CN108029679B - Freezing medium for freezing mononuclear cells - Google Patents
Freezing medium for freezing mononuclear cells Download PDFInfo
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- CN108029679B CN108029679B CN201810082345.4A CN201810082345A CN108029679B CN 108029679 B CN108029679 B CN 108029679B CN 201810082345 A CN201810082345 A CN 201810082345A CN 108029679 B CN108029679 B CN 108029679B
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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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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
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Abstract
The invention discloses a freezing solution for freezing mononuclear cells, which consists of fetal calf serum, dimethyl sulfoxide and a dextran dissolving solution, wherein the fetal calf serum accounts for 45-95%, the dextran dissolving solution accounts for 10-50%, and the dimethyl sulfoxide accounts for 5-10%. The dextran refers to low molecular weight dextran, and the molecular weight of the dextran is 4 ten thousand. The invention uses dextran with low molecular weight to replace part of animal serum, and can effectively reduce the introduction amount of foreign protein. The freezing medium can effectively retain the cell activity when mononuclear cells are directly frozen after being extracted. After the mononuclear cells are subjected to cryopreservation treatment by using the cryopreservation solution disclosed by the invention, if the mononuclear cells are subjected to resuscitation culture at intervals, the cell resuscitation rate can be kept at a higher level, and the differentiation capacity of the cells is still strong.
Description
Technical Field
The invention relates to a freezing medium for freezing mononuclear cells, and belongs to the field of low-temperature freezing of biotechnology.
Background
The immunological activity of human monocytes is closely related to the age of the individual and to the health of the individual. It is a problem how to preserve the mononuclear cells for a longer time, and if the cells are frozen, it is also a challenge whether the cells can be restored and then can maintain the original activity.
The activity and the function of the mononuclear cell can be maintained by cryopreservation, and the method has important significance for the induction culture of the late cells and the direct treatment of patients after the recovery of the cryopreserved cells. The cryopreservation of the mononuclear cells can solve the problem of multiple induction of cells during multiple blood sampling and culture of a patient, and can also preserve the mononuclear cells with the strongest fighting capacity in a human health state, and the mononuclear cells are used for tumor treatment or anti-aging health care treatment after resuscitation and culture.
The main factors influencing the activities of the mononuclear cells after freezing and thawing comprise the modes of freezing and thawing the mononuclear cells and the formula of the mononuclear cell freezing solution, wherein the factors of the freezing solution are dominant. If the mononuclear cells are to be preserved for a long period of time, the mononuclear cells must be stored in a liquid nitrogen environment in order to avoid damage to the cells. The quality of the cell morphology and phenotype of the mononuclear cell after freezing and thawing mainly depends on whether an effective freezing reagent is used and a proper temperature reduction and rewarming procedure is adopted. Dimethyl sulfoxide is the most ideal cell protective agent adopted in the current domestic cryopreservation formula, and the cell cryoprotectant can avoid the formation of ice crystals in cells during freezing and protect cell membranes and organelles from being damaged. Fetal calf serum is also a frequent target in cell cryopreservation formulations because it is a very complex mixture of plasma defibrinated and contains various plasma proteins, polypeptides, fats, carbohydrates, growth factors, hormones, minerals, etc., which are all beneficial for protecting cells. The fetal calf serum has the advantages and the disadvantages, and the biggest disadvantage is that the fetal calf serum introduces animal protein, increases the possibility of being polluted by animal pathogens, and can generate certain influence on the adoptive immune cell therapy of a human body.
Disclosure of Invention
In view of the above prior art, the present invention provides a cryopreservation solution for cryopreserving mononuclear cells. The invention effectively solves the direct cryopreservation problem of the peripheral blood mononuclear cells after extraction, can effectively protect the mononuclear cells from freezing damage, maintains the physiological function and biological characteristics of the recovered mononuclear cells, and ensures that the mononuclear cells have cell proliferation and differentiation capacity after cryopreservation.
The invention is realized by the following technical scheme:
a freezing solution for freezing and storing mononuclear cells comprises fetal calf serum, dimethyl sulfoxide and a dextran dissolving solution, wherein the fetal calf serum accounts for 45-95%, the dextran dissolving solution accounts for 10-50%, and the dimethyl sulfoxide accounts for 5-10%, in percentage by volume;
the dextran dissolving solution is a PBS solution of dextran, and the concentration of the dextran is 0.1-2 g/ml.
Preferably, the fetal calf serum accounts for 70%, the dextran dissolving solution accounts for 20% and the dimethyl sulfoxide accounts for 10%.
The dextran refers to low molecular weight dextran, and the molecular weight of the dextran is about 4 ten thousand.
The invention adopts the dextran dissolving solution to replace part of fetal calf serum, reduces the introduction amount of foreign protein and reduces the possibility of animal pathogen pollution; the dextran is introduced, so that the function of plasma can be well replaced, and the aggregation of red blood cells is hindered.
The preparation method of the freezing medium for freezing the mononuclear cells comprises the following steps: dissolving dextran (solid particles) by using 1 XPBS buffer solution to enable the final concentration to be 0.1-2 g/ml (preferably 0.1g/ml) to obtain dextran dissolving solution; then, the fetal calf serum, the dimethyl sulfoxide and the dextran solution are mixed and mixed evenly to obtain the composite.
The application of the freezing medium for freezing and storing the mononuclear cells in freezing and storing the mononuclear cells.
A cryopreservation method of mononuclear cells comprises the following steps: resuspending freshly extracted mononuclear cells using the above-described cryopreservation solution; the resuspended cells were placed in an ultra-low temperature freezer at-80 ℃ for 24 hours using a temperature programmed cassette NALGENE (Cat. No.: 5100-0001), and then transferred to liquid nitrogen for cryopreservation.
Further, the mononuclear cells are cord blood mononuclear cells, and further, peripheral blood mononuclear cells.
The freezing medium can effectively retain the cell activity when mononuclear cells are directly frozen after being extracted. After the mononuclear cells are subjected to cryopreservation treatment by using the cryopreservation solution disclosed by the invention, if the mononuclear cells are subjected to resuscitation culture at intervals, the cell resuscitation rate can be kept at a higher level, and the differentiation capacity of the cells is still strong.
Drawings
FIG. 1: comparison of the next day survival rate of the number 1-12 frozen cells of the mononuclear cell frozen stock solution after recovery.
FIG. 2: comparison graph of expansion times of the number 1-12 frozen cells of the mononuclear cell frozen stock solution after recovery for 14 days.
Detailed Description
The present invention will be further described with reference to the following examples.
The instruments, reagents, materials and the like used in the following examples are conventional instruments, reagents, materials and the like in the prior art and are commercially available in a normal manner unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like described in the following examples are conventional experimental methods, detection methods, and the like in the prior art.
The instant cell freezing medium is sold in the market and is specially used for long-time low-temperature preservation of cells under the serum-free culture condition. To achieve higher cell recovery rates, the cryopreservation series suggests the use of cells in exponential growth phase. The experiment aim of the invention is to ensure that the cell death rate of freshly extracted mononuclear cells after cryopreservation and recovery is lower, the culture can grow normally after recovery, and the cell phenotype is identical with the expectation. The present invention therefore decided to compare several ready-to-use cell lysates in the assay.
In addition, the formulation of the monocyte cryopreservation solution commonly used by the inventor of the invention, namely 90% FBS + 10% DMSO by volume ratio, is suitable for the related cryopreservation after the in vitro amplification culture is completed after the monocyte extraction. Whether the formula is suitable for direct freezing storage after extraction of the mononuclear cells or not is not verified through experiments.
According to the data, the trehalose is also found to have a non-specific protection effect on various bioactive substances, and can form a unique protective film on the cell surface in a low-temperature environment, so that protein molecules are effectively protected from being inactivated. Trehalose was therefore also added to the comparative screening experiments of the present invention.
First, a batch of ready-to-use cell cryopreservation solution is purchased in the market, and the number of the cell cryopreservation solution is as follows: no. 1 Dayou serum-free cell frozen stock solution (cat No. DWK3CFM0100), No. 2 Homey source serum-free cell frozen stock solution (cat No. TL-501), No. 3 Youkang (cat No. NC1001), No. 4 LiveCyte (cat No. LC-1601), No. 5 ScienceCelCFM (cat No. 0133), No. 6 classical formula 90% FBS + 10% DMSO, No. 7 Cryopressionversion Medium KM Bank II (cat No. 88-702-CB).
Second, after looking up the literature, the inventors configured 5 formulations, 8-12, that may be more suitable for cryopreserving freshly extracted mononuclear cells. The formula 8 is 95% FBS + 5% DMSO by volume, the formula 9 is 40% FBS + 10% DMSO + 50% culture medium by volume, the formula 10 is 70% FBS + 20% dextran solution + 10% DMSO by volume, the formula 11 is 70% FBS + 20% trehalose + 10% DMSO by volume, and the formula 12 is 75% FBS + 20% trehalose + 5% DMSO by volume. The freezing medium reagent formula meeting the requirements is screened out by using various indexes (cell recovery survival rate, cell multiplication factor and the like) of cell growth after the freezing recovery culture of the mononuclear cells by using methods of comparison, elimination and the like. After comparative analysis of experimental results (as shown in fig. 1 and 2), the number 10 monocyte cryopreservation solution (70% FBS + 20% dextran solution + 10% DMSO by volume) of the invention is found to be very superior. After the formula is used, the survival rate of the recovered cryopreserved cells is high, the phenotype of the cells after induction culture is relatively best, and the cell growth speed is high.
The cell culture medium used by the invention is Corning 88-581-CM lymphocyte serum-free medium.
Example 1: preparation of dextran dissolving solution reagent
2g of dextran with 4 ten thousand molecular weight is weighed by an analytical balance, placed in a 50ml centrifugal tube, 20ml of 1 XPBS is sucked by a pipette and dripped into the centrifugal tube, and the dextran is uniformly mixed and placed in a refrigerator at 4 ℃ for standby.
Example 2: preparation of monocyte cryopreservation liquid No. 6 (fetal bovine serum, dimethyl sulfoxide)
Taking 90% fetal calf serum according to the volume percentage, slowly dripping 10% dimethyl sulfoxide, and finally shaking up to prepare the mononuclear cell frozen stock solution. Stored at 4 ℃ for further use.
Example 3: preparation of monocyte cryopreservation solution No. 9 (fetal bovine serum, dimethyl sulfoxide, cell culture Medium)
Taking 40 percent fetal calf serum according to the volume percentage, slowly dripping 10 percent dimethyl sulfoxide, finally adding 50 percent cell culture medium, and shaking up to prepare the mononuclear cell frozen stock solution. Stored at 4 ℃ for further use.
Example 4: preparation of monocyte cryopreservation liquid No. 10 (fetal bovine serum, dimethyl sulfoxide, dextran)
Mixing 70% fetal calf serum and 20% dextran solution (prepared according to the method of example 1 for later use) according to volume percentage, slowly dripping 10% dimethyl sulfoxide, and shaking up to prepare the mononuclear cell freezing solution. Stored at 4 ℃ for further use.
Example 5
The method comprises the steps of extracting fresh single nuclei from one part of cord blood by adopting a process of extracting SOP from the fresh single nuclei of a company of the applicant, counting cells, taking out equivalent cells according to the culture demand of 2 x 10^7/ml, directly and freshly culturing one part of the cells for 14 days to collect data, and performing cryopreservation on the other parts of the cells by using different mononuclear cell freezing solutions, storing the frozen cells in a liquid nitrogen environment for a period of time, and then performing recovery culture for 14 days. All the preparation operations of the freezing solution and the cell freezing operation need to be carried out in the environment of 0 ℃. The number 10 frozen stock solution of the invention is selected by cross-comparing the cell survival rate of the next day after recovery, namely the cell recovery rate, and the expansion multiple of the cells after 14 days.
After the screening experiment is finished, the data of the whole experiment are collated, and in order to more intuitively show the advantages of the invention, namely the number 10 cell freezing solution (20% dextran solution, 70% fetal calf serum and 10% dimethyl sulfoxide), the following two figures are made: fig. 1 and 2.
From the above results, it was found that the cell freezing solutions 1 to 12 were used to freeze the cells, and the cell viability was recovered the next day after many experiments. The cells of the No. 3 frozen stock solution survive without cells, the average value of the cell recovery survival rate of the frozen and recovered No. 1, No. 2, No. 4 and No. 5 frozen stock solutions is lower than that of other groups, and the graph 1 shows that the average value of the cell recovery survival rate of the No. 10 frozen stock solution is the highest. FIG. 2 shows that the mean value of the 14-day expansion fold of resuscitated cultured cells shows that the expansion fold of No. 10 cells is higher than that of other groups.
＊＊＊＊＊
The above examples are provided to those of ordinary skill in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims. All publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each such publication, patent, or patent application were specifically and individually indicated to be incorporated by reference.
Claims (5)
1. A cryopreservation solution for cryopreserving mononuclear cells, comprising: the serum-free biological agent consists of fetal calf serum, dimethyl sulfoxide and a dextran dissolving solution, wherein the fetal calf serum accounts for 70%, the dextran dissolving solution accounts for 20%, and the dimethyl sulfoxide accounts for 10%, in percentage by volume;
the dextran dissolving solution is PBS solution of dextran, and the concentration of dextran is 0.1 g/mL.
2. The cryopreservation solution for cryopreserving mononuclear cells according to claim 1, wherein: the molecular weight of the dextran is 4 ten thousand.
3. The method for preparing a cryopreservation solution for cryopreserving mononuclear cells according to claim 1 or 2, wherein: dissolving dextran with 1 XPBS buffer solution to make the final concentration of the dextran be 0.1-2 g/mL to obtain dextran dissolving solution; then, the fetal calf serum, the dimethyl sulfoxide and the dextran solution are mixed and mixed evenly to obtain the composite.
4. Use of the cryopreservation solution for cryopreserving monocytes according to claim 1 or 2 for cryopreserving monocytes.
5. A cryopreservation method of mononuclear cells is characterized in that: resuspending freshly extracted mononuclear cells using the cryopreservation solution of claim 1 or 2; the resuspended cells were placed in a-80 ℃ ultra-low temperature freezer for 24 hours using a programmed cooling box and then transferred to liquid nitrogen for cryopreservation.
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| CN108849854B (en) * | 2018-07-13 | 2021-02-09 | 深圳市润科生物科技有限公司 | Peripheral blood mononuclear cell cryopreservation method |
| CN109792984B (en) * | 2019-02-01 | 2020-03-31 | 北京健坤禾润科技有限公司 | Cell cryopreservation culture medium for in vitro cell culture and application thereof |
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| CN102763642B (en) * | 2012-08-14 | 2014-04-02 | 郑州赛英科干细胞技术有限公司 | Cryoprotectant and method for cryopreserving placenta amnion and chorion |
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Effective date of registration: 20231008 Address after: 702, Floor 7, Building 8 (Building 9), No. 6, Nanjiang Second Road, the Pearl River Street, Nansha District, Guangzhou, Guangdong 510000 Patentee after: Guangdong Yinfeng Cell Technology Co.,Ltd. Patentee after: YINFENG BIOLOGICAL GROUP Co.,Ltd. Address before: 250101 Export Processing Zone, Jinan High-tech Zone, Shandong Province, 1109 Gangxing Third Road Patentee before: QILU CELL THERAPY TECHNOLOGY CO.,LTD. Patentee before: YINFENG BIOLOGICAL GROUP Co.,Ltd. |
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