CN110331081A - Cell eluant container, cell collect purging method and system - Google Patents
Cell eluant container, cell collect purging method and system Download PDFInfo
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- CN110331081A CN110331081A CN201910493279.4A CN201910493279A CN110331081A CN 110331081 A CN110331081 A CN 110331081A CN 201910493279 A CN201910493279 A CN 201910493279A CN 110331081 A CN110331081 A CN 110331081A
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- 238000000034 method Methods 0.000 title claims abstract description 18
- 238000010926 purge Methods 0.000 title claims abstract description 9
- 239000007788 liquid Substances 0.000 claims abstract description 80
- 239000012528 membrane Substances 0.000 claims abstract description 33
- 239000002504 physiological saline solution Substances 0.000 claims abstract description 30
- 239000006285 cell suspension Substances 0.000 claims abstract description 17
- 238000004321 preservation Methods 0.000 claims abstract description 14
- 238000001816 cooling Methods 0.000 claims abstract description 8
- 239000002699 waste material Substances 0.000 claims description 22
- 239000007787 solid Substances 0.000 claims description 17
- 239000000463 material Substances 0.000 claims description 9
- 239000004695 Polyether sulfone Substances 0.000 claims description 8
- 229920006393 polyether sulfone Polymers 0.000 claims description 8
- 230000015572 biosynthetic process Effects 0.000 claims description 5
- 238000011002 quantification Methods 0.000 claims description 5
- 238000006073 displacement reaction Methods 0.000 claims description 4
- 150000003839 salts Chemical class 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000009736 wetting Methods 0.000 claims description 3
- 230000035479 physiological effects, processes and functions Effects 0.000 claims description 2
- 238000004080 punching Methods 0.000 claims 1
- 239000000725 suspension Substances 0.000 claims 1
- 238000011010 flushing procedure Methods 0.000 abstract description 8
- 230000005779 cell damage Effects 0.000 abstract description 6
- 238000001914 filtration Methods 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 241000204031 Mycoplasma Species 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- 230000003115 biocidal effect Effects 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 4
- 241001430197 Mollicutes Species 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 238000009825 accumulation Methods 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000004062 sedimentation Methods 0.000 description 2
- 239000004743 Polypropylene Substances 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 230000001268 conjugating effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- -1 polypropylene Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
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
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/02—Membranes; Filters
-
- 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
- C12M33/14—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus with filters, sieves or membranes
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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
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/40—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pressure
-
- 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
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/04—Cell isolation or sorting
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
Abstract
The invention discloses cell eluant container, cells to collect purging method and system, and method includes pretreatment；It is equipped with filter membrane in cell eluant container, its inner cavity is divided into epicoele and cavity of resorption, epicoele is made to form negative pressure for negative pressure device so that cell suspension to be rinsed is in upper intracavitary；Upper intracavity liquid is filtered under intracavitary；Physiological saline is added to epicoele, cell is resuspended；After repeated multiple times after liquid complies with standard, simultaneously cell is resuspended in the upward intracavitary cell-preservation liquid for being quantitatively adding pre-cooling, is completed cell and is rinsed.Cell collects rinse-system, including cell eluant container, physiological saline container, cell suspension container, air filter, the first negative pressure device and the second negative pressure device.The beneficial effects of the present invention are: being replaced cell suspension to complete cell enrichment and flushing by way of vacuum suction combined filtering film, it is fast not only to rinse speed, and it is higher while smaller to cellular damage compared to enrichment and flush efficiency for the mode of conventional centrifugal and natural subsidence.
Description
Technical field
The present invention relates to technical field of cell culture, collect purging method in particular to a kind of cell eluant container, cell
And system.
Background technique
With the development of science and technology cell therapy gradually to clinical development, is also pushing cell culture and process to handle skill
The development of art.Cell is after large-scale culture, it is necessary to be enriched with, and by culture solution, the ingredients such as nutrient solution are rinsed only, together
When to keep its activity.Existing cell purging method generallys use the mode of centrifugation and natural subsidence, although centrifugation is imitated
Rate is high but very big to cellular damage, although and natural subsidence damaging cells are small, therefore, how efficiently flush efficiency is very low,
The problem of be enriched with and cleaned to cell and be preferably minimized to cell damage, be urgent need to resolve.
Summary of the invention
It is an object of the invention to overcome cell enrichment existing for existing method and device and flush efficiency low, cell is hurt
The big problem of evil, provides a kind of cell eluant container, including a tank body, and the tank body is interior equipped with above and below being divided into its inner cavity two
The filter of a chamber；The tank body is circumscribed with liquid input circuit, negative pressure access pipeline, waste collection pipeline；
The filter be it is bowl-shape, bottom of bowl is the solid disk of non-porous structure, is disposed with filter membrane on bowl side wall；
The liquid input circuit is in the intracorporal end of tank close to the solid luminaire layout along circle；The negative pressure access pipeline connects
Enter end and be located at top of the tank, the incoming end of the waste collection pipeline is located at tank base.
Further, the liquid input circuit is less than 1CM in the vertical range of the intracorporal end of tank and the solid disk.
Further, the filter membrane is made of polyether sulfone materials, aperture 300nm.
The invention also provides a kind of cells to collect rinse-system, including cell eluant container, physiological saline container, cell
Suspension container, air filter, reversal valve, the first negative pressure device and the second negative pressure device；
Its inner cavity is divided into upper and lower two chambers by the cell eluant container, including a tank body, interior be equipped with of the tank body
Filter；The tank body is circumscribed with liquid input circuit, negative pressure access pipeline, waste collection pipeline；The filter be it is bowl-shape,
Its bottom of bowl is the solid disk of non-porous structure, is disposed with filter membrane on bowl side wall；The liquid input circuit is at the intracorporal end of tank
Portion is close to the solid luminaire layout along circle；The negative pressure access pipeline incoming end is located at top of the tank, the waste collection pipeline
Incoming end is located at tank base；
The physiological saline container, cell suspension container, air filter are connected with reversal valve respectively, and by reversal valve and
Liquid input circuit connects the cell eluant container；
The negative pressure access pipeline is connected with the first negative pressure device, the waste collection pipeline and the second negative pressure device phase
Even.
Further, the filter membrane is made of polyether sulfone materials, and aperture is 200~400nm.
It further, further include cell-preservation liquid container, efflux collection vessel, the first valve and the second valve；It is described thin
Born of the same parents save liquid container and are connected to by reversal valve with liquid input circuit；The efflux collection vessel and waste collection pipeline or the
Two negative pressure devices or cavity of resorption connection；First valve is arranged on negative pressure access tube road；Second valve is arranged in waste liquid
In collecting.
The present invention also proposes that a kind of HepG2 cell collects purging method, includes the following steps:
S1, the pre- physiological saline for being cooled to 0~4 DEG C is added into cell eluant container by pipeline, is arranged with wetting thin
Its cavity is divided into the filter membrane of epicoele and cavity of resorption in born of the same parents' eluant container, and pipeline and cell eluant container is pre-chilled, realizes pre- place
Reason；
S2, cell suspension to be rinsed is added to the epicoele quantification for completing the pretreated cell eluant container of step S1,
And the upper intracavitary negative pressure for forming 100~300mmHg is made so that cell suspension to be rinsed is in upper intracavitary by negative pressure device；
S3, epicoele negative pressure is released, and under being made by negative pressure device the intracavitary negative pressure for forming 100~300mmHg so that on
Intracavity liquid is filtered with the flow velocity of 0.5~3L/min under intracavitary；Meanwhile with the flow velocity of 0.5~3L/min into epicoele 1.3
The pre- physiological saline for being cooled to 0~4 DEG C is added, liquid in epicoele 1.3 is made to keep dynamic equilibrium and continue 1~5min；
S4, stop being added physiological saline into epicoele 1.3 to release the dynamic equilibrium of liquid in epicoele 1.3, until epicoele
After liquid is filtered to cavity of resorption 1.4 in 1.3, cavity of resorption negative pressure is released, it is intracavitary upwards to be quantitatively adding the pre- life for being cooled to 0~4 DEG C
Manage salt water, and make the negative pressure of 100~300mmHg of upper intracavitary formation by negative pressure device, make physiological saline with 0.5~
The flow velocity of 3L/min breaks up epicoele inner cell and cell is resuspended；
S5, step S3 and S4 are repeated, it is intracavitary upwards to be quantitatively adding after being filtered under intracavitary liquid and being complied with standard
It is cooled to 0~4 DEG C of cell-preservation liquid in advance and cell is resuspended, completes cell and rinse.
Further, in the step S4, the physiological saline of pre-cooling is added into upper intracavitary in such a way that varied angle or displacement are set.
The beneficial effects of the present invention are: being replaced cell suspension with complete by way of vacuum suction combined filtering film
At cell enrichment and flushing, it is fast not only to rinse speed, but also be enriched with and rush compared to for the mode of conventional centrifugal and natural subsidence
It washes more efficient while smaller to cellular damage.
Detailed description of the invention
Fig. 1 is the structural schematic diagram that cell of the present invention collects rinse-system.
Fig. 2 is the schematic cross-sectional view of cell eluant container of the present invention.
Fig. 3 is the schematic perspective view of tank body in Fig. 2.
Fig. 4 is the schematic perspective view of filter in Fig. 2.
Fig. 5 is the schematic perspective view of partial cutaway in Fig. 4.
In figure, 1. cell eluant containers；1.1. tank body；1.2. lid；1.3. epicoele；1.4. cavity of resorption；2. the physiology salt water capacity
Device；3. cell suspension container；4. air filter；5. the first negative pressure device；6. the second negative pressure device；7. filter；7.1. solid
Disk；7.2. filter membrane；7.3. side bracket；8. cell-preservation liquid container；10. efflux collection vessel；11. the first valve；
12. the second valve；13. reversal valve；14. negative pressure accesses pipeline；15. waste collection pipeline；16. liquid input circuit.
Specific embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.
Cell eluant container as shown in Figure 2-5, including a tank body are additionally provided with lid 1.2 on tank body 1.1；Tank body
The filter 7 that its inner cavity is divided into two chambers (including epicoele 1.3 and cavity of resorption 1.4) up and down is equipped in 1.1；Tank body 1.1 is circumscribed with
Liquid input circuit 16, negative pressure access pipeline 14, waste collection pipeline 15.
Filter 7 is preferably bowl-shape, and bottom of bowl is the solid disk 7.1 of non-porous structure, is disposed with filter membrane on bowl side wall
7.2；Filter membrane 7.2 is made of polyether sulfone materials, aperture 300nm.Filtering is used to support it is highly preferred that being additionally provided on bowl side wall
The side bracket 7.3 of film 7.2.
End of the liquid input circuit 16 in tank body 1.1 is arranged close to solid disk 7.1；Negative pressure accesses pipeline 14 and accesses
End is located at 1.1 top of tank body, and the incoming end of waste collection pipeline 15 is located at 1.1 bottom of tank body.Negative pressure accesses pipeline 14 and liquid
Intake line 16 is connected to epicoele 1.3, and waste collection pipeline 15 is connected to cavity of resorption 1.4.Liquid input circuit 16 is in tank body 1.1
End and solid disk 7.1 vertical range be less than 1CM.
Tank body 1.1 and lid 1.2 are preferably threadedly coupled, and keep lid 1.2 rotatable, thus the position of liquid input circuit 16
It sets variable, can effectively dispel cell mass.Cell is preferably HepG2 cell.
The cell as shown in Fig. 1~5 collects rinse-system, including cell eluant container 1, physiological saline container 2, cell hang
Liquid container 3, air filter 4, the first negative pressure device 5 and the second negative pressure device 6.
Cell eluant container 1, including a tank body 1.1, tank body 1.1 is interior to be equipped with the mistake that its inner cavity is divided into upper and lower two chambers
Filter；Tank body 1.1 is circumscribed with liquid input circuit 16, negative pressure access pipeline 14, waste collection pipeline 15；Filter be it is bowl-shape,
Its bottom of bowl is the solid disk 7.1 of non-porous structure, is disposed with filter membrane 7.2 on bowl side wall；Liquid input circuit 16 is in tank body 1.1
Interior end is arranged close to solid disk 7.1；Negative pressure access 14 incoming end of pipeline is located at 1.1 top of tank body, waste collection pipeline
15 incoming end is located at 1.1 bottom of tank body；
Physiological saline container 2, cell suspension container 3, air filter 4 are connected with reversal valve 13 respectively, and pass through reversal valve
13 and liquid input circuit 16 connect cell eluant container 1；
Negative pressure access pipeline 14 is connected with the first negative pressure device 5, and waste collection pipeline 15 is connected with the second negative pressure device 6.
It further includes cell-preservation liquid container 8, efflux collection vessel 10, the first valve 11 and that cell, which collects rinse-system,
Two valves 12；Cell-preservation liquid container 8 is connected to by reversal valve 13 with liquid input circuit 16；Efflux collection vessel 10 and useless
Liquid collecting 15 or the connection of the second negative pressure device 6 or cavity of resorption 1.4；The setting of first valve 11 is on negative pressure access pipeline 14；The
Two valves 12 are arranged on waste collection pipeline 15.
Cell collect rinse-system can also include that cell saves container, cell saves container and the first negative pressure device 5 or
Negative pressure accesses pipeline 14 and is connected to.Cell saves container and is used to store the cell (including cell-preservation liquid) for rinsing and completing.
First negative pressure device 5 or the second negative pressure device 6 are negative pressure vacuum pump, if setting structure or the negative-pressure vacuum of selection pump
Model is reasonable, and the first negative pressure device 5 and the second negative pressure device 6 can share the same negative-pressure vacuum pump, and certainly, this is also required to phase
Answer the cooperation of valve and pipeline；First valve 11 or the second valve 12 are preferably solenoid valve, and equally, reversal valve 13 is preferably also electricity
Magnet valve.
The physiological saline of pre-cooling is stored in physiological saline container 2；It is outstanding that cell to be rinsed is stored in cell suspension container 3
Supernatant liquid；Cell-preservation liquid is stored in cell-preservation liquid container 8.Efflux collection vessel 10 is flowed out for collecting from cavity of resorption 1.4
Liquid.
Cell eluant container 1, physiological saline container 2, cell suspension container 3, cell-preservation liquid container 8, cell, which save, to be held
The containers such as device, efflux collection vessel 10 can be tank container, box-shaped container, tubular container of hard etc., be also possible to soft
The bag-like container or cystic container of matter.
As shown in Figure 2,3, cell eluant container 1 selects tank container in the present embodiment comprising tank body 1.1 and lid
1.2；One end of negative pressure access pipeline 14 and liquid input circuit 16 passes through lid 1.2 and seals fixation with it.Lid 1.2 can be excellent
It is selected as transparence, in order to meet different demands, 1.1 size of tank body can be there are many different size of size.
In order to improve flush efficiency, the connection type use of lid 1.2 and tank body 1.1 is threadedly coupled, and reach is answered
Meet lid 1.2 can with respect to tank body 1.1 revolve turn around it is above, due to negative pressure access pipeline 14 and liquid input circuit 16 one end
In lid 1.2 to tank body 1.1, therefore, when lid 1.2 rotates, negative pressure accesses pipeline 14 and liquid input circuit 16
One end can change position, circumferentially rotate relative to tank body 1.1 along it, so that the physiological saline of pre-cooling be made to add in a manner of conjugating and set
Enter in epicoele 1.3, and effectively dispels cell mass under the action of negative pressure.Certainly, this to circumferentially rotate so that negative pressure access tube
Road 14 and liquid input circuit 16 are distorted to a certain extent, and therefore, the circle number of rotation is not easy too long, preferably rotation one
It encloses to two circles, and simultaneously non-standing rotates, for example, rotating clockwise a circle first, the rotation of another mistake hour hands is turned around.
In order to further increase flush efficiency and reduce cellular damage, liquid input circuit 16 is located at one in tank body 1.1
Changeable angle is held, so that the physiological saline for adjusting pre-cooling breaks up the incidence angle of cell.The mode for changing its angle can be
Increase angular transducer on liquid input circuit 16, and below lid 1.2 setting connect with liquid input circuit 16 it is electronic
Push rod pushes 16 end of liquid input circuit by electric pushrod so that the change of its inclination angle, measures its angle by angular transducer
Degree is to realize the regulation to its inclination angle.
Filter membrane 7.2 is made of polyether sulfone materials, and aperture is 200~400nm.Filter membrane 7.2 includes at least one inclination
Face or convex surface.That is the filter membrane 7.2 of the present embodiment can be the film of an out-of-flatness, because the membrane area of smooth film is smaller, and
Cell accumulation is easily caused, physiological saline flowing is also not easy to and realizes therefore the flushing to cell filter membrane 7.2 is arranged extremely
A few inclined surface or convex surface, can effectively promote membrane area, flow convenient for liquid, to improve flush efficiency.It preferably will be more
Piece filter membrane 7.2 is fixed to small top and big bottom shaped hollow round table or net bag-shaped or cup-shaped or funnel-form by the connection of bracket 17
Or bowl-shape filter；Bracket 17 is made of polypropylene material.
The shape of filter can be as shown in Figure 4,5.The inclined surface or convex surface of filter membrane 7.2 can be small top and big bottom
Shaped hollow round table or net bag-shaped or cup-shaped or funnel-form or bowl-shape filter side, filter floor be non-filtered face (i.e.
The solid disk 7.1 of cell eluant container).
In order to verify flushing effect, applicant chooses more parts of volumes and the identical HepG2 cell to be rinsed of sample data is outstanding
Liquid, and conventional centrifugal rinse-system, natural subsidence rinse-system and rinse-system proposed by the present invention is respectively adopted and has done repeatedly
Rinse experiment；Wherein, cell cytopore microcarrier culture, fermentor 14L, culture solution 10L can disposably obtain 1010
Above HepG2 cell.
It this time rinses in experiment, the cell eluant container volume used is 2L, floor space 80cm2Tank body, filter
Bowl volume be about 200cm3, antibiotic residual quantity 94ug/ml, BSA residual quantity 46mg/ml, cell activity before being rinsed through detection
95%.The cell to be rinsed being placed in every time is about 1 × 1010A cell.
This system vacuum magnitude is controlled in 100~300mmHg, and vacuum suction flow control is in 0.5~3L/min, in order to more
Good confirmatory experiment is in 100mmHg as a result, rinsing control negative pressure in experiment, and vacuum suction flow control is in 1L/min, pre-cooling
Temperature is uniformly controlled at 0 DEG C；Experimental result data is as follows:
After being rinsed 3 times using centrifugal elutriation system to cell suspension to be rinsed, liquid in the epicoele 1.3 of cell eluant container 1
Body content data is as follows:
| Washing time | 1 time | 2 times | 3 times |
| It is time-consuming | 5min | 5min | 5min |
| Have sterile | It is sterile | It is sterile | It is sterile |
| Whether there is or not mycoplasmas | Without mycoplasma | Without mycoplasma | Without mycoplasma |
| Antibiotic residual quantity | 2.7ug/ml | 1.7ug/ml | 0.7ug/ml |
| BSA residual quantity | 190ng/ml | 65ng/ml | 39ng/ml |
| Cell activity | 85% | 83% | 80% |
After being rinsed 3 times using natural subsidence rinse-system to cell suspension to be rinsed, the epicoele 1.3 of cell eluant container 1
Interior content liquid data are as follows:
After using this system and aperture being selected to rinse 3 times for the filter membrane 7.2 of 200nm, the epicoele of cell eluant container 1
Content liquid data are as follows: in 1.3
| Washing time | 1 time | 2 times | 3 times |
| It is time-consuming | 5min | 5min | 5min |
| Have sterile | It is sterile | It is sterile | It is sterile |
| Whether there is or not mycoplasmas | Without mycoplasma | Without mycoplasma | Without mycoplasma |
| Antibiotic residual quantity | 2.2ug/ml | 1.6ug/ml | 0.6ug/ml |
| BSA residual quantity | 200ng/ml | 59ng/ml | 38ng/ml |
| Cell activity | 94% | 92% | 91% |
After using this system and aperture being selected to rinse 3 times for the filter membrane 7.2 of 300nm, the epicoele of cell eluant container 1
Content liquid data are as follows: in 1.3
| Washing time | 1 time | 2 times | 3 times |
| It is time-consuming | 5min | 5min | 5min |
| Have sterile | It is sterile | It is sterile | It is sterile |
| Whether there is or not mycoplasmas | Without mycoplasma | Without mycoplasma | Without mycoplasma |
| Antibiotic residual quantity | 2.4ug/ml | 1.8ug/ml | 0.6ug/ml |
| BSA residual quantity | 267ng/ml | 62ng/ml | 39ng/ml |
| Cell activity | 94% | 92% | 91% |
After using this system and aperture being selected to rinse 3 times for the filter membrane 7.2 of 400nm, the epicoele of cell eluant container 1
Content liquid data are as follows: in 1.3
From above-mentioned each table data it is found that filter membrane 7.2 made of polyether sulfone materials selects 200nm, 300nm, 400nm respectively
Aperture, after flushing three times, the endotoxin of liquid, antibiotic, BSA content all obviously subtract in the epicoele 1.3 of cell eluant container 1
It is few.The variation in its aperture only will affect a degree of flushing effect, and the cell activity after rinsing 3 times is much higher than traditional centrifugation
And sedimentation method.The flushing time-consuming and centrifugal process of this system are time-consuming quite, but compared to for sedimentation, when this system is more saved
Between.Therefore, this system is higher compared to cell enrichment for the mode of conventional centrifugal and natural subsidence and flush efficiency, while to thin
Cellular damage is smaller.
Rinse-system is collected for cell proposed by the present invention, the invention proposes a kind of HepG2 for being applicable in the system is thin
Born of the same parents collect purging method, include the following steps:
S1, the pre- physiological saline for being cooled to 0~4 DEG C is added into cell eluant container 1 by pipeline, is arranged with wetting thin
Its cavity is divided into the filter membrane 7.2 of epicoele 1.3 and cavity of resorption 1.4 in born of the same parents' eluant container 1, and pipeline and cell eluant container is pre-chilled
1, realize pretreatment；
S2, it is hanged to 1.3 quantification of the epicoele addition cell to be rinsed for completing the pretreated cell eluant container 1 of step S1
Liquid contains cell, and makes to be formed the negative pressure of 100~300mmHg in epicoele 1.3 so that at cell suspension to be rinsed by negative pressure device
In in epicoele 1.3；
S3,1.3 negative pressure of epicoele is released, and makes the negative pressure of 100~300mmHg of formation in cavity of resorption 1.4 by negative pressure device
So that liquid is filtered with the flow velocity of 0.5~3L/min to cavity of resorption 1.4 in epicoele 1.3；Meanwhile with the stream of 0.5~3L/min
Speed is added into epicoele 1.3 is cooled to 0~4 DEG C of physiological saline in advance, so that liquid is kept dynamic equilibrium and continue 1~
5min；The preferred dynamic equilibrium time is 2min in the present embodiment；
S4, stop being added physiological saline into epicoele 1.3 to release the dynamic equilibrium of liquid in epicoele 1.3, until epicoele
After (90% or more) liquid is filtered to cavity of resorption 1.4 in 1.3,1.4 negative pressure of cavity of resorption is released, to 1.3 quantification of epicoele
The pre- physiological saline for being cooled to 0~4 DEG C is added, and makes the negative pressure of 100~300mmHg of formation in epicoele 1.3 by negative pressure device, makes
Physiological saline breaks up 1.3 inner cell of epicoele with the flow velocity of 0.5~3L/min and cell is resuspended；
S5, step S3 and S4 are repeated, until after being filtered to the liquid in cavity of resorption 1.4 and complying with standard, it is default to epicoele 1.3
Amount is added to be cooled to 0~4 DEG C of cell-preservation liquid and cell is resuspended in advance, is completed cell and is rinsed.
Filter membrane 7.2 is made in this method of polyether sulfone materials, and aperture is preferably 300nm.Filter membrane 7.2 is fixed on cell
In eluant container 1.The setting of filter membrane 7.2 forms or needs to make its at least one inclined surface or convex surface when fixed.This set
The filter membrane 7.2 of form will be helpful to cell flushing, especially can avoid cell accumulation, improve fluid mobility.
In order to improve flush efficiency and cell activity, in step s 4, the physiological saline of pre-cooling is set with varied angle or displacement
Mode be added into epicoele 1.3.For example one end protrudes into tube body adjustable-angle in epicoele 1.3 or the tube body can be with respect to mistake
Filter membrane 7.2 rotates.The specific embodiment that this varied angle or displacement are set is realized by other devices, such as motor, push rod etc.
Component, can also be manually implemented.
The mode that 1.3 negative pressure of epicoele releases in step S3 includes closing the negative pressure device being connected to epicoele 1.3, and make epicoele
1.3 are communicated with the atmosphere by air filter 4；The mode that 1.4 negative pressure of cavity of resorption releases in step S4 includes closing to be connected to cavity of resorption 1.4
Negative pressure device.
Negative pressure device is preferably negative-pressure vacuum pump in this method, if setting structure or the negative-pressure vacuum pump type of selection close
Reason, the negative pressure device being connected to epicoele 1.3 and the negative pressure device being connected to cavity of resorption 1.4 can pump for the same negative-pressure vacuum, when
So, this cooperation for being also required to respective valves (such as triple valve) and pipeline；Valve in this method is preferably solenoid valve.
Claims (8)
1. a kind of cell eluant container, including a tank body (1.1), it is characterised in that: being equipped in the tank body (1.1) will be in it
Chamber is divided into the filter (7) of upper and lower two chambers；The tank body (1.1) is circumscribed with liquid input circuit (16), negative pressure access pipeline
(14), waste collection pipeline (15)；
The filter (7) be it is bowl-shape, bottom of bowl is the solid disk (7.1) of non-porous structure, is disposed with filter membrane on bowl side wall
(7.2)；
End of the liquid input circuit (16) in tank body (1.1) is arranged close to the solid disk (7.1)；The negative pressure
Access pipeline (14) incoming end is located at the top of tank body (1.1), and the incoming end of the waste collection pipeline (15) is located at tank body (1.1)
Bottom.
2. cell eluant container according to claim 1, it is characterised in that: the liquid input circuit (16) is in tank body
(1.1) vertical range of end and the solid disk (7.1) in is less than 1CM.
3. cell eluant container according to claim 1, it is characterised in that: the filter membrane (7.2) is polyether sulfone materials
It is made, aperture 300nm.
4. a kind of cell collects rinse-system, it is characterised in that: including cell eluant container (1), physiological saline container (2), thin
Born of the same parents' suspension container (3), air filter (4), reversal valve (13), the first negative pressure device (5) and the second negative pressure device (6)；
Its inner cavity is divided into up and down by the cell eluant container (1), including a tank body (1.1), interior be equipped with of the tank body (1.1)
The filter (7) of two chambers；The tank body (1.1) is circumscribed with liquid input circuit (16), negative pressure access pipeline (14), waste liquid receipts
Collect pipeline (15)；The filter be it is bowl-shape, bottom of bowl is the solid disk (7.1) of non-porous structure, is disposed on bowl side wall
Filter membrane (7.2)；End of the liquid input circuit (16) in tank body (1.1) is arranged close to the solid disk (7.1)；Institute
It states negative pressure access pipeline (14) incoming end to be located at the top of tank body (1.1), the incoming end of the waste collection pipeline (15) is located at tank
Body (1.1) bottom；
The physiological saline container (2), cell suspension container (3), air filter (4) are connected with reversal valve (13) respectively, and logical
It crosses reversal valve (13) and liquid input circuit (16) connects the cell eluant container (1)；
Negative pressure access pipeline (14) is connected with the first negative pressure device (5), the waste collection pipeline (15) and the second negative pressure
Device (6) is connected.
5. cell according to claim 4 collects rinse-system, it is characterised in that: the filter membrane (7.2) is polyether sulfone
Material is made, and aperture is 200~400nm.
6. cell according to claim 4 collects rinse-system, it is characterised in that: further include cell-preservation liquid container (8),
Efflux collection vessel (10), the first valve (11) and the second valve (12)；The cell-preservation liquid container (8) passes through reversal valve
(13) it is connected to liquid input circuit (16)；The efflux collection vessel (10) and waste collection pipeline (15) or the second negative pressure
Device (6) or cavity of resorption (1.4) connection；First valve (11) setting is in negative pressure access pipeline (14)；Second valve
(12) it is arranged on waste collection pipeline (15).
7. a kind of HepG2 cell collects purging method, characterized by the following steps:
S1, the pre- physiological saline for being cooled to 0~4 DEG C is added into cell eluant container (1) by pipeline, is arranged with wetting in cell
Its cavity is divided into the filter membrane (7.2) of epicoele (1.3) and cavity of resorption (1.4) in eluant container (1), and pipeline and cell punching is pre-chilled
It washes container (1), realizes pretreatment；
S2, it is hanged to epicoele (1.3) the quantification addition cell to be rinsed for completing the pretreated cell eluant container (1) of step S1
Liquid, and the negative pressure that 100~300mmHg is formed in epicoele (1.3) is made so that cell suspension to be rinsed is in upper by negative pressure device
In chamber (1.3)；
S3, epicoele (1.3) negative pressure is released, and makes the negative pressure of 100~300mmHg of formation in cavity of resorption (1.4) by negative pressure device
So that epicoele (1.3) interior liquid is filtered with the flow velocity of 0.5~3L/min to cavity of resorption (1.4)；Meanwhile with 0.5~3L/min
Flow velocity to the physiological saline for being cooled to 0~4 DEG C in advance is added in epicoele (1.3), so that the interior liquid of epicoele (1.3) is kept dynamic equilibrium simultaneously
Continue 1~5min；
S4, stop being added physiological saline into epicoele (1.3) to release the dynamic equilibrium of epicoele (1.3) interior liquid, until epicoele
(1.3) after interior liquid is filtered to cavity of resorption (1.4), cavity of resorption (1.4) negative pressure is released, is added to epicoele (1.3) quantification pre-
It is cooled to 0~4 DEG C of physiological saline, and makes the negative pressure of 100~300mmHg of formation in epicoele (1.3) by negative pressure device, makes physiology
Salt water breaks up epicoele (1.3) inner cell with the flow velocity of 0.5~3L/min and cell is resuspended；
S5, step S3 and S4 are repeated, until after being filtered to the liquid in cavity of resorption (1.4) and complying with standard, it is default to epicoele (1.3)
Amount is added to be cooled to 0~4 DEG C of cell-preservation liquid and cell is resuspended in advance, is completed cell and is rinsed.
8. HepG2 cell according to claim 7 collects purging method, it is characterised in that: in the step S4, pre-cooling
Physiological saline is added into epicoele (1.3) in such a way that varied angle or displacement are set.
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| CN108277152A (en) * | 2018-04-04 | 2018-07-13 | 徐俊 | The sterile liquid changing device of cell recovery and carry out the method that cell recovery changes liquid using the device |
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| CN110331081B (en) | 2022-09-23 |
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Effective date of registration: 20240411 Address after: 430000, West C308, Building C4, Biological Innovation Park, No. 666 Gaoxin Avenue, Donghu New Technology Development Zone, Wuhan City, Hubei Province Patentee after: Wuhan Tonggan Medical Technology Co.,Ltd. Country or region after: China Address before: No. 818, Gaoxin Avenue, Donghu high tech Development Zone, Wuhan, Hubei 430206 Patentee before: WUHAN TOGO MEDITECH Co.,Ltd. Country or region before: China |