CN108094412A - A kind of novel freezing protective agent preserved for cell freezing and its application - Google Patents
A kind of novel freezing protective agent preserved for cell freezing and its application Download PDFInfo
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- CN108094412A CN108094412A CN201810172654.0A CN201810172654A CN108094412A CN 108094412 A CN108094412 A CN 108094412A CN 201810172654 A CN201810172654 A CN 201810172654A CN 108094412 A CN108094412 A CN 108094412A
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- cell
- freezing
- hydrogen bond
- cell suspension
- cryoprotector
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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
Abstract
Disclosure sets forth a kind of novel freezing protective agent preserved for cell freezing and its applications.The frozen solution is made of hydrogen bond donor and hydrogen bond receptor, and the hydrogen bond donor and the hydrogen bond receptor form eutectic eutectic mixture by hydrogen bond association, and the molar ratio of the hydrogen bond donor and the hydrogen bond receptor is 1.3:1~15:1.The cryoprotector can reduce ice crystal in cell freezing preservation and form damage to cell, and this novel freezing preservative agent prepares simple, possesses the advantages that biocompatibility, degradable.
Description
Technical field
The present invention relates to cell freezing protection field more particularly to a kind of novel freezing protections preserved for cell freezing
Agent and application.
Background technology
In order to meet basic research and clinical practice, it is necessary to largely be expanded to cell.But how to extend the goods of cell
The frame phase is the huge bottleneck valency faced in cell research and clinical practice.Freezen protective is to extend its shelf life, realizes the length of cell
Phase stablizes the effective ways preserved.Cell freezing method mainly has sequencing freezing and glass freezing.In recent years, vitrifying was cold
Freeze due to not generating ice crystal during preserving, it is of increased attention.It is a kind of utilization high concentration cryoprotection
The method that the hypervelocity of agent solution freezes organism implements fast cooling and heating in process of cryopreservation, is possible to be implemented as
The glass freezing of work(.But process is difficult to scale.And sequencing freezing can overcome this problem, but the ice crystal of refrigerating process
And solute effect can generate irreversible injury to cell.Simultaneously because the toxicity of cryoprotector, also limits it thin
Clinical practice in born of the same parents' treatment.
Eutectic solvent is usually easy to get by two or three and the component of safety is mutually associated by interaction of hydrogen bond,
Form eutectic mixture.Obtained eutectic solvent is characterized in that fusing point is less than the fusing point of each component.In general, it is low common
Molten solvent is that have very big freeze point depression, and this eutectic solvent has some specific physicals highly useful to bioprocess
Character, and with good biocompatibility and degradation property.
Domestic and international many researchers attempt, by improving cryogenic freezing method, new cryoprotector to be screened, to find
Reliable and stable cell freezing Techniques of preserving.Although having made some progress in these areas, but still without simple, it is reliable and
Cheap method carrys out frozen cell.The present invention provides a kind of new cryoprotectors preserved for cell freezing, have nothing
Malicious safety or easily biological-degradable, it is easy to operate and cheap, with obvious effects, it has a extensive future.
The content of the invention
Limitation according to prior art, the purpose of the present invention is provides a kind of cell novel freezing preservative agent, the present invention
A kind of novel freezing protective agent preserved for cell freezing is provided, is made of hydrogen bond donor and hydrogen bond receptor, the hydrogen bond supplies
Body and the hydrogen bond receptor form cryoprotector by hydrogen bond association, and the cryoprotector is eutectic eutectic mixture,
The molar ratio of the hydrogen bond donor and the hydrogen bond receptor is 1.3:1~15:1.
As improved technical solution, the hydrogen bond donor includes one or both of choline chloride and glycine betaine；Institute
Stating hydrogen bond donor includes the one or more in urea, ethylene glycol, glycerine, glucose, fructose and citric acid.
As improved technical solution, concentration of the cryoprotector in process of cryopreservation between 0.5M~
7M。
As improved technical solution, concentration of the cryoprotector in process of cryopreservation between 1M~
6M。
The present invention also provides a kind of methods using above-mentioned cryoprotector, include the following steps：
Step 1：Obtain cell suspension；
Step 2：Prepare the mixture of cryoprotector and cell suspension；
Step 3：Freezen protective；
Step 4：Defrosting cell suspension；
Step 5：Identification of cell survival rate.
As improved technical solution, the step 1 is：Culture solution is removed after cultured cartilage cell, digestive juice is added to digest
The cartilage cell from culture dish bottom is blown afloat and sucked in centrifuge tube by 3min~5min, and gravity treatment is concentration between 1x107/
The cell suspension of ml~1x108/ml.
As improved technical solution, the step 2 is：It is 2M that frozen solution, which is mixed with cell culture fluid to concentration,
The mixture of~3M.
As improved technical solution, the step 3 is：The mixture of the cryoprotector and cell suspension is slow
Cell freezing pipe is packed into, marks cell and date, -80 DEG C~-60 DEG C preservations.
As improved technical solution, the step 4 is：It takes out the cell freezing pipe and is immediately placed in 37 DEG C of water-bath
Middle defrosting after 10min~15min, takes out the cell freezing pipe and sterilizes and open, suction out the cell suspension, inject from
Heart pipe simultaneously washs, and supernatant is abandoned in centrifugation.
As improved technical solution, the step 5 is：Trypan blue solution dyeing, institute are added in the cell suspension
It is 1 to state the addition volume of trypan blue solution and the volume ratio of the cell suspension:1~1:2, the cell is observed under the microscope
Suspension simultaneously calculates Cell viability.
Advantageous effect
Compared with prior art, the present invention has the following advantages and beneficial effect：
1st, novel freezing protective agent provided by the invention can reduce ice crystal in cell freezing preservation and form damage to cell
Wound, and this novel freezing preservative agent is prepared simply, possesses the advantages that biocompatibility, solidification point is low, degradable；
2nd, novel freezing protective agent provided by the invention is nontoxic to generate toxic action to avoid to cell；
3rd, novel freezing protective agent provided by the invention is capable of being fast degraded, will not long-term existence in cell liquid, can avoid
Cytoactive is had an impact.
Specific embodiment
To make the purpose of the embodiment of the present invention and technical solution clearer, below in conjunction with the embodiment of the present invention to this hair
Bright technical solution is clearly and completely described.Obviously, described embodiment is the part of the embodiment of the present invention, and
The embodiment being not all of.Based on described the embodiment of the present invention, those of ordinary skill in the art are without creative labor
All other embodiments obtained on the premise of dynamic, belong to the scope of protection of the invention.
Those skilled in the art of the present technique are appreciated that unless otherwise defined all terms used herein are (including technology art
Language and scientific terminology) there is the meaning identical with the general understanding of the those of ordinary skill in fields of the present invention.Should also
Understand, those terms such as defined in the general dictionary, which should be understood that, to be had and the meaning in the context of the prior art
The consistent meaning of justice, and unless defined as here, will not be with idealizing or the meaning of overly formal be explained.
Comparative example 1
1. removing culture solution after cultured chondrocytes, add digestive juice 1mL, digest 3min；Add culture medium about 2mL, neutralization disappears
Change liquid, and cell is blown afloat from culture dish bottom, cell is sucked in centrifuge tube, supernatant liquor is abandoned in 800r/min, 5min centrifugation；
Gravity treatment is 1x107The cell suspension of/ml；
2. prepare frozen solution：Ordinary cells cryoprotector (DMSO) is mixed with cell culture fluid to concentration and is
3M；
3. being slowly added same volume frozen solution to cell suspension, cryovial is packed into, cell and date is marked, uses cotton
Flower package cryovial, is put into tulle pouch, places in -80 DEG C of refrigerators；
It 4. taking out cell freezing pipe from refrigerator with long forceps, puts into the stainless steel cup for filling 37 DEG C of warm water, covers immediately
Upper cover is simultaneously shaken frequently, is thawed as early as possible.Cryovial is taken out from 37 DEG C of water-baths after 10min, after alcohol or cotton ball soaked in alcohol disinfection,
Nut is opened, cell suspension is suctioned out with suction pipe, inject centrifuge tube and adds cleaning solution to 10mL, after mixing, 2000r/min, centrifugation
5min abandons supernatant；
5. few cells is taken to carry out Trypan Blue with identification of cell survival rate.The additive amount and cell of trypan blue solution hang
Liquid is 100 μ L/100 μ L.The two is uniformly mixed, stands 3min, draws mixed liquor, micro- Microscopic observation counts, all refractivities
By force without tinter be living cells, colors blue person be dead cell, Cell viability 60%.
Embodiment 1
1. removing culture solution after cultured chondrocytes, add digestive juice 1mL, digest 3min；Add culture medium about 2mL, neutralization disappears
To change liquid, and cell is blown afloat from culture dish bottom, cell is sucked in centrifuge tube, supernatant liquor is abandoned in 800r/min, 5min centrifugation,
Gravity treatment is 1x107The cell suspension of/ml；
2. prepare frozen solution：With choline chloride and urea (1:1.3) frozen solution is configured based on eutectic, altogether
It is 3M that fusant, which is mixed with cell culture fluid to concentration, and the concentration of cryoprotector is 0.5M；
3. being slowly added same volume frozen solution to cell suspension, cryovial is packed into, cell and date is marked, uses cotton
Flower package cryovial, is put into tulle pouch, places in -80 DEG C of refrigerators；
It 4. taking out cell freezing pipe from refrigerator with long forceps, puts into the stainless steel cup for filling 37 DEG C of warm water, covers immediately
Upper cover is simultaneously shaken frequently, and cryovial is taken out from 37 DEG C of water-baths after 10min, after alcohol or cotton ball soaked in alcohol disinfection, opens nut,
Cell suspension is suctioned out with suction pipe, inject centrifuge tube and adds cleaning solution to 10mL, after mixing, 2000r/min centrifuges 5min, abandons
Clear liquid；
5. few cells is taken to carry out Trypan Blue with identification of cell survival rate.The additive amount and cell of trypan blue solution hang
Liquid is 100 μ L/100 μ L.The two is uniformly mixed, stands 2min~3min, draws mixed liquor, micro- Microscopic observation counts, all
Refractivity is by force living cells without tinter, and colors blue person is dead cell, and Cell viability reaches 92%.
Embodiment 2
1. removing culture solution after osteocyte culture, add digestive juice 1mL, digest 5min；Add culture medium about 2mL, neutralize digestion
Liquid, and cell is blown afloat from culture dish bottom, cell is sucked in centrifuge tube, supernatant liquor is abandoned in 800r/min, 5min centrifugation, weight
Elect 1x10 as8The cell suspension of/ml；
2. prepare frozen solution：With glycine betaine and ethylene glycol, glycerine, glucose (1:2:2:2) match somebody with somebody based on eutectic
Frozen solution is put, it is 3M that eutectic, which is mixed with cell culture fluid to concentration, and the concentration of frozen solution is 7M；
3. being slowly added same volume frozen solution to cell suspension, cryovial is packed into, cell and date is marked, uses cotton
Flower package cryovial, is put into tulle pouch, places in -60 DEG C of refrigerators；
It 4. taking out cell freezing pipe from refrigerator with long forceps, puts into the stainless steel cup for filling 37 DEG C of warm water, covers immediately
Upper cover is simultaneously shaken frequently, is thawed as early as possible, and cryovial is taken out from 37 DEG C of water-baths after 15min, after alcohol or cotton ball soaked in alcohol disinfection,
Nut is opened, cell suspension is suctioned out with suction pipe, inject centrifuge tube and adds cleaning solution to 10mL, after mixing, 2000r/min, centrifugation
5min abandons supernatant；
5. few cells is taken to carry out Trypan Blue with identification of cell survival rate.The additive amount and cell of trypan blue solution hang
Liquid is 100 μ L/200 μ L.The two is uniformly mixed, stands 3min, draws mixed liquor, micro- Microscopic observation counts, all refractivities
It is by force living cells without tinter, colors blue person is dead cell, and Cell viability reaches 93%..
Embodiment 3
1. removing culture solution after human pluripotent stem cells culture, add digestive juice 1mL, digest 4min；Add culture medium about 2mL, in
And digestive juice, and cell is blown afloat from culture dish bottom, cell is sucked in centrifuge tube, upper strata is abandoned in 800r/min, 5min centrifugation
Clear liquid；
2. prepare frozen solution：With choline chloride and fructose and citric acid (1:7:8) freezing is configured based on eutectic
Liquid is protected, it is 2.5M that eutectic, which is mixed with cell culture fluid to concentration, and the concentration of cryoprotector is 1M；
3. being slowly added frozen solution to cell, cryovial is packed into, is positioned in liquid nitrogen.
4. taking out cell freezing pipe from liquid nitrogen container, put into the stainless steel cup for filling 37 DEG C of warm water, close the lid immediately
And shake frequently, it thaws as early as possible.After 12min, cryovial is taken out from 37 DEG C of water-baths, after alcohol or cotton ball soaked in alcohol disinfection, is beaten
Nut is opened, cell suspension is suctioned out with suction pipe, inject centrifuge tube and adds cleaning solution to 10mL, after mixing, 2000r/min, centrifugation
5min abandons supernatant.
5. few cells is taken to carry out Trypan Blue with identification of cell survival rate.The additive amount and cell of trypan blue solution hang
Liquid is 100 μ L/150 μ L.The two is uniformly mixed, stands 3min, draws mixed liquor, micro- Microscopic observation counts, all refractivities
It is by force living cells without tinter, colors blue person is dead cell, and Cell viability reaches 95%.
Embodiment 4
1. removing culture solution after cultured chondrocytes, add digestive juice 1mL, digest 5min；Add culture medium about 2mL, neutralization disappears
To change liquid, and cell is blown afloat from culture dish bottom, cell is sucked in centrifuge tube, supernatant liquor is abandoned in 800r/min, 5min centrifugation,
Gravity treatment is 1x108The cell suspension of/ml；
2. prepare frozen solution：With glycine betaine and ethylene glycol (1:8) frozen solution, congruent melting are configured based on eutectic
It is 3M that object, which is mixed with cell culture fluid to concentration, and the concentration of frozen solution is 6M；
3. being slowly added same volume frozen solution to cell suspension, cryovial is packed into, cell and date is marked, uses cotton
Flower package cryovial, is put into tulle pouch, places in -70 DEG C of refrigerators；
It 4. taking out cell freezing pipe from refrigerator with long forceps, puts into the stainless steel cup for filling 37 DEG C of warm water, covers immediately
Upper cover is simultaneously shaken frequently, and after 10min, cryovial is taken out from 37 DEG C of water-baths, after alcohol or cotton ball soaked in alcohol disinfection, opens spiral shell
Cap suctions out cell suspension with suction pipe, injects centrifuge tube and adds cleaning solution to 10mL, after mixing, 2000r/min centrifuges 5min, abandons
Supernatant；
5. few cells is taken to carry out Trypan Blue with identification of cell survival rate.The additive amount and cell of trypan blue solution hang
Liquid is 100 μ L/100 μ L.The two is uniformly mixed, stands 3min, draws mixed liquor, micro- Microscopic observation counts, all refractivities
It is by force living cells without tinter, colors blue person is dead cell, and Cell viability reaches 90%.
Embodiment 5
1. removing culture solution after cell culture, add digestive juice 1mL, digest 3min；Add culture medium 2mL, neutralize digestive juice, and
Cell from culture dish bottom is blown afloat, cell is sucked in centrifuge tube, supernatant liquor is abandoned in 800r/min, 5min centrifugation, and gravity treatment is
1x107The cell suspension of/ml；
2. prepare frozen solution：With glycine betaine and fructose (1:7) frozen solution, eutectic are configured based on eutectic
It is 3M to be mixed with cell culture fluid to concentration, and the concentration of cryoprotector is 3M；
3. being slowly added same volume frozen solution to cell suspension, cryovial is packed into, cell and date is marked, uses cotton
Flower package cryovial, is put into tulle pouch, places in -80 DEG C of refrigerators；
It 4. taking out cell freezing pipe from refrigerator with long forceps, puts into the stainless steel cup for filling 37 DEG C of warm water, covers immediately
Upper cover is simultaneously shaken frequently, and cryovial is taken out from 37 DEG C of water-baths after 12min, after alcohol or cotton ball soaked in alcohol disinfection, opens nut,
Cell suspension is suctioned out with suction pipe, inject centrifuge tube and adds cleaning solution to 10mL, after mixing, 2000r/min centrifuges 5min, abandons
Clear liquid；
5. few cells is taken to carry out Trypan Blue with identification of cell survival rate.The additive amount and cell of trypan blue solution hang
Liquid is 100 μ L/200 μ L.The two is uniformly mixed, stands 3min, draws mixed liquor, micro- Microscopic observation counts, all refractivities
It is by force living cells without tinter, colors blue person is dead cell, and Cell viability reaches 93%.
Embodiment 6
1. removing culture solution after cell culture, add digestive juice 1mL, digest 4min；Add culture medium about 2mL, neutralize digestive juice,
And cell is blown afloat from culture dish bottom, cell is sucked in centrifuge tube, supernatant liquor is abandoned in 800r/min, 5min centrifugation；Gravity treatment
For 1x107The cell suspension of/ml；
2. prepare frozen solution：With glycine betaine and urea (7:1) frozen solution, eutectic are configured based on eutectic
It is 3M to be mixed with cell culture fluid to concentration, and the concentration of cryoprotector is 4M；
3. being slowly added same volume frozen solution to cell suspension, cryovial is packed into, cell and date is marked, uses cotton
Flower package cryovial, is put into tulle pouch, places in -80 DEG C of refrigerators；
It 4. taking out cell freezing pipe from refrigerator with long forceps, puts into the stainless steel cup for filling 37 DEG C of warm water, covers immediately
Upper cover is simultaneously shaken frequently, and after 15min, cryovial is taken out from 37 DEG C of water-baths, after alcohol or cotton ball soaked in alcohol disinfection, opens spiral shell
Cap suctions out cell suspension with suction pipe, injects centrifuge tube and adds cleaning solution to 10mL, after mixing, 2000r/min centrifuges 5min, abandons
Supernatant；
5. few cells is taken to carry out Trypan Blue with identification of cell survival rate.The additive amount and cell of trypan blue solution hang
Liquid is 100 μ L/150 μ L.The two is uniformly mixed, stands 5min, draws mixed liquor, micro- Microscopic observation counts, all refractivities
It is by force living cells without tinter, colors blue person is dead cell, and Cell viability reaches 95%.
It these are only embodiments of the present invention, description is more specific and in detail, but can not therefore be interpreted as pair
The limitation of the scope of the claims of the present invention.It should be pointed out that for those of ordinary skill in the art, the present invention is not being departed from
On the premise of design, various modifications and improvements can be made, these are all belonged to the scope of protection of the present invention.
Claims (10)
1. a kind of novel freezing protective agent preserved for cell freezing, is made of, feature exists hydrogen bond donor and hydrogen bond receptor
In the hydrogen bond donor and the hydrogen bond receptor form cryoprotector by hydrogen bond association, and the cryoprotector is low common
The molar ratio of molten eutectic mixture, the hydrogen bond donor and the hydrogen bond receptor is 1.3:1~15:1.
2. novel freezing protective agent according to claim 1, which is characterized in that the hydrogen bond donor include choline chloride and
One or both of glycine betaine；The hydrogen bond donor is included in urea, ethylene glycol, glycerine, glucose, fructose and citric acid
It is one or more kinds of.
3. novel freezing protective agent according to claim 1, which is characterized in that the cryoprotector is in freezen protective mistake
Concentration in journey is between 0.5M~7M.
4. novel freezing protective agent according to claim 1, which is characterized in that the cryoprotector is in freezen protective mistake
Concentration in journey is between 1M~6M.
A kind of 5. protectant method of novel freezing applied as described in Claims 1 to 4, which is characterized in that including walking as follows
Suddenly：
Step 1：Obtain cell suspension；
Step 2：Prepare the mixture of cryoprotector and cell suspension；
Step 3：Freezen protective；
Step 4：Defrosting cell suspension；
Step 5：Identification of cell survival rate.
6. the application protectant method of novel freezing according to claim 5, which is characterized in that the step 1 is：Culture
Culture solution is removed after cartilage cell, digestive juice is added to digest 3min~5min, the cartilage cell is blown afloat simultaneously from culture dish bottom
It sucks in centrifuge tube, gravity treatment is concentration between 1x107/ ml~1x108The cell suspension of/ml.
7. the application protectant method of novel freezing according to claim 5, which is characterized in that the step 2 is：It will be cold
Freeze protection liquid to be mixed with cell culture fluid to the mixture that concentration is 2M~3M.
8. the application protectant method of novel freezing according to claim 5, which is characterized in that the step 3 is：By institute
The mixture for stating cryoprotector and cell suspension is slowly packed into cell freezing pipe, marks cell and date, -80 DEG C~-60 DEG C
It preserves.
9. the application protectant method of novel freezing according to claim 5, which is characterized in that the step 4 is：It takes out
The cell freezing pipe and being immediately placed in 37 DEG C of water-bath thaws, and after 10min~15min, takes out the cell freezing pipe simultaneously
It sterilizes and opens, suction out the cell suspension, inject centrifuge tube and wash, supernatant is abandoned in centrifugation.
10. the application protectant method of novel freezing according to claim 5, which is characterized in that the step 5 is：
Trypan blue solution dyeing, the addition volume of the trypan blue solution and the volume of the cell suspension are added in the cell suspension
Than for 1:1~1:2, the cell suspension is observed under the microscope and calculates Cell viability.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101338299A (en) * | 2008-07-29 | 2009-01-07 | 山东省农业科学院畜牧兽医研究所 | Vitrification freezing and storing method of a little somatic cells for cloning domestic animal |
| CN101543210A (en) * | 2009-04-30 | 2009-09-30 | 武汉中博生化有限公司 | Device and method for cryopreserving animal cells in batch |
| WO2014055936A1 (en) * | 2012-10-04 | 2014-04-10 | Integenx Inc. | Preservation of biological materials in non-aqueous fluid media |
| CN105392363A (en) * | 2013-03-01 | 2016-03-09 | A·S·戈尔兹伯勒 | Sample fixation and stabilisation |
-
2018
- 2018-03-01 CN CN201810172654.0A patent/CN108094412A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101338299A (en) * | 2008-07-29 | 2009-01-07 | 山东省农业科学院畜牧兽医研究所 | Vitrification freezing and storing method of a little somatic cells for cloning domestic animal |
| CN101543210A (en) * | 2009-04-30 | 2009-09-30 | 武汉中博生化有限公司 | Device and method for cryopreserving animal cells in batch |
| WO2014055936A1 (en) * | 2012-10-04 | 2014-04-10 | Integenx Inc. | Preservation of biological materials in non-aqueous fluid media |
| CN105392363A (en) * | 2013-03-01 | 2016-03-09 | A·S·戈尔兹伯勒 | Sample fixation and stabilisation |
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| Title |
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| ANA GERTRUDES ET.AL.: ""How Do Animals Survive Extreme Temperature Amplitudes? The Role of Natural Deep Eutectic Solvents"", 《ACS SUSTAINABLE CHEMISTRY ＆ ENGINEERING》 * |
| MARIA C. GUTIERREZ ET.AL.: ""Bacteria Incorporation in Deep-eutectic Solvents through Freeze-Drying"", 《ANGEWANDET CHEMIE INTERNATIONAL EDITION》 * |
| 李玲 等: "《细胞生物学实验》", 31 August 2003, 湖南科学技术出版社 * |
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