CN109554297A - A kind of cell dispels device and the cell secondary culture method based on it - Google Patents
A kind of cell dispels device and the cell secondary culture method based on it Download PDFInfo
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- CN109554297A CN109554297A CN201811522958.1A CN201811522958A CN109554297A CN 109554297 A CN109554297 A CN 109554297A CN 201811522958 A CN201811522958 A CN 201811522958A CN 109554297 A CN109554297 A CN 109554297A
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- pipe
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- 238000012136 culture method Methods 0.000 title claims abstract description 10
- 230000002572 peristaltic effect Effects 0.000 claims abstract description 39
- 239000006285 cell suspension Substances 0.000 claims abstract description 37
- 238000007789 sealing Methods 0.000 claims description 13
- 239000007799 cork Substances 0.000 claims description 12
- 241001411320 Eriogonum inflatum Species 0.000 claims description 7
- 239000006143 cell culture medium Substances 0.000 claims description 7
- 239000000463 material Substances 0.000 claims description 6
- 239000007921 spray Substances 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims 1
- 239000007924 injection Substances 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 5
- 238000004113 cell culture Methods 0.000 abstract description 4
- 239000007788 liquid Substances 0.000 description 7
- 238000010586 diagram Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 210000004262 dental pulp cavity Anatomy 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- -1 polyethylene Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
Classifications
-
- 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
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/58—Reaction vessels connected in series or in parallel
-
- 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
Abstract
The invention discloses a kind of cells to dispel device, including the first container, second container, third container, the first hose, the second hose, third hose, the 4th hose and the 5th hose, also discloses a kind of cell secondary culture method that device is dispelled based on cell.The utility model has the advantages that the technical program can guarantee that cell suspension is dispelled processing in a closed environment, reduces the possible pollution of manual operation, the application that biological products, pharmacy etc. are related to cell large-scale culture and dispel can be met.Each peristaltic pump can control the speed of cell suspension piping and druming and be efficiently completed the mixed uniformly task of cell suspension, and cell suspension is enable to be directly used in subsequent spinner culture and bioreactor large-scale culture.The present invention relates to cell culture apparatus and method.
Description
Technical field
The present invention relates to cell culture apparatus and method, in particular to a kind of cell is dispelled device and is passed based on its cell
For cultural method.
Background technique
In the prior art, in industrial massive cell succeeding generations, it may appear that after cell dissociation, cell dispersion is uneven
Even, cell agglomerate, cell is adherent to be unevenly distributed, and amplifies culture with cell so that vitro growth rates are inconsistent, aging falls off
The limited problem of technique.Commercialization large-scale culture cell there is no to dispel device at present.
Being disclosed technology with the most similar one kind of the application patent is: it is a kind of blow and beat cell device (application number:
201520763320.2).The device of the piping and druming cell of the utility model includes 2 Tissue Culture Flasks, agree with cell bottle 2
Bottle stopper and 2 inflating pumps being connect with bottle stopper, 2 bottle stoppers longitudinally through insertion 3 root canals, respectively inflate pump line, spray
Outlet pipe, return pipe；Wherein inflate pump line upper end is connected with inflating pump, and bleed pipe lower end connects a fine needle, return pipe lower end
It is connected with a silica gel hose；The bleed pipe upper end of one bottle stopper, return pipe upper end another bottle stopper with same group respectively
The upper end of return pipe, the upper end of bleed pipe are connected.By alternately opening the inflating pump of 2 bottle stoppers connection, repeated multiple times piping and druming is thin
Born of the same parents reduce cell mass.There are several defects for the device: 1. inflating pumps with inflate pump line and be connected, be not connected with air filter, meeting
It is easy to cause cell contamination, because inflating pump is only to fan the air in gas to 2 cell bottles, it cannot be guaranteed that the cleaning of air.2. dozen
Air pump is not specified oil-free or has oily inflating pump, even if there is oily inflating pump to use air filter, certain harmful substance particles
Or cell bottle can be entered by inflating pump line, certain injury is generated to cell.3. cell liquid is more, since bleed pipe is thin
The cell that syringe needle sprays directly is beaten inside liquid or on liquid level, and impact force weakens, and it is undesirable that cell breaks up effect；Cell liquid
When few, after cell liquid squeezes into another bottle from a bottle, archaeocyte bottle can remain a part of cell on bottom and bottle wall, cause
The reduction of cell number.
Some other patent such as cell tissue liquid blows and beats device (application number: 201510439544.2), a kind of automatic thin automatically
Born of the same parents' suspension blows and beats evenly mixing device and (application number: 201710307386.4) blows and beats cell using liquid-transfering gun, it is thin to be only applicable to laboratory
Born of the same parents' passage, is not suitable for use of large-scale production；A kind of sterile automatic piping and druming device of primary cell (application number:
201620533106.2) and a kind of cell culture apparatus (application number: 201720252959.3) with originally to apply for a patent structure complete
It is complete different.
Summary of the invention
Device is dispelled technical problem to be solved by the invention is to provide a kind of cell and based on its cell secondary culture
Method makes cell suspension that can complete to dispel processing in closed environment.
Used technical solution to solve above-mentioned technical problem:
A kind of cell dispels device, including the first container, second container, third container, the first hose, the second hose,
Three hoses, the 4th hose, the 5th hose and several peristaltic pumps；
The both ends of first hose are connected to third container and the first container respectively, and the both ends of the second hose hold with first respectively
Device is connected to second container, and tee tube, third hose, the 4th hose are equipped between third hose, the 4th hose and the 5th hose
It is connect with one end of the 5th hose with tee tube, the other end of third hose is connected to second container, the other end of the 4th hose
It is connected to second container, the other end and third reservoir of the 5th hose；
The first container, second container, third container, the first hose, the second hose, third hose, the 4th hose and the 5th
Hose is linked to be closed space, is respectively equipped with air filter on the first container, second container and third container, the first container,
Second container and third container pass through air filter respectively and atmosphere keeps air pressure balance；
Peristaltic pump removably cooperates with the first hose, the second hose, the 4th hose or the 5th hose；
Second hose, which is extend into the first container, forms the first return pipe, and third hose, which is extend into second container, forms the
Two return pipes, the first hose, which is extend into third container, forms third return pipe, and the 4th hose extend into second container and formed
Bleed pipe, the inner wall of the nozzle of bleed pipe towards second container.
As an improvement, peristaltic pump include cooperate with the first peristaltic pump of the first hose cooperation, with the second hose it is second compacted
Dynamic pump, the 4th peristaltic pump with the third peristaltic pump of the 4th hose cooperation and with the cooperation of the 5th hose.
As an improvement, the first container, second container and third container be Tissue Culture Flask, the first container, second container and
The oral area of third container is equipped with sealing bottle cork.
It is the first hose, second soft as an improvement, sealing bottle cork is equipped with several stainless pipes through sealing bottle cork
Pipe, third hose, the 4th hose and the 5th hose are extend into each Tissue Culture Flask by stainless pipe, and bleed pipe is hard
One end of material, bleed pipe is connected to stainless pipe, and the other end of bleed pipe is equipped with conical nozzle, the outlet of bleed pipe
It is located at the tip of conical nozzle.
As an improvement, the nozzle of the first return pipe, the second return pipe and third return pipe is respectively protruding into the first container,
The bottom of two containers and third container.
As an improvement, the length of the first return pipe is not less than the height of the first container, the length of the second return pipe is not less than
The height of second container, the length of third return pipe are not less than the height of third container；
The orifice configuration of first return pipe, the second return pipe and third return pipe respectively with the first container, second container and
The bottom surface of third container intracavity is bonded, to reduce the gap between each nozzle and the bottom surface of each container intracavity.
The cell secondary culture method of device is dispelled based on cell, comprising the following steps:
Postdigestive cell suspension is added in step 1 in the first container, is made in the first container using the second peristaltic pump
Cell suspension is entered in second container by the first return pipe and the second hose；
Step 2 clamps the 5th hose using clamping tool, makes the path blockade in the 5th hose；
Step 3 makes the cell suspension in second container pass through the second return pipe, third hose, the using third peristaltic pump
Four hoses and ejection pipe inspection dispel on the inner wall of second container；
Step 4, repeats step 3, and the cell suspension after overspray dispels flows back into second container, continues to follow
Ring, until the cell in cell suspension is fully dispersed uniformly；
Clamping tool in step 2 is unclamped, clamps the 4th hose using clamping tool, make in the 5th hose by step 5
Access conducting, and make the path blockade in the 4th hose；
Step 6 makes the cell suspension in second container pass through the second return pipe, third hose, the using the 4th peristaltic pump
Five hoses enter in third container.
As an improvement, cell culture medium is added into third container before carrying out step 1.
As an improvement, keeping the cell culture medium in third container soft by third return pipe and first using the first peristaltic pump
Pipe enters in the first container, repeats step 1 to step 6.
As an improvement, cell suspension is added into the first container after completing step 6, step 1 is repeated to step
Six.
The utility model has the advantages that the technical program can guarantee that cell suspension is dispelled processing in a closed environment, reduce
The possible pollution of manual operation can meet the application that biological products, pharmacy etc. are related to cell large-scale culture and dispel.Respectively
A peristaltic pump can control the speed of cell suspension piping and druming and be efficiently completed the mixed uniformly task of cell suspension, make thin
Born of the same parents' suspension can be directly used in subsequent spinner culture and bioreactor large-scale culture.
Detailed description of the invention
Following further describes the present invention with reference to the drawings:
Fig. 1 is the structural schematic diagram of the embodiment of the present invention；
Fig. 2 is the structural schematic diagram of the bleed pipe of the embodiment of the present invention；
Fig. 3 is the structural schematic diagram of the nozzle of the first return pipe of the embodiment of the present invention.
Specific embodiment
Referring to figs. 1 to 3, a kind of cell dispels device, including the first container 1, second container 2, third container 3, first are soft
Pipe 4, the second hose 5, third hose 6, the 4th hose 7, the 5th hose 8 and several peristaltic pumps.
The first container 1, second container 2 and third container 3 of the present embodiment are Tissue Culture Flask, and the first container 1, second is held
The oral area of device 2 and third container 3 is equipped with sealing bottle cork.In order to improve the sealing of the first container 1, second container 2 and third container 3
Property, mutually matched screw thread is equipped between sealing bottle cork and the bottleneck of each Tissue Culture Flask, sealing bottle cork can be tightened on cell
In culture bottle.
The first container 1 of the present embodiment is the Tissue Culture Flask of capacity 1L, is used for cell dissociation.Second container 2 is capacity
The Tissue Culture Flask of 10L, for dispelling cell.Third container 3 is the Tissue Culture Flask of capacity 10L, for cultivating cell.
The both ends of first hose 4 are connected to third container 3 and the first container 1 respectively, and the both ends of the second hose 5 are respectively with
One container 1 is connected to second container 2, and tee tube 9, the present embodiment are equipped between third hose 6, the 4th hose 7 and the 5th hose 8
9 material of tee tube be polyethylene, one end of third hose 6, the 4th hose 7 and the 5th hose 8 connect with tee tube 9, third
The other end of hose 6 is connected to second container 2, and the other end of the 4th hose 7 is connected to second container 2, the 5th hose 8 it is another
End is connected to third container 3.
The first container 1, second container 2, third container 3, the first hose 4, the second hose 5, third hose 6, the 4th hose 7
It is linked to be closed space with the 5th hose 8, is respectively equipped with air filter on the first container 1, second container 2 and third container 3
10, the first container 1, second container 2 and third container 3 keep air pressure balance by air filter 10 and atmosphere respectively.This reality
The filter pore size for applying the air filter 10 of example is 0.22 micron.
First hose 4 is equipped with the first peristaltic pump 11, and the second hose 5 is equipped with the second peristaltic pump 12, sets on the 4th hose 7
There is third peristaltic pump 13, the 5th hose 8 is equipped with the 4th peristaltic pump 14.Existing peristaltic pump generally consists of three parts: driving
Device, pump head and hose.The characteristics of peristaltic pump is: fluid is isolated in pump line, Ke Kuaisugenghuan pump line, fluid can drive in the wrong direction, can
With dry operating and maintenance cost it is low.
Second hose 5, which is extend into the first container 1, forms the first return pipe 15, and third hose 6 is extend into second container 2
Form the second return pipe 16, the first hose 4, which is extend into third container 3, forms third return pipe 17, and the 4th hose 7 extend into the
Form bleed pipe 18 in two containers 2, the inner wall of the nozzle of bleed pipe 18 towards second container 2.
The sealing bottle cork of the present embodiment is equipped with several stainless pipes 19 through sealing bottle cork, the first hose 4, second
Hose 5, third hose 6, the 4th hose 7 and the 5th hose 8 are extend into each Tissue Culture Flask by stainless pipe 19.This
The lower end of the stainless pipe 19 of embodiment passes through sealing bottle cork and extend into Tissue Culture Flask from the bottom surface of sealing bottle cork, stretches
Entering length is 3cm.The internal diameter of stainless pipe 19 is 4mm, and outer diameter 5mm, material is 316 stainless steels.
In order to promote the effect that cell dispels, bleed pipe 18 is hard material, and 18 material of bleed pipe of the present embodiment is poly-
One end of ethylene, bleed pipe 18 is connected to stainless pipe 19, as shown in Fig. 2, the shape of bleed pipe 18 is " L " font, is sprayed
The other end of pipe 18 is equipped with conical nozzle 20, and the outlet of bleed pipe 18 is located at the tip of conical nozzle 20, bleed pipe 18
The bore of outlet is not more than 1mm, and the distance between the inner wall of the outlet of bleed pipe 18 apart from second container 2 is not more than 3cm, sprays
The orifice configuration of pipe 18 makes cell suspension accelerate to spray, and is conducive to improve the effect that cell dispels.
In order to reduce cell residue, the nozzle of the first return pipe 15, the second return pipe 16 and third return pipe 17 is stretched respectively
Enter to the bottom of the first container 1, second container 2 and third container 3.The length of first return pipe 15 is not less than the first container 1
Highly, the length of the second return pipe 16 is not less than the height of second container 2, and the length of third return pipe 17 is not less than third container
3 height.The orifice configuration of first return pipe 15, the second return pipe 16 and third return pipe 17 respectively with the first container 1, second
The bottom surface of 3 inner cavity of container 2 and third container is bonded, to reduce the gap between each nozzle and the bottom surface of each container intracavity.
The tip of first return pipe 15 of the present embodiment, the second return pipe 16 and third return pipe 17 is trimmed to 45 ° shown in Fig. 3, has
Conducive to reduction cell residue.The side of the nozzle of first return pipe 15 of the present embodiment, the second return pipe 16 and third return pipe 17
Edge or tip are equipped with notch 21 as shown in Figure 3, are bonded nozzle not exclusively with the bottom surface of the inner cavity of container, can be each in reduction
While gap between nozzle and the bottom surface of each container intracavity, make between nozzle and the bottom surface of inner cavity that there are tiny always
Gap is conducive to draw residual cell as far as possible.Notch 21 as shown in Figure 3 can avoid the inner cavity that nozzle is completely close to container
Bottom surface can prevent the problem of liquid is inhaled not up.
The cell secondary culture method of device is dispelled based on a kind of cell of right, comprising the following steps:
Postdigestive cell suspension is added in step 1 in the first container 1, makes the first container 1 using the second peristaltic pump 12
In cell suspension entered in second container 2 by the first return pipe 15 and the second hose 5.
Step 2 clamps the 5th hose 8 using clamping tool, makes the path blockade in the 5th hose 8.
Step 3 keeps the cell suspension in second container 2 soft by the second return pipe 16, third using third peristaltic pump 13
Pipe 6, the 4th hose 7 and bleed pipe 18 spray on the inner wall for dispelling second container 2.
Step 4, repeats step 3, and the cell suspension after overspray dispels flows back into second container 2, continues
Circulation, until the cell in cell suspension is fully dispersed uniformly.Step 4 is conducive to dispel cell suspension sufficiently, makes cell
Suspension uniformly mixes, and can also reduce the cell residue in bottle wall.
Clamping tool in step 2 is unclamped, clamps the 4th hose 7 using clamping tool, make the 5th hose 8 by step 5
Interior access conducting, and make the path blockade in the 4th hose 7.
Step 6 keeps the cell suspension in second container 2 soft by the second return pipe 16, third using the 4th peristaltic pump 14
Pipe 6, the 5th hose 8 enter in third container 3.
The technical program can guarantee that cell suspension is dispelled processing in a closed environment, and reducing manual operation can
Energy bring pollution, can meet the application that biological products, pharmacy etc. are related to cell large-scale culture and dispel.Each peristaltic pump energy
It enough controls the speed of cell suspension piping and druming and is efficiently completed the mixed uniformly task of cell suspension, keep cell suspension straight
It connects for subsequent spinner culture and bioreactor large-scale culture.
The quantity of the peristaltic pump of the present embodiment be four, but in order to simplify configuration, the technical program also can be used one it is compacted
Dynamic pump is realized.Peristaltic pump removably cooperates with each hose, does not need to open four peristaltic pumps simultaneously in the technical program, because
This can equally realize the technical program by needing successively to cooperate with each hose peristaltic pump by operation.
In order to make the cell suspension in 3 container of third container be suitable for it is subsequent be directly used in cell culture, carry out step 1
Before, the cell culture medium of 1L~10L is added into third container 3.
In order to efficiently use residual cell, make the cell culture medium in third container 3 by the using the first peristaltic pump 11
Three return pipes 17 and the first hose 4 enter in the first container 1, repeat and execute step 1 to step 6.The above operation can make
Cell culture medium again flows through the first container 1, and the residual cell in the first container 1 can be driven to shift, and it is thin to may advantageously facilitate residual
The effective use of born of the same parents.
It, can be repeatedly into the first container 1 after completing step 6 in order to realize that multiple cell dispels in confined space
Cell suspension is added, repeats step 1 to step 6 progress cell and dispels.
Embodiments of the present invention are explained in detail above in conjunction with attached drawing, but the present invention is not limited to above-mentioned embodiment party
Formula, technical field those of ordinary skill within the scope of knowledge, can also be before not departing from present inventive concept
Put that various changes can be made.
Claims (10)
1. a kind of cell dispels device, it is characterised in that: including the first container (1), second container (2), third container (3),
One hose (4), the second hose (5), third hose (6), the 4th hose (7), the 5th hose (8) and several peristaltic pumps；
The both ends of first hose (4) are connected to third container (3) and the first container (1) respectively, the both ends difference of the second hose (5)
It is connected to the first container (1) and second container (2), three is equipped between third hose (6), the 4th hose (7) and the 5th hose (8)
One end of siphunculus (9), third hose (6), the 4th hose (7) and the 5th hose (8) is connect with the tee tube (9), and third is soft
The other end of pipe (6) is connected to second container (2), and the other end of the 4th hose (7) is connected to second container (2), the 5th hose
(8) the other end is connected to third container (3)；
The first container (1), second container (2), third container (3), the first hose (4), the second hose (5), third hose (6),
4th hose (7) and the 5th hose (8) are linked to be closed space, on the first container (1), second container (2) and third container (3)
It is respectively equipped with air filter (10), the first container (1), second container (2) and third container (3) pass through air filter respectively
(10) air pressure balance is kept with atmosphere；
Peristaltic pump removably cooperates with the first hose (4), the second hose (5), the 4th hose (7) or the 5th hose (8)；
Second hose (5) extend into the first container (1) and forms the first return pipe (15), and third hose (6) extend into the second appearance
The second return pipe (16) are formed in device (2), the first hose (4) extend into third container (3) and forms third return pipe (17), the
Four hoses (7) extend into second container (2) and form bleed pipe (18), and the nozzle of bleed pipe (18) is towards second container (2)
Inner wall.
2. a kind of cell according to claim 1 dispels device, it is characterised in that: the peristaltic pump includes and described first
The first peristaltic pump (11) of hose (4) cooperation, the second peristaltic pump (12) and the described 4th with second hose (5) cooperation
The third peristaltic pump (13) of hose (7) cooperation and the 4th peristaltic pump (14) cooperated with the 5th hose (8).
3. a kind of cell according to claim 2 dispels device, it is characterised in that: the first container (1), second container
(2) and third container (3) is Tissue Culture Flask, and the oral area of the first container (1), second container (2) and third container (3) is equipped with close
Seal bottle stopper.
4. a kind of cell according to claim 3 dispels device, it is characterised in that: the sealing bottle cork is equipped with several pass through
Wear the stainless pipe (19) of sealing bottle cork, first hose (4), second hose (5), the third hose (6), institute
It states the 4th hose (7) and the 5th hose (8) to extend into each Tissue Culture Flask by stainless pipe (19), the spray
Outlet pipe (18) is hard material, and one end of bleed pipe (18) is connected to the stainless pipe (19), bleed pipe (18) it is another
End is equipped with conical nozzle (20), and the outlet of bleed pipe (18) is located at the tip of conical nozzle (20).
5. a kind of cell according to claim 4 dispels device, it is characterised in that: first return pipe (15), described
The nozzle of second return pipe (16) and the third return pipe (17) is respectively protruding into the first container (1), second appearance
The bottom of device (2) and the third container (3).
6. a kind of cell according to claim 5 dispels device, it is characterised in that: the length of first return pipe (15)
Not less than the height of the first container (1), the length of second return pipe (16) is not less than the height of the second container (2)
Degree, the length of the third return pipe (17) are not less than the height of the third container (3)；
The orifice configuration of first return pipe (15), the second return pipe (16) and third return pipe (17) respectively with the first container (1),
The bottom surface of second container (2) and third container (3) inner cavity fitting, to reduce between each nozzle and the bottom surface of each container intracavity
Gap.
7. dispelling the cell secondary culture method of device, feature based on a kind of cell described in claim 2 to 6 any one
Be the following steps are included:
Postdigestive cell suspension is added in the first container (1) for step 1, makes the using second peristaltic pump (12)
Cell suspension in one container (1), which enters described second by first return pipe (15) and second hose (5), to be held
In device (2)；
Step 2 clamps the 5th hose (8) using clamping tool, makes the path blockade in the 5th hose (8)；
Step 3 makes the cell suspension in second container (2) pass through second return pipe using the third peristaltic pump (13)
(16), the third hose (6), the 4th hose (7) and the bleed pipe (18) injection dispel the interior of second container (2)
On wall；
Step 4, repeats step 3, and the cell suspension after overspray dispels flows back into second container (2), continues to follow
Ring, until the cell in cell suspension is fully dispersed uniformly；
Clamping tool in step 2 is unclamped, clamps the 4th hose (7) using clamping tool, make the 5th hose (8) by step 5
Interior access conducting, and make the path blockade in the 4th hose (7)；
Step 6, using the 4th peristaltic pump (14) make the cell suspension in second container (2) by the second return pipe (16),
Third hose (6), the 5th hose (8) enter in the third container (3).
8. cell secondary culture method according to claim 7, it is characterised in that: before carrying out the step 1, to
Cell culture medium is added in the third container (3).
9. cell secondary culture method according to claim 8, it is characterised in that: made using first peristaltic pump (11)
Cell culture medium in the third container (3) enters institute by the third return pipe (17) and first hose (4)
It states in the first container (1), repeats the step 1 to the step 6.
10. cell secondary culture method according to claim 7, it is characterised in that: after completing the step 6, to
Cell suspension is added in the first container (1), repeats the step 1 to the step 6.
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