CN109479873A - A kind of adipose tissue saves liquid and its application - Google Patents
A kind of adipose tissue saves liquid and its application Download PDFInfo
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- CN109479873A CN109479873A CN201811558671.4A CN201811558671A CN109479873A CN 109479873 A CN109479873 A CN 109479873A CN 201811558671 A CN201811558671 A CN 201811558671A CN 109479873 A CN109479873 A CN 109479873A
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- adipose tissue
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- sodium citrate
- amino acid
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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
- A01N1/0226—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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- 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
- C12N5/0653—Adipocytes; Adipose tissue
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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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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Abstract
The present invention relates to technical field of tissue culture, discloses a kind of adipose tissue and save liquid and its application.Adipose tissue of the present invention saves the phosphate buffer that liquid includes nonessential amino acid and sodium citrate.The present invention prepares adipose tissue using the phosphate buffer of nonessential amino acid and sodium citrate and saves liquid, maintain the activity of adipose tissue, improve the cell quantity and Cell viability of isolated SVF, it is aggregated simultaneously prevented also from blood constituent extra in adipose tissue, prevent lipochondrion conglomeration, achieve the purpose that efficiently separate SVF, may be used on separating in SVF.
Description
Technical field
The present invention relates to technical field of tissue culture, and in particular to a kind of adipose tissue saves liquid and its application.
Background technique
Adipose tissue is mainly made of the fat cell of a large amount of clusters, is internal maximum " energy depot ", has storage rouge
Fat, keeping body mildly participate in the function of fat metabolism, while fat also participates in energetic supersession, and have and generate heat, maintain body
The effects of temperature, buffer protection and support filling.In addition, it still have the multi-functional cell of multi-lineage potential source it
One, and this multi-functional cell is present in the vascular stroma part of adipose tissue.
Vascular stroma part (SVF) in adipose tissue is an adipose-derived stem cells and vascular endothelial cell, periphery
The group that haemocyte, T cell and other inflammatory cells mutually mix can be divided into mature fat cell in vivo under environment, promote
Into the directional migration of host stem cells Xiang Shouqu, subparticipation composition neovascular endothelium cell facilitates transplant fat group
Knit the foundation of early stage blood supply.
SVF be adipose tissue through enzymic digestion, filter, be centrifuged off fat tissue cell after obtain, the cell viability of SVF
Autologous adipose tissue transplanting success rate and efficiency are directly affected with cell quantity.And the cell viability and cell quantity of SVF and
The quality of adipose tissue has direct relationship, therefore the preservation of adipose tissue is particularly important.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of adipose tissues to save liquid, so that saving through the preservation liquid
The adipose tissue crossed, when separating SVF, SVF cell quantity and Cell viability are higher；
Another object of the present invention is that provide above-mentioned preservation liquid separation SVF in application or in preparative separation
Application in the reagent of SVF.
For achieving the above object, the invention provides the following technical scheme:
A kind of adipose tissue preservation liquid, the phosphate buffer including nonessential amino acid and sodium citrate.
Wherein, the nonessential amino acid is l-Alanine, altheine, L- aspartic acid, L- glycine, L- ammonia
Commercial prod MEM NEAA (stoste 100X, i.e. 10mM, purchased from Sigma public affairs can be used in acid, L-PROLINE and Pidolidone
Department, #M7145), the present invention needs to be diluted to 1X, i.e. 0.1mM in actual use.
Preferably, the phosphate buffer of the sodium citrate is the 1X PBS phosphoric acid buffer of 30-50 μ g/ml sodium citrate
Liquid, the concentration of sodium citrate may be selected to be 30,35,40,45 or 50 μ g/ml in specific application.Preferably, every liter of 1X
PBS phosphoric acid buffer liquid is as follows:
Weigh sodium chloride 8g, potassium chloride 0.2g, disodium hydrogen phosphate 3.63g respectively, potassium dihydrogen phosphate 0.24g is molten
In 900ml ultrapure water, with hydrochloric acid tune pH value to 7.4, water is added to be settled to 1L, autoclave sterilization.
Any adipose tissue for saving liquid is not added, adipose tissue for 24 hours is saved by DMEM culture medium and passes through this
Invention saves the adipose tissue that liquid saves for 24 hours and compares test, counts each group SVF cell quantity and Cell viability, as a result shows
Show the cell quantity of experimental group of the present invention in 4.0*106Left and right, adipose tissue of the survival rate 98% or so, with fresh acquisition
The SVF of (0h) separation is without significant difference；And other two contrast groups SVF state obviously declines；
Meanwhile cellular morphology, the results show that comparing with contrast groups, experimental group adherence rate of the present invention is higher, cell shape
State is in spindle shape, brighter.
Based on this excellent technical effect, the invention proposes the preservation liquid in the application separated in SVF or to prepare
Separate the application in the reagent of SVF.
From the above technical scheme, the present invention prepares rouge using the phosphate buffer of nonessential amino acid and sodium citrate
Fat tissue preserration liquid, maintains the activity of adipose tissue, improves the cell quantity and Cell viability of the SVF of separation, while can also
Blood constituent agglutination extra in adipose tissue is prevented, lipochondrion conglomeration is prevented, achievees the purpose that efficiently separate SVF.
Detailed description of the invention
Fig. 1 show experimental group 1 and saves the SVF cellular morphology that liquid saves adipose tissue 0h (A) and (B) is separated afterwards for 24 hours；
Fig. 2 show contrast groups 1 without using the SVF cell for saving liquid preservation adipose tissue 0h (A) and (B) is separated afterwards for 24 hours
Form；
Fig. 3 show contrast groups 2 and saves the SVF cellular morphology that liquid saves adipose tissue 0h (A) and (B) is separated afterwards for 24 hours.
Specific embodiment
Saving liquid and its application, those skilled in the art the invention discloses a kind of adipose tissue can use for reference in this paper
Hold, is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications are to those skilled in the art
For be it will be apparent that they are considered as being included in the present invention.Preservation liquid of the present invention and its application have passed through reality
Example is applied to be described, related personnel obviously can not depart from the content of present invention, in spirit and scope to preservation as described herein
Liquid and its application are modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
If not otherwise specified, in the comparative test of the specific embodiment of the invention, each experimental group and contrast groups removing are due
Difference is outer, and used adipose tissue and each reagent, experimental enviroment are all the same.
Just a kind of adipose tissue provided by the present invention saves liquid below and its application is described further.
Embodiment 1: it prepares adipose tissue of the present invention and saves liquid
(1) it forms
The phosphoric acid buffer of the sodium citrate of the MEM NEAA nonessential amino acid of 0.1mM and 30,35,40,45 or 50 μ g/ml
Liquid；
(2) preparation method
The preparation method of 1X phosphate buffer: sodium chloride 8g, potassium chloride 0.2g, disodium hydrogen phosphate are weighed respectively
3.63g, potassium dihydrogen phosphate 0.24g are dissolved in 900ml ultrapure water, with hydrochloric acid tune pH value to 7.4, water are added to be settled to 1L, high temperature is high
After pressure sterilizing, 2-8 DEG C is saved backup.
Tissue preserration liquid making method: it is weighed according to the sodium citrate concentration of 30,35,40,45 or 50 μ g/ml a certain amount of
Sodium citrate powder is added in the 1X phosphate buffer of 1L, and after the strainer filtering degerming of utilization 0.22um, is added
100XMEM NEAA nonessential amino acid 10ml, makes its final concentration of 1X, and slight piping and druming mixing is placed on 2-8 DEG C and saves backup.
Embodiment 2: the active comparative test of adipose tissue separation SVF
(1) experimental subjects
The phosphate buffer of the sodium citrate of the MEM NEAA nonessential amino acid of experimental group 1:0.1mM and 40 μ g/ml；
The phosphate buffer of the sodium citrate of the MEM NEAA nonessential amino acid of experimental group 2:0.1mM and 30 μ g/ml；
The phosphate buffer of the sodium citrate of the MEM NEAA nonessential amino acid of experimental group 3:0.1mM and 50 μ g/ml；
Contrast groups 1: without using preservation liquid；
Contrast groups 2:DMEM culture medium；
(2) experimental method
1) fatty acquisition and preservation
The donor at 20~30 years old age, non-communicable disease is chosen, informed consent form and Operation Agreement Letters are signed, is extracted
120ml fat, and fat is divided into four parts, every part of 30ml, liquid is saved except any adipose tissue is added without in contrast groups 1, remaining
Corresponding adipose tissue that 30ml fat and same volume are respectively added in each experimental group and contrast groups 2 saves liquid, and is placed on 4
It degree Celsius saves respectively, the holding time is 0h, for 24 hours；The separation that each time point takes 15ml pure fat to carry out fat stem cell is trained
It supports.
2) isolation and culture of fat stem cell
The fat of each experimental group and contrast groups 1 and 2 is centrifuged, removal adipose tissue saves liquid；Using isometric
For physiological saline to adipose tissue-wash 2 times of all groups, 500g is centrifuged 5min, abandons upper layer oil layer and lower layer's physiological saline；?
Collagenase type I is added in pure fat, digestion 20-30min is carried out to adipose tissue, is resuspended after cleaning centrifugation with serum free medium
Precipitating takes part cell suspension to carry out the detection of cell quantity and Cell viability, as a result table 1；Adjusting inoculum density simultaneously is 8*
105/ ml or so, is seeded in culture dish, and cross is put in carbon dioxide incubator after mixing and cultivates 48h, micro- using being inverted
Mirror carries out observation cell growth status, the result is shown in Figure 1-3.
The detection of table 1 cell quantity and Cell viability
As can be seen from Table 1, save liquid by the present invention and save adipose tissue for 24 hours and separate SVF, 3 experimental groups it is thin
Born of the same parents' quantity is in 4.0-4.6*106, survival rate is in 96-99%, and the SVF separated with the adipose tissue (0h) of fresh acquisition is without significance difference
It is different；And the quantity and motility rate of the SVF of other two contrast groups separation are remarkably decreased.
In addition, comparing it can be seen from the cellular morphology result of Fig. 1-Fig. 3 with contrast groups 1 and contrast groups 2, experimental group 1 is thin
Born of the same parents' adherent rate is higher, and cellular morphology is in spindle shape, brighter.Fig. 1 shows the 0h and cell quantity for 24 hours of experimental group 1 simultaneously
With Cell viability without significant difference, it is consistent with 1 result of table, illustrates that the present invention saves liquid and is remarkably improved SVF separation rate, while right
Cellular morphology is without influence.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (7)
1. a kind of adipose tissue saves liquid, which is characterized in that the phosphate buffer including nonessential amino acid and sodium citrate.
2. saving liquid according to claim 1, which is characterized in that the nonessential amino acid is l-Alanine, L- asparagus fern acyl
Amine, L- aspartic acid, L- glycine, Serine, L-PROLINE and Pidolidone.
3. preservation liquid according to claim 1 or claim 2, which is characterized in that the nonessential amino acid is the nonessential ammonia of 0.1mM
Base acid.
4. saving liquid according to claim 1, which is characterized in that the phosphate buffer of the sodium citrate is 30-50 μ g/ml
The 1X PBS phosphate buffer of sodium citrate.
5. saving liquid according to claim 4, which is characterized in that every liter of 1X PBS phosphoric acid buffer liquid is such as
Under:
Sodium chloride 8g, potassium chloride 0.2g, disodium hydrogen phosphate 3.63g are weighed respectively, and potassium dihydrogen phosphate 0.24g is dissolved in
In 900ml ultrapure water, with hydrochloric acid tune pH value to 7.4, water is added to be settled to 1L, autoclave sterilization.
6. according to claim 1 or the 4 preservation liquid, which is characterized in that the concentration of the sodium citrate be 30,35,40,45 or
50μg/ml。
7. described in claim 1-6 any one preservation liquid separation SVF in application or in the reagent of preparative separation SVF
Using.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811558671.4A CN109479873B (en) | 2018-12-19 | 2018-12-19 | Adipose tissue preservation solution and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811558671.4A CN109479873B (en) | 2018-12-19 | 2018-12-19 | Adipose tissue preservation solution and application thereof |
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| Publication Number | Publication Date |
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| CN109479873A true CN109479873A (en) | 2019-03-19 |
| CN109479873B CN109479873B (en) | 2021-10-01 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115812695A (en) * | 2022-12-30 | 2023-03-21 | 广州赛莱拉干细胞科技股份有限公司 | Bone marrow preservation solution for efficiently obtaining hematopoietic stem cells and using method thereof |
| CN115956556A (en) * | 2021-10-13 | 2023-04-14 | 无锡赛比曼生物科技有限公司 | Adipose tissue transportation and preservation solution |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104839146A (en) * | 2015-05-29 | 2015-08-19 | 广州赛莱拉干细胞科技股份有限公司 | Composition and application thereof, placenta preservative and preparation method of placenta preservative |
| CN106719602A (en) * | 2016-11-30 | 2017-05-31 | 广州赛莱拉干细胞科技股份有限公司 | A kind of method of frozen stock solution and its application with adipose tissue is frozen |
-
2018
- 2018-12-19 CN CN201811558671.4A patent/CN109479873B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104839146A (en) * | 2015-05-29 | 2015-08-19 | 广州赛莱拉干细胞科技股份有限公司 | Composition and application thereof, placenta preservative and preparation method of placenta preservative |
| CN106719602A (en) * | 2016-11-30 | 2017-05-31 | 广州赛莱拉干细胞科技股份有限公司 | A kind of method of frozen stock solution and its application with adipose tissue is frozen |
Non-Patent Citations (1)
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| 冯国旗 等: "《实用复方药物手册》", 31 May 1998, 中国科学技术出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115956556A (en) * | 2021-10-13 | 2023-04-14 | 无锡赛比曼生物科技有限公司 | Adipose tissue transportation and preservation solution |
| CN115812695A (en) * | 2022-12-30 | 2023-03-21 | 广州赛莱拉干细胞科技股份有限公司 | Bone marrow preservation solution for efficiently obtaining hematopoietic stem cells and using method thereof |
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| CN109479873B (en) | 2021-10-01 |
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