CN111172110A

CN111172110A - Culture method of umbilical cord blood CIK cells

Info

Publication number: CN111172110A
Application number: CN201911329973.9A
Authority: CN
Inventors: 孙丹; 唐忆琳; 陈巧林
Original assignee: Zhongke Cell Technology Guangzhou Co Ltd
Current assignee: Zhongke Cell Technology Guangzhou Co Ltd
Priority date: 2019-12-20
Filing date: 2019-12-20
Publication date: 2020-05-19
Anticipated expiration: 2039-12-20
Also published as: CN111172110B

Abstract

The invention provides a culture method of an umbilical cord blood CIK cell, which comprises the following steps: adding the umbilical cord blood nucleus cell suspension into a culture container coated by fibronectin for culture, adding IFN-gamma, IL-2, IL-1a and anti-CD 3 monoclonal antibodies, and continuing to culture to obtain the CIK cells. The method successfully obtains the cord blood CIK with higher appreciation, higher double-positive cell percentage and stronger killing activity, and is a culture method which is worthy of clinical recommendation and can greatly amplify the cord blood CIK.

Description

Culture method of umbilical cord blood CIK cells

Technical Field

The invention relates to the technical field of cell culture, in particular to a culture method of an umbilical cord blood CIK cell.

Background

Cytokine-induced killer Cells (CIK) are immune effector cells mediating the strongest cytotoxic activity, having both the high tumoricidal activity of T lymphocytes and the limiting tumoricidal effect of NK (natural killer) cells, non-Major Histocompatibility Complex (MHC). The CIK cell expresses two membrane protein molecules of CD3 and CD56 at the same time, has the advantages of high proliferation speed, high tumor killing activity, wide tumor killing spectrum and the like, and is considered as a new hope of adoptive immunotherapy of tumors. In the prior art, most CIK cell preparation methods adopt peripheral blood mononuclear cells of patients, IFN-gamma is added firstly, other cytokines such as IL-2, IL-I a and CD3 monoclonal antibodies are added after 24 hours, and the cells are continuously cultured.

With the wide application of cord blood, cord blood becomes an important source for obtaining CIK (cord blood CRX ℃ B-CHK) cells, and the CIK cells of the cord blood have stronger effect and low GVHD incidence rate after transplantation, thereby attracting more and more attention. However, the amplification rate and the killing property of the CIK cells cultured by the existing method are low, and the solution is needed to be solved.

Chinese patent application publication No. CN 108841790 a discloses a method for inducing CIK cells from placenta-derived mononuclear cells, which is derived from placenta, has a large volume, is not easy to collect and transport, and is complicated to extract mononuclear cells.

Chinese patent with application publication number CN 105112371A discloses a method for preparing DC-CIK cells from umbilical cord blood mononuclear cells and a preparation thereof.

The Chinese patent with application publication number CN 105695404A discloses an amplification activation method of CIK lymphocytes, which needs transfection and is complex to operate, and the obtained CIK lymphocytes have low proliferation rate and killing activity.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects and shortcomings of the prior art, and provide a culture method of CIK cells in umbilical cord blood, which can effectively improve the proliferation activity and killing activity of the CIK cells.

The above purpose of the invention is realized by the following technical scheme:

the invention provides a culture method of an umbilical cord blood CIK cell, which comprises the following steps: adding the umbilical cord blood nucleus cell suspension into a culture container coated by fibronectin for culture, adding IFN-gamma, IL-2, IL-1a and anti-CD 3 monoclonal antibodies, and continuing to culture to obtain the CIK cells.

The cord blood nuclear cells are specifically cord blood isolated mononuclear cells.

IFN-gamma, IL-2, IL-1a and anti-CD 3 monoclonal antibodies are all commercially available, or can be prepared by itself, specifically, purified proteins or host cells can be instantly expressed on the surface of cell membranes.

Optionally, the IFN- γ is added within 30min after the umbilical cord blood nuclear cells are added into the culture container coated with fibronectin, specifically, the IFN- γ is added immediately after the cells are inoculated, or within 3min, 5min, 8min, 10min, 12min, 15min, 17min, 20min, 22min, 23min, 25min, 28min, and 30 min.

Optionally, the IL-2, IL-1a and anti-CD 3 monoclonal antibodies are added at 24-26 h after the umbilical cord blood cells are added into the culture container coated with the fibronectin, including but not limited to 24h, 24.1h, 24.2h, 24.3h, 24.4h, 24.5h, 24.6h, 24.7h, 24.8h, 24.9h, 25h, 25.1h, 25.2h, 25.3h, 25.4h, 25.5h, 25.6h, 25.7h, 25.8h, 25.9h and 26 h.

Optionally, IL-15 is further added at 24-26 h after the umbilical cord blood nuclear cells are added into the culture container coated with the fibronectin, including but not limited to 24h, 24.1h, 24.2h, 24.3h, 24.4h, 24.5h, 24.6h, 24.7h, 24.8h, 24.9h, 25h, 25.1h, 25.2h, 25.3h, 25.4h, 25.5h, 25.6h, 25.7h, 25.8h, 25.9h and 26 h. IL-15 is also commercially available.

Optionally, the fibronectin is selected from RetroNectin.

Optionally, when the culture container is coated with the fibronectin, the coating solution contains the fibronectin and a buffer solution.

Optionally, the concentration of the fibronectin in the coating solution is greater than or equal to 6 μ g/mL, and can be 6.25-25 μ g/mL, including but not limited to 6.25 μ g/mL, 7 μ g/mL, 8 μ g/mL, 9 μ g/mL, 10 μ g/mL, 11 μ g/mL, 12.5 μ g/mL, 13 μ g/mL, 14 μ g/mL, 15 μ g/mL, 16 μ g/mL, 17 μ g/mL, 18 μ g/mL, 19 μ g/mL, 20 μ g/mL, 21 μ g/mL, 22 μ g/mL, 23 μ g/mL, 24 μ g/mL, 25 μ g/mL.

Optionally, the buffer is selected from at least one of PBS buffer, normal saline, D-PBS buffer.

Optionally, the IFN- γ is present in the culture medium at a final concentration of 500-1500 IU/mL.

Optionally, the IL-2 is present in the culture medium at a final concentration of 500-1500 IU/mL.

Optionally, the final concentration of IL-1a in the culture medium is 500-1500 IU/mL.

Optionally, the final concentration of IL-15 in the culture medium is 10-30 ng/mL.

Optionally, the anti-CD 3 monoclonal antibody is present in the cell fluid at a final concentration of 30-70 ng/mL.

Alternatively, after suspending cord blood mononuclear cells in a culture medium containing upper plasma obtained by separating cord blood, the cord blood mononuclear cells are added into the culture container coated with fibronectin for culture.

Alternatively, the medium is selected from immune cell culture media including, but not limited to, GT-T551 medium, DKW medium, LONZA medium, and the like. The immune cell culture medium can be purchased from the market.

The invention also provides a composition for culturing the CIK cells in umbilical cord blood, which comprises the following components: fibronectin, IFN-gamma, IL-2, IL-1a, IL-15 and anti-CD 3 monoclonal antibody.

Optionally, the fibronectin is used to coat to a culture vessel.

Optionally, the coating solution for coating the culture vessel contains fibronectin and a buffer solution.

Optionally, the concentration of fibronectin in the coating solution is more than or equal to 6 mug/mL.

Optionally, the final concentration of IFN- γ in the cell fluid is 500-1500 IU/mL.

Optionally, the final concentration of IL-2 in the cell sap is 500-1500 IU/mL.

Optionally, the final concentration of IL-1a in the cell sap is 500-1500 IU/mL.

Optionally, the final concentration of IL-15 in the cell fluid is 10-30 ng/mL.

The invention has the following beneficial effects:

the method adopts the umbilical cord blood mononuclear cells as the original cells for culture, effectively avoids serious rejection reaction easily caused by transfusion of allogeneic CIK cells, and successfully obtains the umbilical cord blood CIK cells with higher proliferation, higher double-positive cell percentage and stronger killing activity than the CIK cells cultured by the traditional method by introducing the specific cell factors. Further, IL-15 is a pleiotropic cytokine, has the functions of activating T cells and mediating the proliferation and survival of the T cells, and is a promising factor for tumor treatment. RetroNectin is a recombinant human fibronectin fragment and has physiological activities of participating in attachment, extension, differentiation and proliferation of cells, and research shows that fibronectin can promote adhesion and proliferation of lymphocytes. According to the invention, RetroNectin coating technology and IL-15 are introduced into a culture system of the cord blood CIK, on one hand, the high amplification performance enables one collected cord blood to be used for treating a plurality of patients, on the other hand, effector cells with higher killing activity can be obtained, and the method is an effective culture method for amplifying the cord blood CIK in large quantity which is worthy of clinical recommendation.

Drawings

FIG. 1 is a graph showing CIK cell proliferation of cord blood of A, B, C, D, E, F groups in the example of the present invention.

FIG. 2 is a graph showing the CIK cell proliferation of cord blood in groups H1 and H2 in the example of the present invention.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

The embodiment of the invention provides a novel preparation method of cord blood CIK cells, which comprises the steps of taking cord blood mononuclear cells, suspending the cord blood mononuclear cells in a culture medium containing upper plasma separated from cord blood, adding a recombinant fibronectin (RetroNectin, RN for short, 12.5 mu g/mL) coated culture flask, adding IFN-gamma (1000IU/mL) in D0 days, adding IL-2(1000IU/mL), IL-1a (1000IU/mL), IL-15(20ng/mL) and anti-CD 3 monoclonal antibody (50ng/mL) in D1 days, and continuing to culture. IL-15 is a pleiotropic cytokine with the function of activating T cells and mediating the proliferation and survival of T cells. In addition, IL-15 can activate, maintain and expand CD8+ memory T cells, but not activate regulatory T lymphocytes (Tregs, with immunosuppressive functions), and IL-15 is a promising factor for tumor therapy. The RetroNectin coating technology is applied to in-vitro culture of the cord blood CIK, and compared with the cord blood CIK induced by the combination of traditional cytokines without RetroNectin, immunocompetent cells with higher proliferation rate and activity and stronger tumor killing property can be obtained. RetroNectin is a recombinant human fibronectin fragment with physiological activities involved in cell attachment, expansion, differentiation and proliferation, and fibronectin can promote lymphocyte adhesion and proliferation. The RetroNectin coating technology is introduced into a culture system of the cord blood CIK, on one hand, the high amplification performance of the RetroNectin coating technology enables one collected cord blood to be used for treating a plurality of patients, on the other hand, effector cells with higher killing activity can be obtained, and the RetroNectin coating technology is an effective culture method for amplifying the cord blood CIK in large quantity which is worthy of clinical recommendation.

In the following examples, the pH of the PBS buffer was 7.4.

In the following examples, D0 refers to the day, i.e., day 0, D1 refers to day 1 after cell culture, and so on for D2, D3, D4, and so on.

Example 1

The method of this example is as follows: cord Blood Mononuclear Cells (CBMC) were extracted from the lymphocyte fraction and counted and then divided into six groups, including A, B, C, D, E, F on average. Group A was cultured by a conventional method without addition of RN, group B was cultured with the RN (final concentration: 6.25. mu.g/mL) coat, group C was cultured with the RN (final concentration: 9.25. mu.g/mL), group D was cultured with the RN (final concentration: 12.5. mu.g/mL), group E was cultured with the RN (final concentration: 15.5. mu.g/mL), group F was cultured with the RN (final concentration: 18.5. mu.g/mL), and the cell viability of each group was examined by trypan blue staining every 3 days and the absolute number of cells was recorded. Each set of cellular immunophenotypes was determined by flow cytometry at days 7, 14, and 21 of culture. MTT test measures the killing rate of each group of cells to K562 tumor cells. Flow cytometry was used to determine the double positive rate of CD3, CD56 in each group of effector cells. After the RN coating concentration with the optimal effect on the CIK of the umbilical cord blood is determined, two groups of experiments are designed, wherein an H1 group is cultured by the RN coating with the determined optimal concentration, IFN-gamma (1000IU/mL) is added in D0 days, IL-2(1000IU/mL), IL-1a (1000IU/mL) and anti-CD 3 monoclonal antibody (50ng/mL) are added in D1 days, and then IL-2(1000IU/mL) is added in each fluid replacement; the H2 group was cultured with the determined optimal concentration of RN coating, IFN-. gamma.was added for D0 days (1000IU/mL), IL-2(1000IU/mL), IL-15(20ng/mL), IL-1a (1000IU/mL), and anti-CD 3 monoclonal antibody (50ng/mL) were added for D1 days, after which IL-2(1000IU/mL) and IL-15(20ng/mL) were added for each fluid replacement, and the effect of IL-15 on CIK in cord blood was studied.

1. Reagent and consumable

PBS, 50mL centrifuge tube, 10mL pipette, 25mL pipette, RetroNectin (from TAKARA), IFN-gamma (from Beijing Homopolium Biotech Co., Ltd.), IL-15, monoclonal antibody CD3 (from Beijing Homopolium Biotech Co., Ltd.), IL-2 (from Beijing Shuanglu pharmaceutical Co., Ltd.), IL-1a (from PEPRO TECH), GT-T551H3 medium (from TAKARA), T175 flask, 0.4% trypan blue stain, 1% paraformaldehyde, BD test tube, gun tip, K562 cells (from Nanjing Korea Biotech Co., Ltd.), 1640 medium, T25 flask, 96 well plate, LDH 25.

2. Instrumentation and equipment

Centrifuge, electric pipette, biosafety cabinet, flow cytometer, vortex shaker, CO₂Incubator, blood cell counter, inverted microscope, manual pipette gun, MTT.

3. Experimental procedure

3.1 coating: taking RetroNectin, preparing coating solutions by using PBS buffer solution, wherein the final concentrations are respectively 6.25 mu g/mL, 9.25 mu g/mL, 12.5 mu g/mL, 15.25 mu g/mL and 18.25 mu g/mL, independently adding each coating solution into each T175 culture bottle, horizontally placing, uniformly mixing, keeping the temperature at 4 ℃, avoiding light and staying overnight.

3.2 blood collection: under the informed consent of the subject, 100mL of umbilical cord blood of a parturient who is naturally delivered is collected.

3.3 isolation of CBMC: the whole blood is centrifuged to take out the plasma, and the plasma is inactivated and centrifuged for standby. Extracting mononuclear cells from cord blood with lymphocyte separation solution, washing with PBS for 3 times, counting cells, and dividing into four groups (A, B, C, D, E, F six groups) with cell number of 5 × 10⁷。

3.4D0 seeding of cells: B. c, D, E, F groups were cultured in RN overnight-coated flasks at the corresponding concentration, the coating solution was removed, PBS was washed twice, CBMC and GT-T551 medium were added, group A cells were prepared and cultured by in vitro induction according to the conventional method, and 10% autologous plasma and IFN- γ (final concentration 1000IU/mL) were added to six groups respectively

The conventional preparation method is that the recombinant fibronectin is not increased, INF-gamma is added to D0, IL-2, IL-1a and anti-CD 3 monoclonal antibody are added after 24 hours, and only IL-2 and fresh culture medium are added after each fluid replacement.

3.5A, B, C, D, E, F six groups D1 were added IL-2(1000IU/ml final concentration), IL-1a (1000IU/ml final concentration), and anti-CD 3 monoclonal antibody (50 ng/ml).

3.6 continuing the culture, when the medium with high cell density turns yellow, supplementing the solution in time, and A, B, C, D, E, F six groups of freshly added medium are all added with IL-2 (the final concentration is 1000 IU/mL).

4. Experimental data acquisition and determination

4.1 determination of CIK cell proliferation Activity

After cell sap was mixed well, A, B, C, D, E, F six groups of CIK cells were extracted, each 1mL, mixed well, stained with trypan blue, the total number of cells was counted with a hemocytometer under an inverted microscope, and then the average value of cells per group was calculated. The cells were extracted every three days and growth curves were plotted according to cell proliferation fold.

4.2 phenotypic assay

The cells of

day

0, 3, 6, 9, 12, and 15 were labeled with flow antibody in combination, each containing 5X 10 cells⁵Individual cellThen, the ratio of CD3+ CD56+ in four groups of cord blood CIKs was detected by using a flow cytometer.

4.3LDH assay for K562 cell killing Activity of two groups of cells

Six groups of cells are collected on 12 th and 15 th days of culture, LDH killing detection is carried out on a 96-well plate by taking tumor cells K562 as target cells, and the cells are placed on an enzyme-labeling instrument to detect OD values after gradual treatment.

5 results

5.1 Effect of retroNectin on CIK proliferation of cord blood

The CIK proliferation curves of the umbilical cord blood were plotted according to the counting results of

days

0, 3, 6, 9, 12 and 15, and the results are shown in FIG. 1, wherein in FIG. 1, the abscissa is the number of cell culture days, the ordinate is the number of cells, the proliferation of the cells is not obvious in the first 3 days, and the proliferation starts to increase greatly on day 4, and it can be seen that the proliferation amount of D, E, F groups is obviously higher than that of A, B, C groups.

5.2 Effect of retroNectin on the percentage of CD3+ CD56+ cells in CIK from cord blood

The phenotype of six groups of cells under different culture times is shown in Table 1, and the proportion of CD3+ CD56+ double positive cells of the group added with retroNectin is obviously higher than that of the group not added with retroNectin. As can be seen from table 2, the percentage of six groups of primary effect CD3+ CD56+ double positive cells increased greatly with the increase of the induction time, and the percentage of D, E, F group was significantly higher than that of the other three groups from day 9. The percentage of the D, E, F double positive cells has no obvious difference

TABLE 1 six cell phenotypes at different incubation times

Percentage of CD3+ CD56+	D0	D3	D6	D9	D12	D15
							Group A	1	5	7.1	18.5	35.8	48.6
Group B	1	5.4	8.7	22.5	40.9	54.1
							Group C	1	5.8	9.2	26.1	45.6	60.4
Group D	1	6.2	9.8	30.4	50.3	68.8
							Group E	1	6.1	9.7	30.3	50.1	68.7
Group F	1	6.2	9.9	30.5	50.4	68.8

5.3 Effect of varying concentrations of RetroNectin on CIK killing Activity in cord blood

Taking K562 as a target cell, and performing effective targeting on the cells in a ratio of 10: 1. 20: 1. 40: 1, performing a killing experiment, wherein table 2 is a statistical table of killing activities of six groups of cells to K562, and it can be seen that the killing activity of D, E, F group to the tumor cell K562 is higher than that of A, B, C group: two groups of cells of 12 and 15 days are collected, and the result shows that the killing activity of D, E, F groups is higher, and the killing activity of D, E, F three groups of CIK has no obvious difference.

The effective target ratio refers to the ratio of the number of effector cells to target cells.

And the killing activity [% ] is (experimental group OD value-effector cell OD value-target cell group OD value)/target cell group OD value.

TABLE 2 statistical table of killing activity of six groups of cells on K562 cells

5.4 conclusion

The experimental results show that under the induction culture of RN coating with the minimum concentration of 12.5 mug/mL, the increment of the CIK of the cord blood is higher, the killing performance is stronger, the dosage of the RN is the minimum, so that 12.5 mug/mL is the optimal concentration of the human recombinant fibronectin acting on the CIK of the cord blood.

12.5 mu g/mL is the saturation concentration of RN, and the increment and killing of cord blood CIK are not obviously changed after the saturation concentration is reached.

6 IL-15 effect on CIK in cord blood was tested under induction of RN coating at an optimal concentration of 12.5. mu.g/mL.

6.1 Experimental procedure

6.1.1 coating: preparing a coating solution from RetroNectin with PBS, adding the coating solution into a T175 culture flask, horizontally placing, uniformly mixing, keeping at 4 ℃, keeping out of the sun, and standing overnight, wherein the final concentration of the RetroNectin in the coating solution is 12.5 mu g/mL.

6.1.2 blood collection: under the informed consent of the subject, 50mL of umbilical cord blood of a parturient who naturally gives birth is collected.

6.1.3 isolation of CBMC: the whole blood is centrifuged to take out the plasma, and the plasma is inactivated and centrifuged for standby. Extracting umbilical cord blood mononuclear cells with lymphocyte separation solution, washing with PBS for 3 times, counting cells, dividing into two groups, labeled as H1 and H2, with cell number of 5 × 10⁷。

6.1.4D0 seeded cells: RN overnight-coated flasks at 12.5. mu.g/mL were taken from groups H1 and H2, respectively, the coating was removed, PBS was washed twice, CBMC and GT-T551 medium were added, and 10% autologous plasma (i.e., the volume of plasma added was 10% of the volume of cytosol) and IFN-. gamma. (final concentration 1000 units/mL, i.e., 1000IU/mL) were added to the two groups of cells, respectively.

6.1.5H1 group D1 was supplemented with IL-2(1000IU/mL), IL-1a (1000IU/mL), anti-CD 3 monoclonal antibody (50ng/mL), and H2 group D1 days were supplemented with IL-2(1000IU/mL final concentration), IL-15(20ng/mL final concentration), IL-1a (1000IU/mL final concentration) and anti-CD 3 monoclonal antibody (50ng/mL final concentration).

6.1.6 when the cell density increased to turn yellow, the culture medium was replenished immediately, IL-2 (final concentration 1000IU/mL) was added to the fresh culture medium in H1 group, and IL-2 (final concentration 1000IU/mL) and IL-15 (final concentration 20ng/mL) were added to the fresh culture medium in H2 group.

6.2 Experimental data acquisition and measurement

6.2.1 measurement of cell proliferation Activity of CIK

After mixing uniformly, two groups of cord blood CIK cells H1 and H2 are extracted, each group is 1mL, mixing uniformly, trypan blue staining is carried out, the total number of the cells is counted by a blood cell counter under an inverted microscope, and then the average value of each group of the cells is calculated. The cells were extracted every three days and growth curves were plotted according to cell proliferation fold.

6.2.2 phenotypic assay

The cells of each group on

days

0, 3, 6, 9, 12 and 15 were labeled with a flow antibody combination, and the number of cells per group was 5X 10⁵Then, the proportion of CD3+ CD56+ double positive cells in two groups of cord blood CIK is detected by using a flow cytometer.

6.2.3LDH method for detecting killing activity of four groups of cells on K562 cells

Two groups of cells are collected on 12 th and 15 th days of culture, tumor cells K562 are taken as target cells, LDH killing detection is carried out on a 96-well plate, and after gradual treatment, the cells are placed on an enzyme labeling instrument for detecting OD values.

6.3 results

6.3.1 Effect of IL-15 on CIK proliferation of cord blood at optimal RN-coating concentration

The CIK proliferation curves of the cord blood were plotted based on the counting results on

days

0, 3, 6, 9, 12 and 15, FIG. 2 shows a cell culture proliferation curve, from which it can be seen that the proliferation of cells was not significant on the first 3 days and a large amount of proliferation was started on day 4, and it can be seen that the proliferation activity of the group of RetroNectin and IL-15, i.e., H2, added at a concentration of 12.5. mu.g/mL was significantly higher than that of the group of H1 without IL-15.

6.3.2 Effect of IL-15 on CD3+ CD56+ in CIK from cord blood at optimal RN-coating concentrations

The proportion of CD3+ CD56+ double positive cells in the H2 group is obviously higher than that in the H1 group without IL-15. As can be seen from table 3, the percentage of the two groups of primary effector CD3+ CD56+ double positive cells increased greatly with the increase of the induction time, and the percentage of the double positive cells in the H2 group was significantly higher than that in the H1 group.

TABLE 3 two sets of cell phenotypes at different incubation times

Double positive of CD3+ CD56+

D0

D3

D6

D9

D12

D15

H1 group

1％

6.1％

9.5％

27.9％

51.2％

68.9％

H2 group

1％

6.3％

11.8％

31.9％

58.6％

77.1％

6.3.3 Effect of IL-15 on cord blood CIK killing Activity at optimal RN coating concentration

Using K562 as a target cell, and performing cell growth by using an effective target ratio of 10: 1. 20: 1. 40: 1, performing a killing experiment, and collecting two groups of cells in12 and 15 days, wherein the results are shown in table 4, and the killing activity of the H2 group on the tumor cell K562 is higher than that of the H1 group, which indicates that the killing activity of the H2 group is higher.

TABLE 4 statistical table of killing activity of two groups of cells on K562

6.4 conclusion

The experimental results show that under the induction culture of RN coating with the concentration of 12.5 mug/mL, the increment of the CIK of the umbilical cord blood added with the IL-15 is higher than that of the CIK without the IL-15, and the killing performance is stronger.

It should be noted that, even if IL-15 is not added, the embodiment of the present invention also provides a CIK cell culture method with simple operation, the proliferation activity and the killing activity of the obtained CIK cell are both at a higher level, and the proliferation activity and the killing activity of the CIK cell can be further improved by adding IL-15.

Example 2

This example was conducted by referring to step 6 of example 1, except that IFN-. gamma.was used at a final concentration of 500IU/mL in the culture system, IL-2 and IL-1a were used at a final concentration of 500IU/mL in the culture system, and the anti-CD 3 monoclonal antibody was used at a final concentration of 30ng/mL in the culture system.

TABLE 5 two sets of cell phenotypes at different incubation times

Double positive of CD3+ CD56+

D0

D3

D6

D9

D12

D15

H1 group

1％

5.8％

9.1％

27.3％

49.8％

66.4％

H2 group

1％

6％

11.5％

30.5％

57.3％

75.3％

As can be seen from Table 5, the cell phenotype of IFN-gamma, IL-2 and IL-1 α in the culture system at a final concentration of 500IU/mL and the cell phenotype of anti-CD 3 monoclonal antibody in the culture system at a final concentration of 30ng/mL were not significantly different from the cell phenotype of IFN-gamma, IL-2 and IL-1 α in the culture system at a final concentration of 1000IU/mL and the cell phenotype of anti-CD 3 monoclonal antibody in the culture system at a final concentration of 50 ng/mL.

TABLE 6 statistical table of killing activity of two groups of cells on K562

As can be seen from Table 6, the final concentrations of IFN-gamma, IL-2 and IL-1 α in the culture system are 500IU/mL, the final concentration of the anti-CD 3 monoclonal antibody in the culture system is 30ng/mL, the final concentrations of the anti-CD 3 monoclonal antibody in the culture system are 1000IU/mL, and the final concentration of the anti-CD 3 monoclonal antibody in the culture system is 50ng/mL, so that the percentage killing activity of the cells on K562 is not obviously different.

Example 3

This example was conducted in accordance with example 1, except that IFN-. gamma.was used at a final concentration of 1500IU/mL in the culture system, IL-2 and IL-1a were used at a final concentration of 1500IU/mL in the culture system, and the anti-CD 3 monoclonal antibody was used at a final concentration of 70ng/mL in the culture system.

TABLE 7 two sets of cell phenotypes at different incubation times

Double positive of CD3+ CD56+

D0

D3

D6

D9

D12

D15

H1 group

1％

6.2％

9.4％

26.9％

50.9％

68.8％

H2 group

1％

6.3％

11.5％

32％

59.1％

76.9％

As can be seen from Table 7, the cell phenotype of IFN-gamma, IL-2 and IL-1 α in the culture system at a final concentration of 1500IU/mL and the cell phenotype of anti-CD 3 monoclonal antibody at a final concentration of 70ng/mL was not significantly different from the cell phenotype of IFN-gamma, IL-2 and IL-1 α at a final concentration of 1000IU/mL and anti-CD 3 monoclonal antibody at a final concentration of 50 ng/mL.

TABLE 8 statistical table of killing activity of two groups of cells on K562 cells

As can be seen from Table 8, the final concentrations of IFN-gamma, IL-2 and IL-1 α in the culture system are 1500IU/mL, the final concentration of the anti-CD 3 monoclonal antibody in the culture system is 70ng/mL, the final concentrations of the anti-CD 3 monoclonal antibody in the culture system are 1000IU/mL, and the final concentration of the anti-CD 3 monoclonal antibody in the culture system is 50ng/mL, so that the percentage killing activity of the cells on K562 cells is not obviously different.

To sum up, after suspending umbilical cord blood mononuclear cells with a culture medium containing upper plasma separated from umbilical cord blood, adding recombinant fibronectin (RetroNectin12.5 mu g/mL) coated culture flasks, adding IFN-gamma (1000IU/mL) in D0 days, and adding IL-2(1000IU/mL), IL-1a (1000IU/mL), IL-15(20ng/mL) and anti-CD 3 monoclonal antibody (50ng/mL) in D1 days can obtain umbilical cord blood CIK with higher proliferation, higher double-positive cell percentage and stronger killing activity, and is a culture method which is clinically recommended and can greatly amplify umbilical cord blood CIK.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A culture method of CIK cells in umbilical cord blood is characterized in that an umbilical cord blood nuclear cell suspension is added into a culture container coated by fibronectin for culture, IFN-gamma, IL-2, IL-1a and anti-CD 3 monoclonal antibodies are added, and continuous culture is carried out to obtain the CIK cells.

2. The culture method according to claim 1, wherein: the IFN- γ is added within 30min after the cord blood cells are added to the fibronectin coated culture vessel.

3. The culture method according to claim 1, wherein: the IL-2, IL-1a and anti-CD 3 monoclonal antibody are added 24-26 h after the umbilical cord blood nuclear cells are added into the culture container coated with the fibronectin.

4. The culture method according to claim 3, wherein: and IL-15 is also added in 24-26 h after the umbilical cord blood nuclear cells are added into the culture container coated with the fibronectin.

5. The culture method according to claim 4, wherein: the final concentration of the IL-15 in the culture system after the addition is 10-30 ng/mL.

6. The culture method according to claim 1, wherein: the fibronectin is selected from RetroNectin;

and/or when the culture container is coated with the fibronectin, the adopted coating solution contains the fibronectin and a buffer solution, and preferably, the concentration of the fibronectin in the coating solution is more than or equal to 6 mu g/mL.

7. The culture method according to claim 1, wherein: the final concentration of the IFN-gamma in the culture system is 500-1500 IU/mL;

and/or the final concentration of the IL-2 in the culture system is 500-1500 IU/mL;

and/or the final concentration of the IL-1a in the culture system is 500-1500 IU/mL;

and/or the final concentration of the anti-CD 3 monoclonal antibody in the culture system is 30-70 ng/mL.

8. The culture method according to claim 1, wherein: suspending umbilical cord blood mononuclear cells by using a culture medium containing umbilical cord blood upper plasma, and then adding the umbilical cord blood mononuclear cells into the culture container coated with the fibronectin for culture.

9. A composition for inducing culture of cord blood CIK cells, which comprises: fibronectin, IFN-gamma, IL-2, IL-1a, anti-CD 3 monoclonal antibody, preferably, IL-15 is also contained.

10. The composition of claim 9, wherein: the fibronectin is used to coat to a culture vessel.