CN107912420A - A kind of cell preservation method, preserve liquid and preserve liquid and preparation method thereof - Google Patents
A kind of cell preservation method, preserve liquid and preserve liquid and preparation method thereof Download PDFInfo
- Publication number
- CN107912420A CN107912420A CN201711031396.6A CN201711031396A CN107912420A CN 107912420 A CN107912420 A CN 107912420A CN 201711031396 A CN201711031396 A CN 201711031396A CN 107912420 A CN107912420 A CN 107912420A
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- China
- Prior art keywords
- cell
- trehalose
- preservation liquid
- liquid
- preservation
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- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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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
-
- 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
Abstract
The invention discloses a kind of cell-preservation liquid, including following material：Albumin, low molecular sodium heparin, trehalose and physiological saline.Beneficial effects of the present invention：Cell-preservation liquid of the present invention, wherein, used albumin can maintain the viscosity and osmotic pressure of solution appropriateness, while can prevent cellular adhesion, maintain cell bio-activity.Heparin sodium can prevent fibrin from generating, and further prevent cellular adhesion.And trehalose is a kind of non-specific natural protective agent; have the function that to protect biological tissue, cell and large biological molecule; it can be very good the bioactivity of cell in the storage and transport of guarantee long period; therefore; the cell-preservation liquid of the present invention plays excellent maintenance effect to the vigor and form of cell, is suitable for long period storage and transport, maintains its bioactivity; and component is simple, preparation method is succinct.
Description
Technical field
The present invention relates to cell to preserve preparation technique field, it particularly relates to a kind of cell preservation method, preserve liquid and
Preserve liquid and preparation method thereof.
Background technology
Preserving liquid will keep therefore how the bioactivity of cell, maintains cell to meet its application clinically
It is crucial that high activity becomes treatment.Before this, people are by the use of physiological saline or glucose solution as protection liquid, but due to lacking
Nutrition, solution isostension wait penetration the reason such as to have differences, cause cell it is long-term preserve or transport in vigor occur low
Under, phenomena such as a large amount of swelling is dead.Then, people constantly improve the compositional system for preserving liquid, with the addition of KCl, sodium citrate, in vain
Albumen, vitamin etc., form and preserve liquid.Although these preserve liquid and maintain cytoactive, group well in a long time
It is cumbersome into complicated component, process for preparation.
Patent publication No. is that the Chinese patent application of CN104542578A discloses a kind of cell-preservation liquid, it includes white egg
In vain, glucose, vitamin C and preservation basal liquid, it is Multiple electrolytes injection and Amino Acid Compound Injection to preserve basal liquid
Mixture.The cell-preservation liquid shortcoming is that the preservation liquid to the bioactivity-protected preferable not enough of cell, preserves cell
Motility rate is not up to gratifying effect.In order to adapt to the needs of clinic, exploitation one kind adapts to transport and improve for a long time thin
The preservation liquid that born of the same parents preserve motility rate is very necessary.
The problem of in correlation technique, not yet propose effective solution at present.
The content of the invention
For the above-mentioned technical problem in correlation technique, the present invention proposes a kind of cell-preservation liquid, living to the biology of cell
Property protection it is preferable, it is satisfactory to preserve Cell viability, adapts to transport for a long time, another aspect of the present invention, additionally provides one kind
Cell preservation method and cell-preservation liquid preparation method.
To realize above-mentioned technical purpose, the technical proposal of the invention is realized in this way：
A kind of cell-preservation liquid, including following material：Albumin, low molecular sodium heparin, trehalose and physiological saline.
To keep cell, adding trehalose, this is a kind of non-specific natural in the bioactivity of long-time storage and transport
Protective agent, trehalose can be formed solely under the severe environmental conditions such as high temperature, high and cold, hyperosmosis and dry dehydration in cell surface
Special protective film, effectively protected protein matter molecule consistency inactivate so that the life process and biological characteristic of the body that sustains life.
The trehalose that have studied detailed British universities Camilo.C etc. gathers DNA restriction enzymes DNA ligase and DNA
The protective effect of synthase, the results showed that, all enzyme samples for adding trehalose drying, preserve 35 days at 70 DEG C or preserve 9 at 37 DEG C
After a month, its vigor and free of losses, remain to accurately block DNA.Institute of microbiology of the Chinese Academy of Sciences of China does using trehalose
Dry preparation, three kinds of diagnostic tool enzymes for human serum cholesterol determination, at room temperature after long-term preservation, active conservation rate all exists
More than 90%, successfully enter in clinical practice.This is the effect that the protective agent of other species is impossible to reach, and is utilized
Stabilizer and protective agent of the trehalose as biological reagents such as diagnostic tool enzymes, can be placed under normal temperature condition and dry and preserve.
Trehalose in the present invention can be as bioactive substances such as blood product, vaccine, lymphocyte, cell tissues
Stabilizer.And at room temperature after long-term preservation, for active conservation rate all more than 90%, this is that the protective agent of other species all can not
It can achieve the effect that, by the use of trehalose as the stabilizer of biological reagent and protective agent, can be placed under normal temperature condition and dry and protect
Deposit, simplify the preparation process of biological reagent.
Further, the concentration of the albumin is 0.01g/ml, and the concentration of low molecular sodium heparin is 2U/ml, trehalose
Concentration be 0.015g/ml, the concentration of physiological saline is 0.9%.
Another aspect of the present invention, there is provided a kind of preparation method of cell-preservation liquid, includes the following steps：
S1. 0.1g/ml trehalose normal saline solutions are configured；
S2. configuration preserves liquid:0.9% physiological saline is drawn, human serum albumin, heparin sodium, step S1 is sequentially added and obtains
Trehalose normal saline solution mix.
Further, the step S1 specifically comprises the following steps：1.0g trehaloses are weighed, add 0.9% physiological saline
10ml, mixes, and crosses 0.22 μm of strainer；The step S2 specifically comprises the following steps：80ml0.9% physiological saline is drawn, successively
Draw the sea that 32 μ l of heparin sodium, the 15ml steps S1 of human serum albumin 5ml, 12500u/2ml that mass volume ratio is 20% are obtained
Algae sugar normal saline solution, mixes, obtains 100ml cell-preservation liquids.
Another aspect of the present invention, additionally provides a kind of cell preservation method, it is protected using any one above-mentioned cell
Liquid storage preserves.
Beneficial effects of the present invention：Cell-preservation liquid of the present invention, wherein, used albumin can make cell
Scattered, non-agglomerate, heparin sodium further ensures that cell non-agglomerate, anticoagulation, and trehalose is a kind of non-specific natural guarantor
Agent is protected, has the function that to protect biological tissue, cell and large biological molecule, can be very good to ensure in long-time storage and transport
The bioactivity of cell, therefore, cell-preservation liquid of the invention play excellent maintenance effect to the vigor and form of cell, can fit
Long-time storage and transport are answered, maintain its biological nature, and component is simple, and preparation method is succinct.
Brief description of the drawings
, below will be to required in embodiment in order to illustrate more clearly of inventive embodiments or technical solution of the prior art
Attached drawing to be used is briefly described.
Fig. 1 is the ratio that total cell is accounted for using the cell-preservation liquid preservation 0h and 24h aim cells described in the embodiment of the present invention
Example；
Fig. 2 is to preserve 0h and 24h cd4+ cells and cd8+ cells point using the cell-preservation liquid described in the embodiment of the present invention
Public sentiment condition；
Fig. 3 is to preserve (the center notes of Tcm in 0h and 24h cd8 cells using the cell-preservation liquid described in the embodiment of the present invention
Recall type T cell, Central Memory T cell) proportion；
Fig. 4 is to preserve Tcm institutes accounting in 0h and 24h cd4 cells using the cell-preservation liquid described in the embodiment of the present invention
Example.
Embodiment
Below in conjunction with specific embodiment, technical scheme is clearly and completely described, it is clear that retouched
The embodiment stated is only part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, originally
Field those of ordinary skill's all other embodiments obtained, belong to the scope of protection of the invention.
Embodiment one
S1. 0.1g/ml trehalose normal saline solutions are configured.
1.0g trehaloses are weighed using balance to be put into 15ml centrifuge tubes.Use 0.9% physiology salt of 10ml pipette, extracts
Water 10ml is added in centrifuge tube.Piping and druming mixes repeatedly.Using 20ml syringe draw solutions, 0.22 μm of strainer is crossed, with degerming.
S2. 100ml cell-preservation liquids are configured.
It is put into using 10ml pipette, extract 80ml physiological saline in 250ml centrifuge tubes, draws 5ml human serum albumins successively
(20% (g/v)), 32 μ l heparin sodiums (12500u/2ml), 15ml trehalose normal saline solutions.It is reverse to mix.
Operation according to embodiment one obtains some groups of cell-preservation liquids, and the 100ml cells obtained through above-mentioned steps are protected
Liquid storage carries out mescenchymal stem cell and Tcm cells (central memory T cells) carry out the test of cell survival rate and conglomeration rate, knot
Fruit difference is as shown in Table 1 and Table 2.
Test result is shown, by cell-preservation liquid made from method of the present invention, to mescenchymal stem cell and Tcm
Cell is preserved, and survival rate is very high, and 24h only has a small amount of loss cell, also, cell conglomeration rate is extremely low, of the present invention
The protective agent that cell-preservation liquid has reached other species is impossible to the effect reached, and more preferable treatment is can reach in clinical practice
Effect, also, the constituent of cell-preservation liquid of the present invention only includes albumin, low molecular sodium heparin, trehalose and life
Brine is managed, component is more simple compared with conventional art, so as to simplify process for preparation, can be preserved for a long time at 2-8 DEG C, non-
Often it is adapted to promote the use of.
1. cell survival rate of table
2. cell conglomeration rate of table
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
With within principle, any modification, equivalent replacement, improvement and so on, should all be included in the protection scope of the present invention god.
Claims (5)
1. a kind of cell-preservation liquid, it is characterised in that including following material：Albumin, low molecular sodium heparin, trehalose and physiology
Brine.
2. a kind of cell-preservation liquid according to claim 1, it is characterised in that the concentration of the albumin is 0.01g/
Ml, the concentration of low molecular sodium heparin is 2U/ml, and the concentration of trehalose is 0.015g/ml, and the concentration of physiological saline is 0.9%.
3. a kind of preparation method of cell-preservation liquid, it is characterised in that include the following steps：
S1. 0.1g/ml trehalose normal saline solutions are configured;
S2. configuration preserves liquid:0.9% physiological saline is drawn, sequentially adds the seaweed that human serum albumin, heparin sodium, step S1 are obtained
Sugared normal saline solution mixes.
4. the preparation method of a kind of cell-preservation liquid according to claim 3, it is characterised in that the step S1 is specifically wrapped
Include following steps：1.0g trehaloses are weighed, add 0.9% physiological saline 10ml, are mixed, cross 0.22 μm of strainer；The step S2 tools
Body includes the following steps：Draw 80ml0.9% physiological saline, successively draw mass volume ratio be 20% human serum albumin 5ml,
The trehalose normal saline solution that 32 μ l of heparin sodium, the 15ml steps S1 of 12500u/2ml is obtained, mixes, obtains 100ml cells
Preserve liquid.
5. a kind of cell preservation method, it is characterised in that any one cell-preservation liquid described in usage right requirement 1 or 2 is protected
Deposit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711031396.6A CN107912420A (en) | 2017-10-27 | 2017-10-27 | A kind of cell preservation method, preserve liquid and preserve liquid and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711031396.6A CN107912420A (en) | 2017-10-27 | 2017-10-27 | A kind of cell preservation method, preserve liquid and preserve liquid and preparation method thereof |
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| Publication Number | Publication Date |
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| CN107912420A true CN107912420A (en) | 2018-04-17 |
Family
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| CN201711031396.6A Pending CN107912420A (en) | 2017-10-27 | 2017-10-27 | A kind of cell preservation method, preserve liquid and preserve liquid and preparation method thereof |
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| CN (1) | CN107912420A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112522190A (en) * | 2020-12-08 | 2021-03-19 | 山东省齐鲁干细胞工程有限公司 | High-survival-rate storage and transportation method for umbilical cord tissues |
| CN112640888A (en) * | 2020-12-30 | 2021-04-13 | 深路医学科技(武汉)有限公司 | Cell preservation solution, preparation method thereof and cell preservation method |
| CN113322232A (en) * | 2021-04-29 | 2021-08-31 | 广东先康达生物科技有限公司 | Preparation method of platelet-rich plasma |
| CN115633675A (en) * | 2021-07-19 | 2023-01-24 | 无锡赛比曼生物科技有限公司 | Preservation solution for preserving adipose-derived mesenchymal stem cells at low temperature for long time |
| CN115777695A (en) * | 2023-02-07 | 2023-03-14 | 成都海默云因医学检验实验室有限公司 | Compound preservation solution suitable for morphological examination of cerebrospinal fluid cells and preparation method thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112522190A (en) * | 2020-12-08 | 2021-03-19 | 山东省齐鲁干细胞工程有限公司 | High-survival-rate storage and transportation method for umbilical cord tissues |
| CN112640888A (en) * | 2020-12-30 | 2021-04-13 | 深路医学科技(武汉)有限公司 | Cell preservation solution, preparation method thereof and cell preservation method |
| CN112640888B (en) * | 2020-12-30 | 2022-05-03 | 深路医学科技(武汉)有限公司 | Cell preservation solution, preparation method thereof and cell preservation method |
| CN113322232A (en) * | 2021-04-29 | 2021-08-31 | 广东先康达生物科技有限公司 | Preparation method of platelet-rich plasma |
| CN113322232B (en) * | 2021-04-29 | 2022-02-18 | 广东先康达生物科技有限公司 | Preparation method of platelet-rich plasma |
| CN115633675A (en) * | 2021-07-19 | 2023-01-24 | 无锡赛比曼生物科技有限公司 | Preservation solution for preserving adipose-derived mesenchymal stem cells at low temperature for long time |
| CN115777695A (en) * | 2023-02-07 | 2023-03-14 | 成都海默云因医学检验实验室有限公司 | Compound preservation solution suitable for morphological examination of cerebrospinal fluid cells and preparation method thereof |
| CN115777695B (en) * | 2023-02-07 | 2023-05-12 | 成都海默云因医学检验实验室有限公司 | Composite preservation solution suitable for cerebrospinal fluid cell morphology examination and preparation method thereof |
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Effective date of registration: 20191128 Address after: 072150 floor 3, West unit, building 12, Zhongguancun innovation base, Baoding, No. 369, Huiyang street, Baoding, Hebei Province Applicant after: Baoding Norway Technology Co., Ltd. Address before: 100022 Jiulong Mountain home, Chaoyang District, Chaoyang District, Beijing, 1 gate 1605 Applicant before: Beijing Union Pharmaceutical Science and Technology Co Ltd |
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Application publication date: 20180417 |