CN107974399A - A kind of cell dissociation device - Google Patents
A kind of cell dissociation device Download PDFInfo
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- CN107974399A CN107974399A CN201711449592.5A CN201711449592A CN107974399A CN 107974399 A CN107974399 A CN 107974399A CN 201711449592 A CN201711449592 A CN 201711449592A CN 107974399 A CN107974399 A CN 107974399A
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- 238000010494 dissociation reaction Methods 0.000 title claims abstract description 44
- 230000005593 dissociations Effects 0.000 title claims abstract description 44
- 230000029087 digestion Effects 0.000 claims abstract description 44
- 239000012530 fluid Substances 0.000 claims abstract description 18
- 239000007788 liquid Substances 0.000 claims abstract description 18
- 238000011084 recovery Methods 0.000 claims abstract description 12
- 239000012535 impurity Substances 0.000 claims abstract description 7
- 238000003756 stirring Methods 0.000 claims description 22
- 230000008676 import Effects 0.000 claims description 16
- 230000001079 digestive effect Effects 0.000 claims description 14
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 11
- 238000002156 mixing Methods 0.000 claims description 9
- 238000004140 cleaning Methods 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 239000000463 material Substances 0.000 claims description 6
- 229910001220 stainless steel Inorganic materials 0.000 claims description 5
- 239000010935 stainless steel Substances 0.000 claims description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 4
- 239000001301 oxygen Substances 0.000 claims description 4
- 229910052760 oxygen Inorganic materials 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 238000009413 insulation Methods 0.000 claims description 3
- 230000001105 regulatory effect Effects 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 2
- 230000014759 maintenance of location Effects 0.000 claims description 2
- 229910000831 Steel Inorganic materials 0.000 claims 1
- 239000010959 steel Substances 0.000 claims 1
- 238000005070 sampling Methods 0.000 abstract description 18
- 238000000034 method Methods 0.000 abstract description 12
- 238000004113 cell culture Methods 0.000 abstract description 11
- 230000008569 process Effects 0.000 abstract description 11
- 238000004064 recycling Methods 0.000 abstract description 3
- 230000000717 retained effect Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 96
- 239000006285 cell suspension Substances 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 239000001963 growth medium Substances 0.000 description 10
- 238000000855 fermentation Methods 0.000 description 9
- 230000004151 fermentation Effects 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 8
- 239000007853 buffer solution Substances 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 239000000203 mixture Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000010008 shearing Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000287828 Gallus gallus Species 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 238000005138 cryopreservation Methods 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
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- 206010040007 Sense of oppression Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
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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
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/08—Homogenizing
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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
- C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
- C12M27/02—Stirrer or mobile mixing elements
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- 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
- C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
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Abstract
The present invention provides a kind of cell dissociation device.The present invention cell dissociation device by digester, be used to support digester stent, digestion top ends of cans form；The digester includes tank body and tank bottom, tank bottom is to collect the cavity of liquid, the bottom of cavity sets leakage fluid dram, tank body and tank bottom junction are horizontally disposed with a dismountable sieve, the sieve is engaged with tank body, microcarrier or impurity agglomerate can be retained, and cell is flowed through to the cavity of tank bottom；Tank body lateral wall sets sampling/recovery port, and sampling/recovery port is located above sieve, for sampling or recycling microcarrier.The cell dissociation device of the present invention can be used for digestion and the microcarrier culture cell dissociation of primary cell; cell and microcarrier or large crumb can be separated in real time in digestion process; substantially reduce the cell dissociation cycle and avoid bottom cell from being pressurized; be conducive to the secondary culture of cell, suitable for industrial massive cell culture.
Description
Technical field
The present invention relates to a kind of cell dissociation device.
Background technology
, it is necessary to which tissue is cut into small pieces in primary cell preparation process, then using digestive pharmaceutical be digested to tissue agglomerate into
And it is digested to individual cells.During this, cell is progressively to dissociate to get off to become single cell suspension from tissue.It is existing
Digestion techniques can not real-time collecting cell and terminate digest, characteristics of cell biology is subject to very havoc, cause cell attachment
Rate is low, it is difficult to survives.
Field of biological product is when preparing cell seed and cell culture, it will usually seeds cells on microcarrier, adopts
Use fermentation tank culture.The above all digestion of cell in cell fermentation incubation.And various fermentations disclosed in the prior art
The leakage fluid dram of tank when the top of the lowermost end of fermentation tank, cell dissociation, is usually washed carefully with suitable cleaning solution
Cellular surface.Cleaning solution discharge is needed first to settle microcarrier, needs more than 30 minutes every time, generally requires washing 3 times, sedimentation
Microcarrier 3 times, it is very time-consuming.After cell washs, addition digestive pharmaceutical digestion.Because fermentation tank inner cell is more, plus hair
The construction of fermentation tank in itself leads to not naked eyes and judges digestion process.More sub-samplings are needed in digestion process, observe cell state.So
And the sample tap of commercially available fermentation tank all be higher than fermentation tank bottom, cause must excessive addition digestive juice increase volume cause
Liquid level can just get sample higher than sample tap.It is well known that excessive digestive juice easily causes cell irreversible infringement, so that
Change characteristics of cell biology, cause cell survival rate during follow-up cultivation low, or even influence the quality of biological products.And
After cell dissociation, microcarrier and cell precipitate can not efficiently separate at the same time, therefore the extremely difficult preparation for being applied to cell seed.
Therefore, just can conveniently be sampled without excessive addition digestive juice there is an urgent need to one kind and need not settle microcarrier just can quick drain
The high efficiency cell digester of solution.
The content of the invention
It is an object of the invention to provide a kind of cell dissociation device.
A kind of cell dissociation device provided by the invention, the cell dissociation device by digester, be used to support digester stent,
Top ends of cans composition is digested, the digester includes tank body and tank bottom, and to collect the cavity of liquid, the bottom of cavity is set tank bottom
Leakage fluid dram, tank body and tank bottom junction are horizontally disposed with a dismountable sieve, and the sieve is engaged with tank body, can will be micro-
Carrier or the retention of impurity agglomerate, make cell flow through to the cavity of tank bottom；Connected below tank body lateral wall and in tank body with tank bottom
The top at place sets sampling/recovery port.
The tank body is cylinder, and the bottom surface of the tank bottom is recessed arc or triangle, tank bottom bottom surface it is most lower
End sets a leakage fluid dram.The leakage fluid dram is used to connect external container or equipment, and leakage fluid dram is connected with external container or equipment
There is tongs on pipeline, for controlling the discharge of liquid.
Those skilled in the art can be according to the sieve for treating the upright suitable aperture of footpath selection of trapped substance.
The material of the digester is glass or stainless steel.
The material of the sieve is stainless steel.
Sampling/the recovery port is used for digestion process sampling, judges digestible degree, it may also be used for recycling microcarrier.
The tank body outer surface is equipped with insulation jacket, and chuck water outlet, chuck lateral wall are equipped with above chuck lateral wall
Lower section is equipped with chuck water inlet.
The digestion top ends of cans is equipped with dismountable agitating paddle, stirring motor and mixing speed adjuster；Digest top ends of cans
Cover when starting to work on digester, agitating paddle is always positioned at the top of the sieve.Mixing speed adjuster is stirred for adjusting
Mix the mixing speed of paddle.Agitating paddle, stirring motor and mixing speed adjuster are connected by signal Input/Output Device.
Further, agitating paddle be independent double-deck stirring, upper strata agitating paddle for washing cell when from important stirring action.Under
For layer agitating paddle to be stirred during cell dissociation, lower floor's agitating paddle has hole, is the damage in order to reduce stirring to cell, stirs
It is streamlined to mix paddle edge.Agitating paddle has certain angle, can reduce the shearing force in whipping process.
The digestion top ends of cans, which is equipped with, treats vitellophag import, cleaning solution or digestive juice import, the import of digestion terminate liquid, pressure
Contracting air intlet, exhaust emissions outlet, pressure gauge connector, spare mouth.
The spare mouth has several, can connect pH regulating devices, dissolved oxygen control device or temperature control equipment.
The beneficial effects of the present invention are：
(1) sieve that the present invention is set in tank body and the tank bottom junction of digester can be with effectively catching tissue agglomerate and micro-
Carrier, realizes solution quick drain, realizes the unicellular separation with tissue agglomerate or microcarrier, autotelic can collect single
One product.For cell seed preparation process, greatly reduce microcarrier and produced not during cell cryopreservation as impurity
Profit influences, and ensure that the purity of cell.
(2) due to there is provided the sieve that can retain microcarrier and cell mass, making whole digestion process without waiting for micro-
Carrier infall process, substantially reduces the cell dissociation cycle, and overcomes and settle what microcarrier was brought for a long time in the prior art
The drawbacks of bottom cell is pressurized, existing bottom cell suffers oppression during reducing bioreactor digestion in site, and dissolved oxygen passes
The problems such as matter is poor, makes cell be in relatively good state, is conducive to cell survival rate height, and promote cell in new system
Quick wall attaching and propagation.
(3) present invention is provided with leakage fluid dram in the lowermost end of digester tank bottom, and separated cell or solution can pass through discharge opeing
Mouth enters next stage container or equipment.Connected drainage mouth can be controlled with having tongs on next stage container or the pipeline of equipment
Liquid discharges.
(4) present invention sets sampling/recovery port, the sampling/return in the top of the outer wall of tank body, tank body and tank bottom junction
Close up since positioned at the lower section of digestion tank, microcarrier and impurity cluster can be recycled while additionally digestive juice is not increased
Block, and can sample at any time to judge digestible degree, whether reach terminal.
(5) double-layer detachable formula agitating paddle of the present invention：It can be sufficiently stirred in digestion process, make digestive juice distribution equal
It is even, reach the homogeneous digestion of cell, overcome digestion in site due to the problem of digestive juice small volume can not be sufficiently stirred.Stirring speed
Degree can be adjusted in real time as needed.Agitating paddle possesses certain angle and streamlined, it is ensured that in digestion process is stirred
Shearing force is small, reduces energy consumption and cost.
(6) cell dissociation device provided by the invention ensures to be in the gnotobasis closed all the time during whole cell dissociation
It is middle to carry out single digestion, reduce the contaminated risk of cell.
Brief description of the drawings
Fig. 1 is cell dissociation device structure chart of the present invention, wherein 1 is leakage fluid dram, 2 be sieve, and 3 be sampling/recovery port, and 4 are
Chuck water inlet, 5 be agitating paddle, and 6 be chuck water outlet, and 7 be digestion top ends of cans, and 8 is support the stent of digester, and 9 be stirring
Motor.
Fig. 2 is the structure diagram of cell dissociation device head cover of the present invention, wherein 10 be head cover handle, 11 be cleaning solution or digestion
Liquid import, 12 is treat vitellophag import, and 13 be digestion terminate liquid import, and 14 be pressure gauge connector, and 15,18 be spare mouth,
16 export for exhaust emissions, and 17 be clamping screw, and 19 be compressed air inlet.
Embodiment
Following embodiments are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment
In the conventional means that are well known to those skilled in the art of used technological means.
1 cell dissociation device of embodiment
As shown in Figure 1, cell dissociation device by digester, be used to support digester stent 8, digestion top ends of cans 7 form, disappear
Changing tank includes tank body and tank bottom, and digester material is stainless steel, and for tank bottom to collect the cavity of liquid, the bottom of cavity sets row
Liquid mouth 1, tank body and tank bottom junction are horizontally disposed with a dismountable sieve 2, and sieve is engaged with tank body, the diameter of sieve with
The diameter of cylindrical tank is identical, can retain microcarrier or impurity agglomerate, cell is flowed through to the cavity of tank bottom；Tank body
Sampling/recovery port 3 is set below lateral wall and in the top of tank body and tank bottom junction.
The bottom surface of tank bottom is recessed arc or triangle, and the bottom of tank bottom bottom surface sets a leakage fluid dram 1.The discharge opeing
Mouth is used to connect external container or equipment, has tongs on the pipeline that leakage fluid dram is connected with external container or equipment, for controlling
The discharge of liquid.
Those skilled in the art can be according to the sieve for treating the upright suitable aperture of footpath selection of trapped substance.
The material of sieve 2 is stainless steel.
Sampling/recovery port 3 is used for digestion process sampling, judges digestible degree, it may also be used for recycling microcarrier.
The tank body outer surface is equipped with insulation jacket, and chuck water outlet 6, chuck lateral wall are equipped with above chuck lateral wall
Lower section is equipped with chuck water inlet 4.
Digestion top ends of cans is equipped with dismountable agitating paddle 5, stirring motor 9 and mixing speed adjuster；Digest top ends of cans lid
When starting to work on digester, agitating paddle is always positioned at the top of sieve.Mixing speed adjuster is used to adjust agitating paddle
Mixing speed.Agitating paddle, stirring motor and mixing speed adjuster are connected by signal Input/Output Device.
Further, agitating paddle stirs for independent double-deck, and agitating paddle edge is streamlined.Agitating paddle has certain angle,
The shearing force in whipping process can be reduced
As shown in Fig. 2, digestion top ends of cans, which is equipped with, treats vitellophag import 12, cleaning solution or digestive juice import 11, digestion eventually
Only liquid import 13, compressed air inlet 19, exhaust emissions outlet 16, pressure gauge connector 14, spare mouth 15,18.
Spare mouth has several, can connect pH regulating devices, dissolved oxygen control device or temperature control equipment.
The cell dissociation device using the present invention of embodiment 2 carries out primary cell (chicken embryo fibroblasts) and prepares
1) chicken embryo leg flesh, chest muscle are chosen in an aseptic environment, remove epidermis, blood vessel and connective tissue.Will tissue be digested
Using 0.02M PBS buffer washes cleans, 1mm is cut into3Fritter, about 1L.
2) by the sterile cell dissociation device (autoclaving) for being transferred to embodiment 1 of tissue to be digested, add isometric
0.25% trypsin solution.The mesh size that the present embodiment is selected is 75 μm.
3) external temperature-controlling system, maintains digestion temperature to be carried out at the same time stirring at low speed at 37 DEG C.
4) when digestion system becomes cloudy or tissue block to be digested becomes rotten shape, collecting cell by bottom leakage fluid dram hangs
Liquid, cell suspension, which is transferred in other container, carries out termination digestion.
5) 0.25% isometric trypsin solution, temperature control (37 DEG C) stirring at low speed are added again into cell dissociation device
(50rpm), continues to digest.
6) repeat step 4) -5) terminate to digestion.
7) cell suspension for collecting two batches merges, then low-speed centrifugal (1000rpm, 15min), removes supernatant.
8) cell mass is resuspended using 199 cell culture mediums containing 10% serum, final volume 1000ml.Calculate thin
Born of the same parents' density is 2.3 × 106Cells/ml and cell survival rate are 97%.
9) seed cells into T175 bottles, cell-seeding-density is 5 × 106Cells/ml, volume of culture 60ml/
Bottle.In CO2Cultivated in incubator, condition of culture is 37 DEG C, 5.0%CO2Concentration.When cell culture 48 is small, cell is completely adherent
Afterwards, original culture medium and not adherent fiber and impurity part are discarded, the culture medium for replacing isometric same composition continues to train
Support.
10) treat that cell growth to fine and close individual layer, carries out had digestive transfer culture.
Embodiment 3 realizes that cell microcarrier suspension culture 14L amplifies to 130L scales using cell dissociation device.
1) cell culture 3-5 days in 14L bioreactors (fermentation tank), treat that cell grows up to fine and close individual layer on microcarrier
Stop cell culture.
2) the cell dissociation device main body of the present invention and subsidiary pipeline are cleaned up and assembled, in high pressure steam sterilization cabinet
121 DEG C of autoclaving 30min of middle progress.After sterilizing, equipment is set to be down to room temperature under laminar flow protection.
3) bioreactor harvest pipeline and cell dissociation device are treated into the sterile connection of vitellophag import, by with cell
Microcarrier and culture medium are transferred to cell dissociation device.The leakage fluid dram of cell dissociation device tank bottom is connected with waste liquid collection vessel at the same time,
While cell and culture medium are transferred to digester, digester bottom discharge tongs is opened, exhausts culture medium in time.The present embodiment
The mesh size of selection is 75 μm.
4) about 1-2L microcarriers are finally obtained, 2 times of volumes of wash buffer are added from cleaning solution import.Open temperature control system
System, maintaining temperature, stirring at low speed (60rpm) washs cell at 37 DEG C.Treat that microcarrier stops stirring after being sufficiently agitated, from tank
Bottom leakage fluid dram exhausts buffer solution.
5) 3 cells are washed up to no culture medium color, finally exhaust lavation buffer solution.
6) 2 times of volume cells digestive juices, temperature control (37 DEG C) stirring at low speed (60rpm) digestion are added.Treat that digestion system becomes muddy
It is turbid or 50% cell is digested from being sampled from sampling/recovery port, received by the leakage fluid dram for digesting pot bottom
Collect cell suspension, cell suspension is transferred in other container and carries out termination digestion.
7) repeat step 6) to sampling observation cells on microcarriers digest completely.
8) cell suspension collected in batches is merged, it is final to obtain 4L cell suspensions (under microscope, cell is uniformly single),
Then low-speed centrifugal, removes supernatant.
9) cell mass is resuspended using cell culture medium, final volume is 3L.Calculate cell density obtain 9.3 ×
106Cells/ml, cell survival rate 96%.
10) according to the required cell density of next stage cell culture, measure appropriate volume born of the same parents' suspension and be transferred to next stage life
Thing reactor carries out cell culture.
11) by microcarrier recovery channel (i.e. 3 samplings/recovery port in Fig. 1) and the sterile connection of next stage bioreactor.
Microcarrier is transferred to next stage bioreactor to be reused.
Embodiment 4 carries out micro-carriers cell culture using bioreactor and the cell dissociation device of the present invention and prepares cell kind
Son
1) cell culture 3-5 days in 40L bioreactors (actual volume of culture is 30L), treat cell on microcarrier
Grow up to fine and close individual layer and stop cell culture, calculate cell density.
2) cell dissociation device main body and subsidiary pipeline are cleaned up and assembled, carried out in high pressure steam sterilization cabinet
121 DEG C of autoclaving 30min.After sterilizing, equipment is set to be down to room temperature under laminar flow protection.
3) bioreactor harvest pipeline and cell dissociation device are treated into the sterile connection of vitellophag import, by with cell
Microcarrier and culture medium are transferred to cell dissociation device.The leakage fluid dram of cell dissociation device tank bottom is connected with waste liquid collection vessel at the same time,
While cell and culture medium are transferred to digester, digester bottom discharge tongs is opened, exhausts culture medium in time.The present embodiment
The mesh size of selection is 75 μm.
4) about 3L microcarriers are finally obtained, 2 times of volumes of wash buffer are added from cleaning solution import.Open temperature-controlling system,
Maintaining temperature, stirring at low speed (60rpm) washs cell at 37 DEG C.Treat that microcarrier stops stirring after being sufficiently agitated, from tank bottom
Portion's leakage fluid dram exhausts buffer solution.
5) 3 cells are washed up to no culture medium color, finally exhaust lavation buffer solution.
6) 2 times of volume cells digestive juices, temperature control (37 DEG C) stirring at low speed (60rpm) digestion are added.Treat that digestion system becomes muddy
Turbid or sampling observation 50% cell is digested, and cell suspension is collected by bottom pipe, cell suspension is transferred to
Termination digestion is carried out in other container.
7) repeat step 6) to sampling observation cells on microcarriers digest completely.
8) cell suspension collected in batches is merged, by cell suspension low-speed centrifugal, removes supernatant.
9) cell mass is resuspended using cell culture medium, freezes density and volume according to the rules by cell suspension point
It is attached in the cryopreservation tube of appropriate size.
10) the cell seed dispensed is positioned in programmed cooling instrument, it is -190 DEG C near from room temperature (25 DEG C).
11) the cell seed frozen is placed in liquid nitrogen container and preserved.
Although above with general explanation, embodiment and experiment, the present invention is described in detail,
But some modifications on the basis of the present invention, can be made to it or are improved, this is apparent to those skilled in the art
's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed
Scope.
Claims (10)
1. a kind of cell dissociation device, by digester, the stent, the digestion top ends of cans that are used to support digester form, it is characterised in that
The digester includes tank body and tank bottom, and for tank bottom to collect the cavity of liquid, the bottom of cavity sets leakage fluid dram, tank body and tank
Bottom junction is horizontally disposed with a dismountable sieve, and the sieve is engaged with tank body, can be by microcarrier or impurity agglomerate
Retention, makes cell flow through to the cavity of tank bottom；Set below tank body lateral wall and in the top of tank body and tank bottom junction and take
Sample/recovery port.
2. cell dissociation device as claimed in claim 1, it is characterised in that the tank body is cylindrical, the bottom of the tank bottom
Face is recessed arc or triangle, and the bottom of tank bottom bottom surface sets a leakage fluid dram.
3. cell dissociation device as claimed in claim 1, it is characterised in that the tank body outer surface is equipped with insulation jacket, folder
Chuck water outlet is equipped with above set lateral wall, chuck water inlet is equipped with below chuck lateral wall.
4. cell dissociation device as claimed in claim 1, it is characterised in that the digestion top ends of cans is equipped with dismountable stirring
Paddle, stirring motor and mixing speed adjuster；Digestion top ends of cans is covered when starting to work on digester, and agitating paddle is always positioned at institute
State the top of sieve.
5. cell dissociation device as claimed in claim 4, it is characterised in that agitating paddle stirs for independent double-deck, agitating paddle edge
To be streamlined；Lower floor's agitating paddle has hole, to reduce the damage to cell.
6. the cell dissociation device as described in claim 1-5 is any, it is characterised in that the digestion top ends of cans, which is equipped with, treats that digestion is thin
Born of the same parents' import, cleaning solution or digestive juice import, the import of digestion terminate liquid, compressed air inlet, exhaust emissions outlet, pressure gauge connection
Mouth, spare mouth.
7. cell dissociation device as claimed in claim 6, it is characterised in that the spare mouth has several, can connect pH tune
Regulating device, dissolved oxygen control device or temperature control equipment.
8. the cell dissociation device as described in claim 1-6 is any, it is characterised in that the material of the digester is for glass or not
Become rusty steel.
9. the cell dissociation device as described in claim 1-6 is any, it is characterised in that the material of the sieve is stainless steel.
10. application of any cell dissociation devices of claim 1-9 in biological products preparation.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113355240A (en) * | 2021-07-02 | 2021-09-07 | 宁波希诺赛生物科技有限公司 | Culture digestion equipment and culture digestion method for full-automatic culture of stem cells |
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Application publication date: 20180501 |