CN103146578B - Transgenic cell culture device - Google Patents
Transgenic cell culture device Download PDFInfo
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- CN103146578B CN103146578B CN201310106352.0A CN201310106352A CN103146578B CN 103146578 B CN103146578 B CN 103146578B CN 201310106352 A CN201310106352 A CN 201310106352A CN 103146578 B CN103146578 B CN 103146578B
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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 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 ", " genetically engineered ", " genetic transformation " that it has often been said are genetically modified synonym.Such as Animal Transgenic is exactly the animal that contains foreign gene in genome, 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 to sperm, ovum or zygote, with reproduction engineering technology, be likely bred as 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 as short, farrowing, raw egg is many and lactation amount is high, meat, fur quality and good processability, and there is the animal varieties of disease resistance.In 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 need to be cultivated as bovine fibroblasts etc. quickly and easily, the material consumption that whole process need is larger, particularly sterilization process, the utensil using is more, needs the article of consumption more, experimental cost also increases thereupon, so the experiment of cells involved all needs a large amount of financial supports, otherwise just cannot complete.Through long-term practical observation, we find that the usage quantity of cell culture apparatus in transgenic experiments process is maximum, are also links the most rambunctious, and a little error sometimes just may cause large-area pollution, brings greater impact to experiment.Therefore the chance that reduces to pollute just becomes extremely important, how designing a kind of transfectional cell that can reduce cell culture apparatus usage quantity and make to cultivate can be in an airtight environment can be shifted and then reduce the contaminated chance of transfectional cell of cultivating and become and be badly in need of a great problem that solves by picking, thereby so utilize in cell culture apparatus, increase the adherent area of Growth of Cells reduce cell culture apparatus usage quantity and add one can be manual the method for picking device, 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 object cell of device for cell attachment growth.
Summary of the invention
Use too much and a normal difficult problem that increases various risks to experiment in transitional cell process repeatedly in the culturing process that goes down to posterity because of cell culture apparatus in order to overcome in Cell Biology Experiment process, the invention provides a kind of transgenic cell culture apparatus, this transgenic cell culture apparatus utilization increases multilayer in cell culture apparatus can be for the growth plate of cell attachment growth, liquid box is set and ensures that every growth plate all can make the cell on it obtain sufficient nutrient solution, arrange can be by the manually operated device of picking of operator outside device simultaneously, thereby reach the object that better completes cell cultures.
The technical solution adopted for the present invention to solve the technical problems is:
Transgenic cell culture apparatus of the present invention, by bottle cap 1, bottleneck 2, bottle 3, bottleneck 4, bottle above 5, bottle below 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, pick device 37, growth plate push-and-pull ring 52 forms, it is characterized in that: described growth plate 10 by a holder layer wall 21, the adherent layer 24 of the liquid of growing, liquid-adsorption layer 23, holder layer 22, liquid drain hole 25, support axis hole 26 and form; The described 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 adherent layer 24 of the saturating liquid of growth, liquid-adsorption layer 23, holder layer 22; The adherent layer 24 of the saturating liquid of described growth is one deck tunica fibrosas, and thick 0.01-0.1 millimeter, has liquid drain hole 25 above, and described liquid-adsorption layer 23 thickness are 0.69-1.9 millimeter, are made up of absorbent cotton; The nutrient solution adsorbing from liquid box 14 is penetrated into the outside surface of the adherent layer of the saturating liquid of growth 24 film by liquid drain hole 25, the cell of inoculation is grown on film outer surface; Described holder layer 22 plastics quality or glass quality, thick 0.3-1 millimeter, leading edge and both side edges are upwards turned up, and form holder layer wall 21; Described support axis hole 26 be on growth plate 10 with the long strip shape hole of back shaft 19 corresponding sections, length is that 3-13 centimetre, width are 2.1-3.1 millimeter; 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, enters 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 1-2 millimeter, glue draught animals place round shape, and diameter is 0.5 centimetre, length is 0.5-1 centimetre; Bottle cap turn ball 28 for diameter be the spheroidal sealing member of 1-1.5 centimetre, formed by shell and endosphere, shell disc, totally 2, the diameter of every is 1-1.5 centimetre, and thickness is 0.5-0.75 centimetre, and available bolt turns ball 28 by bottle cap and is fixed on bottle cap 1 bottom surface, simultaneously two shells are by stacking the right cylinder of formation, and having a diameter in these right cylinder central authorities is the spheroidal space of 0.75-1.25 centimetre; Crossing endosphere central authorities, to have a diameter be the duct of 0.5 centimetre, it is the space that Glass tubing 30 inserts, when Glass tubing 30 inserts behind endosphere duct, suction glue head 27 is enclosed within Glass tubing 30 one end by operator, because endosphere can freely rotate and good seal performance, operator just can control pick device bottle inside 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 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 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 inner inner side of flexible tube sealing 29 0.5-1 centimeters on Glass tubing 30; Unidirectional control valve 32 is positioned at Glass tubing 30 inner ends, in the time that suction glue head 27 is pinched and 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 entering cytostome 35, then by going out cytostome 36 out, and then be placed on specific position; Glass tube neck 33 be Glass tubing 30 ends extend part, base structure, with Glass tubing 30, is then tapered, 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, enters cytostome 35 front, and bore is 1-2 millimeter, and opening direction is consistent with glass tube neck 33 front opening directions; Go out cytostome 36 rear, bore is 2-3 millimeter, opening direction and glass tube neck 33 front opening opposite directions.
?described bottle cap 1 lid mouthful internal surface has the silk that revolves of protrusion, and bottle 3 outside surface have the silk that revolves of evagination, and the two is screwed in together mutually; Bottleneck 2 is openings of bottle 3 free end; Bottle 3 is the outwardly directed one section of cirque structure of bottleneck 4 ends, and internal surface is smooth, and outside surface has the silk that revolves of evagination, and when transgenic cell culture apparatus is placed on horizontal plane, bottle 3 is arranged in the top of bottle 7 axial planes; Bottleneck 4 is the parts that are connected between bottle 3 and bottle 7, lower face length, above short; Bottleneck 4 inner chambers are connected with bottle 3 inner chamber and bottle chamber 8; 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, there is no a bottle wall above, be opening, after being placed on horizontal plane, bottle 7 be above bottle 5 above, be bottle below 6 below, both sides are a bottle sidewall 16, after be a bottle end 15, the interior cavity being surrounded by each face of bottle 7 is bottle chamber 8.
Described pillar stiffener 9 is to be connected in bottle above 5 and the bottle column constructions of 6 centers below, plastics quality or glass quality, comprise between plate, support 18, back shaft 19, growth plate draw-in groove 20; Between plate, support 18 for cylindrical ring set, internal diameter is 2.2-3.2 millimeter, and external diameter is 3-4 millimeter, and length is 2-3 millimeter; Back shaft 19 is the axle of pillar stiffener 9 central authorities, cylindrical, and diameter is 2-3 millimeter; Between upper and lower two plates, supporting slit-shaped between 18, to become length be the growth plate draw-in groove 20 of 2-3 millimeter.
Described liquid permeable plate 11 is the cuboid plates that connect bottle 7 two sides and lower wall, and plastics quality or glass quality, comprise growth plate jack 13 and liquid permeable plate body 17 two portions, long 2.8-6.4 centimetre, and wide 1.6-3.8 centimetre, thickness is 1-3 millimeter; Described growth plate jack 13 is made up of cell cultures growth plate jack 49 and mono-clonal screening growth plate jack 51, to be bathed in duct in liquid box 14 interior nutrient solution after the plug that stretches out of growth plate 10 inserts growth plate jack 13 below, length is 0.5-1 centimetre, width is 0.1-0.3 centimetre, is positioned at the both sides of liquid permeable plate body 17; After plug that growth plate 10 stretches out below inserts growth plate jack 13 and pillar stiffener 9 form the supporting structure of growth plate 10,2-3 millimeter apart up and down between adjacent two growth plate jacks 13.
Described liquid box 14 is box like structure of liquid permeable plate 11 and the formation at the bottle 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, for containing nutrient solution; Liquid box epicoele 12 is liquid permeable plate 11 and the bottle space between 5 above, is the opening of nutrient solution liquid in-out box 14.
While 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 between plate and support 18 at the support axis hole 26 of growth plate 10, insert back shaft 19, make back shaft 19 lower ends be connected to bottle below 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 utilization of transgenic cell culture apparatus increases multilayer in cell culture apparatus can be for the growth plate of cell attachment growth, liquid box is set and ensures that every growth plate all can make the cell on it obtain sufficient nutrient solution, simultaneously arrange can device outside by the manually operated device of picking of operator, thereby reach the object that better completes cell cultures.Transgenic cell culture apparatus is made simple, and diverse in function is workable, with low cost, successful.
brief description of the drawings
Below in conjunction with accompanying drawing, the present invention is further described.
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, 3. bottle head, 4. bottleneck, 5. bottle, 6. bottle, 7. bottle, 8. bottle chamber, 9. pillar stiffener, 10. growth plate, 11. liquid permeable plates, 12. liquid box epicoeles, 13. growth plate jacks, 14. liquid boxes, at the bottom of 15. bottles, 16. bottles of sidewalls, 17. liquid permeable plate bodies, between 18. plates, support, 19. back shafts, 20. growth plate draw-in grooves, 21. holder layer wall, 22. holder layers, 23. liquid-adsorption layers, the adherent layer of the saturating liquid of 24. growth, 25. liquid drain holes, 26. support axis hole, 27. suction glue heads, 28. bottle caps turn ball, 29. flexible tube sealings, 30. Glass tubings, 31. breather cheek valves, 32. unidirectional control valves, 33. glass tube necks, 34. choose cell pipe, 35. enter cytostome, 36. go out cytostome, 37. pick device, 38. trypsin solution grooves, 39. D-hanks liquid tanks, 40. screening and culturing liquid baths, 41. waste liquid tanks, 42. buffering liquid grooves, 43. grown cultures liquid baths, 44. transfection reagent grooves, 45. plasmid grooves, 46. plasmids are hatched liquid bath, 47. transfection reagents are hatched liquid bath, 48. syringe needle jacks, 49. cell cultures growth plate jacks, 50. waste liquid outlet orifices, 51. mono-clonal screening growth plate jacks, 52. growth plate push-and-pull rings.
Embodiment
Embodiment mono-:
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 above 5, bottle below 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, between plate, support 18, 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, enter 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, lid mouthful internal surface has the silk that revolves of protrusion, can with the revolving together with silk is screwed in mutually of a bottle 3 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, and additional liquid injects the opening that the liquid in bottle chamber 8 or bottle chamber 8 flows out.Bottle 3 is the outwardly directed one section of 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 is placed on horizontal plane, liquid flows 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, there is no a bottle wall above, being opening, after being placed on horizontal plane, be above bottle 5 above, be bottle below 6 below, both sides are a bottle sidewall 16, after be a bottle end 15, the cavity being surrounded by each face in it is bottle chamber 8.Pillar stiffener 9 is to be connected in bottle above 5 and bottle 6 center column constructions below, plastics quality or glass quality, comprise between plate, support 18, back shaft 19, growth plate draw-in groove 20.Between plate, support 18 cylindrical ring sets, internal diameter is 2.2-3.2 millimeter, and external diameter is 3-4 millimeter, and length is 2-3 millimeter; Back shaft 19 is the axle of pillar stiffener 9 central authorities, cylindrical, and diameter is 2-3 millimeter; The gap of supporting between upper and lower two plates between 18 forms growth plate draw-in groove 20, for blocking growth plate 10, makes between adjacent two growth plates 10 at a distance of 2-3 millimeter.Liquid permeable plate 11 is the cuboid plates that connect bottle 7 two sides and lower wall, and plastics quality or glass quality, comprise growth plate jack 13 and liquid permeable plate body 17 two portions, long 2.8-6.4 centimetre, and wide 1.6-3.8 centimetre, thickness is 1-3 millimeter.Growth plate jack (13) is made up of cell cultures growth plate jack (49) and mono-clonal screening growth plate jack (51), to be bathed in duct in liquid box (14) interior nutrient solution after the plug that stretches out of growth plate (10) inserts below, 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 2-3 millimeter.Liquid box 14 is box like structure of liquid permeable plate 11 and the formation at the bottle end 15, for containing nutrient solution.Liquid box epicoele 12 is liquid permeable plate 11 and the bottle space between 5 above, 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 one end, top, volume is 5-10 milliliter, inside fill trypsinase, tryptic amount is determined according to the amount of the required trypsin solution of passage, ratio used is 0.25%.In 100 milliliters of D-hanks liquid, add 0.25 gram of trypsinase by mass percent.D-hanks liquid tank 39 volumes are 5-10 milliliter, and interior Sheng is without the balanced salt damping fluid of calcium ions and magnesium ions, pH 8.0.Screening and culturing liquid bath 40 volumes are 15-40 milliliter, and for the needed nutrient solution of splendid attire mono-clonal screening growth plate, it is 400-500 ug/ml that aminoglycoside antibiotics G418, concentration are used in 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 is 5-7, claim again mono-clonal screening growth plate jack 51, each mono-clonal screening growth plate jack 51 length are 0.5-1 centimetre, width is 0.1-0.3 centimetre, is that the growth plate 10 of cultivating monoclonal cell inserts and nutrient solution enters the place of growth plate 10.Waste liquid tank 41 volumes are 20-50 milliliter, be the space of collecting the various waste liquids that produce in 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 1-2 millimeter by a diameter entangles, in order to avoid extraneous bacterium enters with waste liquid and overflows in cell cultivation process.Buffering liquid groove 42 volumes are 20-50 milliliter, interior Sheng PBS phosphate buffered saline buffer.Because in 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 syringe penetrates simultaneously also can rinse preferably and pick the parts that device need to clean.The structure of syringe needle jack 48 is with waste liquid outlet orifice 50, also be 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 1-2 millimeter by a diameter entangles, in order to avoid extraneous bacterium enters and cushions hydrorrhea and goes out in cell cultivation process.Grown cultures liquid bath 43, volume is 15-40 milliliter, for the needed nutrient solution of splendid attire cell cultures growth plate, for example bovine fibroblasts grow required nutrient solution by volume percentage ratio in 90 milliliters of DMEM substratum, add 10 milliliters of standard foetal calf serums, 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 are 0.5-1 centimetre, width is 0.1-0.3 centimetre, that the growth plate 10 of cultivating transgenic cell inserts and nutrient solution enters the place of growth plate 10.Transfection reagent groove 44 volumes are 3-5 milliliter, inside fill PEI polymine transfection reagent, this reagent has been got rid of and in cell cultures, has been used the impact of serum on transfection efficiency, and this reagent transfection efficiency is high, cost is low, reproducible, can be good at being applicable to the transfection research of bovine fibroblasts etc.Plasmid groove 45 volumes are 3-5 milliliter, inside fill containing foreign gene and wishing the plasmid vector that it expresses in host cell of selecting according to genetically modified object, such as the plasmid expression vector of 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 to enzyme and cut processing, become linearizing carrier segments, thereby can be by transfection, be incorporated into better in the genome of bovine fibroblasts, improve the efficiency of green fluorescent protein stably express.It is 3-5 milliliter that plasmid is hatched liquid bath 46 volumes, inside fills DMEM substratum (the Eagle substratum of Dulbecco improvement), is the solution of hatching of plasmid.It is 3-5 milliliter that transfection reagent is hatched liquid bath 47 volumes, inside fills DMEM substratum (the Eagle substratum of Dulbecco improvement), is the solution of hatching of transfection reagent.Growth plate push-and-pull ring 52 is that diameter is 1-5 millimeter is connected to growth plate 10 glass ring above by handle, 3 growth plate push-and-pull rings 52 are set before every growth plate, lay respectively at growth plate 10 above in the middle of and centre between centre and edge, in the time that needs are observed on a certain growth plate 10 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 by asking layer wall 21, the adherent layer 24 of the liquid of growing, liquid-adsorption layer 23, holder layer 22, liquid drain hole 25, support axis hole 26 to form, 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, there are a lot of liquid drain holes 25 above, cell only has several microns of sizes on the one hand, the thickness of 0.01-0.1 millimeter is enough to provide for Growth of Cells the nutrient solution of the degree of depth that can submergence cell, on the other hand, the nutrient solution adsorbing from liquid box 14 in liquid-adsorption layer 23 can also be penetrated into very smoothly continually by liquid drain hole 25 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 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, 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 and the strip hole of back shaft 19 corresponding sections, length is that 3-13 centimetre, width are 2.1-3.1 millimeter, intert after growth plate 10 for back shaft 19, together with supporting 18 between plate, growth plate 10 is fixed on to specific position, and growth plate 10 can form slideway to support axis hole 26 edges and back shaft 19, between slave plate and plate, advance or pull-out, to observe the growing state of cell on growth plate with inverted microscope, or clean dead cell at growth plate 10, pick monoclonal cell operation.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, enters 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 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 Glass tubing outer end in the time 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 specifying.After picking, suction glue head 27 is taken off, other at Glass tubing 30 external port stickup one deck millipore filtrations, to allow the oxygen in outside air enter in culture apparatus, for Growth of Cells utilization at flame.Bottle cap turn ball 28 for diameter be the spheroidal sealing member of 1-1.5 centimetre, formed by shell and endosphere, 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 by bottle cap and is fixed on bottle cap 1 bottom surface, two shells are by stacking the right cylinder of formation simultaneously, having a diameter in these right cylinder central authorities is the spheroidal space of 0.75-1.25 centimetre, so that the endosphere that fixed diameter is 0.75-1.25 centimetre is fixed and can rotates therein in this space.Crossing endosphere central authorities, to have a diameter be the duct of 0.5 centimetre, it is the space that Glass tubing 30 inserts, when Glass tubing 30 inserts behind endosphere duct, suction glue head 27 is enclosed within Glass tubing 30 one end by operator, because endosphere can freely rotate and good seal performance, operator just can control pick device bottle inside 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, whole flexible tube sealing 29 is enclosed within the outside of Glass tubing 30, on the one hand can fixing glass pipe 30, can further seal on the other hand bottleneck 2, prevent that bacterium is in bottle cap turns the gap access to plant ball 28, namely extract out or inject when bottle cap turns ball 28 at Glass tubing 30 and set up barrier one, 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, on wall, there is minimum and is the scale of 1 millimeter, to pick the accurate quantitative liquid of drawing in process, outer end turns ball 28 from bottle cap and passes bottle cap 1, and opening part has millipore filtration to seal, to supply needed oxygen in 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 at the inner inner side of flexible tube sealing 29 0.5-1 centimeters on Glass tubing 30, and structure, with known breather cheek valve, can allow the 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, in the time that suction glue head 27 is pinched and 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 entering cytostome 35, then by going out cytostome 36 out, and then be placed on specific position.Glass tube neck 33 be Glass tubing 30 ends extend part, base structure, with Glass tubing 30, is then tapered, 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, enters cytostome 35 front, and bore is 1-2 millimeter, and opening direction is consistent with glass tube neck 33 front opening directions; Go out cytostome 36 rear, bore is 2-3 millimeter, opening direction and glass tube neck 33 front opening opposite directions.While 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 between plate and support 18 at the support axis hole 26 of growth plate 10, insert back shaft 19, make back shaft 19 lower ends be connected to bottle below 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, transgenic cell culture apparatus can be carried out cell cultures, plasmid transfection and screening function, and in transgenic cell culture apparatus application process, taking bovine fibroblasts as example, through many experiments, preferred concrete operations are as follows:
1. first 1 day of transfection, the bovine fibroblasts of taking the logarithm vegetative period is inoculated on the growth plate 10 of transgenic cell culture apparatus, the cleaning of cell afterwards, digestion and cultivate the PBS phosphate buffered saline buffer, 0.25% trypsinase and the bovine fibroblasts that adopt respectively in the liquid box 14 different slots required nutrient solution of growing, the growing state of observation of cell after 12 hours, while occupying the 70-80% of whole growth plate 10 taking cell attachment area, 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;
2. in liquid box 14 corresponding slot of transgenic cell culture apparatus, fill respectively 100 microlitre DMEM substratum, wherein in 1 groove, fill the PEI transfection reagent of 8 microlitres, in other 1 groove, adding 4 microlitre concentration is the IRES-EGFP plasmid after 1 microgram/microlitre linearization process, in corresponding incubation slot, hatches respectively 5 minutes.Reagent in 2 grooves is mixed fully, and hatch 15 minutes, thereby obtained the mixture of PEI transfection reagent and IRES-EGFP plasmid;
3. in the time that 2. step carries out, get the cell for the treatment of transfection that step is chosen in 1., discard the covering bovine fibroblasts thereon required nutrient solution of growing, adopt the PBS phosphate buffered saline buffer of 2 milliliters to cover cell, and shake gently transgenic cell culture apparatus to clean cell, then discard PBS phosphate buffered saline buffer, and repeat this cleaning step 1 time, on the growth plate 10 for the treatment of transfection, from screening and culturing liquid bath 40, introduce afterwards enough bovine fibroblasts required nutrient solution of growing;
4. adopt step 2. the mixture of the PEI transfection reagent of acquisition and IRES-EGFP plasmid to be dropwise added drop-wise to the cell surface for the treatment of transfection with picking device 37, gently shake the growth plate 10 of transgenic cell culture apparatus on dropping limit, limit.Then be placed in the CO of 37 DEG C
2in cell culture incubator, hatch after 4 hours, discard the mixing solutions of growth plate 10 in transgenic cell culture apparatus, replace it with the required nutrient solution of growing of the bovine fibroblasts in the reagent bottle of 2 milliliters.And in the CO of 37 DEG C
2in cell culture incubator, continue to cultivate;
5. continue to cultivate and discard the covering bovine fibroblasts thereon required nutrient solution of growing after 48 hours, adopt the PBS phosphate buffered saline buffer of 2 milliliters to cover cell, and shake gently transgenic cell culture apparatus to clean cell, then discard 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 after 1-2 minute 2 milliliters of bovine fibroblasts required nutrient solution of growing to stop digestion reaction, and cell piping and druming is mixed, then inoculation is passaged in the growth plate 10 in transgenic cell culture apparatus, continue to add bovine fibroblasts and grow that to make the final liquor capacity in this growth plate 10 be 20 milliliters to required nutrient solution.And in the CO of 37 DEG C
2in cell culture incubator, continue to cultivate;
6. after continuing to cultivate 24 hours, when in growth plate 10 in transgenic cell culture apparatus, cell presents the state of adherent growth, discard the solution being covered on cell, eukaryotic cell screening taking the concentration in 20 milliliters of reagent bottles as 400-500 ug/ml replaces it with aminoglycoside antibiotics G418 solution, to carry out drug screening.Change 1 G418 solution every 2 days, in about 7 days, can under inverted fluorescence microscope, can observe some positive colony cell colonies that send green fluorescence that formed by individual cells.Be gfp transgene bovine fibroblasts strain prepared by this test kit.
More than show 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 in above-described embodiment and specification sheets, describes 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 (5)
1. transgenic cell culture apparatus, 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), pick device (37), growth plate push-and-pull ring (52) composition, it is characterized in that: described 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) composition, the described 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 adherent layer of the saturating liquid of growth (24), liquid-adsorption layer (23), holder layer (22), the adherent layer of the saturating liquid of described growth (24) is one deck tunica fibrosa, and thick 0.01-0.1 millimeter, has liquid drain hole (25) above, and described liquid-adsorption layer (23) thickness is 0.69-1.9 millimeter, is made up of absorbent cotton, the nutrient solution adsorbing from liquid box (14) is penetrated into the outside surface of the adherent layer of the saturating liquid of growth (24) film by liquid drain hole (25), the cell of inoculation is grown on film outer surface, described holder layer (22) plastics quality or glass quality, thick 0.3-1 millimeter, leading edge and both side edges are upwards turned up, and form holder layer wall (21), described support axis hole (26) is the long strip shape hole of upper and back shaft (19) corresponding section of growth plate (10), and length is that 3-13 centimetre, width are 2.1-3.1 millimeter, 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), enters 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 1-2 millimeter, glue draught animals place round shape, and diameter is 0.5 centimetre, length is 0.5-1 centimetre, bottle cap turn ball (28) for diameter be the spheroidal sealing member of 1-1.5 centimetre, formed by shell and endosphere, shell disc, totally 2, the diameter of every is 1-1.5 centimetre, and thickness is 0.5-0.75 centimetre, and available bolt turns ball (28) by bottle cap and is fixed on bottle cap (1) bottom surface, simultaneously two shells are by stacking the right cylinder of formation, and having a diameter in these right cylinder central authorities is the spheroidal space of 0.75-1.25 centimetre, crossing endosphere central authorities, to have a diameter be the duct of 0.5 centimetre, it is the space that Glass tubing (30) inserts, when Glass tubing (30) inserts behind endosphere duct, suction glue head (27) is enclosed within Glass tubing (30) one end by operator, because endosphere can freely rotate and good seal performance, operator just can control pick device bottle inside 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 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 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 inner inner side of the upper flexible tube sealing (29) of Glass tubing (30) 0.5-1 centimeters, unidirectional control valve (32) is positioned at Glass tubing (30) inner end, in the time that suction glue head (27) is pinched and 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 entering cytostome (35), then by going out cytostome (36) out, and then be placed on specific position, glass tube neck (33) be Glass tubing (30) end extend part, the same Glass tubing of base structure (30), is then 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, enters cytostome (35) front, and bore is 1-2 millimeter, and opening direction is consistent with glass tube neck (33) front opening direction, go out cytostome (36) rear, bore is 2-3 millimeter, opening direction and glass tube neck (33) front opening opposite direction.
2. transgenic cell culture apparatus according to claim 1, is characterized in that: described bottle cap (1) lid mouthful internal surface has the silk that revolves of protrusion, and bottle head (3) outside surface has the silk that revolves of evagination, and the two is screwed in together mutually; Bottleneck (2) is the opening of bottle head (3) free end; Bottle head (3) is the outwardly directed one section of cirque structure of bottleneck (4) end, and internal surface is smooth, and outside surface has the silk that revolves of evagination, and when transgenic cell culture apparatus is placed on horizontal plane, bottle head (3) is arranged in the top of bottle (7) axial plane; Bottleneck (4) is the part being connected between bottle head (3) and bottle (7), lower face length, above short; Bottleneck (4) inner chamber is connected with bottle head (3) inner chamber and bottle chamber (8); Described bottle (7) cuboid, 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, there is no a bottle wall above, be an opening, after being placed on horizontal plane, bottle (7) is above bottle (5) above, be below bottle below (6), both sides are a bottle sidewall (16), after be a bottle end (15), the interior cavity being surrounded by each face of bottle (7) is a bottle chamber (8).
3. transgenic cell culture apparatus according to claim 1, it is characterized in that: described pillar stiffener (9) is the column construction that is connected in (6) center below (5) above bottle and bottle, plastics quality or glass quality, comprise support (18), back shaft (19), growth plate draw-in groove (20) between plate; Between plate, supporting (18) is cylindrical ring set, and internal diameter is 2.2-3.2 millimeter, and external diameter is 3-4 millimeter, and length is 2-3 millimeter; Back shaft (19) is the axle of pillar stiffener (9) central authorities, and cylindrical, diameter is 2-3 millimeter; It is the growth plate draw-in groove (20) of 2-3 millimeter that slit-shaped between upper and lower two plates between support (18) becomes length.
4. 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, 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 1-3 millimeter; Described growth plate jack (13) is made up of cell cultures growth plate jack (49) and mono-clonal screening growth plate jack (51), to be bathed in duct in liquid box (14) interior nutrient solution after the plug that stretches out of growth plate (10) inserts growth plate jack (13) below, length is 0.5-1 centimetre, width is 0.1-0.3 centimetre, is positioned at the both sides of liquid permeable plate body (17); After plug that growth plate (10) stretches out below inserts growth plate jack (13) and pillar stiffener (9) form the supporting structure of growth plate (10), 2-3 millimeter apart up and down between adjacent two growth plate jacks (13).
5. 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) composition, be used for containing nutrient solution, liquid box epicoele (12) is liquid permeable plate (11) and the space between (5) above bottle, is the opening of nutrient solution liquid in-out box (14).
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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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| CN1498960A (en) * | 2002-10-31 | 2004-05-26 | 惠普开发有限公司 | Cell culture device |
| CN1814745A (en) * | 2005-12-15 | 2006-08-09 | 中国人民解放军军事医学科学院野战输血研究所 | Cell culturation apparatus |
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| CN102864076A (en) * | 2012-09-26 | 2013-01-09 | 无锡耐思生物科技有限公司 | Cell culture bottle structure |
| CN102947438A (en) * | 2010-05-11 | 2013-02-27 | 阿特利斯公司 | Apparatus and methods for cell culture |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1498960A (en) * | 2002-10-31 | 2004-05-26 | 惠普开发有限公司 | Cell culture device |
| CN1814745A (en) * | 2005-12-15 | 2006-08-09 | 中国人民解放军军事医学科学院野战输血研究所 | Cell culturation apparatus |
| CN101665768A (en) * | 2008-09-05 | 2010-03-10 | 中国人民解放军第三军医大学第二附属医院 | Bioreactor for in vitro culture of neurotrophic factors |
| CN102947438A (en) * | 2010-05-11 | 2013-02-27 | 阿特利斯公司 | Apparatus and methods for cell culture |
| CN102864076A (en) * | 2012-09-26 | 2013-01-09 | 无锡耐思生物科技有限公司 | Cell culture bottle structure |
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