CN112143652A

CN112143652A - Circulation vortex air-lift type cell suspension culture bottle

Info

Publication number: CN112143652A
Application number: CN202011039468.3A
Authority: CN
Inventors: 许常河
Original assignee: Anhui Haotian Biotechnology Co ltd
Current assignee: Anhui Haotian Biotechnology Co ltd
Priority date: 2020-09-28
Filing date: 2020-09-28
Publication date: 2020-12-29

Abstract

The invention discloses a circulating turbulent flow airlift cell suspension culture bottle, and relates to the technical field of biological cell culture. The oxygen bottle comprises a bottle body and a bottle cap, wherein an oxygen tube is arranged at the lower end of the bottle body; the oxygen delivery pipe is positioned right below the oxygen return pipe; the oxygen delivery pipe is communicated with the oxygen return pipe; a main shaft is fixedly arranged at the middle position of the bottom inside the bottle body; turbulent flow rotating wheels are uniformly distributed on the main shaft; a spiral vent pipe is arranged outside the turbulent flow rotating wheel; the bottom end of the spiral vent pipe is communicated with the oxygen therapy pipe; the top end of the spiral vent pipe is communicated with the oxygen return pipe. The oxygen supply device generates oxygen, the oxygen is returned to the oxygen delivery pipe through the oxygen delivery pipe, the spiral vent pipe and the oxygen return pipe to form internal circulation of oxygen supply, and the oxygen is continuously contacted and dissolved with liquid in the cell culture bottle, so that the dissolved oxygen in the culture bottle is improved; the vortex runner rotates and makes cell culture liquid rock, prevents the cell deposit for the cell is in the suspended state all the time.

Description

Circulation vortex air-lift type cell suspension culture bottle

Technical Field

The invention belongs to the technical field of biological cell culture, and particularly relates to a circulating turbulent flow airlift cell suspension culture bottle.

Background

Cell culture refers to a method of simulating the in vivo environment in vitro, allowing it to survive, grow, reproduce and maintain the main structure and function. The cell culture is divided into suspension culture and adherent culture. The plant cells are generally cultured in suspension, and the free single cells or cell clusters are suspended in a liquid culture medium according to a certain cell density and cultured by using a shaking table or a rotating bed.

The cell culture bottle can obtain a large amount of experimental data under the condition of limited manpower, material resources and space. In addition, the shake flask experiment result provides basic growth information and culture process parameters of the cells, and provides technical reference for large-scale cell culture. Therefore, cell culture flasks are widely used in laboratory research and industrial production. However, the conventional cell culture flask still has the following problems:

(1) the growth, development, division, reproduction and other life activities of the cells all need to consume a large amount of oxygen and participate in the tricarboxylic acid cycle process. The traditional culture bottle has small bottle neck and deep and large bottle bottom, and seriously blocks the circulation of gas. The culture bottle rotates on the shaking table along the central axis of the culture bottle, and the fluid is in a laminar flow state, which is not beneficial to mixing and oxygen transfer. At present, researchers often adopt a method of reducing a liquid filling coefficient and increasing a rotating speed to improve an oxygen mass transfer coefficient. However, if the liquid filling factor is too low, a large amount of water in the culture flask evaporates as the culture process proceeds, resulting in an increase in the osmotic pressure of the culture medium and inhibition of cell growth. The higher rotating speed of the shaking table generates high shearing force to cause cell damage or cell death by dissolution; in addition, the long term operation at the limiting speed can greatly shorten the life of the shaking table.

(2) In order to understand the growth metabolic state of the cells, the cells are sampled at variable time to measure the growth parameters of the cells and observe the cell morphology. Traditional culture flask sampling is accomplished by aseptic manipulation techniques at a clean bench. The whole process consumes a great deal of time, manpower and material resources. In addition, the bottle cap and the ventilation packaging paper need to be opened in the sampling process, and sampling devices such as a gun head and a pipette need to be introduced, so that the risk of bacterial contamination is increased.

(3) In order to maintain high growth rate and high cell viability of the cells, a large amount of nutrients needs to be supplemented. The traditional culture bottle has no supplement adding device, and the nutrient supply is insufficient, so that the later-stage cells grow slowly and even tend to crack and die, and finally the cell density and the product expression are influenced.

Therefore, there is a need to design a high-efficiency cell culture flask capable of rapid sampling, feeding and transferring on the premise of meeting the cell growth requirement, especially meeting the dissolved oxygen requirement.

Disclosure of Invention

The invention aims to provide a circulating turbulent air-lift cell suspension culture bottle, which solves the problem of inconvenient oxygen supply of the existing cell culture bottle by arranging an oxygen tube, an oxygen reflux pipe, a turbulent rotating wheel and a spiral vent pipe; the rotation speed of the shaking table easily causes cell death; and the problems of inconvenient sampling and nourishing.

In order to solve the technical problems, the invention is realized by the following technical scheme:

the invention relates to a circulation turbulent flow airlift cell suspension culture bottle, which comprises a bottle body and a bottle cap; the bottle body is in a circular truncated cone shape; the bottle mouth of the bottle body is matched with the bottle cap; an oxygen return pipe, an exhaust pipe and a liquid discharge pipe are arranged at the upper part of the outer side surface of the bottle body; the oxygen return pipe and the liquid discharge pipe are symmetrically arranged around the central axis of the bottle body; the exhaust pipe is arranged at the front end of the bottle body; the exhaust pipe is in a gooseneck shape; the lower end of the bottle body is provided with an oxygen catheter; the oxygen therapy pipe is S-shaped; the oxygen delivery pipe and the oxygen return pipe are positioned on the same side; the oxygen delivery pipe is positioned right below the oxygen return pipe; the oxygen delivery pipe is communicated with the oxygen return pipe; one end of the oxygen delivery pipe is connected with the top of the oxygen supply device; the other end of the oxygen therapy tube extends into the bottle body; a main shaft is fixedly arranged in the middle of the bottom inside the bottle body; turbulent flow rotating wheels are uniformly distributed on the main shaft; a spiral vent pipe is arranged outside the turbulent flow rotating wheel; the pipe wall of the spiral vent pipe is uniformly provided with first vent holes; the bottom end of the spiral vent pipe is communicated with the oxygen therapy pipe; the top end of the spiral vent pipe is communicated with an oxygen return pipe; the oxygen therapy pipe is provided with a first control valve; the first control valve is arranged at one end of the oxygen therapy pipe far away from the bottle body; the oxygen return pipe is provided with a second control valve; a third control valve is arranged on the liquid discharge pipe; the bottle cap is provided with a sampling port and a material supplementing port.

Further, the bottle cap is connected with the bottle mouth in a threaded fit or clamping manner.

Furthermore, the bottle body, the exhaust pipe, the liquid discharge pipe, the oxygen return pipe, the main shaft and the spiral vent pipe are all made of glass materials; the bottle body, the exhaust pipe, the liquid discharge pipe, the oxygen backflow pipe, the main shaft and the spiral vent pipe are of an integrated structure.

Furthermore, the outer side wall of the bottle body is also provided with measuring range scale marks and a handle.

Furthermore, the turbulent flow rotating wheel is rotationally connected with the main shaft; the number of the turbulent flow rotating wheels is 3-5.

Further, a liquid inlet pipe is arranged at the top of the oxygen supply device; a lifting rod is arranged in the middle of the top of the oxygen supply device; one end of the lifting rod is fixedly connected with a catalyst container.

Furthermore, the other end of the lifting rod is movably connected with the limiting rod; a vertical plate is arranged beside the lifting rod; the top end of the vertical plate is provided with a circular sliding chute; one end of the limiting rod is provided with a circular limiting block; the circular limiting block is matched with the circular sliding groove.

Further, the outer side wall of the catalyst container is hollowed out; hydrogen peroxide is filled in the oxygen supply device; the catalyst contained in the catalyst container is manganese dioxide, copper oxide, copper sulfate, ferric oxide or ferric chloride.

The reaction equation of hydrogen peroxide and manganese dioxide is:

2H₂O₂＝MnO₂＝2H₂O+O₂↑

the invention has the following beneficial effects:

1. aiming at the problems that the oxygen supply is inconvenient and the rapid propagation and growth of cells are not facilitated in the existing cell culture process, the invention is provided with the oxygen supply device, the oxygen return pipe, the oxygen conveying pipe and the spiral vent pipe, wherein the oxygen supply device generates oxygen which is returned to the oxygen conveying pipe through the oxygen conveying pipe, the spiral vent pipe and the oxygen return pipe to form internal circulation of oxygen supply, and the oxygen is continuously contacted and dissolved with liquid in a cell culture bottle, so that the dissolved oxygen in the culture bottle is improved.

2. According to the invention, the turbulent flow rotating wheel and the spiral vent pipe are arranged, so that oxygen escapes from the spiral vent hole to drive the turbulent flow rotating wheel to rotate, the turbulent flow rotating wheel rotates to shake the cell culture solution, cell precipitation is prevented, and cells are always in a suspension state.

3. Aiming at the problem that the existing cell culture bottle is inconvenient to sample, supplement and transfer, the bottle cap is provided with the sampling port and the supplement port, and the outer side wall of the bottle body is provided with the liquid discharge pipe, so that the cell culture solution can be conveniently sampled, supplemented and transferred.

Of course, it is not necessary for any product in which the invention is practiced to achieve all of the above-described advantages at the same time.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a schematic structural diagram of a circular turbulent air-lift cell suspension culture flask according to the present invention;

FIG. 2 is another schematic structural view of the present invention;

FIG. 3 is a schematic structural view of a spiral vent hole and a turbulent flow runner according to the present invention;

FIG. 4 is a schematic diagram of the internal circulation of oxygen in a culture flask according to the present invention;

fig. 5 is a schematic structural view of an oxygen supply apparatus according to the present invention.

In the drawings, the components represented by the respective reference numerals are listed below:

1-bottle body, 2-bottle cap, 3-oxygen therapy tube, 4-oxygen reflux tube, 5-liquid discharge tube, 6-gas discharge tube, 7-handle, 8-main shaft, 9-spiral vent tube, 10-turbulent flow rotating wheel, 11-first vent hole, 12-oxygen supply device, 13-liquid inlet tube, 14-lifting rod, 15-catalyst container, 16-limiting rod, 17-vertical plate, 18-circular limiting block, 19-circular sliding groove, 20-first connecting hole, 101-bottle mouth, 201-sampling port, 202-material supplement port, 301-first control valve, 302-gas inlet, 401-second control valve and 501-third control valve.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the description of the present invention, it is to be understood that the terms "upper", "front", "side", "right below", "one end", "middle position", "outside", etc. indicate orientations or positional relationships only for convenience in describing the present invention and simplifying the description, but do not indicate or imply that the referenced components or elements must have a specific orientation, be constructed and operated in a specific orientation, and thus, should not be construed as limiting the present invention.

Referring to fig. 1-5, the present invention relates to a circular turbulent air-lift cell suspension culture bottle, which comprises a bottle body 1 and a bottle cap 2; the bottle body 1 is in a round table shape; the bottle mouth 101 of the bottle body 1 is matched with the bottle cap 2; the bottle cap 2 is connected with the bottle mouth 101 in a threaded fit or clamping manner; an oxygen return pipe 4, an exhaust pipe 6 and a liquid discharge pipe 5 are arranged at the upper part of the outer side surface of the bottle body 1; the oxygen return pipe 4 and the liquid discharge pipe 5 are symmetrically arranged around the central axis of the bottle body 1; the exhaust pipe 6 is arranged at the front end of the bottle body 1; the exhaust pipe 6 is in a gooseneck shape; the lower end of the bottle body 1 is provided with an oxygen catheter 3; the oxygen catheter 3 is S-shaped; the oxygen delivery pipe 3 and the oxygen reflux pipe 4 are positioned at the same side; the oxygen delivery pipe 3 is positioned right below the oxygen return pipe 4; the oxygen delivery pipe 3 is communicated with an oxygen return pipe 4; one end of the oxygen conveying pipe 3 is connected with the top of the oxygen supply device 12; the top of the oxygen supply device 12 is provided with a liquid inlet pipe 13; a lifting rod 14 is arranged at the middle position of the top of the oxygen supply device 12; one end of the lifting rod 14 is fixedly connected with a catalyst container 15; the other end of the lifting rod 14 is movably connected with a limiting rod 16; a vertical plate 17 is arranged beside the lifting rod 14; the top end of the vertical plate 17 is provided with a circular sliding chute 19; one end of the limiting rod 16 is provided with a circular limiting block 18; the round limiting block 18 is matched with the round chute 19; the outer side wall of the catalyst container 15 is hollowed; the oxygen supply device 12 is filled with hydrogen peroxide; the catalyst contained in the catalyst container 15 is manganese dioxide, copper oxide, copper sulfate, ferric oxide or ferric chloride; the other end of the oxygen therapy tube 3 extends into the bottle body 1; a main shaft 8 is fixedly arranged at the middle position of the bottom inside the bottle body 1; turbulent flow rotating wheels 10 are uniformly distributed on the main shaft 8; a spiral vent pipe 9 is arranged outside the turbulent flow rotating wheel 10; the pipe wall of the spiral vent pipe 9 is uniformly provided with first vent holes 11; the bottom end of the spiral vent pipe 9 is communicated with the oxygen therapy pipe 3; the top end of the spiral vent pipe 9 is communicated with the oxygen return pipe 4; the oxygen catheter 3 is provided with a first control valve 301; the first control valve 301 is arranged at one end of the oxygen therapy pipe 3 far away from the bottle body 1; the oxygen return pipe 4 is provided with a second control valve 401; a third control valve 501 is arranged on the liquid discharge pipe 5; the bottle cap 2 is provided with a sampling port 201 and a material supplementing port 202; the bottle body 1, the exhaust pipe 6, the liquid discharge pipe 5, the oxygen return pipe 4, the main shaft 8 and the spiral vent pipe 9 are all made of glass materials; the bottle body 1, the exhaust pipe 6, the liquid discharge pipe 5, the oxygen return pipe 4, the main shaft 8 and the spiral vent pipe 9 are of an integrated structure; the outer side wall of the bottle body 1 is also provided with measuring range scale marks and a handle 7.

One specific application of this embodiment is:

firstly, pouring culture solution and cells to be cultured into a culture bottle, and covering a bottle cap 2; the oxygen tube 3 is connected with an oxygen supply device 12; hydrogen peroxide solution is poured into the oxygen supply device 12, manganese dioxide is put into the catalyst container 15, and the manganese dioxide catalyzes hydrogen peroxide to release oxygen under the lifting rod 14;

opening the first control valve 301; closing the second control valve 401 and the third control valve 501; oxygen enters the bottle body 1 through the oxygen conveying pipe 3 and is fully contacted and dissolved with the culture through the spiral vent pipe 9; lifting the lifting rod 14, and placing the limiting rod 16 on the vertical plate 17; manganese dioxide and hydrogen peroxide with stopped catalysis; closing the first control valve 301; oxygen which is not dissolved in the bottle escapes from the culture solution, the second control valve 401 is opened, and the oxygen flows back to the oxygen conveying pipe 3 from the oxygen return pipe 4, so that internal circulation that the oxygen is dissolved by contacting with the cell culture solution is formed;

in addition, oxygen escapes from the first vent hole 11 in the spiral vent pipe 9 to drive the turbulent runner 10 to rotate, so that no extra energy is consumed; so that the cells are always in suspension.

In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A circulation turbulent flow airlift cell suspension culture bottle comprises a bottle body (1) and a bottle cap (2);

the method is characterized in that:

the bottle body (1) is in a round table shape; the bottle mouth (101) of the bottle body (1) is matched with the bottle cap (2); an oxygen return pipe (4), an exhaust pipe (6) and a liquid discharge pipe (5) are arranged at the upper part of the outer side surface of the bottle body (1);

the oxygen return pipe (4) and the liquid discharge pipe (5) are symmetrically arranged around the central axis of the bottle body (1); the exhaust pipe (6) is arranged at the front end of the bottle body (1); the exhaust pipe (6) is in a gooseneck shape; the lower end of the bottle body (1) is provided with an oxygen catheter (3);

the oxygen therapy pipe (3) is S-shaped; the oxygen delivery pipe (3) and the oxygen return pipe (4) are positioned on the same side; the oxygen delivery pipe (3) is positioned right below the oxygen return pipe (4); the oxygen delivery pipe (3) is communicated with the oxygen return pipe (4); one end of the oxygen conveying pipe (3) is connected with the top of the oxygen supply device (12); the other end of the oxygen therapy pipe (3) extends into the bottle body (1);

a main shaft (8) is fixedly arranged in the middle of the bottom inside the bottle body (1); turbulent flow rotating wheels (10) are uniformly distributed on the main shaft (8);

a spiral vent pipe (9) is arranged outside the turbulent flow rotating wheel (10); the pipe wall of the spiral vent pipe (9) is uniformly provided with first vent holes (11);

the bottom end of the spiral vent pipe (9) is communicated with the oxygen conveying pipe (3); the top end of the spiral vent pipe (9) is communicated with the oxygen return pipe (4);

a first control valve (301) is arranged on the oxygen conveying pipe (3); the first control valve (301) is arranged at one end of the oxygen therapy pipe (3) far away from the bottle body (1); a second control valve (401) is arranged on the oxygen return pipe (4); a third control valve (501) is arranged on the liquid discharge pipe (5); the bottle cap (2) is provided with a sampling port (201) and a material supplementing port (202).

2. A cell suspension culture flask according to claim 1, wherein the cap (2) is connected to the mouth (101) by means of a screw-fit or snap-fit.

3. A circulation turbulence air-lift cell suspension culture flask as claimed in claim 1 or 2, characterized in that the flask body (1), the exhaust pipe (6), the drain pipe (5), the oxygen return pipe (4), the main shaft (8) and the spiral vent pipe (9) are made of glass; the bottle body (1), the exhaust pipe (6), the liquid discharge pipe (5), the oxygen return pipe (4), the main shaft (8) and the spiral vent pipe (9) are of an integrated structure.

4. A cyclically disturbed airlift cell suspension culture flask according to claim 3, wherein the outer side wall of the flask body (1) is further provided with range scale lines and a handle (7).

5. A circularly turbulent air-lift cell suspension culture flask according to claim 1, 2 or 4, wherein the turbulent wheel (10) is rotatably connected to the main shaft (8); the number of the turbulent flow rotating wheels (10) is 3-5.

6. A circulating turbulent air-lift type cell suspension culture flask according to claim 1, wherein the top of the oxygen supply device (12) is provided with an inlet liquid pipe (13); a lifting rod (14) is arranged in the middle of the top of the oxygen supply device (12); one end of the lifting rod (14) is fixedly connected with a catalyst container (15).

7. A circularly disturbed airlift cell suspension culture flask according to claim 6, wherein the other end of the lifting rod (14) is movably connected to a limiting rod (16); a vertical plate (17) is arranged beside the lifting rod (14); the top end of the vertical plate (17) is provided with a circular sliding groove (19).

8. A circulating turbulent air-lift type cell suspension culture flask according to claim 7, wherein one end of the limiting rod (16) is provided with a circular limiting block (18); the round limiting block (18) is matched with the round sliding groove (19).

9. A circulating turbulent air-lift type cell suspension culture flask according to claim 6, wherein the outer side wall of the catalyst container (15) is hollowed out; the catalyst contained in the catalyst container (15) is manganese dioxide, copper oxide, copper sulfate, ferric oxide or ferric chloride.

10. A cyclically disturbed airlift cell suspension culture flask according to claim 6, wherein the oxygen supply means (12) is filled with hydrogen peroxide.