CN111387175A - Cryopreservation solution and cryopreservation method for lymphoid cells - Google Patents
Cryopreservation solution and cryopreservation method for lymphoid cells Download PDFInfo
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- CN111387175A CN111387175A CN202010373141.3A CN202010373141A CN111387175A CN 111387175 A CN111387175 A CN 111387175A CN 202010373141 A CN202010373141 A CN 202010373141A CN 111387175 A CN111387175 A CN 111387175A
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- cryopreservation
- culture medium
- lymphoid cells
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- lymphocyte culture
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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
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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
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
Abstract
The invention relates to the technical field of biology, in particular to a cryopreservation solution and a cryopreservation method for lymphoid cells. The cryopreservation liquid comprises a lymphocyte culture medium and a cryopreservation prefabricated liquid, wherein the cryopreservation prefabricated liquid comprises dextran, human serum albumin, dimethyl sulfoxide and the balance of the lymphocyte culture medium; the volume ratio of the lymphocyte culture medium to the freezing prefabricated liquid is 1: 1. The cryopreservation solution can remarkably improve the cryopreservation efficiency of the lymphoid cells and the survival rate of the cells during resuscitation, and the reagents are relatively cheap and easily available, so that the cost of the cryopreservation solution can be reduced. The freezing method uses the freezing liquid for direct freezing, has simple and easy operation method, and can be used for freezing in large production scale.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a cryopreservation solution and a cryopreservation method for lymphoid cells.
Background
With the rapid development of biotechnology, especially the overall progress of stem cell and immune cell related technologies, the cryopreservation technology and related cryopreservation reagents of human living cells and tissues become important bottlenecks that restrict the cryopreservation efficiency of human living cells and tissues and reduce cell loss.
The existing low-temperature preservation method of the biological sample is mainly divided into two categories of non-vitrification freezing preservation and vitrification freezing preservation. Although the vitrification cryopreservation can provide better low-temperature protection for a biological sample to be cryopreserved theoretically, the used high-concentration cryopreservation protective agent has relatively complex components and high cost, and the related cryopreservation and recovery method has more complicated operation requirements, so that the vitrification cryopreservation is mainly limited to the small-scale experimental research such as the cryopreservation fields of biological fertilized eggs, embryos and the like at present and is not widely put into the production field. The non-vitrification cryopreservation mode has stronger applicability, the cryopreservation and recovery operation methods are relatively simple and easy to implement, and the cost of the cryopreservation protective solution is relatively low, so that the non-vitrification cryopreservation mode is more widely applied.
But aiming at the field of cryopreservation of lymphoid cells, the existing non-vitrification cryopreservation technology still has the technical problems that: on one hand, the lymphoid cells are relatively fragile, the components of the existing non-vitrified cryopreservation liquid are biased to various cell universal types, the pertinence to the lymphoid cells is poor, the cryopreservation efficiency is relatively low, and the cell loss rate during recovery is relatively high; on the other hand, the conventional non-vitrification cryopreservation method is generally implemented by program cooling, the operation is still more complicated, and the large-scale operability is still to be improved.
Disclosure of Invention
The invention provides a cryopreservation solution for lymphoid cells, aiming at the problems of poor pertinence to the lymphoid cells, low cryopreservation efficiency, high cell recovery loss ratio, low cell survival rate and complicated cryopreservation operation of the conventional non-vitrification cryopreservation method.
The invention also provides a cryopreservation method of the lymphoid cells.
In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:
the cryopreservation liquid for the lymphoid cells comprises a lymphocyte culture medium and a cryopreservation prefabricated liquid, wherein the cryopreservation prefabricated liquid comprises the following components in percentage by weight: 1.9-2.1% dextran, 11-13% human serum albumin, 19-21% dimethyl sulfoxide, and the balance lymphocyte culture medium; the volume ratio of the lymphocyte culture medium to the freezing prefabricated liquid is 1: 1.
In the frozen stock solution, the dextran can protect the cell membrane structure and reduce the damage in the freezing process. The cryopreservation system using the dextran in the prior art usually needs higher dextran content to achieve a corresponding protection effect, but the cryopreservation liquid is matched with the components in a specific proportion for use, so that the effects of improving the cryopreservation efficiency and the cell viability can be achieved, and the dosage of the dextran can be obviously reduced.
Dimethyl sulfoxide (DMSO) can prevent damage caused by intracellular fluid ice crystal formation, osmotic pressure changes, cell structural disorders, and the like. However, DMSO has significant cytotoxicity, and simultaneously inhibits cell growth, cells are not suitable for long-term contact in a non-frozen state, and subsequent adverse effects on cells caused by the fact that a large amount of DMSO cannot be effectively eluted in time during recovery are more obvious, and the yield of living cells is possibly influenced. The frozen stock solution can still achieve higher yield of living cells by matching the components in a specific ratio.
The human serum albumin can play a corresponding balance role in the regulation of osmotic pressure, the maintenance of cell membrane structure and the environmental maintenance of cell suspension at low concentration, the content of the human serum albumin is in positive correlation with the freezing effect in a certain range, but the higher the dosage is, the higher the cost is. The frozen stock solution can improve the freezing effect by compounding human serum albumin with other components at the dosage of 5.5-6.5% (w/v).
Compared with PBS, normal saline and other cell culture media which are commonly used in cell freezing medium, the lymphocyte culture medium used in the cell freezing medium can obviously improve the cell resuscitation survival rate and the living cell yield, and the difference is more obvious especially for the frozen cells which are preserved for a long time (more than one month). For example, when the sample is cryopreserved by using the same cryopreservation method and standard recovery operation is performed for about one week, the cell survival rate of the sample preserved by using the cryopreservation solution is higher than 90%, the average survival rate is higher than 95%, the average viable cell yield is higher than 95%, the cell survival rate and the viable cell yield are not obviously reduced, while the cell survival rate of the sample preserved by using PBS cryopreservation solution (PBS is used for replacing lymphocyte culture medium in the scheme) is lower than 85%, the average survival rate is lower than 80%, the average viable cell yield is lower than 80%, and the cell survival rate and the viable cell yield are obviously reduced; when standard recovery operation is carried out after one month, the cell survival rate of the sample preserved by using the frozen stock solution is higher than 85%, the average survival rate is more than 92%, the average viable cell yield is more than 90%, the cell survival rate and the viable cell yield are not obviously reduced, the cell survival rate of the sample preserved by using the PBS frozen stock solution is lower than 70%, the average survival rate is less than 60%, the average viable cell yield is lower than 60%, and the cell survival rate and the viable cell yield are greatly reduced.
The cryopreservation solution is prepared by mixing the components in a specific ratio, so that the loss of cells during recovery can be reduced, the cryopreservation efficiency of the lymphoid cells is remarkably improved, namely the survival rate and the yield of the cells obtained by recovering the cells in the same mode after the cells are frozen for a certain period of time under the same cryopreservation method storage condition, and the convenience degree and the cost of cryopreservation are saved.
The freezing liquid is used for direct freezing, the freezing effect equivalent to program cooling can be achieved, freezing operation can be simplified, the freezing liquid is suitable for large-scale freezing of the lymphoid cell samples, and the samples after freezing can be stored in liquid nitrogen for a long time.
The reagents in the frozen stock solution are relatively cheap and easily obtained, and the frozen stock solution cost can be reduced. The lymphocyte culture medium can be selected from commercial lymphocyte culture medium, such as lymphocyte serum-free culture medium KBM581 from Corning (CORNING).
Preferably, the freezing preservation prefabricated liquid comprises the following components in percentage by weight: 1.95-2.05% of dextran, 11.5-12.5% of human serum albumin, 19.5-20.5% of dimethyl sulfoxide and the balance of lymphocyte culture medium.
Preferably, the freezing preservation prefabricated liquid comprises the following components in percentage by weight: 2% dextran, 12% human serum albumin and 20% dimethyl sulfoxide, the balance being lymphocyte culture medium.
Preferably, the preparation method of the cryopreservation prefabricated liquid comprises the following steps: and uniformly mixing the dextran, the human serum albumin and the lymphocyte culture medium, adding the dimethyl sulfoxide to the mixture while adding the dimethyl sulfoxide to the mixture in an adherent manner, and uniformly mixing the mixture to obtain the dextran.
The embodiment of the invention also provides a cryopreservation method of the lymphoid cells, and the cryopreservation method uses the cryopreservation liquid to cryopreserve the lymphoid cells.
The cryopreservation method can directly perform cryopreservation at the temperature of minus 80 ℃ by using the cryopreservation liquid, can achieve the cryopreservation effect which is nearly equal to the conventional program cooling in the non-vitrification cryopreservation method, has simple and easy operation method, and can be used for large-scale cryopreservation. The lymphoid cells can be stored for a long time after being stored in liquid nitrogen by the cryopreservation method.
Preferably, the specific operations of the cryopreservation method are as follows: and (2) taking the lymphocyte culture medium as a dispersion medium, preparing the lymphoid cells into lymphoid cell suspension, mixing the lymphoid cell suspension with the pre-frozen prefabricated liquid pre-cooled to 2-6 ℃, keeping the mixture at-80 ℃ for 16-18 h, and then storing the mixture in liquid nitrogen. The invention carries out the cryopreservation by combining the cryopreservation liquid with the operation of the cryopreservation method, the cell survival rate and the living cell yield can be maintained to be more than 90 percent, and the cell survival rate and the living cell yield are high during recovery, and both the cell survival rate and the living cell yield exceed 90 percent after the cryopreservation for more than 6 months.
Preferably, the cryopreservation prep solution is pre-cooled to 4 ℃ prior to mixing.
Preferably, the lymphoid cell suspension concentration is 2 × 107～10×107/ml。
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Examples
The embodiment of the invention provides a cryopreservation solution for lymphoid cells, which comprises a lymphocyte culture medium and a cryopreservation prefabricated solution, wherein the weight percentages of the components of the cryopreservation prefabricated solution in each embodiment are shown in table 1.
TABLE 1 weight percentages of the components of the frozen stock solutions in the examples
Wherein the lymphocyte culture medium is lymphocyte serum-free culture medium KBM581 of Corning company.
The preparation method of the frozen prefabricated liquid comprises the following steps: mixing dextran, human serum albumin and lymphocyte culture medium uniformly, adding DMSO while adding wall, and mixing uniformly.
The embodiments of the present invention also provide a method for cryopreserving lymphoid cells by using the cryopreservation solution of embodiments 1 to 5, wherein the method comprises the steps of preparing a cell concentration of 2 × 10 using the lymphocyte culture medium of each embodiment as a dispersion medium7～10×107The lymphoid cell suspension is mixed with a pre-frozen prefabricated liquid pre-cooled to 2-6 ℃, kept in a-80 ℃ environment for 12 hours and then stored in liquid nitrogen. The cryopreservation parameters of the examples are shown in table 2.
TABLE 2 Freeze storage parameters for the examples
Examples of effects
This effect example provides the cryopreservation effect of the above-described embodiment.
The samples stored in liquid nitrogen obtained by the cryopreservation methods of examples 6 to 10 were allowed to stand for a predetermined period of time, and then recovered by a conventional cell recovery procedure, and the yield and survival rate of viable cells were examined, and the results are shown in table 3.
Wherein the yield of the living cells is × 100% of the number of the living cells after resuscitation and elution/the number of the living cells before cryopreservation, and the yield of the living cells is × 100% of the number of the living cells after resuscitation and elution/the total number of the living cells after resuscitation and elution.
TABLE 3 viable cell yield and viability for each example
Note that the data in the same row in the table are shoulder marked with the same letter to indicate no significant difference (P > 0.05).
Comparative example 1
The invention provides a cryopreservation solution for lymphoid cells in each proportion, which comprises a lymphocyte culture medium and a cryopreservation prefabricated solution, wherein the components of the cryopreservation prefabricated solution in each proportion are shown in Table 3. In each proportion, the volume ratio of the lymphocyte culture medium to the freezing prefabricated liquid is 1: 1.
Table 4 comparative example final mass percentage concentrations in frozen stock solutions of the ingredients in the frozen stock solutions
Wherein the lymphocyte culture medium is lymphocyte serum-free culture medium KBM581 of Corning company.
The preparation method of the frozen prefabricated liquid comprises the following steps: mixing the other components except DMSO uniformly, adding DMSO into wall while mixing uniformly to obtain the final product.
The above respective proportions were each subjected to cryopreservation by the cryopreservation method of example 6, and the obtained sample stored in liquid nitrogen was allowed to stand for one week, then revived according to a conventional cell revival procedure, and then the yield and the survival rate of viable cells were examined, and the results are shown in table 5.
TABLE 5 yield and viability of viable cells in various proportions (direct cryopreservation)
The temperature reduction method of comparative examples 7, 9, 13 and 16, in which the yield of viable cells was high, was changed from direct freezing to conventional programmed temperature reduction (4 ℃ C. 10min → -20 ℃ C. 30min → -80 ℃ C. 18 hours), and the obtained sample stored in liquid nitrogen was allowed to stand for one week, then revived according to a conventional cell revival program, and then the yield of viable cells was examined, and the results are shown in Table 6.
TABLE 6 yield of viable cells for each scale (cryopreservation by conventional programmed freezing method)
Comparative example 2
The lymphoid cells were cryopreserved using the cryopreservation solutions of examples 1 to 5, with the same cryopreservation parameters as in table 2, the conventional programmed cryopreservation method (4 ℃ 10min → -20 ℃ 30min → -80 ℃ 18 hours) was changed from the direct cryopreservation method to the cooling method, the obtained sample stored in liquid nitrogen was left for a predetermined time, and then the sample was revived according to the conventional cell revival program, and the yield and survival rate of viable cells were examined, and the results are shown in table 7.
TABLE 7 yield and viability of viable cells for each comparative pair
Note that the same letters in the shoulder marks in the table indicate that the data at the same positions in Table 3 are not significantly different (P > 0.05).
The results show that the cryopreservation solution provided by the invention can reduce the loss of the lymphoid cells during recovery, and obviously improve the yield and the survival rate of the lymphoid cells. In addition, the freezing liquid provided by the invention is used for direct freezing, the freezing effect of the freezing liquid has no obvious difference with the freezing effect of a program cooling freezing mode, but the freezing operation process can be greatly simplified.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (8)
1. The cryopreservation liquid for the lymphoid cells is characterized by comprising a lymphocyte culture medium and a cryopreservation prefabricated liquid, wherein the cryopreservation prefabricated liquid comprises the following components in percentage by weight: 1.9-2.1% dextran, 11-13% human serum albumin, 19-21% dimethyl sulfoxide, and the balance lymphocyte culture medium; the volume ratio of the lymphocyte culture medium to the freezing prefabricated liquid is 1: 1.
2. The cryopreservation solution for lymphoid cells according to claim 1, wherein said cryopreservation preparation comprises the following components by weight percent: 1.95-2.05% of dextran, 11.5-12.5% of human serum albumin, 19.5-20.5% of dimethyl sulfoxide and the balance of lymphocyte culture medium.
3. The cryopreservation solution for lymphoid cells according to claim 2, wherein said cryopreservation preparation comprises the following components by weight percent: 2% dextran, 12% human serum albumin and 20% dimethyl sulfoxide, the balance being lymphocyte culture medium.
4. The cryopreservation solution for lymphoid cells according to any one of claims 1 to 3, wherein said cryopreservation preparation solution is prepared by: and uniformly mixing the dextran, the human serum albumin and the lymphocyte culture medium, adding the dimethyl sulfoxide to the mixture while adding the dimethyl sulfoxide to the mixture in an adherent manner, and uniformly mixing the mixture to obtain the dextran.
5. A method for cryopreserving lymphoid cells, comprising cryopreserving the lymphoid cells with the cryopreservation solution according to any one of claims 1 to 4.
6. The cryopreservation method according to claim 5, characterized by comprising the following specific operations: and (2) taking the lymphocyte culture medium as a dispersion medium, preparing the lymphoid cells into lymphoid cell suspension, mixing the lymphoid cell suspension with the pre-frozen prefabricated liquid pre-cooled to 2-6 ℃, keeping the mixture at-80 ℃ for 16-18 h, and then storing the mixture in liquid nitrogen.
7. The cryopreservation method of claim 6, wherein the cryopreservation pre-solution is pre-cooled to 4 ℃ before mixing.
8. The cryopreservation method of claim 6, wherein the lymphoid cell suspension concentration is 2 × 107～10×107/ml。
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| CN109315386A (en) * | 2018-12-12 | 2019-02-12 | 中南大学湘雅三医院 | A kind of frozen stock solution and cryopreservation methods can be used for candidate stem cell or lymphocyte |
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| CN103210903A (en) * | 2013-05-03 | 2013-07-24 | 新乡医学院 | Cryopreservation solution for preserving congenital intrathoracic kidney (CIK) cells and application thereof |
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Application publication date: 20200710 |