorption layer (23), holder layer (22), the adherent layer of the liquid of growing (24) is one deck tunica fibrosa, thick 0.01-0.1 millimeter, the above has a lot of liquid drain holes (25), the nutrient solution that adsorbs from liquid box (14) in liquid-adsorption layer (23) can successfully be penetrated into the outside surface of the adherent layer of the saturating liquid of growth (24) film by liquid drain hole (25), in order to the growth that is seeded in cell on film outer surface, liquid-adsorption layer (23) is one deck absorbent cotton, and thickness is the 0.69-1.9 millimeter, can adsorb nutrient solution, holder layer (22) plastics quality or glass quality, the preferred glass quality, thick 0.3-1 millimeter, leading edge and both side edges are upwards turned up, and form holder layer wall (21), in order to avoid nutrient solution outwards oozes out, support axis hole (26) and be the strip hole of the upper and back shaft (19) of growth plate (10) corresponding section, length is 3-13 centimetre, width is the 2.1-3.1 millimeter, after being used for the interspersed growth plate (10) of back shaft (19), together with supporting (18) between plate, growth plate (10) is fixed on specific position, and growth plate (10) can advance between slave plate and plate to support axis hole (26) edge and back shaft (19) to form slideway or pull out, so that the growing state of cell on the observation growth plate, perhaps clean dead cell at growth plate (10), pick the operations such as monoclonal cell.
2. transgenic cell culture apparatus according to claim 1, it is characterized in that: described bottle cap (1) profile plastics quality, profile and structure are with known bottle cap, lid mouthful internal surface has the silk that revolves of protrusion, can with bottle head (3) outside surface evagination revolve the silk mutually be screwed in together with, so that bottle cap (1) seals bottleneck (2), prevent that in a bottle chamber (8), liquid flows out; Bottleneck (2) is the opening of bottle head (3) free end, namely adds liquid and injects the opening that the liquid in bottle chamber (8) or bottle chamber (8) flows out; Bottle head (3) is the outwardly directed one section cirque structure of bottleneck (4) end, and internal surface is smooth, and outside surface has the silk that revolves of evagination; Bottleneck (4) is the part that is connected between bottle head (3) and bottle (7), lower face length, above short, make bottle head (3) be arranged in the top of bottle (7) axial plane, in order to avoid after the transgenic cell culture apparatus is placed on horizontal plane, liquid flows out from bottleneck (2), and bottleneck (4) inner chamber is connected with bottle head (3) and bottle chamber (8).
3. transgenic cell culture apparatus according to claim 1, it is characterized in that: described bottle (7) cuboid, long 5-15 centimetre, wide 3-8 centimetre, high 2-5 centimetre, its wall thickness 1-3 millimeter, quality are plastics quality or glass quality, the preferred glass quality, the front does not have a bottle wall, being an opening, after being placed on horizontal plane, is above bottle (5) above bottle (7), the below is bottle following (6), both sides are bottle sidewall (16), and the back is the bottle end (15), and the cavity that is surrounded by each face in bottle (7) is a bottle chamber (8).
4. transgenic cell culture apparatus according to claim 1, it is characterized in that: described pillar stiffener (9) is to be connected in (5) and bottle following (6) center column construction above bottle, plastics quality or glass quality, the preferred glass quality comprises and supports (18), back shaft (19), growth plate draw-in groove (20) between plate; Support (18) cylindrical ring set between plate, internal diameter is the 2.2-3.2 millimeter, and external diameter is the 3-4 millimeter, and length is the 2-3 millimeter; Back shaft (19) is the axle of pillar stiffener (9) central authorities, and cylindrical, diameter is the 2-3 millimeter; The gap of supporting between (18) between the plate of two of up and down forms growth plate draw-in groove (20), is used for blocking growth plate (10), makes between adjacent two growth plates (10) at a distance of the 2-3 millimeter.
5. transgenic cell culture apparatus according to claim 1, it is characterized in that: described liquid permeable plate (11) is the cuboid plate that connects bottle (7) two side and lower wall, plastics quality or glass quality, the preferred glass quality, comprise growth plate jack (13) and liquid permeable plate body (17) two portions, long 2.8-6.4 centimetre, wide 1.6-3.8 centimetre, thickness is the 1-3 millimeter; Growth plate jack (13) is comprised of cell cultures growth plate jack (49) and mono-clonal screening growth plate jack (51), it is the duct that is bathed in after the plug that stretches out later of growth plate (10) inserts in the interior nutrient solution of liquid box (14), length is 0.5-1 centimetre, width is 0.1-0.3 centimetre, be positioned at the both sides of liquid permeable plate body 17, simultaneously also can and pillar stiffener (9) form the supporting structure of growth plate (10), between adjacent two growth plate jacks (13) up and down at a distance of the 2-3 millimeter.
6. transgenic cell culture apparatus according to claim 1, it is characterized in that: described liquid box (14) is the box like structure of the formation between liquid permeable plate (11) and the bottle end (15), by trypsin solution groove (38), D-hanks liquid tank (39), screening and culturing liquid bath (40), waste liquid tank (41), buffering liquid groove (42), grown cultures liquid bath (43), transfection reagent groove (44), plasmid groove (45), plasmid is hatched liquid bath (46), transfection reagent is hatched liquid bath (47), syringe needle jack (48), cell cultures growth plate jack (49), waste liquid outlet orifice (50), mono-clonal screening growth plate jack (51) forms, be used for containing nutrient solution, liquid box epicoele (12) is the space between liquid permeable plate (11) and bottle top (5), is the opening of nutrient solution liquid in-out box (14).
7. transgenic cell culture apparatus according to claim 1 is characterized in that: the described device (37) of picking is turned ball (28), flexible tube sealing (29), Glass tubing (30), breather cheek valve (31), unidirectional control valve (32), glass tube neck (33), chooses cell pipe (34), advances cytostome (35), is gone out cytostome (36) and form by suction glue head (27), bottle cap; Suction glue head (27) is the glue head that commercially available glue head dropper is used, and diameter is 0.5-1 centimetre, and length is 1-2 centimetre, and glue head wall thickness is the 1-2 millimeter, glue draught animals place round shape, and diameter is 0.5 centimetre, length is 0.5-1 centimetre; It is the spheroidal sealing member of 1-1.5 centimetre for diameter that bottle cap turns ball (28), formed by shell and endosphere, the shell disc, totally 2, the diameter of every is 1-1.5 centimetre, thickness is 0.5-0.75 centimetre, available bolt turns ball (28) with bottle cap and is fixed on bottle cap (1) bottom surface, two shells are by stacking the right cylinder of formation simultaneously, in this right cylinder central authorities, a diameter being arranged is the spheroidal space of 0.75-1.25 centimetre, is fixed in this space and can rotates therein so that fixed diameter is the endosphere of 0.75-1.25 centimetre; Crossing endosphere central authorities, a diameter is arranged is the duct of 0.5 centimetre, it is the space that Glass tubing (30) inserts, after Glass tubing (30) inserts the endosphere duct, the operator is enclosed within Glass tubing (30) one ends with suction glue head (27), because endosphere can freely rotate and good seal performance, the operator just can control bottle inside of picking device and be divided into line operate; Flexible tube sealing (29) is the telescopic long tube of plastics matter, and back cut diameter is 0.75-1.25 centimetre, and lower port diameter is 0.5 centimetre, and structure can be stretched with the crook of known milk pipet, and whole flexible tube sealing (29) is enclosed within the outside of Glass tubing (30); Glass tubing (30) is that diameter is the long tube of 0.5 centimetre of glass quality, and total length is whole device inner length, wall thickness 1-2 millimeter, and the outer end turns ball (28) from bottle cap and passes bottle cap (1), and opening part has millipore filtration to seal; The inner is connected to glass tube neck (33) central authorities, and inner chamber communicates with the inner chamber of glass tube neck (33); Breather cheek valve (31) is positioned at the upper flexible inner inboard 0.5-1 centimeters of tube sealing (29) of Glass tubing (30), and structure is with known breather cheek valve, can allow gas unidirectional in device enter Glass tubing (30) chamber; Unidirectional control valve (32) structure is with known unidirectional control valve, be positioned at Glass tubing (30) inner end, when suction glue head (27) is pinched or is unclamped, can and choose cell pipe (34) at glass tube neck (33) and form one suction, allow the cell picked enter and choose cell pipe (34) from advancing cytostome (35), then by going out cytostome (36) out, and then be placed on specific position; Glass tube neck (33) is the part that Glass tubing (30) end extends, and the same Glass tubing of base structure (30) then is tapered, with the diameter of choosing cell pipe (34) connecting place be 0.2-0.3 centimetre, the length of whole glass tube neck (33) is 1-2 centimetre; Choose cell pipe (34) arc, radian is 10-30 °, and length is 1-2 centimetre, advances cytostome (35) front, and bore is the 1-2 millimeter, and opening direction is consistent with glass tube neck (33) front opening direction; Go out cytostome (36) rear, bore is the 2-3 millimeter, opening direction and glass tube neck (33) front opening opposite direction.
8. transgenic cell culture apparatus according to claim 1, it is characterized in that: during described manufacturing transgenic cell culture apparatus, first produce two portions up and down of transgenic cell culture apparatus, then add in the inside of bottom that liquid permeable plate (11) forms liquid box (14), insert growth plate (10), place up and down at the support axis hole (26) of growth plate (10) and support (18) between plate, insert back shaft (19), back shaft (19) lower end is connected on the internal surface of (6) below bottle, then two portions up and down of transgenic cell culture apparatus are heat sealing at together.
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| CN111548938A (en) * | 2020-06-09 | 2020-08-18 | 英诺维尔智能科技（苏州）有限公司 | Novel high-performance culture bottle applicable to suspension and adherent cell mixed culture |
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