CN114600868A - Cell preservation solution and application thereof in preservation of CIK cells - Google Patents
Cell preservation solution and application thereof in preservation of CIK cells Download PDFInfo
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Images
Classifications
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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
- A01N1/0231—Chemically defined matrices, e.g. alginate gels, for immobilising, holding or storing cells, tissue or organs for preservation purposes; Chemically altering or fixing cells, tissue or organs, e.g. by cross-linking, for preservation purposes
-
- 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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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a cell preservation solution, which comprises a basic culture medium and the following components added in the culture medium: potassium dihydrogen phosphate, dextran-40, sodium hyaluronate, herba cistanches polypeptide, Artemisia desertorum polysaccharide, soybean phospholipid, and IL-2. The cell preservation solution provided by the invention is safe and stable in components, provides a stable external environment for CIK cells through the synergistic effect of potassium dihydrogen phosphate, dextran-40, sodium hyaluronate, cistanche polypeptides, artemisia desertorum polysaccharide, soybean phospholipid, IL-2 and other components, helps the CIK cells to keep activity at 2-6 ℃, has the survival rate of the CIK cells of more than 90% after being preserved for 48 hours, still has good killing activity after being preserved, fully meets the use requirements of clinical and scientific research, further reduces the cost, and relieves the treatment burden of patients. The invention also provides an application method of the preservation solution in CIK cell preservation, which is simple and convenient to operate, easy to realize and beneficial to commercial popularization and application.
Description
Technical Field
The invention relates to a cell preservation solution, in particular to a cell preservation solution and application thereof in preservation of CIK cells.
Background
With the increasing threat of malignant tumor to human survival and health, various new drugs and methods for treatment emerge in endlessly, wherein immune cell therapy becomes an important development direction in biological treatment of tumor. Adoptive cellular immunotherapy of tumor is directed to the tumor patients to transfuse immune cells (specific and non-specific) with anti-tumor activity to directly kill tumor or stimulate the immune response of the body to kill tumor cells, and can be used as a supplement for surgery, radiotherapy and chemotherapy to improve the curative effect and the quality of life of the patients, and the quality of immune cells influences the therapeutic effect.
The CIK cell, namely Cytokine-Induced Killer (CIK), is a novel immunocompetent cell, has certain immunological characteristics, and has the advantages of strong antitumor activity of T lymphocytes and non-MHC restricted tumor killing of NK cells. CIK cell has strong proliferation ability and main effect cell CD3+CD56+Can proliferate 1000 times. The killing activity is strong and far superior to that of the traditional LAK cells, cytokine gamma interferon, interleukin 2 and the like. The CIK cell has strong recognition capability on tumor cells, has obvious effect on patients after operation or radiotherapy and chemotherapy, can eliminate residual tiny metastasis focuses, prevent cancer cells from diffusing and relapsing, and improve the immunity of the organism, so the CIK cell is considered as a first-choice scheme of the next generation of tumor adoptive cell immunotherapy.
In the adoptive cellular immunotherapy of tumors, the quantity of CIK cells needed is large, and CIK cells prepared in vitro need to be expanded to be collected from a sufficient number of cells. Usually, autologous peripheral blood or umbilical blood is adopted to prepare CIK, but the CIK cell culture period is long, pollution is easy to occur, and the culture cost is high. Therefore, the preservation of the cultured and collected CIK cells is important, the components are required to be safe and easy to operate, and the activity of the preserved cells is not affected. Therefore, it is necessary to provide a cell preservation solution for realizing the in vitro preservation of CIK cells and reducing the treatment cost of patients.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide the cell preservation solution which has safe components, does not influence the activity of CIK cells and effectively reduces the preservation cost of the cells
The invention also aims to provide the application of the cell preservation solution in preservation of CIK cells.
One of the purposes of the invention is realized by adopting the following technical scheme:
a cell preservation solution comprises a basic culture medium and the following components added in the culture medium: potassium dihydrogen phosphate, dextran-40, sodium hyaluronate, herba cistanches polypeptide, Artemisia desertorum polysaccharide, soybean phospholipid, and IL-2.
Preferably, the final concentration of each component in the culture medium is: 10-15mg/mL of monopotassium phosphate, 405-10 mg/mL of dextran, 8-12 mu g/mL of sodium hyaluronate, 15-20 mu g/mL of cistanche polypeptide, 0.5-1mg/mL of artemisia desertorum polysaccharide, 1-5mg/mL of soybean phospholipid and 300IU/mL of IL-2200-.
Preferably, the final concentration of each component in the culture medium is: 12mg/mL of monopotassium phosphate, 408 mg/mL of dextran, 10 mu g/mL of sodium hyaluronate, 18 mu g/mL of cistanche polypeptide, 0.7mg/mL of artemisia desertorum polysaccharide, 3mg/mL of soybean phospholipid and IL-2250 IU/mL.
Preferably, the molecular weight of the cistanche polypeptide is: 500- & lt1000 Da.
Preferably, the basal medium is RPMI-1640 medium.
The second purpose of the invention is realized by adopting the following technical scheme:
the preservation solution is applied to preservation of CIK cells.
Preferably, the storage temperature is 2-6 ℃.
Preferably, the density of the CIK cells in the preservation solution is 1-6X 107one/mL.
Compared with the prior art, the invention has the beneficial effects that: the invention provides a cell preservation solution which is safe and stable in components, provides a stable external environment for CIK cells through the synergistic effect of potassium dihydrogen phosphate, dextran-40, sodium hyaluronate, cistanche polypeptides, artemisia desertorum polysaccharide, soybean lecithin, IL-2 and other components, helps the CIK cells to keep activity at 2-6 ℃, has the activity rate of more than 90% after being preserved for 48 hours, still has good killing activity after being preserved, fully meets the use requirements of clinical and scientific research, further reduces the cost, and relieves the treatment burden for patients. The invention also provides an application method of the preservation solution in CIK cell preservation, which is simple and convenient to operate, easy to realize and beneficial to commercial popularization and application.
Drawings
FIG. 1 shows the killing activity of CIK cells in example 1, comparative example 1 to comparative example 7 after 60h storage.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict.
Example 1
A cell preservation solution comprises an RPMI-1640 culture medium and the following components added in the culture medium: potassium dihydrogen phosphate, dextran-40, sodium hyaluronate, herba cistanches polypeptide, herba Artemisiae Halodendri polysaccharide, soybean phospholipid, and IL-2; the final concentration of each component in the culture medium is as follows: 12mg/mL of monopotassium phosphate, 408 mg/mL of dextran, 10 μ g/mL of sodium hyaluronate, 18 μ g/mL of cistanche polypeptide with the molecular weight of 500-1000Da, 0.7mg/mL of artemisia desertorum polysaccharide, 3mg/mL of soybean phospholipid and IL-2250 IU/mL.
The application of the cell preservation solution in preservation of CIK cells comprises the following steps: the preservation solution is respectively filled in 2mL cold storage tubes, each tube is 1.5mL, precooled to 2 ℃, and added with CIK cells to be preserved, wherein the cell density is 3 multiplied by 107one/mL, and continuously preserving at 2 ℃.
Example 2
A cell preservation solution comprises an RPMI-1640 culture medium and the following components added in the culture medium: potassium dihydrogen phosphate, dextran-40, sodium hyaluronate, herba cistanches polypeptide, herba Artemisiae Halodendri polysaccharide, soybean phospholipid, and IL-2; the final concentration of each component in the culture medium is as follows: 10mg/mL of monopotassium phosphate, 405 mg/mL of dextran, 8 mu g/mL of sodium hyaluronate, 15 mu g/mL of cistanche polypeptide with the molecular weight of 500-1000Da, 0.5mg/mL of artemisia desertorum polysaccharide, 1mg/mL of soybean phospholipid and 2200 IU/mL of IL.
The application of the cell preservation solution in preservation of CIK cells comprises the following steps: the preservation solution is respectively filled in 2mL cold storage tubes, each tube is 1.5mL, precooled to 4 ℃, and added with CIK cells to be preserved, wherein the cell density is 1 multiplied by 107one/mL, and continuously storing at 4 ℃.
Example 3
A cell preservation solution comprises an RPMI-1640 culture medium and the following components added in the culture medium: potassium dihydrogen phosphate, dextran-40, sodium hyaluronate, herba cistanches polypeptide, herba Artemisiae Halodendri polysaccharide, soybean phospholipid, and IL-2; the final concentration of each component in the culture medium is as follows: 15mg/mL of monopotassium phosphate, 0mg/mL of dextran-4010, 12 mu g/mL of hyaluronic acid, 20 mu g/mL of cistanche polypeptide with the molecular weight of 500-1000Da, 1mg/mL of artemisia desertorum polysaccharide, 5mg/mL of soybean phospholipid and IL-2300 IU/mL.
The application of the cell preservation solution in preservation of CIK cells comprises the following steps: the preservation solution is respectively filled in 2mL cold storage tubes, each tube is 1.5mL, precooled to 6 ℃, and added with CIK cells to be preserved, wherein the cell density is 6 multiplied by 107one/mL, and continuously preserving at 6 ℃.
Comparative example 1
Comparative example 1 provides a cell preservation solution, which is different from example 1 in that: cistanche polypeptides were omitted and the procedure was as in example 1.
Comparative example 2
Comparative example 2 provides a cell preservation solution, which is different from example 1 in that: the cistanche polypeptides were replaced by cistanche glycosides, the rest being the same as in example 1.
Comparative example 3
Comparative example 3 provides a cell preservation solution, which differs from example 1 in that: the above-mentioned polysaccharide was omitted, and the procedure was repeated as in example 1.
Comparative example 4
Comparative example 4 provides a cell preservation solution, which is different from example 1 in that: the soybean lecithin was omitted, and the procedure was the same as in example 1.
Comparative example 5
Comparative example 5 provides a cell preservation solution, which is different from example 1 in that: the amount of the soybean lecithin was adjusted to 3.7mg/mL without the use of Artemisia desertorum polysaccharide, and the rest was the same as in example 1.
Comparative example 6
Comparative example 6 provides a cell preservation solution, which is different from example 1 in that: the soybean phospholipids were omitted, and the amount of artemisia desertorum polysaccharide used was adjusted to 3.7mg/mL, the rest being the same as in example 1.
Comparative example 7
Comparative example 7 provides a cell preservation solution, which is different from example 1 in that: the artemisia desertorum polysaccharide is replaced by the ganoderma lucidum polysaccharide, and the rest is the same as the example 1.
The cell viability of the CIK cells stored in examples 1 to 3 and comparative examples 1 to 7 was calculated by trypan blue staining after 0h, 12h, 24h, 48h and 60h, respectively, the cold storage tube was taken out and rapidly thawed at 37 ℃, the cells were collected by centrifugation and made into a suspension with PBS, 500. mu.L of the cell suspension was placed in an EP tube, 100. mu.L of 0.4% trypan blue staining was added, the dead cells were stained blue under a microscope, and the live cells were colorless and transparent, and the results are shown in Table 1.
TABLE 1
As can be seen from Table 1, the cell viability of the CIK cells preserved in examples 1-3 was over 90% after 48h, and over 85% after 60 h. Compared example 1 omits cistanche polypeptides, the survival rate of the CIK cells after 24h storage is 87.32%, the survival rate of the CIK cells after 60h storage is only 62.19%, and the CIK cells have more apoptosis. Compared with the method for omitting the cistanche polypeptide in the comparative example 1, the method for replacing the cistanche polypeptide with the cistanoside in the comparative example 2 has the advantage that the CIK cell survival rate is improved, but the CIK cell survival rate is 80.38% and is lower than 91.13% in the comparative example 1 after 48 hours of storage compared with the method for omitting the cistanche polypeptide in the comparative example 1. In comparative examples 2 to 6, when one of the artemisia desertorum polysaccharide and the soybean lecithin is omitted or the amount of the remaining component is increased after one of the components is omitted, the cell viability of the CIK is remarkably reduced along with the prolonging of the preservation time. Compared with the comparative examples 2 to 6, the survival rate of the CIK cells is improved to a certain extent by replacing artemisia desertorum polysaccharide with ganoderma lucidum polysaccharide in the comparative example 7, but the survival rate of the CIK cells is not higher than that of the example 1, so that the stable external environment is provided for the CIK cells by adding cistanche desertorum polypeptide, artemisia desertorum polysaccharide, soybean phospholipid and the synergistic effect of potassium dihydrogen phosphate, dextran-40, sodium hyaluronate, IL-2 and other components in the preservation solution, the activity of the CIK cells is maintained at 2-6 ℃, and the survival rate of the CIK cells is still more than 90% after 48 hours of preservation, thereby fully meeting the use requirements of clinical and scientific research.
Evaluation of CIK cellsKilling activity of (2): centrifuging CIK cells stored for 60h, and then resuspending the CIK cells in RPMI-1640 medium at a cell density of 1 × 106each/mL, with K562 cells as target cells, were seeded in 6-well plates at 37 ℃ with 5% CO in an effective target ratio of 40:12The culture box is co-cultured for 24h, simultaneously a target cell control hole and an effector cell control hole are arranged, each group is provided with 3 holes, the absorbance value (A) of each group is determined by a CCK-8 color development method, and the killing activity is ═ A value of the target cell control hole (A value of the target cell experimental hole-A value of the effector cell control hole)]The results are shown in FIG. 1, where A is 100% of the control wells per target cell.
As can be seen from FIG. 1, the killing activity of the CIK cells after storage in example 1 is better. The killing activity of the CIK cells in comparative examples 1 to 7 is reduced in different degrees, the cistanche polypeptides are omitted in the comparative example 1, the cistanche polypeptides are replaced by the cistanche glycosides in the comparative example 2, one of artemisia desertorum polysaccharide and soybean phospholipid is omitted in the comparative examples 2 to 6 respectively or the dosage of the rest components is increased after one of the components is omitted, the artemisia desertorum polysaccharide is replaced by the ganoderma lucidum polysaccharide in the comparative example 7, and the adjusted components have different degrees of influence on the killing activity of the CIK cells.
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.
Claims (8)
1. A cell preservation solution is characterized by comprising a basic culture medium and the following components added in the culture medium: potassium dihydrogen phosphate, dextran-40, sodium hyaluronate, herba cistanches polypeptide, Artemisia desertorum polysaccharide, soybean phospholipid, and IL-2.
2. The cell preservation solution according to claim 1, wherein the final concentrations of the respective components in the medium are: 10-15mg/mL of monopotassium phosphate, 405-10 mg/mL of dextran, 8-12 mu g/mL of sodium hyaluronate, 15-20 mu g/mL of cistanche polypeptide, 0.5-1mg/mL of artemisia desertorum polysaccharide, 1-5mg/mL of soybean phospholipid and 300IU/mL of IL-2200-.
3. The cell preservation solution according to claim 1, wherein the final concentrations of the respective components in the medium are: 12mg/mL of monopotassium phosphate, 408 mg/mL of dextran, 10 mu g/mL of sodium hyaluronate, 18 mu g/mL of cistanche polypeptide, 0.7mg/mL of artemisia desertorum polysaccharide, 3mg/mL of soybean phospholipid and IL-2250 IU/mL.
4. The cell preservation solution according to claim 1, wherein the molecular weight of the cistanche polypeptides is: 500- & lt1000 Da.
5. The cell preservation solution according to claim 1, wherein the basal medium is RPMI-1640 medium.
6. Use of a preservation solution according to any one of claims 1 to 5 for preserving CIK cells.
7. The use of the cell preservation solution according to claim 6, wherein the preservation temperature is 2 to 6 ℃.
8. The use of the cell preservation solution according to claim 6, wherein the density of the CIK cells in the preservation solution is 1-6 x 107One per mL.
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