CN107258766A - A kind of cell freezing method and cells frozen storing liquid - Google Patents
A kind of cell freezing method and cells frozen storing liquid Download PDFInfo
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- CN107258766A CN107258766A CN201710460512.XA CN201710460512A CN107258766A CN 107258766 A CN107258766 A CN 107258766A CN 201710460512 A CN201710460512 A CN 201710460512A CN 107258766 A CN107258766 A CN 107258766A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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Abstract
The embodiment of the present invention provides a kind of cell freezing method and cells frozen storing liquid, it is related to biological technical field, due to being free of serum in the cells frozen storing liquid, it is possible to increase cell recovery rate, make after cell recovery activity and performance height, can meet the need for a variety of different cell long-periods freeze.The cell freezing method specifically includes following two steps：Step 1: the cells frozen storing liquid for being mixed into cell is loaded into container 1, then container 1 is loaded into container 2, vacuum is formed between container 1 and container 2；Step 2: A, using the air-cooled temperature for making container 2 of normal temperature from normal temperature dropping to 4 DEG C, the speed of temperature drop declines 34 DEG C to be per minute；B, the temperature of container 2 is set using refrigerator to drop to 20 DEG C from 4 DEG C, the speed of temperature drop declines 12 DEG C to be per minute；C, the temperature of container 2 is set using refrigerator to drop to 80 DEG C from 20 DEG C, the speed of temperature drop declines 23 DEG C to be per minute；D, container 2 is put into liquid nitrogen container frozen, the temperature of liquid nitrogen container is 196 DEG C.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of cell freezing method and cells frozen storing liquid.
Background technology
Ultra-cryofreezing preservation be cell long-period preserve a kind of effective ways, mainly by reduce the metabolic rate of cell come
Play a protective role.It is a kind of most common cell long-period preserving type that (- 196 DEG C) of liquid nitrogen, which preserves cell, and regulation and control are thin
The effect of the enzyme classes of intracellular growth metabolism is greatly suppressed, and makes the biochemical reaction of cell interior very slow, or even is stopped, from
And the change of cytogenetics character is avoided, cell and tissue will not lose morphogenetic potential.But, frostbite is that ultralow temperature is protected
Protect maximum negative reaction, its reason is the mechanical injuries and permeability damage caused by ice crystal.Current cell cryopreservation is mainly soon
Fast freezing preservation method, i.e. Vitrification.But if not taking suitable cryopreservation methods and suitable cryoprotector, cell is freezing
Easy frost bitten during depositing.It is to improve the important channel for preserving cell to improve frozen stock solution and cryopreservation methods, many researchs at present
All it is to improve frozen stock solution and cryopreservation methods to protect cell to try one's best the situation from freezing process frostbite.
Contain animal derived serum composition in overwhelming majority frozen stock solution at present, and there is virus spread in animal derived serum
Risk；Because animal individual is different, the serum place of production, lot number are different, and very big per quality of lot difference, its composition can not be consistent,
Every batch of experiment of influence or production result；Serum cost is very high, has aggravated production cost.Therefore, the cells frozen storing liquid without serum
It is the trend of development in the future, some units or mechanism research serum-free cell frozen stock solution.Application number CN201610322202.7's
Patent discloses a kind of serum-free cell frozen stock solution, for solve frozen stock solution in-convenience in use, cost is high, cell survival rate is low
Problem.Application number CN201610124499.6 patent discloses a kind of human adipose-derived stem cell serum-free frozen stock solution and its preparation side
Method.
Thawed after the frozen stock solution long-term frozen of existing serum-free, cell recovery rate is low, after recovery under cytoactive and performance
Drop, and can not meet the need for different cell long-periods freeze.
Contain animal derived serum composition in current most of frozen stock solutions, and there is virus spread in animal derived serum
Risk；Because animal individual is different, the serum place of production, lot number are different, and very big per quality of lot difference, its composition can not be consistent, shadow
Ring every batch of experiment or production result；Product safety inspection cost containing the later stage after serum can be very high, has aggravated production cost.Although
Current glass frozen preservation is more satisfactory cryopreservation methods, but it still can form ice crystal damaging cells in During Vitrification in vitro,
And Cell viability is low after recovery, cell proliferating number amount is not enough, the easy aging of cell.
The content of the invention
The embodiments of the invention provide a kind of cell freezing method and cells frozen storing liquid, due to being free of in the cells frozen storing liquid
Serum, it is possible to increase cell recovery rate, makes after cell recovery activity and performance height, can meet what a variety of different cell long-periods froze
Need.
To reach above-mentioned purpose, the embodiment of the present invention is adopted the following technical scheme that：
The embodiment of the present invention provides a kind of cell freezing method, and cell freezing method includes following two steps：
Step 1: the cells frozen storing liquid for being mixed into cell is loaded into container 1, then container 1 is loaded into container 2, wherein, container 1 and appearance
Vacuum is formed between device 2；
Step 2: performing following tetra- steps of A-D according to this to the container 2 obtained by step one：
A, using the air-cooled temperature for making container 2 of normal temperature from normal temperature 4 DEG C are dropped to, wherein, the speed of temperature drop is under per minute
3-4 DEG C of drop；
B, using refrigerator the temperature of container 2 is set to drop to -20 DEG C from 4 DEG C, wherein, the speed of temperature drop is decline 1- per minute
2℃；
C, using refrigerator the temperature of container 2 is set to drop to -80 DEG C from -20 DEG C, wherein, the speed of temperature drop declines to be per minute
2-3℃；
D, container 2 is put into liquid nitrogen container frozen, wherein, the temperature of liquid nitrogen container is -196 DEG C.
Further, step 2 is specifically included：Following tetra- steps of A-D are performed according to this to the container 2 obtained by step one：
A, using the air-cooled temperature for making container 2 of normal temperature from normal temperature 4 DEG C are dropped to, wherein, the speed of temperature drop is under per minute
4 DEG C of drop；
B, using refrigerator the temperature of container 2 is set to drop to -20 DEG C from 4 DEG C, wherein, the speed of temperature drop declines 1 to be per minute
℃；
C, using refrigerator the temperature of container 2 is set to drop to -80 DEG C from -20 DEG C, wherein, the speed of temperature drop declines to be per minute
2℃；
D, container 2 is put into liquid nitrogen container frozen, wherein, the temperature of liquid nitrogen container is -196 DEG C.
Further, cell be epidermal stem cells, fibroblast, keratocyte, fat stem cell, melanocyte,
Buccal mucosa cell or oral cavity gingival cell.
Further, cell is the cell in the 2nd ~ 7 generation.
The embodiment of the present invention also provides a kind of cells frozen storing liquid, and cells frozen storing liquid is applied to as any in claim 1-4
The cell freezing method of one, cells frozen storing liquid includes：DMEM/F12, DMSO, glycerine and serum substitute；
Wherein, contents of the DMSO in cells frozen storing liquid is 2-10% DMEM/F12 volume；Glycerine is in cells frozen storing liquid
Content is 5-30% DMEM/F12 volume；Content of the serum substitute in cells frozen storing liquid is 20-60% DMEM/F12
Volume；
Serum substitute includes HES, glucan, human serum albumin, restructuring human transferrin, people AB serum, epidermis life
The long factor, rh-insulin, progesterone, hydrocortisone, putrescine, sodium selenate and flavones.
Further, the concentration of HES is 0.1-10%W/V, and the concentration of glucan is 0.1-10%W/V, and human blood is white
The concentration of albumen is 0.1-15%W/V, and the concentration of restructuring human transferrin is 1-10%W/V, and the concentration of people's AB serum is 1-10%W/
V, the concentration of EGF is 0.01-10%W/V, and the concentration of rh-insulin is 1-10%W/V, and the concentration of progesterone is
0.01-5%W/V, the concentration of hydrocortisone is 0.01-5%W/V, and the concentration of putrescine is 0.01-5%W/V, and the concentration of sodium selenate is
0.1-5%W/V, the concentration of flavones is 0.01-5%W/V.
Further, content of the glycerine in cells frozen storing liquid is 10-25% DMEM/F12 volume.It is preferred that, glycerine
The volume for the DMEM/F12 that content in cells frozen storing liquid is 15-20%.
Further, content of the serum substitute in cells frozen storing liquid is 30-55% DMEM/F12 volume.It is preferred that
, content of the serum substitute in cells frozen storing liquid is 35-50% DMEM/F12 volume.
It is preferred that, contents of the DMSO in cells frozen storing liquid is 5-7% DMEM/F12 volume.
Further, DMEM/F12 is DMEM nutrient solutions and F12 nutrient solutions with volume ratio 3:1 ratio mixing.
The embodiment of the present invention provides a kind of cell freezing method, and cell freezing method includes following two steps：Step 1:
The cells frozen storing liquid for being mixed into cell is loaded into container 1, then container 1 is loaded into container 2, wherein, formed between container 1 and container 2
Vacuum；Step 2: performing following tetra- steps of A-D according to this to the container 2 obtained by step one：A, using normal temperature is air-cooled make container 2
Temperature drop to 4 DEG C from normal temperature, wherein, the speed of temperature drop declines 3-4 DEG C to be per minute；B, make container 2 using refrigerator
Temperature drop to -20 DEG C from 4 DEG C, wherein, the speed of temperature drop declines 1-2 DEG C to be per minute；C, make container 2 using refrigerator
Temperature drop to -80 DEG C from -20 DEG C, wherein, the speed of temperature drop declines 2-3 DEG C to be per minute；D, container 2 is put into liquid
Frozen in nitrogen tank, wherein, the temperature of liquid nitrogen container is -196 DEG C.Description based on above-described embodiment, due in the cells frozen storing liquid
It is applied widely in the absence of animality virus spread risk without serum.After being thawed by the cell of long-term frozen of the present invention,
Cell recovery rate is up to that cytoactive after more than 90%, recovery is high, non-aging, can meet what a variety of different cell long-periods froze
Need.
Brief description of the drawings
Fig. 1 is that cell freezing method provided in an embodiment of the present invention freezes the cell of 6 months and freezes 6 using existing method
Cellular morphology before and after the recovery of the cell of individual month compares figure；
Fig. 2 is that cell freezing method provided in an embodiment of the present invention is frozen the cell of 6 months and frozen 6 months using existing method
The anabiosis rate of cell compare figure；
Fig. 3 is that cells frozen storing liquid provided in an embodiment of the present invention freezes the cell of 6 months and freezes 6 months thin using existing liquid
Cellular morphology before and after the recovery of born of the same parents compares figure；
Fig. 4 is that cells frozen storing liquid provided in an embodiment of the present invention freezes the cell of 6 months and freezes 6 months thin using existing liquid
The anabiosis rate of born of the same parents compares figure.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is retouched in detail
State.
Embodiment 1
The embodiment of the present invention provides a kind of cell freezing method, and the cell freezing method includes following two steps：
Step 1: the cells frozen storing liquid for being mixed into cell is loaded into container 1, then the container 1 is loaded into container 2, wherein, it is described to hold
Vacuum is formed between device 1 and the container 2；
Step 2: performing following tetra- steps of A-D according to this to the container 2 obtained by step one：
A, using the air-cooled temperature for making the container 2 of normal temperature from normal temperature 4 DEG C are dropped to, wherein, the speed of temperature drop is every point
Clock declines 3-4 DEG C；
B, using refrigerator the temperature of the container 2 is set to drop to -20 DEG C from 4 DEG C, wherein, the speed of temperature drop is under per minute
1-2 DEG C of drop；
C, using refrigerator the temperature of the container 2 is set to drop to -80 DEG C from -20 DEG C, wherein, the speed of temperature drop is per minute
Decline 2-3 DEG C；
D, the container 2 is put into liquid nitrogen container frozen, wherein, the temperature of the liquid nitrogen container is -196 DEG C.
In a kind of possible implementation, by epidermal stem cells, fibroblast, fat stem cell, melanocyte,
The primary cell of Buccal mucosa cell is mixed into frozen stock solution, is respectively charged into after 1ml centrifuge tubes and is sealed；Frozen stock solution is by DMEM/
F12, DMSO, glycerine and serum substitute composition, the serum substitute is formed sediment by ethoxy, glucan, human serum albumin, restructuring
Human transferrin, people AB serum, EGF, rh-insulin, progesterone, hydrocortisone, putrescine, sodium selenate, Huang
Ketone, dexamethasone composition.
Then the 1ml centrifuge tubes that will be equipped with primary cell are fitted into 15ml centrifuge tubes and seal closed, with cold wind with 3 DEG C of drop
Warm speed is cooled to 4 DEG C；
4 DEG C of 15ml centrifuge tubes are put to refrigerator 180 minutes, -20 DEG C are down to 1 DEG C of rate of temperature fall；
Then -20 DEG C of 15ml centrifuge tubes are put rapidly to -80 DEG C of refrigerators and frozen 8 hours；
- 196 DEG C of liquid nitrogen is finally moved directly to freeze.
It is in alternatively possible implementation, epidermal stem cells, fibroblast, fat stem cell, melanin is thin
Born of the same parents, the primary cell of Buccal mucosa cell are mixed into frozen stock solution, are respectively charged into after 1ml centrifuge tubes and are sealed；Frozen stock solution by
DMEM/F12, DMSO, glycerine and serum substitute composition, the serum substitute is formed sediment by ethoxy, glucan, the white egg of human blood
In vain, restructuring human transferrin, people AB serum, EGF, rh-insulin, progesterone, hydrocortisone, putrescine, selenium
Sour sodium, flavones, dexamethasone composition.
Then the 1ml centrifuge tubes that will be equipped with primary cell are fitted into 15ml centrifuge tubes and seal closed, with cold wind with 4 DEG C of drop
Warm speed is cooled to 4 DEG C；
4 DEG C of 15ml centrifuge tubes are put to refrigerator 90 minutes, -20 DEG C are down to 2 DEG C of rate of temperature fall；
Then -20 DEG C of 15ml centrifuge tubes are put rapidly to -80 DEG C of refrigerators and freezes 12 lab scales；
- 196 DEG C of liquid nitrogen is finally moved directly to freeze.
It should be noted that the cell of 6 months will be frozen by cryopreservation methods provided in an embodiment of the present invention, by direct
Place in 40 DEG C of waters and recover, observation freezes the preceding form with cell after recovery.Cell with freezing 6 months using existing method
Compare, it will be seen from figure 1 that the cryopreservation methods of the application present invention, the cell before freezing is non-with freezing the cellular morphology after 6 months
It is often close, have almost no change, illustrate that cryopreservation methods of the present invention can play a very good protection freezing process to cell.
Meanwhile, cryopreservation methods provided in an embodiment of the present invention and existing conventional cryopreservation methods are frozen into 6 months thin
Born of the same parents, directly place in 42 DEG C of waters and recover 3 minutes, and the anabiosis rate of cell after observation recovery, specific experiment result is shown in Fig. 2.Pass through
Fig. 2 has found that cryopreservation methods cell recovery rate of the present invention is substantially better than existing cryopreservation methods.
The embodiment of the present invention provides a kind of cell freezing method, and cell freezing method includes following two steps：Step 1:
The cells frozen storing liquid for being mixed into cell is loaded into container 1, then container 1 is loaded into container 2, wherein, formed between container 1 and container 2
Vacuum；Step 2: performing following tetra- steps of A-D according to this to the container 2 obtained by step one：A, using normal temperature is air-cooled make container 2
Temperature drop to 4 DEG C from normal temperature, wherein, the speed of temperature drop declines 3-4 DEG C to be per minute；B, make container 2 using refrigerator
Temperature drop to -20 DEG C from 4 DEG C, wherein, the speed of temperature drop declines 1-2 DEG C to be per minute；C, make container 2 using refrigerator
Temperature drop to -80 DEG C from -20 DEG C, wherein, the speed of temperature drop declines 2-3 DEG C to be per minute；D, container 2 is put into liquid
Frozen in nitrogen tank, wherein, the temperature of liquid nitrogen container is -196 DEG C.Description based on above-described embodiment, due in the cells frozen storing liquid
It is applied widely in the absence of animality virus spread risk without serum.After being thawed by the cell of long-term frozen of the present invention,
Cell recovery rate is up to that cytoactive after more than 90%, recovery is high, non-aging, can meet what a variety of different cell long-periods froze
Need.
Embodiment 2
The embodiment of the present invention provides a kind of cells frozen storing liquid, and the cells frozen storing liquid is applied to such as any one institute in embodiment 1
The cell freezing method stated, the cells frozen storing liquid includes：DMEM/F12, DMSO, glycerine and serum substitute；
Wherein, contents of the DMSO in the cells frozen storing liquid is 2-10% DMEM/F12 volume；The glycerine
The volume for the DMEM/F12 that content in the cells frozen storing liquid is 5-30%；The serum substitute is in the cell
The volume for the DMEM/F12 that content in frozen stock solution is 20-60%；
The serum substitute includes HES, glucan, human serum albumin, restructuring human transferrin, people AB serum, table
Skin growth factor, rh-insulin, progesterone, hydrocortisone, putrescine, sodium selenate and flavones.
Further, the concentration of the HES is 0.1-10%W/V, and the concentration of the glucan is 0.1-10%W/
V, the concentration of the human serum albumin is 0.1-15%W/V, and the concentration of the restructuring human transferrin is 1-10%W/V, the people
The concentration of AB serum is 1-10%W/V, and the concentration of the EGF is 0.01-10%W/V, the rh-insulin's
Concentration is 1-10%W/V, and the concentration of the progesterone is 0.01-5%W/V, and the concentration of the hydrocortisone is 0.01-5%W/V, institute
The concentration for stating putrescine is 0.01-5%W/V, and the concentration of the sodium selenate is 0.1-5%W/V, and the concentration of the flavones is 0.01-5%
W/V。
In a kind of possible implementation, cells frozen storing liquid provided in an embodiment of the present invention is by following proportional arrangement：10%
DMEM/F12 volumes DMSO, the glycerine of 30% DMEM/F12 volumes, the serum substitute and body of 50%DMEM/F12 volumes
The DMEM/F12 of product ratio 10%, wherein serum substitute proportion of composing is as follows：
10%（W/V）HES, 0.1%（W/V）Glucan, 15%（W/V）Human serum albumin, 5%（W/V）Recombined human turns iron egg
In vain, 10%（W/V）People AB serum, 0.01%（W/V）EGF, 10%（W/V）Rh-insulin, 5%（W/V）Progesterone,
5%（W/V）Hydrocortisone, 0.01%（W/V）Putrescine, 5%（W/V）Sodium selenate, 0.01%（W/V）Flavones is constituted.
The epidermal stem cells of equivalent amount, fibroblast, fat stem cell, melanocyte are mixed into above-mentioned preparation
After frozen stock solution, liquid nitrogen cryopreservation is recovered 2 minutes after 6 months in 42 DEG C of waters, and observation freezes form such as Fig. 3 of front and rear cell.
From figure 3, it can be seen that with the frozen stock solution of the present invention, the cell before freezing is very close with freezing the cellular morphology after 6 months, several
Do not change, illustrate that frozen stock solution of the present invention serves protective effect well to freezing for cell.
In alternatively possible implementation, cells frozen storing liquid provided in an embodiment of the present invention is by following proportional arrangement：
The DMSO of 2%DMEM/F12 volumes, the glycerine of 30% DMEM/F12 volumes, the blood serum substituting of 20%DMEM/F12 volumes
The DMEM/F12 of thing and volume ratio 48%；Wherein serum substitute proportion of composing is as follows：
0.1%（W/V）HES, 10%（W/V）Glucan, 10%（W/V）Human serum albumin, 1%（W/V）Recombined human turns iron egg
In vain, 10%（W/V）People AB serum, 0.5%（W/V）EGF, 10%（W/V）Rh-insulin, 0.01%（W/V）It is pregnant
Ketone, 0.01%（W/V）Hydrocortisone, 0.5%（W/V）Putrescine, 0.01%（W/V）Sodium selenate, 5%（W/V）Flavones is constituted.
Epidermal stem cells, fibroblast, fat stem cell, melanocyte, keratocyte, oral cavity gingival cell are mixed
After the frozen stock solution and commercial serum-free frozen stock solution that enter above-mentioned preparation, liquid nitrogen cryopreservation is after 6 months, with identical conventional method for resuscitation 40
DEG C recovery 2 minutes, cell recovery rate result is specifically shown in Fig. 4.
You need to add is that, epidermal stem cells, fibroblast, fat stem cell, melanocyte are mixed into above-mentioned system
After standby frozen stock solution and commercial serum-free frozen stock solution, liquid nitrogen cryopreservation after being recovered with identical method for resuscitation, is passed after 6 months
It is commissioned to train foster, observation cell starts aging generation, and concrete outcome is shown in Table 1.
Table 1
| The present invention | It is commercialized serum-free frozen stock solution | |
| Epidermal stem cells | P15 | P9 |
| Fibroblast | P21 | P10 |
| Fat stem cell | P15 | P12 |
| Melanocyte | P18 | P9 |
| Mucous membrane of mouth model | P24 | P20 |
Pass through table 1, it can be seen that it is substantially big that the cell that the frozen stock solution that the present invention is provided freezes starts aging generation after recovery
In commercialization serum-free frozen stock solution, illustrate the frozen stock solution that provides of the present invention freeze after cell activity or performance better than being commercialized
Serum-free frozen stock solution.
Supplement is also needed to, existing conventional cryopreservation is frozen into cell use after 6 months with provided in an embodiment of the present invention
The method for resuscitation recovery that the embodiment of the present invention is mentioned, then with commercialization serum-free DMEM/F-12 continuous passage cultures, daily more
Liquid, observation cell starts the number of days of aging, and specific experiment the results are shown in Table.
Table 2
| The present invention | Existing cryopreservation methods | |
| Epidermal stem cells | 10th day | 6th day |
| Fibroblast | 12nd day | 7th day |
| Fat stem cell | 18th day | 10th day |
| Melanocyte | 14th day | 9th day |
| Mucous membrane of mouth model | 24th day | 21st day |
Pass through table 2, it can be seen that the number of days that the cell that the cryopreservation methods that the present invention is provided freeze starts aging after recovery is obvious
It is later than the activity or performance of the cell after existing cryopreservation methods, i.e. the explanation cryopreservation methods of the invention provided freeze better than existing
The cell of cryopreservation methods.
It is described above, the only embodiment of the application, but the protection domain of the application is not limited thereto, and it is any
Change or replacement in the technical scope that the application is disclosed, should all cover within the protection domain of the application.Therefore, this Shen
Protection domain please should be based on the protection scope of the described claims.
Claims (9)
1. a kind of cell freezing method, it is characterised in that the cell freezing method includes following two steps：
Step 1: the cells frozen storing liquid for being mixed into cell is loaded into container 1, then the container 1 is loaded into container 2, wherein, it is described to hold
Vacuum is formed between device 1 and the container 2；
Step 2: performing following tetra- steps of A-D according to this to the container 2 obtained by step one：
A, using the air-cooled temperature for making the container 2 of normal temperature from normal temperature 4 DEG C are dropped to, wherein, the speed of temperature drop is every point
Clock declines 3-4 DEG C；
B, using refrigerator the temperature of the container 2 is set to drop to -20 DEG C from 4 DEG C, wherein, the speed of temperature drop is under per minute
1-2 DEG C of drop；
C, using refrigerator the temperature of the container 2 is set to drop to -80 DEG C from -20 DEG C, wherein, the speed of temperature drop is per minute
Decline 2-3 DEG C；
D, the container 2 is put into liquid nitrogen container frozen, wherein, the temperature of the liquid nitrogen container is -196 DEG C.
2. cell freezing method according to claim 1, it is characterised in that the step 2 is specifically included：To step one
The container 2 of gained performs following tetra- steps of A-D according to this：
A, using the air-cooled temperature for making the container 2 of normal temperature from normal temperature 4 DEG C are dropped to, wherein, the speed of temperature drop is every point
Clock declines 4 DEG C；
B, using refrigerator the temperature of the container 2 is set to drop to -20 DEG C from 4 DEG C, wherein, the speed of temperature drop is under per minute
1 DEG C of drop；
C, using refrigerator the temperature of the container 2 is set to drop to -80 DEG C from -20 DEG C, wherein, the speed of temperature drop is per minute
Decline 2 DEG C；
D, the container 2 is put into liquid nitrogen container frozen, wherein, the temperature of the liquid nitrogen container is -196 DEG C.
3. cell freezing method according to claim 1 or 2, it is characterised in that the cell is epidermal stem cells, into fibre
Tie up cell, keratocyte, fat stem cell, melanocyte, Buccal mucosa cell or oral cavity gingival cell.
4. cell freezing method according to claim 3, it is characterised in that the cell is the cell in the 2nd ~ 7 generation.
5. a kind of cells frozen storing liquid, it is characterised in that the cells frozen storing liquid is applied to such as any one institute in claim 1-4
The cell freezing method stated, the cells frozen storing liquid includes：DMEM/F12, DMSO, glycerine and serum substitute；
Wherein, contents of the DMSO in the cells frozen storing liquid is 2-10% DMEM/F12 volume；The glycerine
The volume for the DMEM/F12 that content in the cells frozen storing liquid is 5-30%；The serum substitute is in the cell
The volume for the DMEM/F12 that content in frozen stock solution is 20-60%；
The serum substitute includes HES, glucan, human serum albumin, restructuring human transferrin, people AB serum, table
Skin growth factor, rh-insulin, progesterone, hydrocortisone, putrescine, sodium selenate and flavones.
6. cells frozen storing liquid according to claim 5, it is characterised in that the concentration of the HES is 0.1-10%
W/V, the concentration of the glucan is 0.1-10%W/V, and the concentration of the human serum albumin is 0.1-15%W/V, the recombined human
The concentration of transferrins is 1-10%W/V, and the concentration of the people AB serum is 1-10%W/V, the concentration of the EGF
For 0.01-10%W/V, the concentration of the rh-insulin is 1-10%W/V, and the concentration of the progesterone is 0.01-5%W/V, institute
The concentration for stating hydrocortisone is 0.01-5%W/V, and the concentration of the putrescine is 0.01-5%W/V, and the concentration of the sodium selenate is
0.1-5%W/V, the concentration of the flavones is 0.01-5%W/V.
7. the cells frozen storing liquid according to claim 5 or 6, it is characterised in that the glycerine is in the cells frozen storing liquid
Content for 10-25% the DMEM/F12 volume.
8. the cells frozen storing liquid according to claim 5 or 6, it is characterised in that the serum substitute freezes in the cell
The volume for the DMEM/F12 that content in liquid storage is 30-40%.
9. the cells frozen storing liquid according to claim 5 or 6, it is characterised in that the DMEM/F12 be DMEM nutrient solutions and
F12 nutrient solutions are with volume ratio 3:1 ratio mixing.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108739795A (en) * | 2018-05-30 | 2018-11-06 | 广州沙艾生物科技有限公司 | A kind of fat stem cell frozen stock solution and its cryopreservation methods |
| CN109699634A (en) * | 2019-01-31 | 2019-05-03 | 和携科技(北京)有限公司 | A kind of Cryopreservation and method for resuscitation of mescenchymal stem cell |
| CN111066777A (en) * | 2019-12-19 | 2020-04-28 | 镇江雷音再生医学科技有限公司 | Corneal lens ultralow-temperature long-term storage method |
| CN112544613A (en) * | 2020-12-25 | 2021-03-26 | 武汉睿健医药科技有限公司 | Pluripotent stem cell cryopreservation liquid, application thereof and cryopreservation method |
| CN114586769A (en) * | 2020-12-07 | 2022-06-07 | 精准生技股份有限公司 | Serum-free cell cryopreservation liquid for cell cryopreservation and cryopreservation method thereof |
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Citations (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2680019Y (en) * | 2003-11-21 | 2005-02-23 | 李治国 | Biologically active substance conservation device |
| CN103548815A (en) * | 2013-11-18 | 2014-02-05 | 王意 | Programmed cell freezing box with direct-in liquid nitrogen |
| CN203776007U (en) * | 2014-04-02 | 2014-08-20 | 新乡学院 | Liquid nitrogen container for long-term storage of cells and biological materials |
| CN104770363A (en) * | 2015-04-21 | 2015-07-15 | 广州赛莱拉干细胞科技股份有限公司 | Cryopreservation solution and cryopreservation method for mesenchymal stem cells |
| CN205082557U (en) * | 2015-10-04 | 2016-03-16 | 北京弘润天源生物技术股份有限公司 | High -efficient constant temperature cell cryopreserving box |
| CN105638642A (en) * | 2016-01-27 | 2016-06-08 | 上海润泉生物技术有限公司 | Immune cell cryopreservation solution and application thereof |
| CN205449637U (en) * | 2016-04-12 | 2016-08-10 | 刘德庆 | Tissue piece storage cases |
| CN105850982A (en) * | 2016-04-29 | 2016-08-17 | 绍兴市人民医院 | Novel tissue cryopreservation liquid |
| CN105941389A (en) * | 2016-05-03 | 2016-09-21 | 上海安集协康生物技术股份有限公司 | Animal derived serum-free cell freezing medium |
| CN106148163A (en) * | 2015-04-22 | 2016-11-23 | 王金 | A kind of biomaterial storage device and application thereof |
| CN106359368A (en) * | 2016-09-30 | 2017-02-01 | 广州赛莱拉干细胞科技股份有限公司 | Cell cryoprotectant and cryopreservation method |
| CN106417253A (en) * | 2016-09-30 | 2017-02-22 | 广州赛莱拉干细胞科技股份有限公司 | Cryopreservation liquid and method for skeletal muscle stem cells |
| CN106477175A (en) * | 2016-11-24 | 2017-03-08 | 徐伟强 | Gas phase liquified nitrogen biology holding vessel |
| CN108739795A (en) * | 2018-05-30 | 2018-11-06 | 广州沙艾生物科技有限公司 | A kind of fat stem cell frozen stock solution and its cryopreservation methods |
| CN108820581A (en) * | 2018-07-12 | 2018-11-16 | 山前（珠海）医疗科技有限公司 | A kind of biological sample storage system |
| CN109601529A (en) * | 2019-02-25 | 2019-04-12 | 安徽古井贡酒股份有限公司 | A kind of cell cryopreservation and method for resuscitation of simplicity |
| CN111088153A (en) * | 2019-11-19 | 2020-05-01 | 嘉兴聚力检测技术服务有限公司 | Novel freezing tube |
| CN214190915U (en) * | 2020-12-24 | 2021-09-14 | 山东省海洋生物研究院 | Sperm cryopreservation box and inner box taking-out device |
| CN215836776U (en) * | 2021-09-27 | 2022-02-18 | 上海轩一生物科技有限公司 | Animal liver cell freezing tube with high survival rate |
-
2017
- 2017-06-18 CN CN201710460512.XA patent/CN107258766A/en active Pending
Patent Citations (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2680019Y (en) * | 2003-11-21 | 2005-02-23 | 李治国 | Biologically active substance conservation device |
| CN103548815A (en) * | 2013-11-18 | 2014-02-05 | 王意 | Programmed cell freezing box with direct-in liquid nitrogen |
| CN203776007U (en) * | 2014-04-02 | 2014-08-20 | 新乡学院 | Liquid nitrogen container for long-term storage of cells and biological materials |
| CN104770363A (en) * | 2015-04-21 | 2015-07-15 | 广州赛莱拉干细胞科技股份有限公司 | Cryopreservation solution and cryopreservation method for mesenchymal stem cells |
| CN106148163A (en) * | 2015-04-22 | 2016-11-23 | 王金 | A kind of biomaterial storage device and application thereof |
| CN205082557U (en) * | 2015-10-04 | 2016-03-16 | 北京弘润天源生物技术股份有限公司 | High -efficient constant temperature cell cryopreserving box |
| CN105638642A (en) * | 2016-01-27 | 2016-06-08 | 上海润泉生物技术有限公司 | Immune cell cryopreservation solution and application thereof |
| CN205449637U (en) * | 2016-04-12 | 2016-08-10 | 刘德庆 | Tissue piece storage cases |
| CN105850982A (en) * | 2016-04-29 | 2016-08-17 | 绍兴市人民医院 | Novel tissue cryopreservation liquid |
| CN105941389A (en) * | 2016-05-03 | 2016-09-21 | 上海安集协康生物技术股份有限公司 | Animal derived serum-free cell freezing medium |
| CN106359368A (en) * | 2016-09-30 | 2017-02-01 | 广州赛莱拉干细胞科技股份有限公司 | Cell cryoprotectant and cryopreservation method |
| CN106417253A (en) * | 2016-09-30 | 2017-02-22 | 广州赛莱拉干细胞科技股份有限公司 | Cryopreservation liquid and method for skeletal muscle stem cells |
| CN106477175A (en) * | 2016-11-24 | 2017-03-08 | 徐伟强 | Gas phase liquified nitrogen biology holding vessel |
| CN108739795A (en) * | 2018-05-30 | 2018-11-06 | 广州沙艾生物科技有限公司 | A kind of fat stem cell frozen stock solution and its cryopreservation methods |
| CN108820581A (en) * | 2018-07-12 | 2018-11-16 | 山前（珠海）医疗科技有限公司 | A kind of biological sample storage system |
| CN109601529A (en) * | 2019-02-25 | 2019-04-12 | 安徽古井贡酒股份有限公司 | A kind of cell cryopreservation and method for resuscitation of simplicity |
| CN111088153A (en) * | 2019-11-19 | 2020-05-01 | 嘉兴聚力检测技术服务有限公司 | Novel freezing tube |
| CN214190915U (en) * | 2020-12-24 | 2021-09-14 | 山东省海洋生物研究院 | Sperm cryopreservation box and inner box taking-out device |
| CN215836776U (en) * | 2021-09-27 | 2022-02-18 | 上海轩一生物科技有限公司 | Animal liver cell freezing tube with high survival rate |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108739795A (en) * | 2018-05-30 | 2018-11-06 | 广州沙艾生物科技有限公司 | A kind of fat stem cell frozen stock solution and its cryopreservation methods |
| CN109699634A (en) * | 2019-01-31 | 2019-05-03 | 和携科技(北京)有限公司 | A kind of Cryopreservation and method for resuscitation of mescenchymal stem cell |
| CN111066777A (en) * | 2019-12-19 | 2020-04-28 | 镇江雷音再生医学科技有限公司 | Corneal lens ultralow-temperature long-term storage method |
| CN114586769A (en) * | 2020-12-07 | 2022-06-07 | 精准生技股份有限公司 | Serum-free cell cryopreservation liquid for cell cryopreservation and cryopreservation method thereof |
| CN112544613A (en) * | 2020-12-25 | 2021-03-26 | 武汉睿健医药科技有限公司 | Pluripotent stem cell cryopreservation liquid, application thereof and cryopreservation method |
| CN115943949A (en) * | 2022-12-28 | 2023-04-11 | 博生吉安科细胞技术有限公司 | Cell cryopreservation solution and preparation method and application thereof |
| CN115943949B (en) * | 2022-12-28 | 2024-04-02 | 博生吉安科细胞技术有限公司 | Cell freezing solution and preparation method and application thereof |
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