CN110463686A - A kind of cell-preservation liquid that can effectively save cell for a long time - Google Patents
A kind of cell-preservation liquid that can effectively save cell for a long time Download PDFInfo
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- CN110463686A CN110463686A CN201910611073.7A CN201910611073A CN110463686A CN 110463686 A CN110463686 A CN 110463686A CN 201910611073 A CN201910611073 A CN 201910611073A CN 110463686 A CN110463686 A CN 110463686A
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- cell
- preservation liquid
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- effectively save
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- 239000007788 liquid Substances 0.000 title claims abstract description 48
- 238000004321 preservation Methods 0.000 title claims abstract description 41
- 239000004094 surface-active agent Substances 0.000 claims abstract description 11
- RCEAADKTGXTDOA-UHFFFAOYSA-N OS(O)(=O)=O.CCCCCCCCCCCC[Na] Chemical compound OS(O)(=O)=O.CCCCCCCCCCCC[Na] RCEAADKTGXTDOA-UHFFFAOYSA-N 0.000 claims description 6
- 239000004615 ingredient Substances 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 2
- 230000003213 activating effect Effects 0.000 claims 2
- 239000003795 chemical substances by application Substances 0.000 claims 2
- 230000000694 effects Effects 0.000 abstract description 21
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 abstract description 9
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 abstract description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 abstract description 8
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 abstract description 5
- 229960000583 acetic acid Drugs 0.000 abstract description 5
- 229960001484 edetic acid Drugs 0.000 abstract description 5
- 244000005700 microbiome Species 0.000 abstract description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 5
- 239000012362 glacial acetic acid Substances 0.000 abstract description 4
- 230000012010 growth Effects 0.000 abstract description 4
- 239000001103 potassium chloride Substances 0.000 abstract description 4
- 235000011164 potassium chloride Nutrition 0.000 abstract description 4
- 239000011780 sodium chloride Substances 0.000 abstract description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 abstract description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 abstract description 3
- 239000001110 calcium chloride Substances 0.000 abstract description 3
- 229910001628 calcium chloride Inorganic materials 0.000 abstract description 3
- 230000006378 damage Effects 0.000 abstract description 3
- 239000008367 deionised water Substances 0.000 abstract description 3
- 229910021641 deionized water Inorganic materials 0.000 abstract description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 abstract description 3
- 230000000813 microbial effect Effects 0.000 abstract description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 abstract description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 abstract description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 abstract description 3
- 229930006000 Sucrose Natural products 0.000 abstract description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 abstract description 2
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 239000005720 sucrose Substances 0.000 abstract description 2
- 241000894006 Bacteria Species 0.000 description 10
- 210000003296 saliva Anatomy 0.000 description 7
- 150000002500 ions Chemical class 0.000 description 6
- 102000039446 nucleic acids Human genes 0.000 description 6
- 108020004707 nucleic acids Proteins 0.000 description 6
- 150000007523 nucleic acids Chemical class 0.000 description 6
- 239000002504 physiological saline solution Substances 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 235000015097 nutrients Nutrition 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 239000006916 nutrient agar Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 101710163270 Nuclease Proteins 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 230000000845 anti-microbial effect Effects 0.000 description 2
- 244000144987 brood Species 0.000 description 2
- 230000003139 buffering effect Effects 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 240000000111 Saccharum officinarum Species 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 238000005660 chlorination reaction Methods 0.000 description 1
- 238000005202 decontamination Methods 0.000 description 1
- 230000003588 decontaminative effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization 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
- A01N1/02—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
- A01N1/0215—Disinfecting agents, e.g. antimicrobials for preserving living parts
-
- 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
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)
- Biophysics (AREA)
- Physiology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a kind of cell-preservation liquids that can effectively save cell for a long time, form as follows: disodium hydrogen phosphate 0.1-3g, potassium dihydrogen phosphate 0.1-4.1g, potassium chloride 0.01-2.2g, sodium chloride 0.6-1g, calcium chloride 0.001-0.01g, glacial acetic acid 0.1-7g, ethylenediamine tetra-acetic acid 1-16g, trishydroxymethylaminomethane 0.001-0.003g, sucrose 0.1-0.24g, surfactant 0.98-2.6g, deionized water is settled to 100mL, pH6.8-7.6.The invention has the beneficial effects that: cell-preservation liquid provided by the invention can maintain the activity of cell in a longer period of time, therefore can effectively save cell for a long time；When saving cell using cell-preservation liquid provided by the invention, under the longer holding time, cell-preservation liquid can still inhibit microbial growth, avoid destruction of the cell by microorganism.
Description
Technical field
The present invention relates to a kind of cell-preservation liquids, and in particular to a kind of cell preservation that can effectively save cell for a long time
Liquid belongs to medical biotechnology field.
Background technique
Cell is the most basic unit of life, all has application value at many aspects.For cell and cell product, with
Unlike common biological products and drug: it needs to maintain cell activity, and maintaining cell activity is a greatly challenge.
Physiological saline has the characteristics that safe and convenient in clinical application, but cell is in physiological saline under activity
The speed of drop is very fast, and when 10h is saved in physiological saline, the loss of activity of cell just reaches as many as 30%.It can
See, physiological saline is only suitable for the of short duration preservation of cell.
Currently, had some cell-preservation liquids on the market, but cell saves the time one saved in liquid at these
As within 30d, although longer than the time saved in physiological saline, still have larger gap with actual demand.
Summary of the invention
To solve the deficiencies in the prior art, cell can be effectively saved for a long time the purpose of the present invention is to provide a kind of
Cell-preservation liquid.
In order to achieve the above objectives, the present invention adopts the following technical scheme that:
A kind of cell-preservation liquid that can effectively save cell for a long time, which is characterized in that the group of aforementioned cells preservation liquid
At as follows:
Preferably, what aforementioned surfactants were selected is polysorbas20 or lauryl sodium sulfate.
Preferably, the ingredient of aforementioned cells preservation liquid is as follows:
Wherein, what surfactant was selected is polysorbas20.
It lives, it is preferable that the ingredient that aforementioned cells save liquid is as follows:
Wherein, what surfactant was selected is lauryl sodium sulfate.
Or, it is preferable that the ingredient that aforementioned cells save liquid is as follows:
Wherein, what surfactant was selected is polysorbas20.
The invention has the beneficial effects that:
(1) glacial acetic acid is antimicrobial, is had to multiple-microorganism (including bacterium, brood cell, virus etc.) efficient, quick
Bactericidal effect make thin so cell-preservation liquid provided by the invention can prevent the microbial growths such as bacterium, brood cell, virus
Born of the same parents are not destroyed, so as to effectively save cell for a long time；
(2) EDTA can inhibit the activity of nuclease, and nucleic acid is prevented to be degraded by enzymes, so cell-preservation liquid provided by the invention
It can prevent the degradation of nucleic acid；
(3) calcium chloride, potassium chloride, sodium chloride are cells with nutrient ion, and nutrient ions cooperate other reagents to make together
With can preferably maintain cell activity；
(4) trishydroxymethylaminomethane is buffering pair, and suitable buffering is to can keep to the maximum extent the form of cell complete
It is whole, it is allowed to sufficiently suspend and disperse；
(5) through testing, after cell-preservation liquid provided by the invention saves Stomatocyte 10h at normal temperature, Stomatocyte
Activity remain to reach 99.9% or more, loss of activity must be lacked, preservation effect be significantly better than physiological saline；
(6) through testing, the time that cell-preservation liquid provided by the invention saves Stomatocyte at normal temperature can be extended to
730d is even longer, and when saving to 730d, the activity of Stomatocyte remains to reach 99.9% or more, and loss of activity is few, protects
It deposits effect and is substantially better than existing cell-preservation liquid.
Specific embodiment
Below in conjunction with detailed description of the invention by specific embodiments.
One, the composition of cell-preservation liquid
Disodium hydrogen phosphate 0.1-3g, potassium dihydrogen phosphate 0.1-4.1g, potassium chloride 0.01-2.2g, sodium chloride 0.6-1g, chlorination
Calcium 0.001-0.01g, glacial acetic acid 0.1-7g, ethylenediamine tetra-acetic acid 1-16g, trishydroxymethylaminomethane 0.001-0.003g, sugarcane
Sugared 0.1-0.24g, surfactant 0.98-2.6g, deionized water are settled to 100mL, pH6.8-7.6.
Surfactant: for nonionic detergent, polysorbas20 or lauryl sodium sulfate be can be.
Two, cell-preservation liquid is prepared
According to following table be formulated each reagent of precise, then reagent is dissolved with a small amount of deionized water, spend again later from
Sub- water is settled to 100mL, shakes up, and finally adjusts pH value of solution, that is, cell-preservation liquid is prepared.
In cell-preservation liquid provided by the invention:
(1) disodium hydrogen phosphate, potassium dihydrogen phosphate: solution nutrition fortifier can be used to increase effects of ion intensity, be
The preservation of cell provides enough nutrient ions；
(2) calcium chloride, potassium chloride, sodium chloride are cells with nutrient ion, and nutrient ions cooperate other reagents to make together
With can preferably maintain cell activity；
(3) glacial acetic acid: antimicrobial makes enzyme lose activity by oxidation, causes microorganism dead, and ice vinegar
Acid has the characteristic of acid, when preparing cell-preservation liquid, damages microorganism by changing intracellular pH value, plays high
Effect, quick bactericidal effect cooperate other reagents to be used together to maintain ambient stable, so that cell provided by the invention
The integrality of cell amplifying nucleic acid can be kept for a long time at normal temperature by saving liquid, and can be avoided the growth of the microorganisms such as bacterium；
(4) ethylenediamine tetra-acetic acid (EDTA): chelating agent can inhibit the activity of nuclease, nucleic acid prevented to be degraded by enzymes, favorably
In the stabilization of nucleic acid；
(5) trishydroxymethylaminomethane: buffer can keep to the maximum extent the form of cell complete, be allowed to sufficiently outstanding
Floating and dispersion；
(6) sucrose: energy source is provided for cell；
(7) polysorbas20 and lauryl sodium sulfate: surfactant has stronger decontamination, permeance property, can be with film egg
White hydrophobic part combines, and makes memebrane protein and UF membrane, to obtain nucleic acid.
Two, preservation effect is verified
1, cell activity
Test method: acquiring saliva with spittle collector, and then saliva is put in respectively to 3 groups of cells provided by the invention protected
It in liquid storage, is put in 37 DEG C and saves at room temperature, the volume ratio of saliva and cell-preservation liquid is 1:1, is then taken in different time sections
10uL mixed liquor puts the number that living cells is counted on cell counter, and statistical result is as follows:
(1) it is stored in the 1st group of cell-preservation liquid
(2) it is stored in the 2nd group of cell-preservation liquid
| 37℃ | Room temperature | |
| 4h | 1.4×106 | 1.4×106 |
| 12h | 1.4×106 | 1.4×106 |
| 48h | 1.4×106 | 1.4×106 |
| 30d | 1.4×106 | 1.4×106 |
| 90d | 1.4×106 | 1.4×106 |
| 180d | 1.3998×106 | 1.3999×106 |
| 240d | 1.3998×106 | 1.3999×106 |
| 540d | 1.398×106 | 1.3985×106 |
| 730d | 1.398×106 | 1.3985×106 |
(3) it is stored in the 3rd group of cell-preservation liquid
It can be seen that: cell-preservation liquid provided by the invention can maintain the activity of cell in a longer period of time, therefore
Cell can be effectively saved for a long time.
2, liquid biocidal property is saved
Test method: acquiring saliva with spittle collector, and then saliva is put in respectively to 3 groups of cells provided by the invention protected
In liquid storage, the volume ratio of saliva and cell-preservation liquid is 1:1, then takes 10uL mixed liquor in different time sections, mixed liquor is connect
Culture sees whether long bacterium in kind to nutrient agar plate, does positive control with the nutrient agar panel of inoculation 10uL saliva, uses
The nonvaccinated nutrient agar panel of blank does negative control, and observation result is as follows:
| 1d | 90d | 180d | 260d | 540d | 730d | |
| 1st group | It is sterile | It is sterile | It is sterile | It is sterile | It is sterile | It is sterile |
| 2nd group | It is sterile | It is sterile | It is sterile | It is sterile | It is sterile | It is sterile |
| 3rd group | It is sterile | It is sterile | It is sterile | It is sterile | It is sterile | It is sterile |
| Positive control | Long bacterium | Long bacterium | Long bacterium | Long bacterium | Long bacterium | Long bacterium |
| Negative control | It is sterile | It is sterile | It is sterile | It is sterile | It is sterile | It is sterile |
It can be seen that: when saving cell using cell-preservation liquid provided by the invention, under the longer holding time, cell
Microbial growth can still be inhibited by saving liquid, avoid destruction of the cell by microorganism.
It should be noted that the above embodiments do not limit the invention in any form, it is all to use equivalent replacement or equivalent change
The mode changed technical solution obtained, falls within the scope of protection of the present invention.
Claims (6)
1. the cell-preservation liquid that one kind can effectively save cell for a long time, which is characterized in that the composition of the cell-preservation liquid
It is as follows:
2. the cell-preservation liquid according to claim 1 that can effectively save cell for a long time, which is characterized in that the table
What face activating agent was selected is polysorbas20.
3. the cell-preservation liquid according to claim 1 that can effectively save cell for a long time, which is characterized in that the table
What face activating agent was selected is lauryl sodium sulfate.
4. the cell-preservation liquid according to claim 1 that can effectively save cell for a long time, which is characterized in that described thin
The ingredient that born of the same parents save liquid is as follows:
Wherein, what surfactant was selected is polysorbas20.
5. the cell-preservation liquid according to claim 1 that can effectively save cell for a long time, which is characterized in that described thin
The ingredient that born of the same parents save liquid is as follows:
Wherein, what surfactant was selected is lauryl sodium sulfate.
6. the cell-preservation liquid according to claim 1 that can effectively save cell for a long time, which is characterized in that described thin
The ingredient that born of the same parents save liquid is as follows:
Wherein, what surfactant was selected is polysorbas20.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910611073.7A CN110463686A (en) | 2019-07-08 | 2019-07-08 | A kind of cell-preservation liquid that can effectively save cell for a long time |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910611073.7A CN110463686A (en) | 2019-07-08 | 2019-07-08 | A kind of cell-preservation liquid that can effectively save cell for a long time |
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| Publication Number | Publication Date |
|---|---|
| CN110463686A true CN110463686A (en) | 2019-11-19 |
Family
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910611073.7A Pending CN110463686A (en) | 2019-07-08 | 2019-07-08 | A kind of cell-preservation liquid that can effectively save cell for a long time |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114190367A (en) * | 2021-12-29 | 2022-03-18 | 松山湖材料实验室 | Frozen stock solution, preparation method thereof and application thereof in immune cells |
| CN114190366A (en) * | 2021-12-29 | 2022-03-18 | 松山湖材料实验室 | Frozen stock solution, preparation method thereof and application thereof in normal human liver cells |
| CN114223651A (en) * | 2021-12-29 | 2022-03-25 | 松山湖材料实验室 | Frozen stock solution, preparation method thereof and application thereof in human renal epithelial cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114190367A (en) * | 2021-12-29 | 2022-03-18 | 松山湖材料实验室 | Frozen stock solution, preparation method thereof and application thereof in immune cells |
| CN114190366A (en) * | 2021-12-29 | 2022-03-18 | 松山湖材料实验室 | Frozen stock solution, preparation method thereof and application thereof in normal human liver cells |
| CN114223651A (en) * | 2021-12-29 | 2022-03-25 | 松山湖材料实验室 | Frozen stock solution, preparation method thereof and application thereof in human renal epithelial cells |
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Application publication date: 20191119 |