CN108410806A - A kind of storage liquid of people's lung mescenchymal stem cell - Google Patents
A kind of storage liquid of people's lung mescenchymal stem cell Download PDFInfo
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- CN108410806A CN108410806A CN201810273783.9A CN201810273783A CN108410806A CN 108410806 A CN108410806 A CN 108410806A CN 201810273783 A CN201810273783 A CN 201810273783A CN 108410806 A CN108410806 A CN 108410806A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
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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
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- 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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/998—Proteins not provided for elsewhere
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Abstract
The present invention relates to a kind of storage liquid of people's lung mescenchymal stem cell, specifically, the storage liquid of people's lung mescenchymal stem cell of the present invention keeps peptide to form by basic culture solution and activity.What the storage liquid energy was enough greatly improved people's lung mescenchymal stem cell freezes safety and stability, so that Cell viability improves.
Description
Technical field
The present invention relates to stem cells technology fields, and in particular to a kind of storage liquid of people's lung mescenchymal stem cell.
Background technology
Mescenchymal stem cell (mesenchymal stem cells, MSC) is a group separated from marrow at first
Stem cell with certain self-renewing and multi-lineage potential.It can be to bone, cartilage, fat, muscle under specific inductive condition
Etc. a variety of interstitial system cell differentiations.Since these cells can finally be divided into interstitial system cell, filled between being named as
Matter stem cell.Marrow is considered as the main residential place of MSC.Many scholars are in the histoorgan other than marrow in recent years
Isolate almost all tissues of body such as MSC, including muscle, fat, bony process matter, pancreas, lungs, thymus gland, umbilical cord and placenta
And organ.In vitro and in vivo experiment, which demonstrates MSC and its noble cells, can give the support of hematopoietic cell structurally and functionally,
With hematopoiesis adjustment effect.MSC has the advantages that foreign gene can stablize expression after being easy to foreign gene transfection and transfection, also
Good gene therapy vector is can be used as, disease treatment is carried out.MSC becomes grinding for organizational project and bone-marrow transplantation cell therapy
Study carefully hot spot.
Type of the mescenchymal stem cell for clinical application research is mainly mesenchymal stem cell and umbilical cord mesenchyma
Stem cell, this two classes cell all have the characteristics that the common low immunogenicity of mescenchymal stem cell, especially the latter become apparent.Navel
Institutional framework is simple in band, and in addition to main big blood vessel, remaining causes umbilical cord mesenchymal stem cells based on connective tissue
Substantially not with antigen contact, so immunogenicity is very low, this significantly increases the possibility of clinical application.
But it is newest studies have shown that compared with allosome stem cell, autologous stem cells treatment more has superiority, such as avoids
Infection risk that may be present, and potential immunological rejection etc..Autologous stem cells detach and culture generally has several ways
Diameter, if the stem cell of marrow detaches, the stem cell separation of Cord blood and the stem cell separation of tissue and organ specificity.There is research
Reparation after display injury of lungs is mainly derived from the stem cell in lung tissue.The stem cell of tissue in situ is adjusting inflammatory reaction,
Tissue repair, fibrosis control, revascularization etc. play an important role.Therefore, it for the treatment of pulmonary disease, is directly separated
Go out lung stem cell cultivated after adoptive therapy pulmonary disease have special significance.
The stem cell of isolated lung tissue generally requires first to isolate lung tissue in zoopery at present, shreds, and grinds, training
It supports, continues to cultivate after flow cytometer screening, then carry out the identification of stem cell again.Process is complicated, it is often more important that needs handle
Lung shreds.Another method is to detach lung original position stem cell by alveolar wass.This method is in zoopery and will
Hope person is explored with it.The result of study of zoopery shows the getable stem cell population of simple bronchoalveolar lavage
Seldom, great inconvenience is caused to carrying out experimental study.If lungs can be pre-processed, alveolar wass is then carried out, hence it is evident that carry
If high original position stem cell separative efficiency, it is not only an important new technique, and have potential important clinical application
Value.
A kind of method of extraction lung mescenchymal stem cell is provided in CN102559591A, including：At intraalveolar administration angle
Then cell plastid growth factor-2 (KGF-2) or keratinocyte growth factor (KGF) carry out lung to be enriched with lung mescenchymal stem cell
Lavation is steeped, will be purified after the lung mescenchymal stem cell separation in the irrigating solution of gained, obtain lung mescenchymal stem cell.But
Mescenchymal stem cell only has been prepared in this method, but can not be steady in a long-term storage, to facilitate follow-up use.Cause
How this, quickly and efficiently prepare hot spot of the mescenchymal stem cell of high-quality for clinical and scientific research to be current stem cell and grind
Study carefully one of content.By being limited by technical merit, at present prepared by mescenchymal stem cell and storage process is still studied not enough
Thoroughly, without specific standard criterion production procedure, it is difficult to meet extensive demand, continue to be improved.
Invention content
The present invention is directed to problems of the prior art, provides a kind of maintenance lung mescenchymal stem cell original function, together
When can keep freezing active storage of cells liquid well again, be used for cryopreserved human lung mescenchymal stem cell.
The present invention also provides a kind of methods for the storage liquid preparing people's lung mescenchymal stem cell.
The purpose that the invention is realized by the following technical scheme：
The acquisition of Activity of Antifreeze peptide：Laboratory early period of the present invention constructs active peptide screening library platform, is done for different
Cell, which can screen to obtain, can specifically be directed to the active peptide that different stem cells plays maximum activity characteristic.Early period passes through library
Screening obtains and is directed to peptide of the lung mescenchymal stem cell with preferable Activity of Antifreeze with high activity, and sequence is respectively such as
SEQ ID NO：1 or SEQ ID NO：2 or SEQ ID NO：Active peptide shown in 3 forms.The active peptide is capable of the thorn of specificity
Swash people's lung mescenchymal stem cell and generate antifreeze protein and collagen, so that the structure of cell at low ambient temperatures can
Ensure that better organelle stability then achievees the effect that maintain cell low temperature integrality.
A kind of storage liquid of people lung mescenchymal stem cell keeps peptide to form by basic culture solution and activity.
Wherein cell activity peptide is by SEQ ID NO：1 or SEQ ID NO：2 or SEQ ID NO：Active peptide group shown in 3
At.The stimulation people lung mescenchymal stem cell that the active peptide is capable of specificity generates antifreeze protein and collagen, to make
Cell structure can ensure at low ambient temperatures better organelle stability then reach maintain cell low temperature it is complete
The effect of property.
Wherein basic culture solution composition is as follows：Sucrose 1.2%, human serum albumin 0.5%, Dextran 40 O.3%, paddy Guang
O.1%, O.2%, trehalose 0.4%, l-GLUTAMINE 0.5%, glycerine 15% (V/V), 1640 is sterile for glutamine for sweet peptide
Culture medium surplus.Solid component degree indicates the content of how many gram in 100mL culture mediums, liquid group in the present invention
Liquid component containing how many mL in the culture medium of the percentage expression 100mL divided.
Preferably, it is 0.3%- that activity, which keeps the additive amount of peptide, in the storage liquid of people's lung mescenchymal stem cell
0.4%.
The present invention also provides a kind of preparation methods of the storage liquid of people's lung mescenchymal stem cell, include the following steps：It presses
It is detached according to the method for the prior art and obtains people's lung mescenchymal stem cell, stem cell is incubated at 1640 aseptic culture mediums and activity
In peptide, in 37 DEG C, 5%CO2Under conditions of cultivate 2-3 days, cell is then transferred to the basic culture solution added with active peptide
Middle culture, in 30 DEG C, 5%CO2Under conditions of cultivate 12h, then in liquid nitrogen directly freezed preserve.
Compared with the existing technology, beneficial effects of the present invention are：The present invention people's lung mescenchymal stem cell storage liquid by
Basic culture solution and active peptide composition.The present invention does not have special excitor substance to lead to unordered point of people's lung mescenchymal stem cell
Change, especially activity keeps the addition of peptide, and people's lung mescenchymal stem cell can be greatly improved freezes safety and stabilization
Property, so that Cell viability improves, freeze the regular growth with obvious effects that is better than and store liquid, it is dry thin to can be used for people's lung mesenchyma
The long-term preservation of born of the same parents and application.
Specific implementation mode
Below in conjunction with specific embodiment, the present invention will be described in detail.But be not considered as limiting the invention,
As long as other can realize the mode of effect of the present invention, all within the scope of the present invention.
The preparation of embodiment 1, people's lung mescenchymal stem cell
People's lung mescenchymal stem cell is commercial products, and concrete ways can be purchased by the fine bio tech ltd of upper sea ice
It buys, brand：Sciencell, article No.：7540.By the commercial product of purchase according to operation instructions carry out amplification cultivation, breeding compared with
After mostly, with cellular immunofluorescence method into the identification of pedestrian's lung mescenchymal stem cell；Fluidic cell immunofluorescence technique marks Oct-4
Positive cell identified, final to obtain lung mescenchymal stem cell, label CD13, CD29, CD44, CD90, the CD105,
CD166, HLA-ABC are the positive, meet the requirement of lung mescenchymal stem cell.
The preparation of 2 storage of cells liquid of embodiment
Artificial synthesized SEQ ID NO：1 or SEQ ID NO：Active peptide shown in 2.
The configuration of basic culture solution：Sucrose 1.2%, human serum albumin 0.5%, dextrorotation are added in 1640 aseptic culture mediums
Sugared acid anhydride 40O.3%, glutathione O.1%, glutamine O.2%, trehalose 0.4%, l-GLUTAMINE 0.5%, glycerine
15% (V/V), using 1640 aseptic culture medium constant volumes to 1L.
It is 0.4% that activity, which keeps the additive amount of peptide, in the basic culture solution.
Compare the preparation of culture solution 1：TC199 containing human serum albumins matches respectively to be prepared containing 0.01%EDTA,
The preservation liquid of 75AXaIU/ml Low-molecular-weight Heparins Calciums (referring to the content of patent CN101210232B).
Compare the preparation of culture solution 2：Membrane protective agent 1%：By sucrose O.5%, macromolecule sugar acid anhydride -500O.5% groups
At DMSO 8%；Cell settlement stabilizer 25%：It is made of methylcellulose 4000CP 15%, hydroxyethyl starch 10%；It is anti-
Oxidant is O.2%：By vitamin C O.1%, O.1% glutathione form；Cytotrophy agent 1.0%, by glutamine
O.4%, O.6% Sodium Pyruvate forms；PBS buffer solution, surplus.
3 people's lung mesenchymal stem cell cryopreserving 1 of embodiment
People's lung mescenchymal stem cell that embodiment 1 is prepared, is incubated at 1640 aseptic culture mediums and SEQ ID NO：1
Active peptide 0.4% in, in 37 DEG C, 5%CO2Under conditions of cultivate 3 days, collect cell, embodiment 2 basic culture solution+
SEQ ID NO：It is cultivated in 1 active peptide 0.4%, in 30 DEG C, 5%CO2Under conditions of cultivate 12h, liquid nitrogen directly freezed preserves
.
4 people's lung mesenchymal stem cell cryopreserving 2 of embodiment
People's lung mescenchymal stem cell that embodiment 1 is prepared, is incubated at 1640 aseptic culture mediums and SEQ ID NO：2
Active peptide 0.4% in, in 37 DEG C, 5%CO2Under conditions of cultivate 2 days, collect cell, embodiment 2 basic culture solution+
SEQ ID NO：It is cultivated in 2 active peptide 0.4%, in 30 DEG C, 5%CO2Under conditions of cultivate 12h, liquid nitrogen directly freezed preserves
.
5 people's lung mesenchymal stem cell cryopreserving 3 of embodiment
People's lung mescenchymal stem cell that embodiment 1 is prepared, is incubated at 1640 aseptic culture mediums and SEQ ID NO：3
Active peptide 0.4% in, in 37 DEG C, 5%CO2Under conditions of cultivate 2 days, collect cell, embodiment 2 basic culture solution+
SEQ ID NO：It is cultivated in 3 active peptide 0.4%, in 30 DEG C, 5%CO2Under conditions of cultivate 12h, liquid nitrogen directly freezed preserves
.
6 people's lung mesenchymal stem cell cryopreserving (control) of embodiment
People's lung mescenchymal stem cell that embodiment 1 is prepared, is incubated at 1640 aseptic culture mediums, in 37 DEG C, 5%
CO2Under conditions of cultivate 2 days, collect cell, respectively the control culture solution 1 of embodiment 2 and control culture solution 2 in cultivate, in
30 DEG C, 5%CO2Under conditions of cultivate 12h, liquid nitrogen directly freezed preserves.
The recovery of 7 frozen cell of embodiment and survival rate test
Cell cryopreservation is recovered after 4 weeks, and each Group groups recover 3, and taking out cryopreservation tube from liquid nitrogen is directly placed into 37
In DEG C warm water, and shaking frequently makes it melt as early as possible in a short time, centrifuges 5min in the centrifuge of 720RPM after thawing, abandons
1mL cell culture fluids (1640 aseptic culture medium) are added in each tube for supernatant, count and count work and are averaged.Specifically, thin
Born of the same parents count and the assay method of Cell viability is：1, pancreatin digests attached cell, prepares single cell suspension, and makees appropriate dilution；
2, it dyes：Cell suspension and 0.4% trypan blue solution are with 9:1 mixing mixing (final concentration of 0.04%)；3, it counts：In 3min
It is interior, difference living cell counting and dead cell；4, under the microscope, dead cell is dyed to apparent blue, and it is in nothing that living cells, which refuses dye,
Color transparence.5, cell viability is counted：Living cell rate (%)=total viable cell/(total viable cell+dead cell sum) ×
100%.The motility rate distribution measured is as shown in table 1：
1 each group motility rate result of table
| Group number | Motility rate (100%) |
| Freeze 1 | 99.30 |
| Freeze 2 | 98.96 |
| Freeze 3 | 99.07 |
| Control 1 | 76.53 |
| Control 2 | 80.26 |
Known to Cell viability experimental result as shown in Table 1：The addition of storage liquid provided by the invention significantly improves multiple
The motility rate of Soviet Union's cell.With significantly effect.
By the cell proliferation after freezing, amplification carries out Analytical Chemical Experiment, and the lung mescenchymal stem cell of culture to four generations is digested
At unicellular, by cell quantity 1:It is inoculated in Matrigel after 1 mixing, the mesenchyma using the embodiment 2 of not glycerol adding is dry
Cell culture medium is cultivated, and uses alveolar culture medium (Celprogen, article No. after 2 days instead：M36075-05S), concrete operations are
It is single cell suspension that pancreatin, which digests mescenchymal stem cell, is resuspended with culture medium after counting, and thaw with isometric 4 DEG C
Matrigel is mixed, and is inoculated in 24 orifice plates, is placed in 37 DEG C of cell incubators, after twenty minutes, waits for that Matrigel solidifies, culture is added
Base is inoculated with 15 mescenchymal stem cells of 5X per hole, is inoculated with after being mixed with 50 μ L Matrigel, after being inoculated with successfully, changes liquid, body every other day
After outer culture 1 day, cell aggregation is at class alveolar shape, after 2 days, it is seen that alveolar is formed, and alveolar grows to maximum after 7 days.This is fully
Illustrate, the stem cell after present invention storage still maintains higher bioactivity, can normal differentiation to become corresponding differentiation thin
Born of the same parents have preferable application prospect.
Several embodiments of the invention above described embodiment only expresses, the description thereof is more specific and detailed, but simultaneously
Cannot the limitation to the scope of the claims of the present invention therefore be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention
Protect range.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Sequence table
<110>The Luoyang bio tech ltd Xuan Zhi
<120>A kind of storage liquid of people's lung mescenchymal stem cell
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> PRT
<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 1
His Ala Ala Phe Glu Trp His Lys Ser Lys His Tyr His Trp Ser Ala
1 5 10 15
Arg Cys Thr Ser Trp Tyr His His
20
<210> 2
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<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
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Trp Arg Pro Ser Gly Ala Trp Lys Asp Trp Val Ser Ser Lys Gln Phe
1 5 10 15
Pro Tyr Pro Arg Lys Asn Asp Leu Phe
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<213>Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 3
Tyr Lys Gly Ser Tyr Tyr Phe Cys Gln Lys Ile Asn Lys Val Pro Tyr
1 5 10 15
Pro Leu Ile Glu Cys Val Pro Ala
20
Claims (5)
1. a kind of people's lung mescenchymal stem cell stores liquid, it is characterised in that wherein containing can keep lung mescenchymal stem cell active
Stable component.
2. a kind of people's lung mescenchymal stem cell stores liquid, it is made of basic culture solution and polypeptide, wherein basic culture solution is matched
It is set to：Sucrose 1.2%, human serum albumin 0.5%, Dextran 40 are added in 1640 aseptic culture mediums O.3%, gluathione
Peptide O.1%, glutamine O.2%, trehalose 0.4%, l-GLUTAMINE 0.5%, glycerine 15% (V/V), using 1640 nothings
Bacterium culture medium constant volume is to 1L；The additive amount of polypeptide is 0.3%-0.4% in the basic culture solution, and wherein the sequence of polypeptide is such as
SEQ ID No：Shown in 1-3 is any.
3. a kind of cryopreservation methods of people's lung mescenchymal stem cell, include the following steps：Lung mescenchymal stem cell is incubated at 1640
In aseptic culture medium and active peptide, in 37 DEG C, 5%CO2Under conditions of cultivate 2 days, then cell is transferred to added with polypeptide
Basic culture solution in cultivate, in 30 DEG C, 5%CO2Under conditions of cultivate 12h, then in liquid nitrogen directly freezed preserve；Its
Middle basic culture solution component is as shown in the basic culture solution in claim 2.
4. method as stated in claim 3, wherein the additive amount of polypeptide is 0.3% in the basic culture solution.
5. method as stated in claim 3, wherein the additive amount of polypeptide is 0.4% in the basic culture solution.
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| CN201810273783.9A CN108410806A (en) | 2018-03-29 | 2018-03-29 | A kind of storage liquid of people's lung mescenchymal stem cell |
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| CN111838130A (en) * | 2020-05-28 | 2020-10-30 | 深圳华大基因细胞科技有限责任公司 | Protective solution and preparation method and application thereof |
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| CN111838130A (en) * | 2020-05-28 | 2020-10-30 | 深圳华大基因细胞科技有限责任公司 | Protective solution and preparation method and application thereof |
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Application publication date: 20180817 |