CN106661555B - Efficient amplification method for autologous CIK cells - Google Patents
Efficient amplification method for autologous CIK cells Download PDFInfo
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Abstract
Provides a high-efficiency amplification method of autologous CIK cells, which comprises the following steps: separating peripheral blood mononuclear cells, preparing platelet aggregation release substances, and culturing autologous CIK cells. The method uses platelet aggregation releaser to replace common animal derived serum, and can also add nutrients of known components such as human serum albumin to promote rapid proliferation of CIK cells.
Description
Technical Field
The invention relates to the technical field of animal immune cell culture, in particular to a high-efficiency amplification method of autologous CIK cells.
Background
In the conventional autologous CIK cell culture process, a large amount of animal-derived serum such as fetal calf serum, new bovine serum, calf serum and the like is required to be used for essential nutrients in the cell culture process. Nowadays, most laboratories still culture cells with animal-derived serum, but with the decrease of blood source and the increasing price of serum, and the pressure from animal protection tissues, the use of animal-derived serum is increasingly limited, and in addition, the complex components in animal-derived serum can cause more uncertain effects on cultured cells, and can not accurately show the required biological characteristics.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides a high-efficiency amplification method of autologous CIK cells, and solves the defect that the animal-derived serum in the existing CIK cell amplification method can bring uncertain influence on immune reaction.
The technical scheme for solving the problems comprises the following steps: provides a high-efficiency amplification method of autologous CIK cells, which comprises the following steps:
s100, separation of peripheral blood mononuclear cells: separating peripheral blood mononuclear cells from the blood of the patient, and suspending for later use;
s200, preparation of platelet aggregation releaser: separating to obtain platelets and resuspending the platelets with phosphate buffer to obtain resuspended platelets; adding 80-150 mu mol/L adenosine diphosphate into the resuspended platelets, incubating and culturing for 2-4 hours in an incubator, centrifuging for 20-25 min at 1800-2000 g, and harvesting supernatant components which are directly used as a solution containing platelet aggregation release substances, or performing freeze-drying treatment to obtain the platelet aggregation release substances;
s300, culturing autologous CIK cells: carefully paving the peripheral blood mononuclear cells into a culture bottle for culture, supplementing 5-20% of serum-free culture medium containing the platelet aggregation release during culture, and adding cell factors for induction until the autologous CIK cells are harvested.
In the efficient autologous CIK cell amplification method provided by the present invention, the step S100 further includes:
s101, preparation of whole blood: extracting venous blood of a tumor patient, and adding an anticoagulant into the venous blood to obtain anticoagulated whole blood for later use;
s102, separating peripheral blood mononuclear cells from autologous plasma: separating peripheral blood mononuclear cells from autologous plasma by using a blood cell separator or lymphocyte separation solution to obtain a crude product of the peripheral blood mononuclear cells;
s103, processing of peripheral blood mononuclear cells: washing the crude peripheral blood mononuclear cell product twice by using normal saline or a serum-free culture medium, centrifuging the crude peripheral blood mononuclear cell product for 3-7 minutes at the temperature of 20 ℃ at 500g, and then re-suspending the crude peripheral blood mononuclear cell product by using a proper amount of the serum-free culture medium to obtain the peripheral blood mononuclear cell for later use.
In the method for efficiently amplifying autologous CIK cells, the phosphate buffer used in the step S200 is Ca-containing2+、Mg2+Phosphate buffer of (4).
In the high-efficiency amplification method of autologous CIK cells provided by the invention, the contents of platelet-derived growth factors, transforming growth factors, insulin-like growth factors and epidermal growth factors in the platelet aggregation releaser are all more than 150 ng/g.
In the method for efficiently amplifying autologous CIK cells provided by the invention, the method for separating and obtaining platelets in the step S200 comprises the following steps:
taking citric acid anticoagulated whole blood or platelet rich plasma, and carefully layering the citric acid anticoagulated whole blood or platelet rich plasma on the upper layer of the platelet separating solution; centrifuging for 20-25 min at 800-1200 g, and sucking a platelet layer between lymphocyte separation liquid and plasma.
In the high-efficiency amplification method of autologous CIK cells, provided by the invention, the platelet separation solution is prepared from polysucrose, diatrizoic acid and deionized water, the density is 1.060g/L, the osmotic pressure is 280mosm/L, and the platelet separation solution is obtained after filtration and sterilization by a 0.22-micron filter.
In the efficient autologous CIK cell amplification method provided by the present invention, the step S300 further includes:
s301, adjusting cell concentration: adjusting the cell concentration of peripheral blood mononuclear cells to 1 × 10 with serum-free culture medium6～2×106Each/ml, after being mixed evenly, the mixture is carefully paved into a culture bottle;
s302, pre-culture: adding 600-1400U/ml IFN-gamma and 0.5-2% platelet aggregation release in parts by weight into a culture bottle, and culturing for 24-48 h;
s303, continuous culture: after being supplemented with IL2 of 600U/ml-1400U/ml, IL1a of 600U/ml-1400U/ml and anti-CD3 of 60 ng/ml-150 ng/ml, the culture is continued, sampling and counting are carried out every other day, fresh serum-free culture medium containing 0.5-2% by mass of platelet aggregation release substances is supplemented, the culture density of the cells is adjusted to 1 x 106～2×106And (4) per ml until the autologous CIK cells are harvested.
In the efficient autologous CIK cell amplification method provided by the invention, when the step S303 is supplemented with a fresh serum-free culture medium containing 0.5-2% by mass of platelet aggregation release substances, the fresh serum-free culture medium further contains 1-2% by mass of human serum albumin, 0.05-0.2% of recombinant human transferrin or ferric citrate and 0.05-0.1% of phytohemagglutinin.
The implementation of the invention has the following beneficial effects: in the amplification process of the CIK cells, platelet aggregation and release substances are used for replacing common animal-derived serum, and nutrients of known components such as human serum albumin and the like can be added to promote the rapid proliferation of the CIK cells, so that the uncertainty and the danger of the CIK cells caused by heterogeneous additives are avoided, and in addition, the proliferation speed, the phenotype, the in vitro killing performance and the like of the CIK cells can be obviously improved.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a flow chart of a preferred embodiment of the method for efficiently amplifying autologous CIK cells of the present invention;
FIG. 2(a) is an inverted microscopic observation image of CIK cells cultured in example 1;
FIG. 2(b) is an inverted microscopic observation image of CIK cells cultured in example 2;
FIG. 2(c) is a diagram showing the results of inverted microscope observation of CIK cells cultured in example 3;
FIG. 2(d) is an inverted microscopic observation image of CIK cells cultured in comparative example 1;
FIG. 2(e) is an inverted microscopic observation image of CIK cells cultured in comparative example 2.
Detailed Description
The technical solutions in the prior art and the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is apparent that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
In the conventional CIK cell culture and amplification process, a large amount of animal-derived serum such as fetal bovine serum, new bovine serum, calf serum and the like is still used, but uncertain influences caused by reduction of blood sources and complex components increasingly limit the use of the animal-derived serum, more particularly, complex antigen active substances in the animal-derived serum easily cause a lot of uncertain interferences to CIK cell culture, possibly cannot reflect the action of specific stimulators and cause other unknown immune reactions, and a large amount of workload needs to be paid to the examination of the interferences or the influences in experimental research or practical application, thereby hindering the working progress and even being incapable of continuous development.
The invention has the main innovation points that the platelet aggregation releaser is used for replacing animal-derived serum in the CIK cell culture process, the platelet aggregation releaser of the platelet aggregation releaser contains trace low-molecular nutrients and various growth factors for promoting the growth of CIK cells, so that the CIK cells are not only beneficial to mass harvest, but also the uncertainty and the danger of xenogenous additives are avoided, and the cultured CIK cells are obviously superior to fetal calf serum culture media and serum-free culture media in the aspects of cell proliferation, phenotype, in-vitro killing and the like.
FIG. 1 shows a flow chart of a preferred embodiment of the method for efficiently amplifying autologous CIK cells of the invention, as shown in FIG. 1, comprising the following steps:
s100, separation of peripheral blood mononuclear cells: separating peripheral blood mononuclear cells from the blood of the patient, and suspending for later use; after incubation culture of Peripheral Blood Mononuclear Cells (PBMCs), suspension cells and adherent cells are changed according to growth conditions, and CIK cells (cytokine-induced killer cells) are obtained by inducing the suspension cells.
S200, preparation of platelet aggregation releaser: separating to obtain platelets and resuspending the platelets with phosphate buffer to obtain resuspended platelets; adding 80-150 mu mol/L adenosine diphosphate into the resuspended platelets, incubating and culturing for 2-4 hours in an incubator, centrifuging for 20-25 min at 1800-2000 g, and harvesting supernatant components which are directly used as a solution containing platelet aggregation release substances, or performing freeze-drying treatment to obtain the platelet aggregation release substances.
S300, culturing autologous CIK cells: carefully paving the peripheral blood mononuclear cells into a culture bottle for culture, supplementing 5-20% of serum-free culture medium containing the platelet aggregation release during culture, and adding cell factors for induction until the autologous CIK cells are harvested. In the step, the platelet aggregation releaser is mainly used for replacing animal-derived serum in the conventional method, and trace low-molecular nutrients and various growth factors contained in the platelet aggregation releaser are fully utilized to promote the growth of lymphocytes and improve the killing performance in vitro.
In another preferred embodiment of the present invention, in addition to the above steps, the step S100 further includes:
s101, preparation of whole blood: venous blood of a tumor patient is extracted, and anticoagulant is added to prepare anticoagulated whole blood for later use. On-site drawing to prepare anticoagulated whole blood, or direct refrigerated anticoagulated whole blood, the refrigeration process had no significant effect on the results.
S102, separating peripheral blood mononuclear cells from autologous plasma: separating peripheral blood mononuclear cells from autologous plasma by using a blood cell separator or a lymphocyte separation solution to obtain a crude product of the peripheral blood mononuclear cells. The blood cell separator can directly obtain crude products of peripheral blood mononuclear cells, and the process of separating peripheral blood mononuclear cells by using the lymphocyte separation liquid is slightly different due to different manufacturers of the lymphocyte separation liquid, and the general process is as follows: adding about one third of human peripheral blood lymphocyte separating medium into a centrifuge tube, carefully spreading the anticoagulated whole blood of a tumor patient on the upper layer of the lymphocyte separating medium, centrifuging (for example, 20 ℃, 800g for 20 minutes), and then carefully sucking the PBMCs layer by using a Pasteur pipette or a pipette to obtain the crude peripheral blood mononuclear cells.
S103, processing of peripheral blood mononuclear cells: washing the crude peripheral blood mononuclear cells with normal saline or a serum-free culture medium twice, centrifuging the crude peripheral blood mononuclear cells for 3-7 minutes at the temperature of 20 ℃ at 500g, and then resuspending the crude peripheral blood mononuclear cells with a proper amount of the serum-free culture medium to obtain the peripheral blood mononuclear cells for later use.
In another preferred embodiment of the present invention, the preparation method of the platelet aggregation release substance in step S200 includes incubation culture after separating platelets, which specifically includes:
separating to obtain platelets and re-suspending with phosphate buffer solution to obtain re-suspended platelets, wherein the phosphate buffer solution has 2 kinds, one kind does not contain calcium and magnesium ions, and the other kind contains calcium and magnesium ions, and the invention preferably uses Ca-containing solution2+、Mg2+The phosphate buffer solution can better promote the incubation and culture of the platelets and promote the release of cytokines in the platelets. Adding 80-150 mu mol/L adenosine diphosphate into the resuspended platelets, and incubating and culturing in an incubator for 2-4 hours at the culture temperature of 37 ℃. During the incubation culture process, the platelets aggregate and release cytokines in the platelets, and the growth promoting capacity of the platelet aggregation releaser on lymphocytes is improved. And after the incubation culture is finished, centrifuging for 20-25 min at 1800-2000 g, removing platelet membranes, fibrin, cell debris and the like, and harvesting supernatant components. In particular, the supernatant component may be used as it is as a solution containing a platelet aggregation releasing substance, or may be subjected to a freeze-drying treatment to obtain a platelet aggregation releasing substance.
In another preferred embodiment of the present invention, the method for separating and obtaining platelets in step S200 is density centrifugation, which specifically comprises:
taking citric acid anticoagulated whole blood or platelet rich plasma, and carefully layering the citric acid anticoagulated whole blood or platelet rich plasma on the upper layer of the platelet separating solution; centrifuging for 20-25 min at 800-1200 g, and sucking a platelet layer between lymphocyte separation liquid and plasma. The platelet separating medium is mainly prepared from polysucrose, diatrizoic acid and deionized water, has the density of 1.060g/L and the osmotic pressure of 280mosm/L, and is obtained after filtering and sterilizing by a 0.22-micron filter. The density centrifugal separation can remove the red blood cells, white blood cells and most of plasma components in the plasma, and the high-purity platelets are obtained. Of course, the above method for separating and obtaining platelets is only exemplary and not limiting, and the methods for separating and obtaining platelets in the prior art can also be used in the present invention as long as the platelets obtained are of sufficient purity and remain intact without breaking.
In another preferred embodiment of the present invention, step S300 further includes:
s301, adjusting cell concentration: adjusting the cell concentration of peripheral blood mononuclear cells to 1 × 10 with serum-free culture medium6～2×106Each/ml, after being mixed evenly, the mixture is carefully paved into a culture bottle;
s302, pre-culture: adding 600-1400U/ml IFN-gamma and 0.5-2% platelet aggregation release in parts by weight into a culture bottle, and culturing for 24-48 h; using CO2The pre-culture in the incubator can be carried out under the condition of 37 deg.C and CO2The concentration was 5%.
S303, continuous culture: after being supplemented with IL2 (interleukin-2) of 600U/ml to 1400U/ml, IL1a (interleukin-1 a) of 600U/ml to 1400U/ml and anti-CD3(CD3 monoclonal antibody) of 60ng/ml to 150ng/ml, the culture is continued, samples are taken every other day for counting, fresh serum-free culture medium containing 0.5 to 2 mass percent of platelet aggregation release substances is supplemented, and the culture density of the cells is adjusted to 1 multiplied by 106～2×106And (4) per ml until the autologous CIK cells are harvested. In the step, a fresh serum-free culture medium containing 0.5-2% of platelet aggregation release substances in parts by mass is supplemented to supplement nutrients required by cell synthesis, but the number is not suitable for frequent use, and the fresh serum-free culture medium also contains 1-2% of human serum albumin in parts by mass, 0.05-0.2% of recombinant human transferrin or ferric citrate in parts by mass and 0.05-0.1% of phytohemagglutinin in parts by mass. The platelet lysate is rich in growth factors for maintaining lymphocyte exponential growth, nutrients with no or little amount in a basic culture medium and main low-molecular nutrients, and can completely replace animal-derived serum as nutrient substances for lymphocyte culture by matching with human serum albumin, recombinant human transferrin or ferric citrate which is easily utilized by lymphocytes.
Example 1
Separation of peripheral blood mononuclear cells:
separating peripheral blood mononuclear cells from autologous plasma by using a blood cell separator or lymphocyte separation solution to obtain a crude product of the peripheral blood mononuclear cells; washing the crude product of the peripheral blood mononuclear cells twice by using normal saline, centrifuging the crude product for 3 minutes at the temperature of 20 ℃ at 500g, and then resuspending the crude product by using a proper amount of serum-free culture medium to obtain the peripheral blood mononuclear cells for later use.
Preparation of platelet aggregation release:
dissolving sucrose and diatrizoic acid into deionized water, adjusting the density to 1.060g/L and the osmotic pressure to 280mosm/L, and filtering and sterilizing by a 0.22-micron filter for later use. Taking citric acid anticoagulated whole blood or platelet rich plasma, and carefully layering the citric acid anticoagulated whole blood or platelet rich plasma on the upper layer of the platelet separating solution; centrifuging at 800g for 25min, and sucking a platelet layer between lymphocyte separation liquid and plasma;
separating to obtain platelets, resuspending the platelets with a phosphate buffer solution to obtain resuspended platelets, adding 80 mu mol/L adenosine diphosphate into the resuspended platelets, incubating and culturing for 4 hours in an incubator, centrifuging for 25min at 1800g, and harvesting supernatant components, wherein the supernatant components are directly used as a solution containing platelet aggregation release substances, or performing freeze-drying treatment to obtain the platelet aggregation release substances.
Culturing the autologous CIK cells:
adjusting the cell concentration of peripheral blood mononuclear cells to 1 × 10 with serum-free culture medium6Each/ml, after being mixed evenly, the mixture is carefully paved into a culture bottle;
adding 600U/ml IFN-gamma and 0.5% platelet aggregation release in parts by mass into a culture bottle, and culturing for 24 h;
culturing is continued after being supplemented with 1400U/ml IL2, 600U/ml IL1a and 60ng/ml anti-CD3, sampling and counting every other day, fresh serum-free medium containing 2% by mass of platelet aggregation release is supplemented, the culture density of the cells is adjusted to 1X 106And (4) cell per ml until the autologous CIK cells are harvested.
Example 2
Separation of peripheral blood mononuclear cells:
separating peripheral blood mononuclear cells from autologous plasma by using a blood cell separator or lymphocyte separation solution to obtain a crude product of the peripheral blood mononuclear cells; washing the crude peripheral blood mononuclear cells twice by using a serum-free culture medium, centrifuging the crude peripheral blood mononuclear cells for 7 minutes at the temperature of 20 ℃ at 500g, and then re-suspending the crude peripheral blood mononuclear cells by using a proper amount of the serum-free culture medium to obtain the peripheral blood mononuclear cells for later use.
Preparation of platelet aggregation release:
dissolving sucrose and diatrizoic acid into deionized water, adjusting the density to 1.060g/L and the osmotic pressure to 280mosm/L, and filtering and sterilizing by a 0.22-micron filter for later use. Taking citric acid anticoagulated whole blood or platelet rich plasma, and carefully layering the citric acid anticoagulated whole blood or platelet rich plasma on the upper layer of the platelet separating solution; centrifuging at 1200g for 20min, and sucking a platelet layer between lymphocyte separation liquid and plasma;
separating to obtain platelets, resuspending the platelets with a phosphate buffer solution to obtain resuspended platelets, adding 150 mu mol/L adenosine diphosphate into the resuspended platelets, incubating and culturing for 2 hours in an incubator, centrifuging for 20min at 2000g, and harvesting supernatant components which are directly used as a solution containing platelet aggregation release substances, or performing freeze-drying treatment to obtain the platelet aggregation release substances.
Culturing the autologous CIK cells:
adjusting the cell concentration of peripheral blood mononuclear cells to 2X 10 by serum-free culture medium6Each/ml, after being mixed evenly, the mixture is carefully paved into a culture bottle;
1400U/ml IFN-gamma and 2% platelet aggregation release in parts by mass are added into a culture bottle, and the mixture is cultured for 48 hours;
the culture is continued after being supplemented with 600U/ml IL2, 1400U/ml IL1a and 150ng/ml anti-CD3, sampling and counting are carried out every other day, fresh serum-free culture medium containing 0.5 percent of platelet aggregation releaser, 2 percent of human serum albumin, 0.2 percent of recombinant human transferrin and 0.05 percent of phytohemagglutinin in mass portion is supplemented, and the culture density of the cells is adjusted to 2 x 106And (4) cell per ml until the autologous CIK cells are harvested.
Example 3
Separation of peripheral blood mononuclear cells:
separating peripheral blood mononuclear cells from autologous plasma by using a blood cell separator or lymphocyte separation solution to obtain a crude product of the peripheral blood mononuclear cells; washing the crude product of the peripheral blood mononuclear cells twice by using normal saline or a serum-free culture medium, centrifuging the crude product for 4 minutes at the temperature of 20 ℃ at 500g, and then re-suspending the crude product by using a proper amount of the serum-free culture medium to obtain the peripheral blood mononuclear cells for later use.
Preparation of platelet aggregation release:
dissolving sucrose and diatrizoic acid into deionized water, adjusting the density to 1.060g/L and the osmotic pressure to 280mosm/L, and filtering and sterilizing by a 0.22-micron filter for later use. Taking citric acid anticoagulated whole blood or platelet rich plasma, and carefully layering the citric acid anticoagulated whole blood or platelet rich plasma on the upper layer of the platelet separating solution; centrifuging at 1000g for 22min, and sucking a platelet layer between lymphocyte separation liquid and plasma;
separating to obtain platelets, resuspending the platelets with a phosphate buffer solution to obtain resuspended platelets, adding 100 mu mol/L adenosine diphosphate into the resuspended platelets, incubating and culturing for 3 hours in an incubator, centrifuging for 23min at 1000g, and harvesting supernatant components, wherein the supernatant components are directly used as a solution containing platelet aggregation release substances, or performing freeze-drying treatment to obtain the platelet aggregation release substances.
Culturing the autologous CIK cells:
adjusting the cell concentration of peripheral blood mononuclear cells to 1.5 × 10 with serum-free culture medium6Each/ml, after being mixed evenly, the mixture is carefully paved into a culture bottle;
adding 900U/ml IFN-gamma and 1% platelet aggregation release in parts by mass into a culture bottle, and culturing for 36 h;
culturing after adding IL2 of 1000U/ml, IL1a of 900U/ml and anti-CD3 of 100ng/ml, sampling every other day, counting, adding fresh serum-free culture medium containing 0.7% platelet aggregation release, 1% human serum albumin, 0.05% ferric citrate and 0.1% phytohemagglutinin, adjusting the cell culture density to 2 × 106And (4) cell per ml until the autologous CIK cells are harvested.
Effect verification
In comparative example 1, 10% fetal bovine serum 1640 was used, and in comparative example 2, AIM-V serum-free medium was used for CIK cell culture under exactly the same conditions as in examples 1 to 3.
1 cell proliferation status
In the case of identical culture conditions, after 14 days of culture, trypan blue staining detection or a cell counter was used: the standard is that the viable cells are above 80%.
FIG. 2 shows the images of cultured CIK cells observed by an inverted microscope, and FIGS. 2(a) to 2(e) show the CIK cells cultured in examples 1, 2 and 3, comparative example 1 and 2, respectively, and it is clear that the surface morphology of the CIK cells cultured by the culturing method of the present invention is significantly better than that of the CIK cells cultured by the culturing method of the present invention.
Experimental counts included total cell number, CD8+, cell viability.
The total cell count in example 1, example 2, example 3, comparative example 1 and comparative example 2 was 1.03X 1010、1.12×1010、1.21×1010、0.89×1010、0.80×1010；
The CD8+ values in example 1, example 2, example 3, comparative example 1 and comparative example 2 were 0.73X 1010、0.84×1010、0.97×1010、0.50×1010、0.51×1010；
The cell viability in example 1, example 2, example 3, comparative example 1 and comparative example 2 was 98.4%, 97.5%, 99.5%, 95.1% and 92.7%, respectively.
The counting results of the comparative experiments show that the CIK cells cultured by the culture method are obviously superior to those of the comparative examples in the aspects of total cell number, CD8+ and cell survival rate.
2 flow cytometry is used for detecting the expression of molecules such as CD8+, CD3+, CD8+ IFN-gamma + and the like on the surface of the cell.
The detection objects comprise CD8+, CD3+, CD8+ IFN-gamma + and CD107 a.
CD8+ in example 1, example 2, example 3, comparative example 1, comparative example 2 were 70.6%, 75.2%, 80.5%, 56.4%, 64.2%, respectively;
the CD3+ values in example 1, example 2, example 3, comparative example 1 and comparative example 2 are 90.5%, 92.1%, 92.4%, 90.2% and 88.2%, respectively;
the CD8+ IFN-. gamma. + in example 1, example 2, example 3, comparative example 1, comparative example 2 was 45.6%, 55.2%, 43.8%, 21.4%, 19.3%, respectively;
the CD107a values in example 1, example 2, example 3, comparative example 1 and comparative example 2 were 20.1%, 17.2%, 15.3%, 8.2% and 9.3%, respectively.
By comparison, the CIK cells cultured by the culture method of the invention are obviously superior to the comparative examples in the expression of cell surface molecules.
3 cell killing experiment: taking DC-CIK cells as effector cells, taking tumor cells (which can be primary tumor cells or tumor cell strains) as target cells, adding the effector cells and the target cells into a 96-well U-shaped plate according to the ratio of 10:1, 20:1 and 30:1(E: T is the number ratio of the effector cells to the target cells), wherein each well contains 1 × 10 target cells4The final volume is 200ml, and 3 multiple holes are arranged. After 4h of culture, centrifugally sucking culture supernatant, detecting the killing rate of effector cells to target cells by using a Lactate Dehydrogenase (LDH) kit, and setting blank control, target cell control and effector cell control. The blank well control was subtracted from the number of each well and the average of duplicate wells was determined. Cytotoxic activity of effector cells was calculated according to the following formula, and expressed as a killing rate (%):
killing rate ═ target cell control value- (experimental well value-effector cell control value)/target cell control value × 100%.
According to the method, the killing rates of the autologous tumor specific cells obtained by the complete culture medium prepared in examples 1-3 and the culture medium obtained in comparative examples 1-2 on the hepatoma cell line HEPG2 and B-cell leukemia K562 are respectively detected. The experiment was repeated 6 times and the average was taken.
The experimental results include cell killing experimental results of liver cancer HEPG2 and leukemia K562 as target cells under three conditions of E: T10: 1, E: T20: 1 and E: T30: 1.
When T is 10:1, the killing results of liver cancer HEPG2 in example 1, example 2, example 3, comparative example 1 and comparative example 2 are 35.4%, 30.5%, 35.7%, 30.7% and 25.6%, respectively; the killing results of leukemia K562 in example 1, example 2, example 3, comparative example were 28.2%, 35.1%, 40.0%, 28.5%, 26.5%, respectively;
when the E: T is 20:1, the killing results of the liver cancer HEPG2 in example 1, example 2, example 3, comparative example 1 and comparative example 2 are 51.5%, 46.4%, 52.5%, 45.7% and 40.5%, respectively; the killing results of leukemia K562 in example 1, example 2, example 3, comparative example were 52.4%, 55.8%, 60.4%, 38.2%, 40.5%, respectively;
when the E: T is 30:1, the killing results of the liver cancer HEPG2 in example 1, example 2, example 3, comparative example 1 and comparative example 2 are 68.2%, 72.5%, 80.1%, 60.1% and 55.8%, respectively; the results of killing of leukemia K562 in example 1, example 2, example 3, and comparative example were 72.5%, 78.6%, 81.2%, 57.5%, and 65.0%, respectively.
4 before harvesting cells, taking a small amount of culture to culture bacteria and fungi, and detecting mycoplasma, chlamydia and endotoxin (standard: negative of etiological detection, endotoxin is less than 5 Eu).
The detection shows that all indexes in the embodiments 1-3 of the invention and the comparative embodiment do not exceed the standard.
Claims (8)
1. An efficient amplification method of autologous CIK cells is characterized by comprising the following steps:
s100, separation of peripheral blood mononuclear cells: separating peripheral blood mononuclear cells from the blood of the patient, and suspending for later use;
s200, preparation of platelet aggregation releaser: separating to obtain platelets and resuspending the platelets with phosphate buffer to obtain resuspended platelets; adding 80-150 mu mol/L adenosine diphosphate into the resuspended platelets, incubating and culturing for 2-4 hours in an incubator, centrifuging for 20-25 min at 1800-2000 g, and harvesting supernatant components which are directly used as a solution containing platelet aggregation release substances, or performing freeze-drying treatment to obtain the platelet aggregation release substances;
s300, culturing autologous CIK cells: carefully paving the peripheral blood mononuclear cells into a culture bottle for culture, supplementing 5-20% of serum-free culture medium containing the platelet aggregation release during culture, and adding cytokines IL2, IL1a and anti-CD3 for induction until the autologous CIK cells are harvested.
2. The method for efficiently expanding autologous CIK cells according to claim 1, wherein the step S100 further comprises:
s101, preparation of whole blood: extracting venous blood of a tumor patient, and adding an anticoagulant into the venous blood to obtain anticoagulated whole blood for later use;
s102, separating peripheral blood mononuclear cells from autologous plasma: separating peripheral blood mononuclear cells from autologous plasma by using a blood cell separator or lymphocyte separation solution to obtain a crude product of the peripheral blood mononuclear cells;
s103, processing of peripheral blood mononuclear cells: washing the crude peripheral blood mononuclear cell product twice by using normal saline or a serum-free culture medium, centrifuging the crude peripheral blood mononuclear cell product for 3-7 minutes at the temperature of 20 ℃ at 500g, and then re-suspending the crude peripheral blood mononuclear cell product by using a proper amount of the serum-free culture medium to obtain the peripheral blood mononuclear cell for later use.
3. The method for efficiently amplifying autologous CIK cells according to claim 1, wherein the phosphate buffer used in step S200 is Ca-containing2+、Mg2+Phosphate buffer of (4).
4. The method for efficiently amplifying autologous CIK cells according to claim 1, wherein the contents of platelet-derived growth factor, transforming growth factor, insulin-like growth factor and epidermal growth factor in the platelet aggregation release are all more than 150 ng/g.
5. The method for efficiently amplifying autologous CIK cells according to claim 1, wherein the method for separating and obtaining platelets in step S200 comprises:
taking citric acid anticoagulated whole blood or platelet rich plasma, and carefully layering the citric acid anticoagulated whole blood or platelet rich plasma on the upper layer of the platelet separating solution; centrifuging for 20-25 min at 800-1200 g, and sucking a platelet layer between lymphocyte separation liquid and plasma.
6. The method for efficiently amplifying autologous CIK cells according to claim 5, wherein the platelet separation fluid is prepared from polysucrose, diatrizoic acid and deionized water, the density is 1.060g/L, the osmotic pressure is 280mosm/L, and the platelet separation fluid is obtained after filtration sterilization by a 0.22 μm filter.
7. The method for efficiently expanding autologous CIK cells according to claim 1, wherein the step S300 further comprises:
s301, adjusting cell concentration: adjusting the cell concentration of peripheral blood mononuclear cells to 1 × 10 with serum-free culture medium6～2×106Each/ml, after being mixed evenly, the mixture is carefully paved into a culture bottle;
s302, pre-culture: adding 600-1400U/ml IFN-gamma and 0.5-2% platelet aggregation release in parts by weight into a culture bottle, and culturing for 24-48 h;
s303, continuous culture: after being supplemented with IL2 of 600U/ml-1400U/ml, IL1a of 600U/ml-1400U/ml and anti-CD3 of 60 ng/ml-150 ng/ml, the culture is continued, sampling and counting are carried out every other day, fresh serum-free culture medium containing 0.5-2% by mass of platelet aggregation release substances is supplemented, the culture density of the cells is adjusted to 1 x 106～2×106And (4) per ml until the autologous CIK cells are harvested.
8. The method for efficiently amplifying autologous CIK cells according to claim 7, wherein in step S303, when supplementing a fresh serum-free medium containing 0.5-2% by mass of platelet aggregation release, the fresh serum-free medium further contains 1-2% by mass of human serum albumin, 0.05-0.2% by mass of recombinant human transferrin or ferric citrate, and 0.05-0.1% by mass of phytohemagglutinin.
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