CN109362714A - A kind of adipose mesenchymal stem cell preservation solution and preparation method and application thereof - Google Patents
A kind of adipose mesenchymal stem cell preservation solution and preparation method and application thereof Download PDFInfo
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- CN109362714A CN109362714A CN201811553563.8A CN201811553563A CN109362714A CN 109362714 A CN109362714 A CN 109362714A CN 201811553563 A CN201811553563 A CN 201811553563A CN 109362714 A CN109362714 A CN 109362714A
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- stem cell
- mesenchymal stem
- preservation solution
- adipose
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- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 52
- 239000003761 preservation solution Substances 0.000 title claims abstract 17
- 238000002360 preparation method Methods 0.000 title abstract description 12
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 81
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 29
- 239000012091 fetal bovine serum Substances 0.000 claims abstract 8
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 10
- 239000002609 medium Substances 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 2
- 210000000130 stem cell Anatomy 0.000 abstract description 10
- 230000003833 cell viability Effects 0.000 abstract description 4
- 239000007788 liquid Substances 0.000 description 31
- 239000012894 fetal calf serum Substances 0.000 description 21
- 210000004027 cell Anatomy 0.000 description 11
- 239000011550 stock solution Substances 0.000 description 11
- 238000004321 preservation Methods 0.000 description 10
- 239000001963 growth medium Substances 0.000 description 8
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 7
- 238000005138 cryopreservation Methods 0.000 description 7
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000010899 nucleation Methods 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 210000004504 adult stem cell Anatomy 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009087 cell motility Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000000151 deposition Methods 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 238000007443 liposuction Methods 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
Classifications
-
- 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
- A01N1/10—Preservation of living parts
- A01N1/12—Chemical aspects of preservation
- A01N1/122—Preservation or perfusion media
- A01N1/125—Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators
-
- 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
- A01N1/10—Preservation of living parts
- A01N1/12—Chemical aspects of preservation
- A01N1/122—Preservation or perfusion media
- A01N1/126—Physiologically active agents, e.g. antioxidants or nutrients
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明提供了一种脂肪间充质干细胞保存液及其制备方法和应用。本发明提供的脂肪间充质干细胞保存液，包括胎牛血清和DMSO，结果发现，本发明提供的保存液作为脂肪间充质干细胞保存液与现有的保存液相比，在脂肪间充质干细胞冻存复苏培养后，细胞活率以及相应的生物学特性均得到了较好的保护。The invention provides an adipose mesenchymal stem cell preservation solution and a preparation method and application thereof. The adipose-derived mesenchymal stem cell preservation solution provided by the present invention includes fetal bovine serum and DMSO. It is found that the preservation solution provided by the present invention, as an adipose-derived mesenchymal stem cell preservation solution, is more effective in adipose-derived mesenchymal stem cells than the existing preservation solution. After the stem cells are cryopreserved, revived and cultured, the cell viability and corresponding biological characteristics are well protected.
Description
Technical field
The present invention relates to stem cell field more particularly to a kind of fat mesenchymal stem cell save liquid and preparation method thereof and
Using.
Background technique
Adult stem cell derives from adult tissue, avoids being related to ethics problem, while being also that one kind is able to carry out self more
New and Multidirectional Differentiation cell thus there is broader research and application prospect.Fat mesenchymal stem cell is to be present in fat
Stem cell in tissue, the potential with Multidirectional Differentiation.2001, fat mesenchymal stem cell was extracted from people's liposuction for the first time
Adipose tissue suspension in separate obtain, it is only necessary to extracting a small amount of fat mesenchymal stem cell can nurture in a short time
A large amount of stem cell is used to carry out transfer operation.
Clinically, when the donor of cell transplantation usually has different from receptor, differently property, it is difficult to ensure that i.e. from using,
And sufficient mescenchymal stem cell quantity is determined whether one of an important factor for capable of reaching clinical efficacy.Currently, that cultivates is dry
Cell needs cell cryopreservation, and cell cryopreservation, which refers to, to be temporarily disengaged from growth conditions even if cell and save cell for a long time,
And maintain the original biological characteristics of cell, for need when recovery tested or treated.As it can be seen that one kind is filled between capable of guaranteeing
The cryopreservation methods of the moderately good biological characteristics of matter stem cell are particularly important.
The fat mesenchymal stem cell cryopreservation methods of laboratory routine are with containing 5% or 10%DMSO and human seralbumin at present
The frozen stock solution of albumen, with certain freezing be put into liquid nitrogen after rate drops to -80 DEG C from room temperature or liquid nitrogen gas phase in.However, this
A method is that basis freezes candidate stem cell and the method for lymphocyte is improved, is not to freeze fat mesenchymal stem cell
The best approach.
Summary of the invention
In view of this, technical problem to be solved by the present invention lies in provide a kind of fat mesenchymal stem cell save liquid and
Preparation method and application, after the recovery of fat mesenchymal stem cell that the preservation liquid of offer of the invention freezes the motility rate of cell and
Corresponding biological characteristics can be protected preferably.
The present invention provides a kind of fat mesenchymal stem cells to save liquid, including fetal calf serum and DMSO.
Preferably, the DMSO is 5~30 parts by weight, and the fetal calf serum is 50~90 parts by weight.
Preferably, the DMSO is 10~20 parts by weight, and the fetal calf serum is 70~80 parts by weight.
Preferably, it further includes Lonza DMEM culture medium that the fat mesenchymal stem cell, which saves liquid,.
Preferably, the fetal calf serum is 50~80 parts by weight, and the DMSO is 5~10 parts by weight, the Lonza
DMEM culture medium is 20~40 parts by weight.
Preferably, the fetal calf serum is 60~70 parts by weight.
The present invention also provides the preparation methods that a kind of fat mesenchymal stem cell saves liquid, comprising:
DMSO is slowly added into fetal calf serum, fat mesenchymal stem cell is obtained and saves liquid.
The present invention also provides the preparation methods that a kind of fat mesenchymal stem cell saves liquid, comprising:
DMSO is slowly added into the mixed liquor of fetal calf serum and Lonza DMEM culture medium, it is dry to obtain fat mesenchymal
Cell-preservation liquid.
Liquid, which is saved, as preparation the present invention also provides a kind of fat mesenchymal stem cell of the present invention saves fat
The application of the preservation liquid of mescenchymal stem cell.
Compared with prior art, liquid and preparation method thereof is saved the present invention provides a kind of fat mesenchymal stem cell and answer
With, fat mesenchymal stem cell provided by the invention saves liquid, including fetal calf serum and DMSO, the experimental results showed that, the present invention
The preservation liquid of offer saves liquid and existing preservation liquid phase ratio as fat mesenchymal stem cell, and fat mesenchymal stem cell freezes
After depositing recovery culture, Cell viability and corresponding biological characteristics have obtained preferable protection.
Specific embodiment
The present invention provides a kind of fat mesenchymal stem cells to save liquid, including fetal calf serum and DMSO.Wherein, described
DMSO is preferably 5~30 parts by weight, more preferably 10~20 parts by weight；The fetal calf serum is preferably 50~90 parts by weight, more
Preferably 70~80 parts by weight.
In the present invention, fat mesenchymal stem cell of the present invention saves liquid, and it is also preferable to include Lonza DMEM cultures
Base, when it includes fetal calf serum, DMSO and Lonza DMEM culture medium that fat mesenchymal stem cell of the present invention, which saves liquid,
The each component content is preferred are as follows: the fetal calf serum is 50~80 parts by weight, and the DMSO is 5~10 parts by weight, described
Lonza DMEM culture medium is 20~40 parts by weight；The more preferably described fetal calf serum is 60~70 parts by weight；Most preferably tire
Cow's serum is 70 parts by weight, DMSO is 10 parts by weight and Lonza DMEM culture medium is 20 parts by weight.
The present invention also provides the preparation methods that a kind of fat mesenchymal stem cell saves liquid, comprising: slowly adds DMSO
Enter into fetal calf serum, obtains fat mesenchymal stem cell and save liquid.The present invention does not have particular/special requirement, ability to mixed mode
Field technique personnel can select suitable mixed method according to common knowledge.Preferably, DMSO is also slowly added by the present invention
In the mixed liquor of fetal calf serum and Lonza DMEM culture medium, obtains fat mesenchymal stem cell and save liquid.
Liquid, which is saved, as preparation the present invention also provides a kind of fat mesenchymal stem cell of the present invention saves fat
The application of the preservation liquid of mescenchymal stem cell.
The present invention provides a kind of fat mesenchymal stem cells to save liquid and its preparation method and application, provided by the invention
Fat mesenchymal stem cell saves liquid, including fetal calf serum and DMSO, and preservation liquid provided by the invention is as fat mesenchymal rouge
Fat mesenchyme stem cell preserving fluid and existing preservation liquid phase ratio, after fat mesenchymal stem cell cryopreservation resuscitation culture, cell
Motility rate and corresponding biological characteristics have obtained preferable protection.
It is clearly and completely described below in conjunction with the technical solution of the embodiment of the present invention, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
Embodiment 1
All kinds of frozen stock solutions are sterile grade product, and ready-to-use, big composition component first adds, and DMSO is slowly added to, and avoid office
Portion's overheat, 4 DEG C save backup.
Specific formula such as 1~table of table 3:
Table 1, formula one
| DMSO | Human serum albumins | DMEM/F12 |
| 10wt% | 10wt% | 80wt% |
Table 2, formula two
| DMSO | FBS (fetal calf serum) |
| 20wt% | 80wt% |
Table 3, formula three
| DMSO | Lonza DMEM culture medium | FBS |
| 10wt% | 20wt% | 70wt% |
After fat mesenchymal stem cell is added to configured frozen stock solution, by 1 DEG C/min, -80 DEG C are cooled to, is then moved to
- 196 DEG C of liquid nitrogen preservations.Then the fat mesenchymal stem cell frozen is recovered and is cultivated, specific recovery incubation step is as follows:
1) it is added in 50mL centrifuge tube with the complete medium solution that pipette draws 10 times of frozen stock solution volumes, rapidly
Cell suspension is transferred in 50mL centrifuge tube, gently piping and druming mixes, and 500g is centrifuged 5min.
2) cell precipitation is resuspended with complete medium, adjustment cell-seeding-density is (5-8) x104Cell/mL adds 1 μ
G/mL EGF to final concentration of 10ng/mL, is inoculated with after mixing well.
3) 5%CO is transferred into 10cm culture dish with the cell suspension of pipette inoculation 10mL2, 37 DEG C, saturated humidity
To be cultivated in 95% carbon dioxide incubator.
Cultivation results are shown in Table 4
Table 4
As can be seen from the table, it relative to traditional formula one, formula two and is formulated between the fat that three frozen stock solution freezes
Mesenchymal stem cells after cryopreservation resuscitation culture, preferably protected by Cell viability and corresponding biological characteristics.
Embodiment 2
According to the recipe configuration frozen stock solution of table 5, and after fat mesenchymal stem cell is added to configured frozen stock solution, press
1 DEG C/min, -80 DEG C are cooled to, then moves to -196 DEG C of liquid nitrogen preservations.Then the fat mesenchymal stem cell frozen is recovered and is trained
It supports, specific step is as follows for culture of recovering:
1) it is added in 50mL centrifuge tube with the complete medium solution that pipette draws 10 times of frozen stock solution volumes, rapidly
Cell suspension is transferred in 50mL centrifuge tube, gently piping and druming mixes, and 500g is centrifuged 5min.
2) cell precipitation is resuspended with complete medium, adjustment cell-seeding-density is (5-8) x104Cell/mL adds 1 μ
G/mL EGF to final concentration of 10ng/mL, is inoculated with after mixing well.
3) 5%CO is transferred into 10cm culture dish with the cell suspension of pipette inoculation 10mL2, 37 DEG C, saturated humidity
To be cultivated in 95% carbon dioxide incubator.
Cultivation results are shown in Table 5, and table 5 is that the frozen stock solution that component is identical, constituent content is different freezes fat mesenchymal stem cell
It recovers afterwards the result of culture.
Table 5
As can be seen from Table 5, the difference of fetal calf serum content freezes effect to frozen stock solution and has a great impact, when FBS's
Content be greater than 50% when, the fat mesenchymal stem cell that obtained frozen stock solution freezes after cryopreservation resuscitation culture, Cell viability and
Corresponding biological characteristics are preferably protected.
The above description of the embodiment is only used to help understand the method for the present invention and its core ideas.It should be pointed out that pair
For those skilled in the art, without departing from the principle of the present invention, the present invention can also be carried out
Some improvements and modifications, these improvements and modifications also fall within the scope of protection of the claims of the present invention.
Claims (8)
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109938010A (en) * | 2019-03-20 | 2019-06-28 | 江苏瑞思坦生物科技有限公司 | A kind of human adipose mesenchymal stem cells transport liquid and preparation method thereof |
| CN112841176A (en) * | 2021-01-28 | 2021-05-28 | 暨南大学 | Long-term cryopreservation medium for adipose-derived stem cells and preparation method and application thereof |
| CN113331176A (en) * | 2021-06-01 | 2021-09-03 | 北京戴域生物技术有限公司 | Mesenchymal stem cell cryopreservation liquid |
| CN115997755A (en) * | 2022-12-21 | 2023-04-25 | 宋芸娟 | A kind of preservation method of adipose-derived mesenchymal stem cells |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109938010A (en) * | 2019-03-20 | 2019-06-28 | 江苏瑞思坦生物科技有限公司 | A kind of human adipose mesenchymal stem cells transport liquid and preparation method thereof |
| CN112841176A (en) * | 2021-01-28 | 2021-05-28 | 暨南大学 | Long-term cryopreservation medium for adipose-derived stem cells and preparation method and application thereof |
| CN113331176A (en) * | 2021-06-01 | 2021-09-03 | 北京戴域生物技术有限公司 | Mesenchymal stem cell cryopreservation liquid |
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| CN115997755A (en) * | 2022-12-21 | 2023-04-25 | 宋芸娟 | A kind of preservation method of adipose-derived mesenchymal stem cells |
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Application publication date: 20190222 |