CN109401960A - The high efficiency cell culture bottle of built-in disturbing flow device - Google Patents
The high efficiency cell culture bottle of built-in disturbing flow device Download PDFInfo
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- CN109401960A CN109401960A CN201710702957.4A CN201710702957A CN109401960A CN 109401960 A CN109401960 A CN 109401960A CN 201710702957 A CN201710702957 A CN 201710702957A CN 109401960 A CN109401960 A CN 109401960A
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- culture bottle
- flow device
- high efficiency
- disturbing flow
- cell culture
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/08—Flask, bottle or test tube
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/38—Caps; Covers; Plugs; Pouring means
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
- C12M27/18—Flow directing inserts
- C12M27/20—Baffles; Ribs; Ribbons; Auger vanes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/04—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Sustainable Development (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Clinical Laboratory Science (AREA)
- Molecular Biology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention discloses a kind of high efficiency cell culture bottles of built-in disturbing flow device comprising: culture bottle body；The valve protection cap being tightly connected with the culture bottle body, is provided with an at least venthole, at least a sample tap, at least a material-feeding port and an at least transfer port on the valve protection cap；It is set to the intrinsic disturbing flow device of the culture bottle, is gradually decreased from the center line of the culture bottle body to the direction of the culture bottle side wall, and the bottom surface of the disturbing flow device and the culture bottle forms 5~45 ° of inclination angle.Built-in disturbing flow device component in high efficiency cell culture bottle provided by the invention, and there is bottleneck unlimited greatly and the design of aeration valve protection cap, good ventilation and mixed effect can be provided, oxygen transfer is high-efficient.High efficiency cell culture bottle provided by the invention can in-situ sampling, sampling is simple, quickly, and changing tradition sampling need to complete in sterile working in ultra-clean work.
Description
Technical field
The invention belongs to field of biotechnology, more specifically to a kind of high efficiency cell culture of built-in disturbing flow device
Bottle.
Background technique
Tissue Culture Flask can obtain a large amount of experimental data under conditions of limited human and material resources, space.In addition, shaking
Bottle experimental result provides the basic growth information of cell and culture process parameter, provides technology ginseng for mass cell culture
It examines.Therefore, Tissue Culture Flask is widely used in laboratory research and industrialized production.However traditional Tissue Culture Flask is still deposited
In following problems:
(1) vital movements such as growth, development, division, breeding of cell are both needed to consume a large amount of oxygen, participate in tricarboxylic acids and follow
Ring process.Traditional culture bottle bottleneck is small and bottom of bottle is deep and big, seriously blocks the circulation of gas.Culture bottle is on shaking table along training
It supports bottle central axis to rotate, flow morphology is laminar state, is unfavorable for mixing and oxygen transmitting.Currently, researchers are normal
Oxygen mass transfer coefficients are improved using reducing dress liquid coefficient, proposing high-revolving method.However, if dress liquid coefficient is too low, with cultivating
The progress of journey, the moisture in culture bottle largely evaporate, and the osmotic pressure of culture medium is caused to increase, and inhibit cell growth.It is higher to shake
Bed revolving speed generates high shear force, causes cell damage or dissolution dead；In addition, the operating of long-time limit speed can be greatly shortened and be shaken
The service life of bed.
(2) it is the growth metabolism state for understanding cell, needs not timing that cell growth parameter(s) is measured by sampling, observes cell shape
State.Traditional culture bottle sampling is to be completed in superclean bench by aseptic technique.Whole process need to expend largely
Time, man power and material.In addition, sampling process need to open valve protection cap and ventilation wrapping paper, and pipette tips, pipette need to be introduced etc. and taken
Sampling device increases microbiological contamination risk.
(3) to maintain cell Seedling height rate and high cell viability, a large amount of nutriment need to be supplemented.Traditional culture bottle
Without feed supplement adding set, nutrition supply deficiency leads to later period cell slow growth, or even is intended to cracking death, final to influence carefully
Born of the same parents' density and product expression amount.
(4) traditional culture bottle inner wall welding baffle or the concave-convex formation baffle of bottle body itself, this undoubtedly increases culture bottle
The difficulty of manufacturing process.The difficult point of traditional culture bottle processing is accurately design size, the location and shape of baffle,
And welding baffle component complex process in bottle.In addition, setting fixed baffle in culture bottle, the difficulty of cleaning is undoubtedly also increased, it is special
It is not that the stickum that cell culture secretion generates is difficult to clean.
Therefore, it is necessary to which especially under the premise of meeting dissolved oxygen demand, designing one kind can meeting cell growth demand
Fast sampling, feed supplement, transfer high efficiency cell culture bottle.
Summary of the invention
The main purpose of the present invention is to provide a kind of high efficiency cell culture bottles of built-in disturbing flow device, to overcome existing skill
The deficiency of art.
To achieve the goals above, technical scheme is as follows:
The embodiment of the invention provides a kind of high efficiency cell culture bottles of built-in disturbing flow device, comprising:
Cultivate bottle body；
The valve protection cap being tightly connected with the culture bottle body is provided with an at least venthole on the valve protection cap, at least one takes
Sample mouth, at least a material-feeding port and at least a transfer port；
It is set to the intrinsic disturbing flow device of the culture bottle, from the center line of the culture bottle body to the culture
Bottle side wall direction gradually decrease, and the disturbing flow device when the high efficiency cell culture bottle is stood on horizontal plane with it is described
Horizontal plane forms 5~45 ° of inclination angle.
As one of preferable preferred embodiment, the disturbing flow device includes a plurality of baffle mechanisms, the baffle mechanism
It is fixedly connected by connecting rod with lower fixed ring, and a plurality of baffle mechanisms are intervally installed, the lower fixed ring passes through rib
Muscle is connect with upper fixed ring, and fixed ring, lower fixed ring are suitable with the side wall of the culture bottle body, bottom wall swelling respectively
Match.
Further, a plurality of baffle mechanisms are recessed in being separated to form a plurality of first on the bottom wall of culture bottle body
Place and one second recess, first recess extend radially from culture bottle body central line to sidewall direction, and described second is recessed
Locate to be arranged around culture bottle body central line, first recess is connected to the second recess.
Further, the baffle mechanism includes preceding guide face, trailing face, upper side and bottom faces, the upper side with
The preceding guide face connects to forming leading edge, and the upper side connects to forming hangover side, the upper side and bottom with the trailing face
Portion face connects to forming upper edge, and the preceding guide face and trailing face connect to forming inclined side, the leading edge and hangover side respectively with
The horizontal plane forms the first inclination angle and the second inclination angle in the same size and contrary.
Further, first inclination angle, the second inclination angle are 30~60 °.
Further, the angle formed between the upper side and horizontal plane is 5~45 °, and the inclined side and water
The angle formed between plane is 10~175 °.
Further, the cross section of first recess is two trapezoidal, adjacent with one first recess baffle mechanisms
In, the hangover side of a baffle mechanism and the leading edge of another baffle mechanism respectively constitute the trapezoidal side, and institute
The endpoint on the hangover side for the baffle mechanism stated is connect to shape with the endpoint of the leading edge of another baffle mechanism
At the upper bottom of the trapezoidal cross-section and bottom.
As one of preferable preferred embodiment, the culture bottle body include sequentially connected bottleneck, bottle main body and
The upper end of bottom of bottle, the bottleneck forms bottleneck, and the bottleneck is provided with external screw thread adjacent to the bottle opening, sets in the valve protection cap
It is equipped with the internal screw thread to match with institute external screw thread.
Preferably, the culture bottle body bottom wall is connect with side wall by the circle turning seamlessly transitted or cambered surface.
Preferably, the height of the culture bottle body bottom wall is slightly above the round turning or cambered surface.
Preferably, the culture bottle body lower sidewall is parallel with culture bottle body central axis, favours in the middle part of side wall
Central axis, tilt angle are 1~80 °, and more preferably 5~45 °, side wall upper part also favours central axis, tilt angle
It is 1~80 °, more preferably 5~45 °.
Preferably, the culture bottle body is conical flask.
As one of preferable preferred embodiment, it is described culture bottle body bottleneck diameter close to the bottleneck length
Degree.
As one of preferable preferred embodiment, ring type seal, the ring type seal position are equipped on the inside of the valve protection cap
Between the bottleneck edge and bottle cap of the culture bottle body, to provide hydraulic seal.
As one of preferable preferred embodiment, the material-feeding port or transfer port are connect with solenoid valve or peristaltic pump, institute
It states solenoid valve or peristaltic pump is electrically connected with control device.
As one of preferable preferred embodiment, the side wall of the culture bottle body indicates graduation mark.
Compared with prior art, advantages of the present invention at least that:
(1) can in-situ sampling, sampling is simple, quickly, and changing tradition sampling need to complete in sterile working in ultra-clean work.
(2) oxygen transfer is high-efficient, built-in disturbing flow device in high efficiency cell culture bottle provided by the invention, and has big open wide
Bottleneck and the design of aeration valve protection cap, can provide good ventilation and mixed effect.
(3) transfer efficiency is high, and compared with traditional transfer process, the valve protection cap of high efficiency cell culture bottle provided by the invention has
Transfer port saves the time that liquid is transferred to bioreactor, and transfer time is only a few minutes, reduces the probability of spilling,
And it can one man operation.
(4) the bottled liquid coefficient of high efficiency cell culture provided by the invention is high, and highest may be up to 60%.
(5) excellent amplification performance easily can be amplified to large scale from small size (125ml) high efficiency cell culture bottle
(5L) high efficiency cell culture bottle.
Detailed description of the invention
It is right with reference to the accompanying drawings and detailed description in order to illustrate more clearly of structure of the invention feature and technical essential
The present invention is described in detail.
Fig. 1 is the structural schematic diagram of high efficiency cell culture bottle body provided in an embodiment of the present invention；
Fig. 2 is the top view of high efficiency cell culture bottle body provided in an embodiment of the present invention；
Fig. 3 is the structural schematic diagram of disturbing flow device provided in an embodiment of the present invention；
Fig. 4 is the structural schematic diagram of valve protection cap provided in an embodiment of the present invention；
Fig. 5 is the sampling schematic diagram of high efficiency cell culture bottle provided in an embodiment of the present invention；
Fig. 6 is the feed supplement schematic diagram of high efficiency cell culture bottle provided in an embodiment of the present invention；
Fig. 7 is that the culture solution of high efficiency cell culture bottle provided in an embodiment of the present invention shifts schematic diagram (force of gravity method)；
Fig. 8 is that the culture solution of high efficiency cell culture bottle provided in an embodiment of the present invention shifts schematic diagram (peristaltic pump transfer
Method)；
Fig. 9 is the cell in 250ml high efficiency cell culture bottle provided in an embodiment of the present invention and 5L high efficiency cell culture bottle
Number, cell activity comparison schematic diagram.
Description of symbols: 1- bottleneck, 2- bottleneck edge, 3- bottleneck, 4- side wall, 5- side wall, 6- side wall, 7- flow-disturbing dress
It sets, 7a- leading edge, 7b- hangover side, 7c- upper edge, 7d- inclined side, 8- external thread structure, at 9- side wall and bottom corners,
10- bottom of bottle, the first recess of 11-, the second recess of 12-, the upper fixed ring of 13-, 14- rib, 15- connecting rod, fixed ring under 16-, 17-
Venthole, 18- sample tap, 19- valve protection cap outside, 20- valve protection cap inside, 21- material-feeding port, 22- transfer port, 23- syringe, 24- take
Sample mouth seals, probe tube in 25- bottles, 26- feed supplement pump, the outer feed supplement pipe of 27-250ml feed supplement bottle bottle, in 28-250ml feed supplement bottle bottle
Feed supplement pipe, 29-250ml feed supplement bottle, 30- personal computer, feed supplement pipe in 31-5L culture bottle bottle, 32-5L high efficiency cell culture
Bottle, the outer transfer pipe of 33-250ml culture bottle bottle, 34-250ml high efficiency cell culture bottle, transfer pipe in 35-250ml culture bottle bottle,
36- culture solution shifting pump, 37-5L high efficiency cell culture bottle.
Specific embodiment
Below with reference to the attached drawing in the present embodiment, the technical solution in embodiment specifically, clearly and completely retouch
It states.
As shown in figures 1-4, high efficiency cell culture bottle provided in this embodiment includes the culture bottle body and bottle being tightly connected
There are internal whorl line in lid, valve protection cap inside 20, cooperate with the external thread structure 8 of culture bottle bottleneck, and 19 be planar rings on the outside of valve protection cap
Shape, the top of valve protection cap have a venthole 17, one or more ports (sample tap 18, material-feeding port 21, any in transfer port 22
One or more).
20 on the inside of valve protection cap, a ring type seal is equipped on the outside of annular valve protection cap 19.Preferably, ring type seal
19 inside on the outside of the adjacent valve protection cap of external margin, the internal edge of ring type seal is adjacent annular flange (under bottle cap), loop method
19 are connected with ring type seal formation on the outside of blue and valve protection cap.Preferably, ring type seal is made of elaxtic seal, such as
Natural rubber, buna (artificial synthetic rubber).When bottle cap and bottleneck are clamped, sealing ring is located at bottleneck edge 2 and bottle
Between lid, hydraulic seal is formed.Preferably, to avoid the threaded connection between bottle cap and bottleneck excessively tight or skidding, outside valve protection cap
Long enough is answered in side 19, and when the adjacent ring type seal in bottleneck edge 2,3 terminal abutment of bottleneck main body ends flange.
Preferably, sealing ring includes O-ring seal or D type sealing ring.Alternative to be, sealing passes through bonding or groove
Mode connect, the size of the size of sealing ring and groove is coincide.Sealing preferably can be single layer sealing, be also possible to double-deck close
Envelope is multi-layer sealed, is preferably spaced at a certain distance between each layer sealing.
21 mouthfuls of the feed supplement end or transfer port 22 are connect with solenoid valve or peristaltic pump 26, peristaltic pump 36, computer control
Solenoid valve or peristaltic pump 26 processed, the switch of peristaltic pump 36 or operating, enter culture bottle to block or close feed liquid.The time of feed supplement
It can manually be set by computer 30 with feed supplement amount.Preferably, venthole 17 should (venthole be most suitable straight using appropriate size
Diameter is 1.6mm), i.e., to meet liquid can flow continually out from culture solution transfer port 22 and sample tap 18, and avoid gas from blocking,
Liquid in bottle is avoided to splash out and reduce microbiological contamination risk from venthole.
In actual use, growth medium is injected into culture bottle by the open ports of bottleneck, and big unlimited bottleneck makes
Must operate it is simpler, venthole allow gas be freely accessible to culture bottle, it is ensured that liquid continuously flows.
Alternatively, the indication wire on lid can prompt how user unscrews bottle cap.When in culture bottle
When liquid is lower than the adjoiner of bottle cap 30 and culture bottle, it is ensured that liquid is stayed open lower than graduation mark and venthole 17.
The culture bottle body further include bottom of bottle 10, bottle main body (bottle main body includes side wall 4,5,6), bottleneck 2,
Bottleneck 1 and built-in disturbing flow device component 7.2.5L culture bottle base diameter is 50mm, a height of 28cm, the long 75mm of bottleneck, diameter
For 75mm.Preferably, culture bottle is made of glass, polymer material, such as polypropylene.Preferably, culture bottle body can be with
The mode of injection molding or blow molding is selected, and can be abandoned, reduces and bring pollution is recycled.
The culture bottle body is in conical, cylindrical, spherical, the square bodily form, cuboid, but not limited to this, it goes back
It can be other shapes.Preferably, culture bottle body is cultivated between the bottom wall 10 of bottle body and side wall 6 and side in cone
By the circle turning of smooth transition or cambered surface connection between wall 4,5,6, bottle body bottom wall 10 is slightly raised, when culture bottle liquid amount compares
When low, the fluid in culture bottle can be flowed along the corner of bottle body bottom wall 10.The side wall 6 and culture bottle central axis of bottle body are flat
Row；Side wall 5 favours central axis, and tilt angle is 1~80 °, it is preferred that tilt angle is 5~45 °；Side wall 4 also favours
Central axis, tilt angle are 1~80 °, it is preferred that tilt angle is 5~45 °.The side-walls of culture bottle body may also set up
Liquid level line, and indicate corresponding culture bottle volume.
The bottleneck 2 is located at the end of bottle side wall 4, cylindrical, rectangular, but is also not necessarily limited to other shapes, preferably
Be cylindrical.3 edge of bottleneck is provided with external thread structure 8.External thread structure 8 can be single line, a plurality of line, can also be with
For continuous lines or interrupt line, preferably a plurality of interrupt line.The form of screw thread is not limited to bolt, flanged joint and other connections
Mode.Flange is located at screw thread position below, and extends to outside bottleneck, and bottleneck 2 is small enough in one-handed performance.Bottleneck 2
Diameter and its length are changeable, and label can print to turning 9 or culture bottle side wall 4,5,6.
Such as Fig. 1, in preferred example, culture bottle includes bottom of bottle 10, several disturbing flow devices 7 built in culture bottle.It is preferred that
, the bottom wall 10 of culture bottle is connect by way of circle turning with culture bottle side wall 4,5,6；But alternative, culture bottle side
Wall 4,5,6 is tapered, with one or more gradients close to central axis.Culture bottle side wall 4,5,6 connects bottleneck 2.2 side of bottleneck
There is a unlimited bottleneck 1 at edge.Side wall 6 and centerline axis parallel, side wall 5 slowly favour central axis, and side wall 5 rapidly favours
Central axis.With the change of the structure snd size of disturbing flow device 7, the outer wall dimension for cultivating bottle container is also changed correspondingly.
The elastic material (such as silica gel or rubber) with auto-reset function can be used in disturbing flow device 7, and no bullet can also be used
Property stainless steel material or hard plastic material (such as polycarbonate, polyester, fluoropolymer, acrylic acid, PET, polyvinyl chloride,
Any one in ABS, PC, PS, GAG, acrylic and polyolefin or two or more combinations).Above-mentioned material should be resistant to not
High-temperature sterilization processing of 121 DEG C lower than the second recess, γ ray sterilization, β ray sterilizing, the processing of chemical reagent sterilization.
Disturbing flow device includes upper fixed ring 13, lower fixed ring 16, rib 14, baffle, connecting rod 15, upper and lower fixed ring 13,
16 are linked into an integrated entity by rib 14, upper fixed ring 13 and lower fixed ring 16 respectively with the side wall of culture bottle 4,5,6 and bottom wall 10
Adaptation, baffle are connect by connecting rod 15 with lower fixed ring 16, and are located in the closed circular space of lower fixed ring 16.
Disturbing flow device 7 plays the role of ventilation mixing, but not damaged cell.The design of disturbing flow device 7 should reduce shearing
Power enhances mixed effect.Therefore, when using culture bottle, revolving speed is set as particular value, and built-in disturbing flow device can get preferable
Admixture, lesser shearing force avoids the breakage of cell wall.
In specific example, the bottle body bottom wall 10 of pie is divided into the first recess 11, the second recess by built-in disturbing flow device 7
12, the first recess 11, the second recess 12 is extended outwardly by center line.The shape of baffle be rectangle, parallelogram, square,
One or more of trapezoidal, triangle, preferably rectangle.The number of baffle is changeable, preferably even number.Such as Fig. 2-
Culture bottle bottom is divided into six recesses by six disturbing flow devices 7 shown in 3, the angle between two adjacent the first recesses 11
It is 60 °.Certainly, using four disturbing flow devices 7 be also it is feasible, culture bottle bottom can be divided into four by four stream devices 7
First recess 11, the angle between two adjacent the first recesses 11 is 90 °.
Culture bottle is along a direction continuous rotation, and preferably clockwise, each baffle of disturbing flow device 7 is along same
Sample prescription is to rotation.In actual moving process, it is furnished with corresponding equipment, the direction of rotation is controlled by operator.Each flow-disturbing dress
7 baffle is set to be made of preceding guide face, trailing face, upper side and bottom surface, any two of them homogeneously connects between face, it is described on
Portion face connects to forming leading edge 7a with the preceding guide face, and the upper side connects to forming hangover side 7b with the trailing face, leading
In 7a and hangover 7b respectively the slanted angle between horizontal plane be 30~60 ° (bottom wall 10 of culture bottle be located at level
On face), more preferably, slanted angle is 30~40 °.The rotation speed of culture bottle is 20~300rpm, it is preferred that rotation speed is
80~200rpm.Certainly, higher rotation speed can also be used, but included angle need to be adjusted, cause cell broken to avoid shearing
Damage.Therefore, the design of disturbing flow device 10 need to consider rotation speed, to maintain low-shearing force, to avoid cell damage.
Leading edge 7a and hangover side 7b extend to center line, radially distribute.Middle section 7c is generally planar, but
It can be slightly convex.Preferably, the bottle body bottom wall 10 of pie is divided into the first recess 11, the second recess 12 by disturbing flow device 7, but
Shape is changeable.When culture bottle container is rotated with certain speed, the inclination of baffle leading edge 7a and hangover side 7b and horizontal plane is pressed from both sides
Angle can avoid cell damage.After cell infection, cell is collected.Stirring slightly influences ventilation, because the formation of bubble influences carefully
The breakage of cell wall.If speed of agitator increases, in order to reduce shearing force, angle may reduce.
Interval between each baffle of disturbing flow device 7 also influences the ventilation and stirring of fluid.First recess 11 it is transversal
Face be it is trapezoidal, in two baffles adjacent with one first recess 11, the hangover side 7b of baffle and with before another baffle
Guide margin 7a respectively constitutes the side of the trapezoidal cross-section, and the endpoint on the hangover side of a baffle is leading with another baffle
The endpoint connection on side is to form the trapezoidal upper bottom and bottom, and nature of going to the bottom is located at the bottom wall 10 of bottle body.Preferably,
Leading edge 7a and hangover side 7b are tilted with same degree, but contrary, so that fluid lightly flows through leading edge 7a, are bypassed
Upper edge 7c, through hangover side 7b outflow.Fluid bypasses the first recess 11, reaches next leading edge 7a, flows through next top
Side 7c is flowed out to next first recess 11 through next hangover side 7b, is recycled with this, so as to cause lumpy fluid
Flowing is conducive to inflation, and will not damaged cell wall.
Culture bottle is rotated around center line, and the fluid velocity of center line is minimum.With increase, fluid with a distance from central axis
Speed linearity increases.Frictional force between fluid and culture bottle container can avoid fluid and be rotated with a certain speed.(the radiation of turning 9
Shape farthest) at fluid-flow rate be greater than centerline fluid-flow rate.
At neighbouring turning 9, the flowing velocity at disturbing flow device 7 is minimum；Flowing at centerline, disturbing flow device 7
Speed highest.Preferably, the disturbing flow device at neighbouring turning 9 successfully incorporates bottom wall 10 and turning 9.Preferably, flow-disturbing fills
Setting the distance between 7 and turning 9 D is center line to the 1/3 of turning outer peripheral lines distance.In specific example, disturbing flow device with turn
The distance between angle D about 25mm.
When culture bottle is with the rotation of 80~180rpm rotation speed, the upper side of disturbing flow device favours 5~12 ° of horizontal plane,
In specific example, preferred tilt angle is 9 °, and preferred rotation speed is 80~100rpm.
Neighbouring centerline, the flowing velocity highest of disturbing flow device 7.The distance between disturbing flow device 7 and central axis are
The 1/8 of container diameter.Disturbing flow device 7 is tilted to the direction far from center line, and about 20~50 ° of folder is formed with horizontal plane
Angle, it is preferred that angle is 38 °.Preferably, because right angle mode connection shearing force it is big, thus avoid inclined side 7d, upper edge 7c,
It is connected with right angle mode in turn to avoid cellular damage, in design generally to justify turning between leading edge 7a, hangover side 7b
Mode connects.
In a specific example, disturbing flow device 7 includes six baffles, spacing about 5~8mm between each baffle.It is training
Bottle radius 2/3, neighbouring corner 9 are supported, upper edge 7c is merged with bottle body bottom wall 10.Six cheese disturbing flow devices 7 are divided into six
The first recess of rectangle 11, and second recess 12 is formed in centerline.
When in use, culture bottle is equipped with the growth medium of 1/3 volume, is totally submerged disturbing flow device 7.Preferably, edge
Central line measurement, the height of disturbing flow device 7 and horizontal plane bottom is about the 1/3 of fluid level.More optimizedly, disturbing flow device
Height is about the 1/5~1/10 of fluid level.
Culture bottle is fixed on shaking table.Side wall 4, side wall 45 are consistent with the central axis of fixture, with the rotation of 80~180rpm speed
Turn.Fluid wave flows through disturbing flow device 7 and plays the role of stirring, mixing, inflation, avoids cell wall damaged.
As shown in figure 5, the sampling method of the high efficiency cell culture bottle 34,32 of built-in disturbing flow device 7 is as follows:
1) the sample tap sealing 24 at sampling port 18 is unscrewed；
2) it is sampled after connecting syringe 23 and sampling port 18, wherein probe tube 25 is connected to sampling port 18 in bottle；
3) the sample tap sealing 24 at sampling port 18 is screwed.
As shown in fig. 6, following (the high efficiency cell culture of the feed process of the high efficiency cell culture bottle 32 of built-in disturbing flow device 7
Bottle 34 is identical):
1) by the feed supplement silica gel of feed supplement pipe 31 and 250ml feed supplement bottle 29 in the 5L culture bottle bottle in high efficiency cell culture bottle 32
The sterile welding of pipe 27；
2) feed supplement silicone tube 27 is mounted on feed supplement to pump in 26 pump heads, and feed supplement silicone tube 27 and 250ml feed supplement bottle bottle
Interior feed supplement pipe 28 is connected to；
3) opening/closing time of the revolving speed of adjustment feed supplement pump 26 and time or solenoid valve, and feed supplement valve is opened, according to cell
Upgrowth situation starts feed supplement.
Culture solution in the high efficiency cell culture bottle 34 of built-in disturbing flow device 7 is transferred to another high efficiency cell culture bottle 37
Method include following methods:
A) force of gravity method (as shown in Figure 7)
A1 transfer pipe in the 250ml culture bottle bottle in Tissue Culture Flask 34 is connect with transfer silicone tube 33), and to turn
Move silicone tube 33 and the sterile welding of high efficiency cell culture bottle 37；
A2) Tissue Culture Flask 34 is inverted or is adjusted to suitable position；
A3 it) opens liquid and shifts valve, culture solution is transferred to completely to efficient Tissue Culture Flask 37.
B) peristaltic pump transfer method (as shown in Figure 8)
B1 transfer pipe in the 250ml culture bottle bottle in Tissue Culture Flask 34 is connect with transfer silicone tube 33), and to turn
Move silicone tube 33 and the sterile welding of high efficiency cell culture bottle 37；
B2) transfer silicone tube 33 is mounted in the pump head of culture solution shifting pump 36；
B3) revolving speed and the time of culture solution shifting pump 36 are set, and open liquid transfer valve, culture solution is transferred to completely
To efficient Tissue Culture Flask 37.
Below in conjunction with specific embodiment and attached drawing, the present invention will be further described in detail.
Embodiment 1
To evaluate, the efficient culture performance of high efficiency cell culture bottle, dress liquid coefficient, (contamination rate, single take operability
Sample is time-consuming, single inoculation transfer is time-consuming), PK-15 cell and Chinese hamster ovary celI are cultivated, every kind of cell cultivates 50 bottles, culture
Condition is as follows:
The condition of culture of PK-15 cell: (1) 5L high efficiency cell culture bottle: is seeded to from 250ml high efficiency cell culture bottle 34
High efficiency cell culture bottle 32, inoculum density are 0.6 × 106Cells/ml, 2g/L the mounting medium (Cytodex- of GE company production
1), 37 DEG C, 5%CO2Culture.Using 26 stream of feed supplement pump plus supplemented medium is added every 1d.When culture is to 4d, collect thin
Born of the same parents.(2) regular growth culture bottle: it is seeded to 5L regular growth culture bottle from 250ml regular growth culture bottle, inoculum density is
0.6×106Cells/ml, 2g/L mounting medium (Cytodex-1 of GE company production), 37 DEG C, 5%CO2Culture.Every 1d benefit
Manually add supplemented medium.When culture is to 4d, cell is collected.
The condition of culture of Chinese hamster ovary celI: (1) 5L high high efficiency cell culture bottle: is seeded to from 250ml high efficiency cell culture bottle 34
Tissue Culture Flask 32 is imitated, inoculum density is 0.8 × 106Cells/ml, 37 DEG C, 5%CO2Culture.When glucose exhausts, feed supplement
Pump 26 adds supplemented medium and glucose.When culture is to 5d, cultivation temperature is adjusted to 32 DEG C.When culture is to 14d, collect thin
Born of the same parents.(2) regular growth culture bottle: it is seeded to 5L regular growth culture bottle from 250ml regular growth culture bottle, inoculum density is
0.8×106Cells/ml, 37 DEG C, 5%CO2Culture.When glucose exhausts, supplemented medium and glucose are manually added.Training
When supporting to 5d, cultivation temperature is adjusted to 32 DEG C.When culture is to 14d, cell is collected.
When sampling and inoculation transfer, calculating single sampling is time-consuming and single inoculation is time-consuming；At the end of culture, it is measured by sampling most
Whole average cell number and observation microbiological contamination situation result simultaneously calculate contamination rate.
Sample 1000rpm is centrifuged 15min, discards supernatant, and 0.1% crystal violet dye liquor, 37 DEG C of water-bath 1h, using blood are added
Ball count plate measures the number of cells in the sample.And observe cell microbiological contamination situation, the calculation formula of contamination rate are as follows: contamination rate (%)=dye
Bacterium bottle number/(non-microbiological contamination bottle number+microbiological contamination bottle number).
The culture performance and the operability rate of exchange of 1 high efficiency cell culture bottle of table and regular growth culture bottle
The PK-15 cell of high-efficient culture bottle culture and the cell density of Chinese hamster ovary celI are up to 53.6% than routine culture respectively
With 37.8%.High-efficient culture bottle not only can guarantee high-density cell growth, moreover it is possible to increase the liquid amount of shaking flask, the training of 5L high efficiency cell
The dress liquid coefficient for supporting bottle may be up to 60%, and the dress liquid coefficient of routine culture bottle is only 30%.High-efficient culture bottle and routine culture
The mode of off normal sampling and in-situ sampling, single sampling difference time-consuming 6.9~7.6min and 2.1~2.2min is respectively adopted in bottle.
High-efficient culture bottle is inoculated with by the way of feed supplement pump transfer, and single inoculation time-consuming is only the half of routine culture bottle inoculation time,
And it largely avoided microbiological contamination.
Embodiment 2
Investigate amplification performance of the HEK-239 cell in high efficiency cell culture bottle.The condition of culture of cell: from 250ml high
Effect Tissue Culture Flask 34 is seeded to 5L high efficiency cell culture bottle 32, and inoculum density is 0.5 × 106Cells/ml, 37 DEG C, 5%CO2
Culture.Supplemented medium is added every 1d.When culture is to 7d, cell is collected.Cell number and cell activity are measured by sampling daily.
The method that cytoactive detection uses propidium iodide (PI) dyeing.When frustule dyes, PI storage is added into sample
(0.2 μm of membrane filtration, saves standby liquid at 4 DEG C, and ultimate density is 10 μm of ol/L, is incubated for 20min under room temperature in dark.
PI dyestuff can not mark living cells, and dead cell PI dyestuff intracellular is emitted red fluorescence after 488nm laser excitation, be led to by FL2
Road (585nm) receives, and the channel FL2 at least detects 10000 cells.The analysis flow velocity of sample is 60 μ l/min, loading volume
For 10 μ l.
As shown in Figure 9, the cell growth change trend of HEK-239 cell is similar in 250ml with 5L high-efficient culture bottle, just
Phase slow growth tends to stablize when cultivating 6d subsequently into fast growing period, when cultivating 7d cell number reach 4.6 ×
106cells/ml.Should the result shows that, HEK-239 cell obtains almost the same growth effect in 250ml and 5L high-efficient culture bottle
Fruit.
Above-mentioned specific embodiment, only technical concept and structure feature to illustrate the invention, it is therefore intended that allow and be familiar with this
The stakeholder of item technology can implement accordingly, but above said content is not intended to limit protection scope of the present invention, all foundations
Any equivalent change or modification made by Spirit Essence of the invention, should all fall under the scope of the present invention.
Claims (10)
1. a kind of high efficiency cell culture bottle of built-in disturbing flow device, characterized by comprising:
Cultivate bottle body；
The valve protection cap being tightly connected with the culture bottle body, be provided on the valve protection cap an at least venthole, an at least sample tap,
An at least material-feeding port and at least a transfer port；
It is set to the intrinsic disturbing flow device of the culture bottle, from the center line of the culture bottle body to the culture bottle side
The direction of wall gradually decreases, and the disturbing flow device when the high efficiency cell culture bottle is stood on horizontal plane with the level
Face forms 5~45 ° of inclination angle.
2. the high efficiency cell culture bottle of built-in disturbing flow device according to claim 1, it is characterised in that: the disturbing flow device
Including a plurality of baffle mechanisms, the baffle mechanism is fixedly connected by connecting rod with lower fixed ring, and a plurality of baffle mechanisms
Be intervally installed, the lower fixed ring is connect by rib with upper fixed ring, fixed ring, lower fixed ring respectively with institute
State side wall, the bottom wall swelling adaptation of culture bottle body.
3. the high efficiency cell culture bottle of built-in disturbing flow device according to claim 2, it is characterised in that: described is a plurality of
Baffle mechanism in culture bottle body bottom wall on be separated to form a plurality of first recesses and one second recess, first recess edge
Radial to extend from culture bottle body central line to sidewall direction, second recess is arranged around culture bottle body central line, institute
The first recess is stated to be connected to the second recess.
4. the high efficiency cell culture bottle of built-in disturbing flow device according to claim 3, it is characterised in that: the baffle mechanism
Including preceding guide face, trailing face, upper side and bottom faces, the upper side connects to forming leading edge with the preceding guide face, it is described on
Portion face connects to forming hangover side with the trailing face, and the upper side and bottom faces connect to forming upper edge, the preceding guide face and
Trailing face connects to forming inclined side, the leading edge and hangover side formed respectively with the horizontal plane it is in the same size and contrary
The first inclination angle and the second inclination angle.
5. the high efficiency cell culture bottle of built-in disturbing flow device according to claim 4, it is characterised in that: first inclination
Angle, the second inclination angle are 30~60 °.
6. the high efficiency cell culture bottle of built-in disturbing flow device according to claim 4, it is characterised in that: the upper side with
The angle formed between horizontal plane is 5~45 °, and the angle formed between the inclined side and horizontal plane is 10~175 °.
7. the high efficiency cell culture bottle of built-in disturbing flow device according to claim 3, it is characterised in that: first recess
Cross section be it is trapezoidal, in two baffle mechanisms adjacent with one first recess, the hangover side of baffle mechanism and another
The leading edge of baffle mechanism respectively constitutes the trapezoidal side, and the endpoint on the hangover side of a baffle mechanism with
The endpoint connection of the leading edge of another baffle mechanism is to form the upper bottom and bottom of the trapezoidal cross-section.
8. the high efficiency cell culture bottle of built-in disturbing flow device according to claim 1, it is characterised in that: the culture bottle sheet
Body includes sequentially connected bottleneck, bottle main body and bottom of bottle, and the upper end of the bottleneck forms bottleneck, and the bottleneck is adjacent to the bottle
Opening is provided with external screw thread, and the internal screw thread to match with institute external screw thread is provided in the valve protection cap.
9. the high efficiency cell culture bottle of built-in disturbing flow device according to claim 1, it is characterised in that:
The culture bottle body bottom wall is connect with side wall by the circle turning seamlessly transitted or cambered surface；
Preferably, the height of the culture bottle body bottom wall is slightly above the round turning or cambered surface；
Preferably, the culture bottle body lower sidewall is parallel with culture bottle body central axis, favours center in the middle part of side wall
Axis, tilt angle are 1~80 °, and preferably 5~45 °, side wall upper part also favours central axis, tilt angle is 1~
80 °, preferably 5~45 °；
Preferably, the culture bottle body is conical flask.
10. the high efficiency cell culture bottle of built-in disturbing flow device according to claim 1, it is characterised in that: the culture bottle
Length of the diameter of the bottleneck of ontology close to the bottleneck；And/or ring type seal, the ring are equipped on the inside of the valve protection cap
Shape sealing ring is located between the bottleneck edge and bottle cap of the culture bottle body, to provide hydraulic seal；And/or it is described
Material-feeding port or transfer port are connect with solenoid valve or peristaltic pump, and the solenoid valve or peristaltic pump are electrically connected with control device；And/or
The side wall of the culture bottle body indicates graduation mark.
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Cited By (1)
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| CN109810899A (en) * | 2019-04-09 | 2019-05-28 | 苏州赛普生物科技有限公司 | Suspending cell culture bottle |
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| CN109401960B (en) | 2024-10-18 |
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