CN105394029B - A kind of cells frozen storing liquid for leukemia treating - Google Patents
A kind of cells frozen storing liquid for leukemia treating Download PDFInfo
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- CN105394029B CN105394029B CN201610001794.2A CN201610001794A CN105394029B CN 105394029 B CN105394029 B CN 105394029B CN 201610001794 A CN201610001794 A CN 201610001794A CN 105394029 B CN105394029 B CN 105394029B
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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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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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Abstract
The invention provides a kind of cells frozen storing liquid and preparation method thereof.The cells frozen storing liquid includes the w/v of dimethyl sulfoxide (DMSO) 5%, 0.5% w/v low-density lipoproteins, 1% w/v trehalose, 1% w/v lecithin, polypeptide 1% w/v, bFGF 1% w/v, the w/v of vitamin E 1%, finally with DMEM culture mediums and hyclone according to 1:1 ratio is settled to 100ml.The cells frozen storing liquid that the present invention is provided is used to freeze culture cell, in particular improves the survival rate after freeze-stored cell recovery.With preferable application prospect.
Description
Technical field
The present invention relates to histocyte culture technique field, and in particular to a kind of cells frozen storing liquid and preparation method thereof and should
With.
Background technology
Stem cell（stem cell）It is the multipotential cell that a class has the of self-replication capacity.Under certain condition, it can
To be divided into a variety of functioning cells.Stage of development according to residing for stem cell is divided into embryonic stem cell and adult stem cell.According to
The potentiality of development of stem cell is divided into three classes：Myeloid-lymphoid stem cell, multipotential stem cell and unipotent stem cell（Specially can stem cell）.Stem cell
It is a kind of inabundant differentiation, still jejune cell, the potential function with the various histoorgans of regeneration and human body, medical field claims
For " general-purpose cell ".In all nerve fibers such as brain, spinal cord, the daughter cell species that different NSC types is produced
Difference, distribution is also different.Although the cell will be widely used in clinical treatment field using extensive, it is originated and quantity
Demand far can not still be met.Therefore the external extensive amplification of NSC, and the cell after amplification is freezed
Preserve, be the urgent demand of those skilled in the art.
Leukaemia is to endanger one of important tumour of the mankind, in leukemia treating and research, dry thin for leukaemia
The transient demand amount of born of the same parents is very big, and traditional separation method can not possibly be obtained at once, therefore, and batch is stored, and application is taken out together
It is necessary, but conventional storage method effect is bad, and the method freezed has significant limitation.Because cell
The process frozen can significantly change the thermodynamics of cell, chemically and physically environment, while with the danger of biological injury.Influence
Freezing the factor of efficiency mainly has：Cell concentration, freezing rate and frozen stock solution.At present, the frozen stock solution that stem cell cryopreserving is used
There are two classes containing serum and serum-free, because serum has the shortcomings that complicated component, quality are unstable, expensive, serum-free
Freeze turns into development trend with resuscitation technique.Cord blood stem cell relies primarily on anti frozen liquid dimethyl sulfoxide (DMSO) (DMSO) and serum
Protection.Commonly used cell-protecting also has HES 0ES), polyethylene glycol（PEG) etc..But, DMSO can be to cell
Toxic action is produced, irreversible damage is caused to the stem cell frozen.To obtain the stem cell of convenient sources, carry out effective
Stem cell cryopreserving method is the urgent demand in this area, sets up a kind of prolonged cold and preserves the method for stem cell for promoting nerve
The grinding of stem cell makes internal disorder or usurp and using being significant.
The content of the invention
Liquid and corresponding preparation method are preserved the technical problem to be solved in the present invention is to provide a kind of cell freezing and are made
Use method.
Technical scheme is as follows：
A kind of cell freezing preserves liquid, it is characterised in that be made up of following each component：It is characterized in that containing in freezing liquid storage:
The w/v of dimethyl sulfoxide (DMSO) 5%, 0.5% w/v low-density lipoproteins, 1% w/v trehalose, 1% w/v lecithin, the w/ of polypeptide 1%
The w/v of v, bFGF 1%, the w/v of vitamin E 1%, finally with DMEM culture mediums and hyclone according to 1:1 ratio is settled to
100ml, the sequence such as SEQ ID NO of the polypeptide：Shown in 1-16 is any.The polypeptide has applicant to pass through the substantial amounts of of early stage
Library immunoscreening is obtained, and the polypeptide has the function of protecting cells from damage.Its title and sequence are as follows：BX-1、BX-3、
BX-4、BX-6、BX-7、BX-9、BX-11、BX-12、BX-14、BX-15、BX-17、BX-19、BX-22、BX-23、BX-25；Its
It is corresponding in turn in SEQ ID NO：1-15.
In the frozen stock solution of the cell of the present invention, trehalose and vitamin E and polypeptide, which have, clearly resists external wound
Damage of the evil to cell, particularly polypeptide, the also damage with ice crystal when protecting cells from freezing to cell.
Present invention also offers a kind of preparation method of the frozen stock solution of cell, will each component mixing shake up.
Present invention also offers a kind of nerve cell cryopreservation methods, comprise the following steps:
A. prepared by frozen stock solution:Described cells frozen storing liquid is prepared, by each component according to conventional operating method by each component
It can be obtained according to the mixing of corresponding ratio；
B. prepared by cell suspension:Culture grows into one bottle of the nerve cell of individual layer, and 0.20% trypsin solution digests 4 minutes,
Trypsin solution is abandoned, DMEM nutrient solution 15ml are added, gently being blown and beaten with suction pipe makes cell uniform, 4000 revs/min is centrifuged, abandons supernatant, plus
Enter step A frozen stock solution 2ml, be mixed, insert in sterile cryopreservation tube；
C. freeze:Cryopreservation tube is first frozen into 30min at 4 DEG C, -30 DEG C is then placed in and freezes lh, -80 °C is placed into and freezes
3h, finally moves into liquid nitrogen and preserves；
D. cell recovery:Take out cryopreservation tube and be quickly placed into 37 DEG C of water-baths, concussion is melted completely up to cell suspension, Ran Houyong
5 times of DMEM liquid dilutions, 1000r/min centrifugation 5min, centrifugation removes supernatant, repeated 3 times.The cell suspended concentration is
108Cell/ml-1010Cell/ml.
Beneficial effects of the present invention:
The cells frozen storing liquid of the present invention, recovery cell survival rate is up to more than 99%, compared with answering using regular growth frozen stock solution
Soviet Union's survival rate is obviously improved, and there is no the loss of cell.
The stem cell cryopreserving liquid of the present invention can preserve stem cell for a long time, and cytoactive does not change, it is ensured that cell
Biological activity.
Nerve cell cryopreservation methods of the present invention, operation is simple and feasible, affordable, with preferable practical value.
Embodiment
The preparation of the cells frozen storing liquid of embodiment 1
By the w/v of dimethyl sulfoxide (DMSO) 5%, 0.5% w/v low-density lipoproteins, 1% w/v trehalose, 1% w/v lecithin,
Polypeptide 1% w/v, bFGF 1% w/v, the w/v of vitamin E 1%, finally with DMEM culture mediums and hyclone according to 1:1 ratio
Example is settled to 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 1.
The preparation of the cells frozen storing liquid of embodiment 2
By the w/v of dimethyl sulfoxide (DMSO) 5%, 0.5% w/v low-density lipoproteins, 1% w/v trehalose, 1% w/v lecithin,
Polypeptide 1% w/v, bFGF 1% w/v, the w/v of vitamin E 1%, finally with DMEM culture mediums and hyclone according to 1:1 ratio
Example is settled to 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 2.
The preparation of the cells frozen storing liquid of embodiment 3
By the w/v of dimethyl sulfoxide (DMSO) 5%, 0.5% w/v low-density lipoproteins, 1% w/v trehalose, 1% w/v lecithin,
Polypeptide 1% w/v, bFGF 1% w/v, the w/v of vitamin E 1%, finally with DMEM culture mediums and hyclone according to 1:1 ratio
Example is settled to 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 3.
The preparation of the cells frozen storing liquid of embodiment 4
By the w/v of dimethyl sulfoxide (DMSO) 5%, 0.5% w/v low-density lipoproteins, 1% w/v trehalose, 1% w/v lecithin,
Polypeptide 1% w/v, bFGF 1% w/v, the w/v of vitamin E 1%, finally with DMEM culture mediums and hyclone according to 1:1 ratio
Example is settled to 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 4.
The preparation of the cells frozen storing liquid of embodiment 5
By the w/v of dimethyl sulfoxide (DMSO) 5%, 0.5% w/v low-density lipoproteins, 1% w/v trehalose, 1% w/v lecithin,
Polypeptide 1% w/v, bFGF 1% w/v, the w/v of vitamin E 1%, finally with DMEM culture mediums and hyclone according to 1:1 ratio
Example is settled to 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 5.
The preparation of the cells frozen storing liquid of embodiment 6
By the w/v of dimethyl sulfoxide (DMSO) 5%, 0.5% w/v low-density lipoproteins, 1% w/v trehalose, 1% w/v lecithin,
Polypeptide 1% w/v, bFGF 1% w/v, the w/v of vitamin E 1%, finally with DMEM culture mediums and hyclone according to 1:1 ratio
Example is settled to 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 6.
The preparation of the cells frozen storing liquid of embodiment 7
By the w/v of dimethyl sulfoxide (DMSO) 5%, 0.5% w/v low-density lipoproteins, 1% w/v trehalose, 1% w/v lecithin,
Polypeptide 1% w/v, bFGF 1% w/v, the w/v of vitamin E 1%, finally with DMEM culture mediums and hyclone according to 1:1 ratio
Example is settled to 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 7.
The preparation of the cells frozen storing liquid of embodiment 8
By the w/v of dimethyl sulfoxide (DMSO) 5%, 0.5% w/v low-density lipoproteins, 1% w/v trehalose, 1% w/v lecithin,
Polypeptide 1% w/v, bFGF 1% w/v, the w/v of vitamin E 1%, finally with DMEM culture mediums and hyclone according to 1:1 ratio
Example is settled to 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 8.
The preparation of the cells frozen storing liquid of embodiment 9
By the w/v of dimethyl sulfoxide (DMSO) 5%, 0.5% w/v low-density lipoproteins, 1% w/v trehalose, 1% w/v lecithin,
Polypeptide 1% w/v, bFGF 1% w/v, the w/v of vitamin E 1%, finally with DMEM culture mediums and hyclone according to 1:1 ratio
Example is settled to 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 9.
The preparation of the cells frozen storing liquid of embodiment 10
By the w/v of dimethyl sulfoxide (DMSO) 5%, 0.5% w/v low-density lipoproteins, 1% w/v trehalose, 1% w/v lecithin,
Polypeptide 1% w/v, bFGF 1% w/v, the w/v of vitamin E 1%, finally with DMEM culture mediums and hyclone according to 1:1 ratio
Example is settled to 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 10.
The preparation of the cells frozen storing liquid of embodiment 11
By the w/v of dimethyl sulfoxide (DMSO) 5%, 0.5% w/v low-density lipoproteins, 1% w/v trehalose, 1% w/v lecithin,
Polypeptide 1% w/v, bFGF 1% w/v, the w/v of vitamin E 1%, finally with DMEM culture mediums and hyclone according to 1:1 ratio
Example is settled to 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 11.
The preparation of the cells frozen storing liquid of embodiment 12
By the w/v of dimethyl sulfoxide (DMSO) 5%, 0.5% w/v low-density lipoproteins, 1% w/v trehalose, 1% w/v lecithin,
Polypeptide 1% w/v, bFGF 1% w/v, the w/v of vitamin E 1%, finally with DMEM culture mediums and hyclone according to 1:1 ratio
Example is settled to 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 12.
The preparation of the cells frozen storing liquid of embodiment 13
By the w/v of dimethyl sulfoxide (DMSO) 5%, 0.5% w/v low-density lipoproteins, 1% w/v trehalose, 1% w/v lecithin,
Polypeptide 1% w/v, bFGF 1% w/v, the w/v of vitamin E 1%, finally with DMEM culture mediums and hyclone according to 1:1 ratio
Example is settled to 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 13.
The preparation of the cells frozen storing liquid of embodiment 14
By the w/v of dimethyl sulfoxide (DMSO) 5%, 0.5% w/v low-density lipoproteins, 1% w/v trehalose, 1% w/v lecithin,
Polypeptide 1% w/v, bFGF 1% w/v, the w/v of vitamin E 1%, finally with DMEM culture mediums and hyclone according to 1:1 ratio
Example is settled to 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 14.
The preparation of the cells frozen storing liquid of embodiment 15
By the w/v of dimethyl sulfoxide (DMSO) 5%, 0.5% w/v low-density lipoproteins, 1% w/v trehalose, 1% w/v lecithin,
Polypeptide 1% w/v, bFGF 1% w/v, the w/v of vitamin E 1%, finally with DMEM culture mediums and hyclone according to 1:1 ratio
Example is settled to 100ml, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 15.
Comparative example 1
70ml MEM cell culture fluids, 20ml calf serum, I0ml dimethyl sulfoxide (DMSO) is measured respectively, is mixed to prepare
Regular growth frozen stock solution.
Comparative example 2
By the w/v of dimethyl sulfoxide (DMSO) 5%, 0.5% w/v low-density lipoproteins, 1% w/v trehalose, 1% w/v lecithin,
The w/v of bFGF 1%, the w/v of vitamin E 1%, finally with DMEM culture mediums and hyclone according to 1:1 ratio is settled to
100ml。
The compliance test result of the frozen stock solution of embodiment 16
Cells frozen storing liquid prepared by above example 1-15 and comparative example 1-2, carries out cell by the following method respectively
Freeze and recovering experiment.
Cell cryopreservation process:
Culture grows into the human leukemia stem cell of individual layer, its cell density about 6*109Individual/ml, adds pH7.0 PBS
Wash cell surface once.
Cell is digested 4 minutes with 0.20% trypsin solution, trypsin solution is abandoned, DMEM nutrient solutions 15ml is added, it is light with suction pipe
Featheriness, which is beaten, makes cell uniform, centrifuges 4000 revs/min, abandons supernatant, adds embodiment 1-3 and the frozen stock solution prepared by comparative example 1
2ml, is mixed, and inserts in sterile cryopreservation tube；
Freezing:Cryopreservation tube is first frozen into 30min at 4 DEG C, -30 DEG C is then placed in and freezes lh, -80 DEG C is placed into and freezes
3h, finally moves into liquid nitrogen and preserves；Freeze respectively 1 week, 4 months, 8 months.Every group of 3 repetitions.
Cell recovery:Take out cryopreservation tube and be quickly placed into 37 DEG C of water-baths, concussion until cell suspension melts completely, then with 5
Times DMEM liquid dilution, 1000r/min centrifugation 5min, centrifugation removes supernatant, repeated 3 times, calculates cell survival rate.As a result such as
Under：
| 1 pericyte survival rate（100%） | 4 months cell survival rates（100%） | 8 months cell survival rates（100%） | |
| The frozen stock solution of embodiment 1 | 99.9% | 99.6% | 96.3% |
| The frozen stock solution of embodiment 2 | 99.9% | 99.8% | 96.5% |
| The frozen stock solution of embodiment 3 | 99.9% | 99.8% | 96.4% |
| The frozen stock solution of embodiment 4 | 99.9% | 99.6% | 96.2% |
| The frozen stock solution of embodiment 5 | 99.9% | 99.7% | 96.3% |
| The frozen stock solution of embodiment 6 | 99.9% | 99.7% | 96.5% |
| The frozen stock solution of embodiment 7 | 99.9% | 99.8% | 96.3% |
| The frozen stock solution of embodiment 8 | 99.9% | 99.8% | 96.4% |
| The frozen stock solution of embodiment 9 | 99.9% | 99.6% | 96.2% |
| The frozen stock solution of embodiment 10 | 99.9% | 99.6% | 96.1% |
| The frozen stock solution of embodiment 11 | 99.9% | 99.7% | 96.2% |
| The frozen stock solution of embodiment 12 | 99.9% | 99.6% | 96.3% |
| The frozen stock solution of embodiment 13 | 99.9% | 99.7% | 96.1% |
| The frozen stock solution of embodiment 14 | 99.9% | 99.7% | 96.2% |
| The frozen stock solution of embodiment 15 | 99.9% | 99.6% | 96.3% |
| The frozen stock solution of comparative example 1 | 85.3% | 84.0% | 55.4% |
| The frozen stock solution of comparative example 2 | 90.4% | 88.4% | 77.3% |
The freeze-stored cell of 8 months is taken, cell differentiation culture is carried out, wherein described cell can be normally carried out differentiation, point
Change efficiency and reach 91.3%, and comparative example 1 takes out cell its differentiation rate can only achieve 59.2%, illustrate cytoactive by very
Big influence.
Sequence table
Brightness during 110 Pan ＞ of ＜
A kind of cells frozen storing liquids for leukemia treating of the ＞ of ＜ 120
〈160〉15
〈210〉1
〈211〉 19
〈212〉PRT
The ＞ artificial sequences of ＜ 213
〈400〉BX-1
WRFHSSWRKMGHYLHDDWK
〈210〉2
〈211〉 18
〈212〉PRT
The ＞ artificial sequences of ＜ 213
〈400〉BX-3
PSEWWQYFGRKPYGLLGG
〈210〉3
〈211〉 17
〈212〉PRT
The ＞ artificial sequences of ＜ 213
〈400〉BX-4
DKGQPKIEKGWREKSAS
〈210〉4
〈211〉 19
〈212〉PRT
The ＞ artificial sequences of ＜ 213
〈400〉BX-6
MDMDKQEWRWWSEQCYYSG
〈210〉5
〈211〉 18
〈212〉PRT
The ＞ artificial sequences of ＜ 213
〈400〉BX-7
WWQRHLVWSPWHALTFCP
〈210〉6
〈211〉 17
〈212〉PRT
The ＞ artificial sequences of ＜ 213
〈400〉BX-9
GAMQMASDMSQLQWDAA
〈210〉7
〈211〉 19
〈212〉PRT
The ＞ artificial sequences of ＜ 213
〈400〉BX-11
VFMTPAKWCENTWHPPQRN
〈210〉8
〈211〉 18
〈212〉PRT
The ＞ artificial sequences of ＜ 213
〈400〉BX-12
LGMITHPISLDMIPSGKG
〈210〉9
〈211〉 17
〈212〉PRT
The ＞ artificial sequences of ＜ 213
〈400〉BX-14
QGMWGSGSHKFITKTDQ
〈210〉10
〈211〉 19
〈212〉PRT
The ＞ artificial sequences of ＜ 213
〈400〉BX-15
HDDKHIIWTRMPMGWKAQT
〈210〉11
〈211〉 18
〈212〉PRT
The ＞ artificial sequences of ＜ 213
〈400〉BX-17
KSQARHNRHFNAYGQFSP
〈210〉12
〈211〉 17
〈212〉PRT
The ＞ artificial sequences of ＜ 213
〈400〉BX-19
PVQPQQGWGNQECMAAR
〈210〉13
〈211〉 19
〈212〉PRT
The ＞ artificial sequences of ＜ 213
〈400〉BX-22
HWDHEFNRSRSFRQGVCSA
〈210〉14
〈211〉 18
〈212〉PRT
The ＞ artificial sequences of ＜ 213
〈400〉BX-23
RWLAWFRRNTQRQEWHRW
〈210〉15
〈211〉 17
〈212〉PRT
The ＞ artificial sequences of ＜ 213
〈400〉BX-25
LLVGELREGMKGYWTML
Claims (4)
1. a kind of cells frozen storing liquid, it is characterised in that be made up of following each component：Dimethyl sulfoxide (DMSO) 5%w/v, 0.5%w/v is low
Density lipoprotein, 1%w/v trehalose, 1%w/v lecithin, polypeptide 1%w/v, bFGF 1%w/v, vitamin E 1%w/v,
Finally with DMEM culture mediums and hyclone according to 1:1 ratio is settled to 100ml, and the peptide sequence is SEQ ID NO:1
It is shown.
2. the preparation method of the cells frozen storing liquid described in claim 1, it is characterised in that the group including mixing the formula rate
Point, obtain described frozen stock solution.
3. application of the cells frozen storing liquid in survival rate after improving freeze-stored cell recovery described in claim 1.
4. a kind of polypeptide, it is characterised in that：Sequence such as SEQ ID NO:Shown in 1.
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| CN106417258A (en) * | 2016-10-15 | 2017-02-22 | 成都育芽科技有限公司 | Freezing solution and freezing method for cardiac stem cell |
| CN106900695A (en) * | 2017-04-01 | 2017-06-30 | 北京焕生汇生物技术研究院 | A kind of mankind's inductive pluripotent stem cells are special to freeze reagent and cryopreservation methods |
| CN107156111A (en) * | 2017-06-30 | 2017-09-15 | 陈印平 | A kind of candidate stem cell cell cryopreservation agent |
| CN107232182A (en) * | 2017-06-30 | 2017-10-10 | 陈印平 | A kind of mesenchymal stem cells MSCs cell cryopreservation agent |
| CN108244097B (en) * | 2018-01-10 | 2018-11-09 | 暨赛再生医学科技有限公司 | A kind of neural stem cell preservation liquid of the C17.2 of long-term preservation overexpression bFGF |
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| CN102228016B (en) * | 2011-04-18 | 2012-07-18 | 上海安集协康生物技术有限公司 | Cryopreservation and resuscitation method of neural stem cells |
| CN103467586B (en) * | 2013-09-25 | 2015-04-15 | 福州大学 | Antifreeze polypeptide for protecting bacteria from low temperature freeze-thawing damage and preparation method thereof |
| CN105052892A (en) * | 2015-07-20 | 2015-11-18 | 山东省齐鲁干细胞工程有限公司 | Cryoprotectant free of DMSO (dimethyl sulfoxide) and ovary cryopreservation method |
| CN105123671B (en) * | 2015-07-24 | 2017-10-13 | 广州赛莱拉干细胞科技股份有限公司 | A kind of cells frozen storing liquid, using and immunocyte cryopreservation methods |
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| CN107027742B (en) | 2019-01-18 |
| CN105394029A (en) | 2016-03-16 |
| CN107027742A (en) | 2017-08-11 |
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