CN103548815B - Programmed cell freezing box with direct-in liquid nitrogen - Google Patents
Programmed cell freezing box with direct-in liquid nitrogen Download PDFInfo
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- CN103548815B CN103548815B CN201310576212.XA CN201310576212A CN103548815B CN 103548815 B CN103548815 B CN 103548815B CN 201310576212 A CN201310576212 A CN 201310576212A CN 103548815 B CN103548815 B CN 103548815B
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 title claims abstract description 46
- 239000007788 liquid Substances 0.000 title claims abstract description 27
- 229910052757 nitrogen Inorganic materials 0.000 title claims abstract description 23
- 238000007710 freezing Methods 0.000 title abstract description 24
- 230000008014 freezing Effects 0.000 title abstract description 24
- 239000000463 material Substances 0.000 claims abstract description 13
- 229910001220 stainless steel Inorganic materials 0.000 claims abstract description 10
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000000741 silica gel Substances 0.000 claims abstract description 8
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 8
- 238000007789 sealing Methods 0.000 claims abstract description 7
- 229920001903 high density polyethylene Polymers 0.000 claims abstract description 6
- 239000004700 high-density polyethylene Substances 0.000 claims abstract description 6
- 239000004033 plastic Substances 0.000 claims abstract description 6
- 229920003023 plastic Polymers 0.000 claims abstract description 6
- 238000004519 manufacturing process Methods 0.000 claims abstract description 4
- 238000005476 soldering Methods 0.000 claims abstract description 4
- 239000010935 stainless steel Substances 0.000 claims abstract description 4
- 238000001816 cooling Methods 0.000 claims description 22
- 230000008595 infiltration Effects 0.000 claims description 3
- 238000001764 infiltration Methods 0.000 claims description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 abstract description 14
- 238000013461 design Methods 0.000 abstract description 2
- 239000000945 filler Substances 0.000 abstract description 2
- 239000010963 304 stainless steel Substances 0.000 abstract 2
- 229910000589 SAE 304 stainless steel Inorganic materials 0.000 abstract 2
- 239000006260 foam Substances 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 59
- 239000000243 solution Substances 0.000 description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 14
- 230000006378 damage Effects 0.000 description 12
- 238000000034 method Methods 0.000 description 11
- 239000013078 crystal Substances 0.000 description 10
- 230000009467 reduction Effects 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 230000004083 survival effect Effects 0.000 description 6
- 238000005138 cryopreservation Methods 0.000 description 5
- 230000001681 protective effect Effects 0.000 description 5
- 229920002274 Nalgene Polymers 0.000 description 4
- 210000000170 cell membrane Anatomy 0.000 description 4
- 230000003834 intracellular effect Effects 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 210000003463 organelle Anatomy 0.000 description 3
- 239000002953 phosphate buffered saline Substances 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 108010019160 Pancreatin Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000005842 biochemical reaction Methods 0.000 description 2
- 239000012620 biological material Substances 0.000 description 2
- 230000005779 cell damage Effects 0.000 description 2
- 230000030833 cell death Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000018044 dehydration Effects 0.000 description 2
- 238000006297 dehydration reaction Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 101150032953 ins1 gene Proteins 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 229940055695 pancreatin Drugs 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 108010022579 ATP dependent 26S protease Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 102100023374 Forkhead box protein M1 Human genes 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 101000907578 Homo sapiens Forkhead box protein M1 Proteins 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 101100221606 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) COS7 gene Proteins 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000012930 cell culture fluid Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000006059 cover glass Substances 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
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- 210000003743 erythrocyte Anatomy 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention relates to a programmed cell freezing box with direct-in liquid nitrogen, which belongs to the technical field of biomedicines. The programmed cell freezing box comprises a box body and a box cover, wherein a plurality of pipe grooves are formed in the box body; a main body of an outer layer of the box body is made of 304 stainless steel; a seal part in the box body is made from silica gel; the pipe grooves are made from white high-density polyethylene materials; fillers in the pipe grooves are foam plastics. A specific manufacturing process for the programmed cell freezing box comprises the following steps: forming a stainless steel tailless vacuum main body, prepared by carrying out vacuumizing as well as soldering and sealing on the 304 stainless steel at the temperature about 1000 DEG C, on the outer layer of the box body, tightly connecting the box body with the box cover through a screw thread, and arranging the silica gel seal ring on the box cover to prevent permeation of liquid nitrogen. The programmed cell freezing box has three advantages that (1) the cell temperature can be directly reduced to be -196 DEG C and cells do not need to be transferred and can be suitably cryopreserved for a long time; (2) isopropanol is not required and the box belongs to lossless permanently-usable products; (3) layered design is adopted, so that the cell temperature can be lower than -80 DEG C even if the box is taken out for a short time (30 minutes).
Description
Technical field
The present invention relates to a kind of liquid nitrogen direct-insert programmed cell cooling box, belong to field of biomedicine technology.
Background technology
At biological technical field, lower than under the condition of ultralow temperature of-70 DEG C, the biochemical reaction of organism cell interior is extremely slow, even stops.Therefore, take suitable method that biomaterial is down to ultralow temperature, vital movement can be made to be fixed on certain one-phase and not aging death.When in a suitable approach frozen biomaterial being returned to normal temperature, the biochemical reaction of its inside can recover normal.So-called freezen protective; exactly culture in vitro thing or bioactive materials are suspended in and are added with or do not add in the solution of cryoprotector; being down to subzero a certain temperature with certain freezing rate (is generally the condition of ultralow temperature lower than-70 DEG C; can preserve for a long time for-196 DEG C), and at this temperature to the process that it is preserved for a long time.Microorganism, zooblast, plant cell or the organ of culture in vitro can carry out frozen, and recover under proper condition.Water can freeze under lower than the condition of zero degree.If be suspended in by cell in pure water, along with the reduction of temperature, the moisture of intraor extracellular all can freeze, and the ice crystal formed can cause the destruction of cell membrane and organelle and cause cell death.This cellular damage caused because cell interior freezes is called the damage of intracellular ice crystal.If suspended in the solution by cell, along with the reduction of temperature, the moisture of outside can be first icing, thus electrolyte concentration in the solution do not frozen is raised.If to be exposed to by cell in the solution of high like this solute and overlong time, on cell membrane, lipid molecular can be damaged, and cell just seepage occurs, and when rewarming, therefore large quantity of moisture can enter in cell, causes cell death.This cellular damage caused because preserving solute concentration rising in solution is called solute damage or claims solution damage.When temperature declines further, intraor extracellular is all icing, produces ice crystal damage.If but add cryoprotector in the solution, then can damage from solute damage and ice crystal by Cell protection.Because cryoprotector is easily with the water molecules in solution; thus reduction freezing point, reduce the formation of ice crystal, and reduce electrolytical concentration in the solution that do not freeze by its molar concentration; cell is damaged from solute, and cell is able to preserve under condition of ultralow temperature.
Liquid nitrogen temperature (-196 DEG C) is freezen protective temperature best at present.-196 DEG C time, the vital movement of cell almost stops completely, but after recovery, the 26S Proteasome Structure and Function of cell is intact.If refrigerating process is proper, general biological sample all can preserve more than 10 years at-196 DEG C.
Freezing rate refers to the speed of cooling, is directly connected to refrigerating effect.Can be there is following change in cell: when cell is chilled to-5 DEG C, reduce the freezing point of solution because being added with cryoprotector in solution in refrigerating process, and intraor extracellular solution freezes not yet; When being chilled between-5 ~-15 DEG C, freezing and still keeping non-icing condition in cell first appears in Extracellular solution.The hydrone that the hydrone do not frozen in cell can freeze in solution than extracellular part has higher chemical energy.Consequently, ICW, in order to the balance with ECW maintenance chemical energy, can flow to extracellular.Chilling rate is different, and ICW situation is outwardly not identical yet: if chilling rate is slow, ICW exosmoses many, cell dehydration, volume-diminished, and intracellular solutes concentration increases, and can not freeze in cell; If chilling rate is fast, ICW does not have time enough to exosmose, and result, along with the decline of temperature, intracellular ice occurs; If chilling rate is (namely supper-fast freezing) quickly, then the ice crystal formed in cell is very little or do not freeze and be vitreousness (glass freezing).Luyet(1973) confirm that solidifying of liquid can be divided into two kinds of forms: one is crystallization, and the molecule in solution is ordered arrangement; Another kind of situation is noncrystal i.e. vitrifying, and the molecule in liquid is disordered state, keeps the state before not solidifying.Since different chilling rates can make, in cell, different physiological change occurs, also different damages can be produced to cell.When chilling rate is crossed slow, cell dehydration is serious, and cell volume significant shrinkage, loses activity when exceeding to a certain degree.Chilling rate is excessively slow simultaneously, Extracellular solution part also can be caused to freeze, thus in the solution that extracellular is not frozen, solute concentration increases, and produces solute damage.When chilling rate is too fast, ICW has little time to exosmose, and can be formed and comparatively arrive ice crystal, causes the destruction of cell membrane and organelle, produces intracellular ice crystal damage.Supper-fast glass freezing is ideal freezing method concerning cell survival.Intraor extracellular is that vitrifying is solidified, and is formed or form very little ice crystal without ice crystal, cell membrane and organelle is unlikely causes damage, and cell also can not expose for a long time and impaired in the solute of high concentration.
The suitableeest freezing rate of different cell is different.The suitableeest freezing rate of mouse marrow stem cell, yeast, human red blood cell is respectively 1.6 DEG C of min, 7 DEG C/min and 200 DEG C/min.The suitableeest freezing rate between cell and cell can at 1.6 DEG C ~ 300 DEG C/min, therefore before carrying out freezen protective to a kind of cell, first need to measure its suitableeest freezing rate, to ensure to obtain the highest freeze survival rate.
At present, existing main flow program temperature reduction box on market, or claim cell cryopreservation box or claim gradient cooling freezing storing box, be the 5100-0001 produced by Nalgene company of the U.S., belong to import brand series products.This program temperature reduction box or claim cell cryopreservation box to provide accurately and the cooling rate of good reproducibility for successful Cell Cryopreservation: the freezing rate of-1 DEG C/min cools to-80 DEG C.Belong to consumptive material class, need to add medicine isopropyl alcohol, isopropyl alcohol belongs to volatilization consumptive material.Can frozen 18 solencytes.And can only-80 DEG C be chilled to, have certain influence as transferred to liquid nitrogen cell.
Summary of the invention
The object of the present invention is to provide a kind of liquid nitrogen direct-insert programmed cell cooling box, to adopt rational freezing storing box material and special structure, ensure suitable temperature lowering curve.
To achieve these goals, technical scheme of the present invention is as follows.
A kind of liquid nitrogen direct-insert programmed cell cooling box, comprise box body and lid, be provided with multiple tube seat in box body, wherein, box body outer layers body material adopts 304 stainless steels, and box body inner seal liner adopts silica gel, and tube seat adopts white high-density polyethylene material; Tube seat inside stuffing adopts foamed plastics.
The concrete manufacture craft of above-mentioned liquid nitrogen direct-insert programmed cell cooling box is, box body skin is stainless steel anury vacuum main body, under nearly 1000 DEG C of conditions, 304 stainless steels are vacuumized and soldering and sealing and making, box body and lid pass through threaded closure, and on lid, added silica gel sealing ring, effectively can prevent the infiltration of liquid nitrogen.
This beneficial effect of the invention is: the present invention has three advantages: (1) directly can cool to-196 DEG C, need not shift, and is applicable to Long-term Cryopreservation cell; (2) without the need to isopropyl alcohol, belong to lossless and forever make articles for use; (3) hierarchical design, gets (30min) and goes out to ensure that cell temperature is lower than-80 DEG C even if of short duration.
Accompanying drawing explanation
Fig. 1 is cooling box structural section figure in the embodiment of the present invention.
Fig. 2 is cooling box structure vertical view in the embodiment of the present invention.
Fig. 3 is cooling box temperature lowering curve figure in the embodiment of the present invention.
Fig. 4 uses Ins-1 cell-line survival rate comparison diagram after apparatus of the present invention cell recovery in the embodiment of the present invention.
Fig. 5 uses Cos-7 cell-line survival rate comparison diagram after apparatus of the present invention cell recovery in the embodiment of the present invention.
Description of symbols in figure: 1, lid; 2, box body; 3, box body is outer; 4, tube seat; 5, screw thread.
Embodiment
Below in conjunction with drawings and Examples, the specific embodiment of the present invention is described, better to understand the present invention.
Embodiment
Liquid nitrogen direct-insert programmed cell cooling box as shown in Figure 1 and Figure 2, comprise box body 2 and lid 1, be provided with multiple tube seat 4 in box body 2, outer 3 material of main parts of box body adopt 304 stainless steels, box body inner seal liner adopts silica gel, and tube seat 4 adopts white high-density polyethylene material; Filler adopts foamed plastics.The concrete manufacture craft of above-mentioned liquid nitrogen direct-insert programmed cell cooling box is, box body outer 3 is stainless steel anury vacuum main body, under nearly 1000 DEG C of conditions, 304 stainless steels are vacuumized and soldering and sealing and making, box body 2 is closely sealed by screw thread 5 with lid 1, and silica gel sealing ring has been added on lid 1, effectively can prevent the infiltration of liquid nitrogen.Tube seat 4 portion of material is white high density polyethylene (HDPE), and inner foamed plastics light weight heat insulating ability of filling is better.
Fig. 3 is the temperature lowering curve (being repeated the result of 5 times) adopting this cooling box test gained.Need Superfreezing except a few cell is frozen, the frozen key of most cells is that cell is linear 20 DEG C to-80 DEG C declines, and cooling rate is at 1-10 DEG C/min.In the present embodiment, it is approximately the cooling rate of 2.6-2.7 DEG C/min that cell declines linear at 20 DEG C to-80 DEG C.Meet the frozen requirement of most cell.Employing liquid nitrogen cryogenics probe cooling cassette interior record in liquid nitrogen, liquid nitrogen temperature (about-196 DEG C) can be dropped to from room temperature, as shown in Figure 3.
Can continue below to illustrate and adopt test effect acquired by this device, concrete process of the test and result as follows:
(1) cell chulture:
1, medium (4 DEG C of preservations, use before in 37 DEG C of water-baths incubation):
INS-1 cell-line: 1) RPMI 1640 (D-glucose containing 11mM); 2) Fetal Bovine Serum (deactivation)+10ml; 3) (100mM makes cell increase endogenous CO to Sodium Pyruvate
2generation) add 1%; 4) β-Mercaptoethanol (scavenger of free radical) adds 0.1%;
COS7 cell-line: the DMEM perfect medium of standard, add 10% serum and 1% Pen .-Strep dual anti-;
2、Poly-L-Lydine:0.01mg/ml；
3,0.25% Trypsin-EDTA(pancreatin) (-20 DEG C of preservations are dissolved naturally in room temperature before using);
4, phosphate buffered saline(PBS) (PBS) (4 DEG C of preservations, use before in 37 DEG C of water-baths incubation);
137mmol/L NaCl (8g); 2.7mmol/L KCl (0.2g); 10mmol/L Na
2hPO
4(1.44g); 2mmol/L KH
2pO
4(0.24g); In 1L tri-distilled water, add four kinds of compositions, adjust pH 7.4, osmotic pressure 300mOsm, filter and preserve.
(2) cell cryopreservation:
1, cryopreserving liquid: 7%DMSO; 13% FBS serum; 80% medium; Liquid nitrogen direct-insert programmed cell cooling box; Nalgene 5100-0001 program temperature reduction box: add 250ml isopropyl alcohol;
2, material:
(1) well-grown cultured cell;
(2) fresh culture;
（3）DMSO (Sigma D-2650) ；
(4) aseptic plastic freezen protective pipe (Nalgene 5000-0020);
（5）0.4 ％ w/v trypan blue (GibcoBRL 15250-061) ；
(6) counting chamber and cover glass;
(7) constant speed cooler (KRYO 10 Series II);
3, step:
(1) half amount or full dose medium is changed before freezing proxima luce (prox. luc), observation of cell growth situation.
(2) stoste in blake bottle is outwelled by frozen beginning, washes 2 times with 5mlPBS.
(3) add 1ml pancreatin, be placed on vitellophag in 37 DEG C of cell culture incubators, add 5ml cell culture fluid and stop digestion, and it is sucked completely in 15ml centrifuge tube;
(4) the centrifugal 1000rpm.5min. of 15ml centrifuge tube abandons supernatant, adds 10ml Cryosreservation solution, makes cell concentration control as 1-5 x 106 cells/ml, mix, be sub-packed in and indicate in complete freezen protective pipe, 1 ml/vial, and the cell suspending liquid that takes a morsel does pollution detection.
(5) traditional cold freezing method for storing: cool to-80 DEG C with the 5100-0001 program temperature reduction box that Nalgene company produces, preserve in manual transfer to liquid nitrogen.
(6) freezing method (new method) in the present embodiment: directly frozen with liquid nitrogen direct-insert programmed cell cooling box.
Ins-1 and Cos-7 cell-line survival rate comparison diagram (being repeated the result of 5 times) after cell recovery as shown in Figure 4, Figure 5, the method applied in the present invention is more efficient than traditional method, and survival rate is higher.
The above is the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications are also considered as protection scope of the present invention.
Claims (2)
1. a liquid nitrogen direct-insert programmed cell cooling box, comprise box body and lid, be provided with multiple tube seat in box body, it is characterized in that: described box body outer layers body material adopts 304 stainless steels, described box body inner seal liner adopts silica gel, and described tube seat adopts white high-density polyethylene material; Described tube seat inside stuffing adopts foamed plastics.
2. liquid nitrogen direct-insert programmed cell cooling box according to claim 1, it is characterized in that: the concrete manufacture craft of described cooling box is, box body skin is stainless steel anury vacuum main body, under nearly 1000 DEG C of conditions, 304 stainless steels are vacuumized and soldering and sealing and making, box body and lid pass through threaded closure, and on lid, added silica gel sealing ring, prevent the infiltration of liquid nitrogen.
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| CN201310576212.XA CN103548815B (en) | 2013-11-18 | 2013-11-18 | Programmed cell freezing box with direct-in liquid nitrogen |
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| CN201310576212.XA CN103548815B (en) | 2013-11-18 | 2013-11-18 | Programmed cell freezing box with direct-in liquid nitrogen |
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| CN103548815B true CN103548815B (en) | 2015-06-10 |
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| CN107258766A (en) * | 2017-06-18 | 2017-10-20 | 广东博溪生物科技有限公司 | A kind of cell freezing method and cells frozen storing liquid |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4377077A (en) * | 1981-07-15 | 1983-03-22 | Biotech Research Laboratories, Inc. | Device and method for controlled freezing of cell cultures |
| US4455842A (en) * | 1981-07-15 | 1984-06-26 | Biotech Research Laboratories, Inc. | Device and method for controlled freezing of cell cultures |
| WO2005066559A2 (en) * | 2004-01-08 | 2005-07-21 | Bernhard Sixt | Transport container for keeping frozen material chilled |
| CN203040527U (en) * | 2013-02-25 | 2013-07-10 | 中国水产科学研究院黑龙江水产研究所 | Cell freezing and storing box |
| CN103202286A (en) * | 2013-04-11 | 2013-07-17 | 天津开发区合普工贸有限公司 | Electronic program-controlled const-speed cooling and freezing device |
-
2013
- 2013-11-18 CN CN201310576212.XA patent/CN103548815B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4377077A (en) * | 1981-07-15 | 1983-03-22 | Biotech Research Laboratories, Inc. | Device and method for controlled freezing of cell cultures |
| US4455842A (en) * | 1981-07-15 | 1984-06-26 | Biotech Research Laboratories, Inc. | Device and method for controlled freezing of cell cultures |
| WO2005066559A2 (en) * | 2004-01-08 | 2005-07-21 | Bernhard Sixt | Transport container for keeping frozen material chilled |
| CN203040527U (en) * | 2013-02-25 | 2013-07-10 | 中国水产科学研究院黑龙江水产研究所 | Cell freezing and storing box |
| CN103202286A (en) * | 2013-04-11 | 2013-07-17 | 天津开发区合普工贸有限公司 | Electronic program-controlled const-speed cooling and freezing device |
Non-Patent Citations (1)
| Title |
|---|
| 程方杰.实验23 不锈钢真空钎焊设备与工艺实验.《材料成型与控制实验教程 焊接分册》.2011, * |
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