CN106973889A - A kind of cells frozen storing liquid of leukemia treating - Google Patents

A kind of cells frozen storing liquid of leukemia treating Download PDF

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Publication number
CN106973889A
CN106973889A CN201710410715.8A CN201710410715A CN106973889A CN 106973889 A CN106973889 A CN 106973889A CN 201710410715 A CN201710410715 A CN 201710410715A CN 106973889 A CN106973889 A CN 106973889A
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storing liquid
cell
polypeptide
cells frozen
frozen storing
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CN106973889B (en
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潘时辉
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Nanjing Sansheng Biotechnology Ltd By Share Ltd
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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
    • 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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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Dentistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Environmental Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention provides a kind of cells frozen storing liquid and preparation method thereof.The cells frozen storing liquid includes dimethyl sulfoxide (DMSO) 5%w/v, 0.5%w/v 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.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

A kind of cells frozen storing liquid of leukemia treating
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) 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 energy 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: Dimethyl sulfoxide (DMSO) 5%w/v, 0.5%w/v 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, 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 be respectively LD-1, LD-3, LD-4, LD-5, LD-8, LD-9, LD-10, LD-13, LD-15, LD-16, LD-21, LD-23, LD-25, LD-27, LD-28, LD-30, its It is corresponding in turn in SEQ ID NO：1-16.
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 points Clock, abandons trypsin solution, adds DMEM nutrient solution 15ml, and gently being blown and beaten with suction pipe makes cell uniform, centrifuges 4000 revs/min, abandons supernatant, Step A frozen stock solution 2ml are added, is mixed, inserts 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 DEG 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%, relatively using regular growth frozen stock solution Recovery 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 dimethyl sulfoxide (DMSO) 5%w/v, 0.5%w/v low-density lipoproteins, 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 100ml is settled to, 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 dimethyl sulfoxide (DMSO) 5%w/v, 0.5%w/v low-density lipoproteins, 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 100ml is settled to, 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 dimethyl sulfoxide (DMSO) 5%w/v, 0.5%w/v low-density lipoproteins, 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 100ml is settled to, 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 dimethyl sulfoxide (DMSO) 5%w/v, 0.5%w/v low-density lipoproteins, 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 100ml is settled to, 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 dimethyl sulfoxide (DMSO) 5%w/v, 0.5%w/v low-density lipoproteins, 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 100ml is settled to, 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 dimethyl sulfoxide (DMSO) 5%w/v, 0.5%w/v low-density lipoproteins, 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 100ml is settled to, 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 dimethyl sulfoxide (DMSO) 5%w/v, 0.5%w/v low-density lipoproteins, 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 100ml is settled to, 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 dimethyl sulfoxide (DMSO) 5%w/v, 0.5%w/v low-density lipoproteins, 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 100ml is settled to, 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 dimethyl sulfoxide (DMSO) 5%w/v, 0.5%w/v low-density lipoproteins, 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 100ml is settled to, 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 dimethyl sulfoxide (DMSO) 5%w/v, 0.5%w/v low-density lipoproteins, 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 100ml is settled to, 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 dimethyl sulfoxide (DMSO) 5%w/v, 0.5%w/v low-density lipoproteins, 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 100ml is settled to, 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 dimethyl sulfoxide (DMSO) 5%w/v, 0.5%w/v low-density lipoproteins, 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 100ml is settled to, 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 dimethyl sulfoxide (DMSO) 5%w/v, 0.5%w/v low-density lipoproteins, 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 100ml is settled to, 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 dimethyl sulfoxide (DMSO) 5%w/v, 0.5%w/v low-density lipoproteins, 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 100ml is settled to, 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 dimethyl sulfoxide (DMSO) 5%w/v, 0.5%w/v low-density lipoproteins, 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 100ml is settled to, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 15.
The preparation of the cells frozen storing liquid of embodiment 16
By dimethyl sulfoxide (DMSO) 5%w/v, 0.5%w/v low-density lipoproteins, 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 100ml is settled to, wherein the sequence of the polypeptide such as SEQ ID NO：Shown in 16.
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 dimethyl sulfoxide (DMSO) 5%w/v, 0.5%w/v low-density lipoproteins, 1%w/v trehalose, 1%w/v lecithin, 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。
The compliance test result of the frozen stock solution of embodiment 17
Cells frozen storing liquid prepared by above example 1-16 and Comparative Example I I, carries out cell jelly by the following method respectively Deposit and recovering experiment.
Cell cryopreservation process:
Culture grows into the human leukemia stem cell of individual layer, and about 6*109/ml of its cell density adds pH7.0 PBS Wash cell surface once.
Cell is digested 4 minutes with 0.20% trypsin solution, trypsin solution is abandoned, DMEM nutrient solution 15ml is added, uses suction pipe Gently piping and druming 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：
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 91.5%, and comparative example 1 takes out cell its differentiation rate can only achieve 59.4%, illustrate that cytoactive is received very Big influence.
Sequence table
Brightness during 110 Pan ＞ of ＜
A kind of cells frozen storing liquids of leukemia treating of the ＞ of ＜ 120
〈160〉16
〈210〉1
〈211〉 19
〈212〉PRT
The ＞ artificial sequences of ＜ 213
〈400〉LD-1
SPRKGTHHTPKRPHKMSFH
〈210〉2
〈211〉 18
〈212〉PRT
The ＞ artificial sequences of ＜ 213
〈400〉LD-3
TGNMHHLCVDLVRWEMRK
〈210〉3
〈211〉 17
〈212〉PRT
The ＞ artificial sequences of ＜ 213
〈400〉LD-4
PRQSYQREEAESTCMEG
〈210〉4
〈211〉 16
〈212〉PRT
The ＞ artificial sequences of ＜ 213
〈400〉LD-5
QGSFLRPVPARVLQDW
〈210〉5
〈211〉19
〈212〉PRT
The ＞ artificial sequences of ＜ 213
〈400〉LD-8
SWLRHADKRFRKRRSPTVI
〈210〉6
〈211〉 18
〈212〉PRT
The ＞ artificial sequences of ＜ 213
〈400〉LD-9
QKHIDPRNMHPIMLASMK
〈210〉7
〈211〉 17
〈212〉PRT
The ＞ artificial sequences of ＜ 213
〈400〉LD-10
SSHRYKSYQGFLVDWTW
〈210〉8
〈211〉 16
〈212〉PRT
The ＞ artificial sequences of ＜ 213
〈400〉LD-13
TWQHQRERQSWIPPVW
〈210〉9
〈211〉 19
〈212〉PRT
The ＞ artificial sequences of ＜ 213
〈400〉LD-15
APPMAVLFKLCRQGWGTQM
〈210〉10
〈211〉 18
〈212〉PRT
The ＞ artificial sequences of ＜ 213
〈400〉LD-16
SYPHWHRTLEFPIHCDAT
〈210〉11
〈211〉 17
〈212〉PRT
The ＞ artificial sequences of ＜ 213
〈400〉LD-21
FCVLYGTSHVPIGWEKA
〈210〉12
〈211〉 16
〈212〉PRT
The ＞ artificial sequences of ＜ 213
〈400〉LD-23
WSYQHPYRCRKERDSW
〈210〉13
〈211〉 19
〈212〉PRT
The ＞ artificial sequences of ＜ 213
〈400〉LD-25
DSGEDSTPIEHQSGYKCPY
〈210〉14
〈211〉 18
〈212〉PRT
The ＞ artificial sequences of ＜ 213
〈400〉LD-27
HYYRQKMPHCFPMRKLEW
〈210〉15
〈211〉 17
〈212〉PRT
The ＞ artificial sequences of ＜ 213
〈400〉LD-28
DLNTRSSIGDKTTAMMS
〈210〉16
〈211〉 16
〈212〉PRT
The ＞ artificial sequences of ＜ 213
〈400〉LD-30
WEQSIRPSCGATSQFD

Claims (5)

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, the w/ of vitamin E 1% V, finally with DMEM culture mediums and hyclone according to 1:1 ratio is settled to 100ml.
2. cells frozen storing liquid as claimed in claim 1, it is characterised in that：The peptide sequence is SEQ ID NO:2-16 is any It is shown.
3. the preparation method of the cells frozen storing liquid described in claim any one of 1-2, it is characterised in that including mixing the formula The component of ratio, obtains described frozen stock solution.
4. application of the cells frozen storing liquid in survival rate after improving freeze-stored cell recovery described in claim any one of 1-2.
5. a kind of polypeptide, it is characterised in that：Sequence such as SEQ ID NO:Shown in 2-16 is any.
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