CN107306939B - Cell cryopreservation liquid for dendritic cells - Google Patents
Cell cryopreservation liquid for dendritic cells Download PDFInfo
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- CN107306939B CN107306939B CN201710635539.8A CN201710635539A CN107306939B CN 107306939 B CN107306939 B CN 107306939B CN 201710635539 A CN201710635539 A CN 201710635539A CN 107306939 B CN107306939 B CN 107306939B
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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
- A01N1/0205—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
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Abstract
The invention discloses a cell cryopreservation solution for dendritic cells, which is prepared by adding an effective amount of cryopreservation protective agent, cell stabilizing agent and anti-settling agent into an RPMI-1640 culture solution and uniformly mixing, wherein the cryopreservation protective agent is dimethyl sulfoxide, and the cell stabilizing agent is human gamma globulin; the anti-settling agent is poly-L-lysine, and the molecular weight range of the poly-L-lysine is 3-7 ten thousand; the volume percentage concentration of the dimethyl sulfoxide is 1-3%. When the cell cryopreservation solution is used for cryopreserving DCs, the cryopreservation recovery rate and the immunocompetence of the DCs can be ensured, and the cryopreservation recovery rate and the immunocompetence are not obviously reduced within 1 year of cryopreservation; human gamma globulin is a cell stabilizer and can maintain the immunocompetence of DCs; poly-L-lysine is an anti-settling agent, so that the human gamma globulin is prevented from settling to the bottom of a frozen stock solution in the storage process, and meanwhile, the poly-L-lysine has the function of a partial freezing protective agent and is also beneficial to maintaining the immunocompetence of DCs.
Description
Technical Field
The invention belongs to the field of cell culture, and particularly relates to a cell cryopreservation solution for dendritic cells.
Background
A large amount of mature Dendritic Cells (DCs) are usually needed clinically, but the content of natural DCs in an organism is low, so that the requirement of clinical application is difficult to meet, and the DCs can be obtained in a large amount only by in vitro culture. Mature DCs need a large amount of cytokines to maintain, are high in cost and easy to pollute, and are not beneficial to clinical application of the DCs, so that the prepared DCs can be frozen and preserved, and can be repeatedly used when needed.
In order to maintain the recovery rate and immunological activity of cryopreserved DCs, it is extremely important to select an appropriate cryopreservation method.
At present, the freezing preservation method of DCs is to adopt two-step cooling of freezing preservation solution containing 10% dimethyl sulfoxide (DMSO) and 20% Fetal Calf Serum (FCS). This approach suffers from two significant drawbacks:
firstly, FCS has complex components, can introduce pollutants and pathogenic factors into DCs, and is not easy to control safety; at present, the imported frozen stock solution does not contain FCS (such as Cellbanker cell frozen stock solution produced by Japan ZENOAQ company), but has high price and high application cost, and limits the use of the frozen stock solution in clinic, especially in developing countries and underdeveloped countries;
secondly, although DMSO is a commonly used cryopreservation protective agent, the cryopreservation protective agent does not represent that DMSO does not cause cryopreservation damage to cells (the cytotoxicity of DMSO is inhibited at a deep low temperature, the action is fast during recovery, dimethyl sulfoxide is washed away as soon as possible, otherwise serious toxicity to cells is caused; however, the speed is fast again, the cytotoxicity cannot be avoided), and the damage limits further improvement of recovery rate; unless new agents are developed that can replace DMSO or reduce the proportion of DMSO.
In order to reduce the freezing cost of the DCs and break the monopoly of imported freezing solution, the applicant is dedicated to develop the DCs freezing solution with independent property rights, and the cost is reduced on the premise of ensuring the recovery rate and the immunocompetence of the DCs freezing.
Disclosure of Invention
The invention aims to reduce the freezing cost of DCs, break the monopoly of imported freezing solution, provide economical, practical and efficient DCs freezing solution, and reduce the cost on the premise of ensuring the recovery rate and the immunocompetence of DCs freezing.
The invention is realized by the following technical scheme:
a cell cryopreservation liquid based on RPMI-1640 culture liquid is prepared by adding effective amount of cryopreservation protective agent, cell stabilizer and anti-settling agent into RPMI-1640 culture liquid, and mixing uniformly, wherein the cryopreservation protective agent is dimethyl sulfoxide, and the cell stabilizer is human gamma globulin; the anti-settling agent is poly-L-lysine, and the molecular weight range of the poly-L-lysine is 3-7 ten thousand; the volume percentage concentration of the dimethyl sulfoxide is 1-3%.
Preferably, the mass concentration of the human gamma globulin is 8-12 mug/mL.
Preferably, the mass concentration of the poly-L-lysine in the cell freezing medium is 20-30 mug/mL.
Preferably, the cell cryopreservation solution further contains an antibiotic.
Preferably, the antibiotic is gentamicin at a concentration of 50U/mL.
The application of the cell cryopreservation liquid in the aspect of cryopreservation of dendritic cells.
The invention has the advantages that:
when the cell cryopreservation solution provided by the invention is used for cryopreserving DCs, the cryopreservation recovery rate and the immunocompetence of the DCs can be ensured, and the cryopreservation recovery rate and the immunocompetence are not obviously reduced within 1 year of cryopreservation; in the cell cryopreservation solution, dimethyl sulfoxide is used as a cryopreservation protective agent, so that the damage of low temperature to DCs is reduced; human gamma globulin is a cell stabilizer and can maintain the immunocompetence of DCs; poly-L-lysine is an anti-settling agent, so that the human gamma globulin is prevented from settling to the bottom of a frozen stock solution in the storage process, meanwhile, the poly-L-lysine has the function of a partial freezing protective agent, and the volume concentration of DMSO can be reduced to 1-3% from 10-20% in the prior art and still can be prevented from being damaged by low temperature on DCs due to the existence of the poly-L-lysine; in addition, poly-L-lysine also helps maintain the immunological activity of DCs. The freezing effect of the cell freezing solution on DCs provided by the invention is basically consistent with that of Cellbanker cell freezing solution produced by the Japanese ZENOAQ company, but the cost is only about 1/10 of the imported freezing solution.
Drawings
FIG. 1 is a graph of the cryopreserved recovery (%) of DCs after 3, 6, 9 and 12 months;
FIG. 2 shows the killing (%) of DC-CIK against K562.
Detailed Description
In order to better explain the technical solution of the present invention, the following is further described with reference to specific embodiments. The experimental materials not particularly emphasized in the examples are all conventional experimental materials, and belong to the category which is easily obtained by a person skilled in the art.
Example 1: preparation of cell cryopreservation solution
And (3) reagent sources:
RPMI-1640 medium, Gibco;
dimethylsulfoxide, carbofuran technologies ltd;
human gamma globulin (CAS number: 9007-83-4), Shanghai Fengshi Biotech, Inc.;
poly-L-lysine (MW3-7 ten thousand, a0057-250mg), shanghai shi feng biotechnology limited.
Preparation of cell frozen stock solution:
the preparation method comprises the steps of taking 100mLRPMI-1640 culture solution as a solvent, taking dimethyl sulfoxide, human gamma globulin and poly-L-lysine as solutes, and mixing the solvent and the solutes. The method specifically comprises the following steps: the 98mLRPMI-1640 culture solution and 2mL of dimethyl sulfoxide are mixed firstly, and then human gamma globulin and poly-L-lysine are added, and the final concentrations of the human gamma globulin and the poly-L-lysine are respectively 10 and 25 mu g/mL.
After being prepared, the mixture is stored in a refrigerator at 4 ℃.
Wherein poly-L-lysine can prevent human gamma globulin from settling. In one embodiment, the inventors used a frozen stock solution of cells without poly-L-lysine as a control, and stored it in a refrigerator at 4 ℃ and measured the concentration of gamma globulin in 3 samples uniformly from the liquid surface to the bottom of the flask after 30 days, so that the concentration of gamma globulin in the samples at the middle height was about 5 times the concentration at the liquid surface and the concentration of gamma globulin in the samples at the bottom of the flask was about 11 times the concentration at the liquid surface. And the cell culture solution added with poly-L-lysine has no sedimentation, and the concentrations at three heights have no obvious difference after standing for 6 months at 4 ℃.
Of course, to avoid contamination, appropriate amounts of antibiotics, such as 50U/mL gentamicin, may also be added to the cell culture.
And preparing a comparison frozen stock solution for later use, wherein the comparison frozen stock solution is only added with poly-L-lysine compared with the cell frozen stock solution.
Example 2: proliferation culture, cryopreservation, resuscitation rate and immune activity detection of DCs
Experimental materials and methods
1. Preparation of DCs and CIK
Culturing mononuclear cell by taking 20ml of peripheral blood of heparin anticoagulated healthy volunteers, centrifuging with lymphocyte separation liquid density gradient (2400 × g, 20min), and gently sucking off-white layer of mononuclear fine cellsThe cells were placed in a 15ml centrifuge tube, washed 3 times (2000 × g, 5min) by centrifugation in RPMI-1640(Gibco) medium, the cells were suspended in a serum-free medium UltraCULTURETM (Beijing Whitbytechnology) with an adjusted cell density of 1 × 105Adding into a cell culture flask, and standing at 37 deg.C and 5% CO2After 2h of incubation in the incubator, the cell flasks were gently shaken.
Induction of CIK cells by pipetting the cells suspended in the above cell flask into another flask and adjusting the density to 1 × 106And/ml, inducing CIK cells with UltraCULTURETM containing IL-21000U/ml, CD3-McAb100ng/ml, changing the solution half every 2-3d, and collecting CIK on day 12 for use.
Induction of DCs cells: adherent cells in the above cell flasks were cultured with UltraCURTURETM containing 100ng/ml hGM-CSF and 1000U/ml IL-4, and the medium was changed half-way for 2-3d, and induced DCs cells were collected by day 12.
Viable cell numbers were counted using trypan blue staining. One part of the cells was assayed for killing activity and the other part was cryopreserved.
2. Cryopreservation and resuscitation of DCs
DCs were diluted to 3.5 × 10 using the inventive and comparative frozen stocks prepared in example 1 (extemporaneous ready mix) respectively5And/ml, each of the 4 tubes is divided into 4 tubes, and the tubes are placed in a low-temperature refrigerator at minus 80 ℃ for cooling for 12 hours and then placed in liquid nitrogen at minus 196 ℃ for storage. Taking out 1 count from liquid nitrogen respectively in 3, 6, 9 and 12 months, rapidly putting into 40 deg.C water bath for rewarming, sucking out cell suspension immediately after cell thawing, centrifuging and washing with RPMI-1640(Gibco) culture solution, and detecting DCs resuscitation rate by trypan blue staining.
After the DCs are recovered, the DCs are cultured for 2d by using an UltraCULTURETM serum-free medium containing 100ng/ml hGM-CSF and 1000U/ml IL-4, and the DCs are collected for later use.
3. Method for detecting killing activity of DC-CIK by MTT method
Mixing CIK cultured on day 12 and DCs cultured for 2d after recovery at ratio of 6:1, and adjusting cell number to 1 × 106/ml, culturing with UltraCULTURETM containing IL-2(1000U/ml) and hGM-CSF (100ng/ml) for 4d, i.e., co-cultured DC-CIK, and adjusting the density to 4 × 105Perml, add 96-well cell culture plates containing K562 cellsThe results were used as experimental groups (effective target ratio 20: 1). Additional DC-CIK alone and K562 cells alone were added, 5 replicates per group, as controls. Placing at 37 ℃ and 5% CO2Culturing for 48h in incubator, centrifuging, discarding supernatant, adding 20 μ l MTT (5mg/ml) per well, standing at 37 deg.C and 5% CO2Culturing in an incubator for 4h, centrifuging, removing supernatant, adding 100 μ l of dimethyl sulfoxide to each well, and shaking to dissolve. Measuring D value with a 570nm wavelength by a microplate reader after 15 min. The killing rate of DC-CIK was calculated as follows:
the killing rate (%) - [ 1- (experimental group D-DC alone-CIK group D)/K562 cell alone D ] x 100%.
Second, experimental results
1. Rate of recovery of DCs
The recovery rates of DCs in 3, 6, 9 and 12 months by using the cryopreservation solution of the invention prepared in example 1 and the comparative cryopreservation solution as cryopreservation protective agents are shown in Table 1 and FIG. 1. Compared with the contrast cryopreservation solution, the cryopreservation solution has higher recovery rate and better cryopreservation protection effect, which shows that 2% concentration of DMSO is not enough to effectively avoid cell cryopreservation injury in the absence of poly-L-lysine.
TABLE 1 Freeze recovery Rate (%) -of DCs after 3, 6, 9 and 12 months
| 3 month | 6 month | 9 month | 12 month | |
| The invention relates to a freezing solution | 97.6±2.3 | 96.8±2.5 | 96.1±2.9 | 95.2±2.6 |
| Contrast freezing solution | 91.5±3.7 | 85.7±4.3 | 79.4±3.8 | 70.9±4.1 |
2. Killing activity of DC-CIK on K562
The killing rate of K562 cells by CIK cultured on day 12 and DCs co-cultured for 2d after 3, 6, 9 and 12 months of recovery from cryopreservation is shown in Table 2 and FIG. 2. Compared with the contrast frozen stock solution, the frozen stock solution has higher killing rate on K562 cells and better maintenance of the immunocompetence of DCs, which shows that poly-L-lysine is also helpful for maintaining the immunocompetence of the frozen DCs.
TABLE 2 killing rate (%) of DC-CIK to K562
| 3 month | 6 month | 9 month | 12 month | |
| The invention relates to a freezing solution | 30.9±3.5 | 29.3±3.9 | 29.5±3.6 | 28.9±3.8 |
| Contrast freezing solution | 31.7±3.9 | 28.8±4.1 | 23.2±3.6 | 18.5±4.2 |
The embodiments can prove that the cell cryopreservation solution provided by the invention can ensure the cryopreservation recovery rate and the immunocompetence of DCs when used for cryopreserving DCs, and the cryopreservation recovery rate and the immunocompetence are not obviously reduced within 1 year of cryopreservation; in the cell cryopreservation solution, dimethyl sulfoxide is used as a cryopreservation protective agent, so that the damage of low temperature to DCs is reduced; human gamma globulin is a cell stabilizer and can maintain the immunocompetence of DCs; poly-L-lysine is an anti-settling agent, so that the human gamma globulin is prevented from settling to the bottom of a frozen stock solution in the storage process, meanwhile, the poly-L-lysine has the function of a partial freezing protective agent, and the volume concentration of DMSO can be reduced to 1-3% from 10-20% in the prior art and still can be prevented from being damaged by low temperature on DCs due to the existence of the poly-L-lysine; in addition, poly-L-lysine also helps maintain the immunological activity of DCs.
The above embodiments are only used to further explain the technical solutions of the present invention, and it should be understood by those skilled in the art that any simple replacement or modification may not depart from the present invention, and the scope of the present invention is not limited to the above specific embodiments.
Claims (1)
1. An application of cell frozen stock solution based on RPMI-1640 culture solution in the aspect of dendritic cell freezing is disclosed, wherein the cell frozen stock solution is prepared by adding effective amounts of freezing protective agent, cell stabilizer and anti-settling agent into the RPMI-1640 culture solution and uniformly mixing; the cryopreservation protective agent is dimethyl sulfoxide; the cell stabilizer is human gamma globulin; the anti-settling agent is poly-L-lysine, and the molecular weight range of the poly-L-lysine is 3-7 ten thousand; the cell freezing solution is prepared by the following method: firstly, mixing 98mL of RPMI-1640 culture solution with 2mL of dimethyl sulfoxide, and then adding human gamma globulin and poly-L-lysine, wherein the final concentrations of the human gamma globulin and the poly-L-lysine are respectively 10 and 25 mu g/mL.
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