CN113519504A - Serum-free protein-free freezing medium for direct liquid nitrogen freezing - Google Patents
Serum-free protein-free freezing medium for direct liquid nitrogen freezing Download PDFInfo
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- CN113519504A CN113519504A CN202110846550.5A CN202110846550A CN113519504A CN 113519504 A CN113519504 A CN 113519504A CN 202110846550 A CN202110846550 A CN 202110846550A CN 113519504 A CN113519504 A CN 113519504A
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- free
- cryopreservation
- glutamine
- liquid nitrogen
- serum
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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
-
- 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 relates to the technical field of cell preservation, in particular to a serum-free protein-free cryopreservation liquid for direct liquid nitrogen cryopreservation. The serum-free protein-free freezing solution for direct liquid nitrogen freezing comprises a PBS buffer solution containing a penetration protective agent, a cell membrane protective agent and a cell stabilizer and an additive, wherein the volume percentage of the penetration protective agent in the PBS buffer solution is 5-10%, the mass-to-volume ratio of the cell membrane protective agent is 1-3%, and the mass-to-volume ratio of the cell stabilizer is 1-6%; the additive comprises L-glutamine, glutamine dipeptide and sodium pyruvate. The cryopreservation liquid is matched with a special cryopreservation tube, cells can be cryopreserved in liquid nitrogen in one step, the cryopreservation liquid is suitable for batch operation, the survival rate and the wall adhesion rate are better compared with those of cells cryopreserved by a traditional cryopreservation liquid after resuscitation, and the cells can be preserved for a long time.
Description
Technical Field
The invention relates to the technical field of cell preservation, in particular to a serum-free protein-free cryopreservation liquid for direct liquid nitrogen cryopreservation.
Background
Cell cryopreservation is a technique for storing cells in a low-temperature environment for a long time by reducing cell metabolism, and is one of the main methods for cell preservation. The cells are stored in liquid nitrogen at the temperature of-196 ℃ by using a freezing technology at a low temperature, so that the cells can be temporarily separated from a growth state and the cell characteristics can be stored, and the cells are recovered for experiments when needed. When the cells are frozen, the freezing point of the solution can be lowered by adding the freezing protective agent into the culture medium, and in addition, water in the cells can permeate out under the condition of slow freezing, so that the formation of ice crystals is reduced, and the cells are prevented from being damaged.
The traditional frozen stock solution needs to adopt a slow freezing and fast melting method in the cooling process to better ensure the survival of cells, the standard freezing speed is-1 to-2 ℃/min, the speed can be increased when the temperature is lower than-25 ℃, and the frozen stock solution can be directly put into liquid nitrogen (-196 ℃) after the temperature is lower than-80 ℃. In the process, various expensive low-temperature refrigerator equipment is needed, and a program cooling box is needed, so that the time period is long, the operation is complex, the batch freezing operation is not facilitated, and the process is easy to make mistakes; if placed directly in liquid nitrogen, all cells will die. The other one is 'quick freezing and quick melting', called vitrification cryopreservation, although liquid nitrogen can be directly placed for cryopreservation, the operation flow of the freezing liquid is very complex, and the used cryopreservation liquid has high concentration, great damage to cells and extremely limited application range, and cannot be popularized in a large range.
Based on this, the inventor provides a new cryopreservation tube structure through research, specifically, refer to the utility model patent with application date of 2021, 7, 13 and application number of 202121595540.0, and the inventor further uses the existing cryopreservation liquid to perform experiments, and finds that the survival rate and the adherence rate of the cells after recovery are still not ideal. As is known, the survival rate and the anchorage rate of cells are basic indexes for evaluating the effect of cryopreserved cells, and the improvement of the survival rate and the anchorage rate of cells has important significance for subsequent experimental research.
Disclosure of Invention
In order to further improve the survival rate and the anchorage rate of frozen cells, the invention aims to provide a novel serum-free protein-free frozen stock solution for direct liquid nitrogen freezing, which is characterized by comprising a PBS (phosphate buffer solution) buffer solution and an additive, wherein the PBS buffer solution contains a penetration protective agent, a cell membrane protective agent and a cell stabilizer, the volume percentage of the penetration protective agent in the PBS buffer solution is 5-10%, the mass-to-volume ratio of the cell membrane protective agent is 1-3%, and the mass-to-volume ratio of the cell stabilizer is 1-6%; the additive comprises L-glutamine, glutamine dipeptide and sodium pyruvate.
The cryopreservation liquid does not contain serum and protein, does not have the risk of virus and mycoplasma infection, is convenient to prepare, has small batch difference, is matched with a special cryopreservation tube to be used for cryopreservation of cells, has small damage to the cells, and can ensure that the activity and the anchorage rate of the recovered cells can reach more than 85 percent.
In one embodiment of the present invention, the molar concentration of L-glutamine is 1 to 4mM, the molar concentration of glutamine-alanine dipeptide is 0.5 to 2mM, and the molar concentration of sodium pyruvate is 0.5 to 2 mM.
In one embodiment of the present invention, the osmotic protective agent is one of dimethyl sulfoxide, glycerol, acetamide and ethylene glycol.
In one embodiment of the present invention, the cell membrane protective agent is one or both of trehalose and sucrose.
In one embodiment of the present invention, the cell stabilizer is one or both of hydroxyethyl starch and anhydrosugar.
In one embodiment of the present invention, the anhydrosugar is anhydrosugar 4 million.
In one embodiment of the invention, the frozen stock solution further comprises reduced glutathione, and the mass volume ratio of the reduced glutathione is 0.5-1%.
As an embodiment of the invention, the frozen stock solution comprises a PBS buffer solution containing acetamide, trehalose and hydroxyethyl starch and an additive, wherein the mass volume percentage of acetamide in the PBS buffer solution is 5%, the mass volume ratio of trehalose is 2.5%, and the mass volume ratio of hydroxyethyl starch is 3%; the additive comprises L-glutamine, glutamine propyl dipeptide, sodium pyruvate and reduced glutathione, wherein the molar concentration of the L-glutamine is 2mM, the molar concentration of the glutamine propyl dipeptide is 1mM, the molar concentration of the sodium pyruvate is 1mM, and the mass volume ratio of the reduced glutathione is 0.8%.
The frozen stock solution provided by the invention has clear components, does not contain serum and protein, has stable batches, and can avoid the influence of animal-derived protein on subsequent experiments; the freezing solution is matched with a special freezing tube, cells can be frozen in liquid nitrogen in one step, batch operation is suitable, expensive instruments are not involved, and cost is saved; the frozen stock solution has the advantages of generally low content of each component, small damage to cells, low toxicity, better survival rate and adherence rate compared with the conventional frozen stock solution after recovery, and capability of preserving the cells for a long time.
Detailed Description
The present invention is further described in detail below with reference to specific examples so that those skilled in the art can more clearly understand the present invention.
The following examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention. All other embodiments obtained by a person skilled in the art based on the specific embodiments of the present invention without any inventive step are within the scope of the present invention.
In the examples of the present invention, all the raw material components are commercially available products well known to those skilled in the art, unless otherwise specified; in the examples of the present invention, unless otherwise specified, all technical means used are conventional means well known to those skilled in the art.
Example 1
The embodiment provides a serum-free protein-free freezing medium for direct liquid nitrogen freezing, which specifically comprises the following components:
2.5 percent of trehalose,
anhydrosugar 4 million (manufacturer: Shanghai Yuan Ye, cat # S14110) 2%,
3 percent of hydroxyethyl starch,
DMSO 5％，
0.8 percent of reduced glutathione,
2mM of L-glutamine (L-glutamine),
1mM of the glutamine propyl dipeptide,
1mM of sodium pyruvate is added into the solution,
the balance being PBS.
Example 2
The embodiment provides a serum-free protein-free freezing medium for direct liquid nitrogen freezing, which specifically comprises the following components:
2.5 percent of trehalose,
4 ten thousand of 2 percent of anhydrosugar,
3 percent of hydroxyethyl starch,
5 percent of acetamide,
0.8 percent of reduced glutathione,
2mM of L-glutamine (L-glutamine),
1mM of the glutamine propyl dipeptide,
1mM of sodium pyruvate is added into the solution,
the balance being PBS.
Example 3
The embodiment provides a serum-free protein-free freezing medium for direct liquid nitrogen freezing, which specifically comprises the following components:
2.5 percent of trehalose,
4 ten thousand of 2 percent of anhydrosugar,
3 percent of hydroxyethyl starch,
5 percent of acetamide,
2mM of L-glutamine (L-glutamine),
1mM of the glutamine propyl dipeptide,
1mM of sodium pyruvate is added into the solution,
the balance being PBS.
Example 4
The embodiment provides a serum-free protein-free freezing medium for direct liquid nitrogen freezing, which specifically comprises the following components:
1 percent of trehalose,
4 ten thousand 3 percent of anhydrosugar,
1 percent of hydroxyethyl starch,
DMSO 8％，
0.8 percent of reduced glutathione,
1.5mM of L-glutamine (glutamine),
1mM of the glutamine propyl dipeptide,
1mM of sodium pyruvate is added into the solution,
the balance being PBS.
Example 5
The embodiment provides a serum-free protein-free freezing medium for direct liquid nitrogen freezing, which specifically comprises the following components:
2.5 percent of trehalose,
4 ten thousand of 2 percent of anhydrosugar,
3 percent of hydroxyethyl starch,
DMSO 5％，
1 percent of reduced glutathione,
4mM of L-glutamine (glutamine),
2mM of the glutamine propyl dipeptide (2 mM),
0.5mM of sodium pyruvate is added,
the balance being PBS.
Example 6
The embodiment provides a serum-free protein-free freezing medium for direct liquid nitrogen freezing, which specifically comprises the following components:
2.5 percent of trehalose,
8 million of anhydrosugar (manufacturer: Shanghai-derived leaves) 2%,
3 percent of hydroxyethyl starch,
5 percent of acetamide,
0.8 percent of reduced glutathione,
m-glutamine (2 mM) and (2 mM),
1mM of the glutamine propyl dipeptide,
1mM of sodium pyruvate is added into the solution,
the balance being PBS.
Comparative example 1
The comparative example provides a serum-free and protein-free frozen stock solution for direct liquid nitrogen freezing, which comprises the following specific components:
5 percent of trehalose,
8 ten thousand of 2 percent of anhydrosugar,
3 percent of hydroxyethyl starch,
DMSO 3％，
0.8 percent of reduced glutathione,
2mM of L-glutamine (L-glutamine),
1mM of the glutamine propyl dipeptide,
1mM of sodium pyruvate is added into the solution,
the balance being PBS.
Comparative example 2
The comparative example adopts the traditional frozen stock solution, and comprises the following specific components:
DMSO 10％，
FBS 80％，
10% DM/F12 medium.
Examples of the experiments
(1) Taking mouse bone marrow mesenchymal stem cells and 293T cells, digesting, collecting and centrifuging to obtain cell precipitates, respectively using the freezing solutions in examples 1-6 and comparative examples 1-2 to resuspend the precipitates, putting the suspension into a freezing tube special for freezing by liquid nitrogen (the freezing tube in the background technology), directly putting the freezing tube into liquid nitrogen for preservation, taking out the freezing tube after 30 days of preservation, putting the freezing tube into a water bath at 37 ℃ according to the freezing tube recovery process for dissolution and recovery, inoculating the cell into a 6-well plate by using a special culture medium, taking 50-100 mu L of the cells, performing trypan blue staining to count the cell viability, and observing the cell adherence rate at 24h, wherein the results are shown in the following table 1.
(2) Taking mouse bone marrow mesenchymal stem cells and 293T cells to obtain precipitates, re-suspending the cells in a common freezing tube by using freezing solutions in examples 1-6 and comparative examples 1-2 according to a traditional freezing method, placing the freezing tube in a special gradient cooling box, placing the cooling box in a refrigerator at 4 ℃ for 30min, transferring the box to a refrigerator at minus 80 ℃ for 24h, picking out the freezing tube and transferring the tube to liquid nitrogen for storage, taking out the freezing tube after storing for 30 days, picking out the freezing tube according to the traditional freezing tube and transferring the tube to liquid nitrogen for storage, taking out the freezing tube after storing for 30 days, recovering according to the traditional flow, inoculating the tube in a 6-well plate by using a special culture medium, taking 50-100 mu L of the cells, performing trypan blue staining to count on the cell viability, observing the cell anchorage rate at 24h, and obtaining the result shown in the following table 1.
TABLE 1 results of cell viability and adherence rates
From the above results, it can be seen that:
when the cryopreservation liquid and the common cryopreservation tube are used for cryopreservation, the staining survival rate and the adherence rate of cells after recovery are low and can only reach 70% at most;
when a specific cryopreservation tube is adopted in cooperation, the staining survival rate and the adherence rate of the cryopreserved cells of the cryopreservation solution in the embodiments 1-6 can both reach more than 85%, wherein the optimal staining survival rate can reach 92% and the optimal adherence rate can reach 96%;
when the components in the application are adjusted, the dyeing survival rate and the adherence rate of the composition are both obviously reduced, which is probably because different components have different effects on cells and the concentration of each component has important influence on the cells;
from the results of examples 2 and 3, it was found that glutathione is mainly used as a nutrient component, and the staining viability and adherence after cell recovery are not greatly affected in the present frozen stock solution.
The inventor also finds that the staining survival rate and the adherence rate after cell recovery can only reach 40% at most when the cryopreservation solution described in the embodiments 1-6 is directly put into liquid nitrogen for cryopreservation (a cryopreservation tube is not adopted).
The cryopreservation liquid provided by the invention can be specifically used for a cryopreservation tube which is developed by an inventor, the cryopreservation tube and the cryopreservation tube are matched for use, cells can be cryopreserved in liquid nitrogen in one step, the cryopreservation liquid is suitable for batch operation, the damage to the cells is small, and the cells can keep good survival rate and adherence after recovery.
It should be noted that the above examples are only for further illustration and description of the technical solution of the present invention, and are not intended to further limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment, and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. The serum-free and protein-free frozen stock solution for direct liquid nitrogen freezing is characterized by comprising a PBS (phosphate buffer solution) and an additive, wherein the PBS comprises a penetration protective agent, a cell membrane protective agent and a cell stabilizer, the volume percentage of the penetration protective agent in the PBS is 5-10%, the mass-to-volume ratio of the cell membrane protective agent is 1-3%, and the mass-to-volume ratio of the cell stabilizer is 1-6%; the additive comprises L-glutamine, glutamine dipeptide and sodium pyruvate.
2. The serum-free and protein-free cryopreservation liquid for direct liquid nitrogen cryopreservation according to claim 1, wherein the molar concentration of L-glutamine is 1-4 mM, the molar concentration of glutamine-glutamine dipeptide is 0.5-2 mM, and the molar concentration of sodium pyruvate is 0.5-2 mM.
3. The serum-free and protein-free cryopreservation liquid for direct liquid nitrogen cryopreservation according to claim 1, wherein the osmoprotectant is one of dimethyl sulfoxide, glycerol, acetamide and ethylene glycol.
4. The serum-free and protein-free cryopreservation solution for direct liquid nitrogen cryopreservation according to claim 1, wherein the cell membrane protective agent is one or two of trehalose and sucrose.
5. The serum-free and protein-free cryopreservation liquid for direct liquid nitrogen cryopreservation according to claim 1, wherein the cell stabilizer is one or both of hydroxyethyl starch and anhydrosugar.
6. The serum-free and protein-free cryopreservation liquid for direct liquid nitrogen cryopreservation of claim 5, wherein the anhydrosugar is anhydrosugar 4 million.
7. The serum-free and protein-free cryopreservation liquid for direct liquid nitrogen cryopreservation according to any one of claims 1 to 6, wherein the cryopreservation liquid further comprises reduced glutathione, and the mass volume ratio of the reduced glutathione is 0.5-1%.
8. The serum-free and protein-free freezing medium for direct liquid nitrogen freezing as claimed in claim 1, which comprises PBS buffer solution containing acetamide, trehalose and hydroxyethyl starch and additive, wherein the PBS buffer solution contains 5% by volume of acetamide, 2.5% by mass and 3% by mass of trehalose and 3% by mass and volume of hydroxyethyl starch;
the additive comprises L-glutamine, glutamine propyl dipeptide, sodium pyruvate and reduced glutathione, wherein the molar concentration of the L-glutamine is 2mM, the molar concentration of the glutamine propyl dipeptide is 1mM, the molar concentration of the sodium pyruvate is 1mM, and the mass volume ratio of the reduced glutathione is 0.8%.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202110846550.5A CN113519504A (en) | 2021-07-26 | 2021-07-26 | Serum-free protein-free freezing medium for direct liquid nitrogen freezing |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114698629A (en) * | 2022-05-11 | 2022-07-05 | 江苏真旺农业生物科技有限公司 | Cell storage device and cell storage method for optimizing cell preservation |
| CN115777689A (en) * | 2022-11-09 | 2023-03-14 | 武汉赛维尔生物科技有限公司 | Serum-free and protein-free non-programmed cell cryopreservation solution |
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| CN101971796A (en) * | 2010-05-26 | 2011-02-16 | 赛业（广州）生物科技有限公司 | Nonprogrammed cell frozen stock solution free of proteins |
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| CN107047542A (en) * | 2017-06-23 | 2017-08-18 | 友康恒业生物科技（北京）有限公司 | A kind of serum-free cell frozen stock solution |
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2021
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Patent Citations (4)
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| US20060183102A1 (en) * | 2005-02-14 | 2006-08-17 | Young Henry E | Serum-free reagents for the isolation, cultivation, and cryopreservation of postnatal pluripotent epiblast-like stem cells |
| CN101971796A (en) * | 2010-05-26 | 2011-02-16 | 赛业（广州）生物科技有限公司 | Nonprogrammed cell frozen stock solution free of proteins |
| CN101983562A (en) * | 2010-05-26 | 2011-03-09 | 赛业（广州）生物科技有限公司 | Cell freezing solution without complicated procedures for freezing |
| CN107047542A (en) * | 2017-06-23 | 2017-08-18 | 友康恒业生物科技（北京）有限公司 | A kind of serum-free cell frozen stock solution |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114698629A (en) * | 2022-05-11 | 2022-07-05 | 江苏真旺农业生物科技有限公司 | Cell storage device and cell storage method for optimizing cell preservation |
| CN114698629B (en) * | 2022-05-11 | 2023-08-25 | 黑龙江国智生物工程有限公司 | Cell storage device and cell storage method for optimizing cell preservation |
| CN115777689A (en) * | 2022-11-09 | 2023-03-14 | 武汉赛维尔生物科技有限公司 | Serum-free and protein-free non-programmed cell cryopreservation solution |
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Application publication date: 20211022 |