CN111424012A

CN111424012A - Treatment method for feeder cell proliferation removing capacity for NK cell culture

Info

Publication number: CN111424012A
Application number: CN202010238642.0A
Authority: CN
Inventors: 张道强; 刘传杰; 宫琪; 葛淑娟
Original assignee: Weihai Central Hospital
Current assignee: Weihai Central Hospital
Priority date: 2020-03-30
Filing date: 2020-03-30
Publication date: 2020-07-17

Abstract

The invention provides a method for treating the proliferation removing capacity of feeder cells for NK cell culture, which is to treat the feeder cells by mitomycin C. The method for operating the deproliferation of the feeder cells has the advantages of low cost, simple operation and convenient popularization and use; the feeder cells treated by the method have intact morphology, can better stimulate the amplification of NK cells, so that the prepared NK cells have high purity and killing activity, and when the NK cells are detected and cultured for 14 days, the purity of the NK cells reaches more than 94 percent, and the amplification times reach more than 720; when the effective target ratio is 5:1, the killing activity of the NK cells on K562 cells is more than 60 percent; the feeder cells treated by the method are almost completely killed by NK cells in the 4 th day of culture, have no later-stage residue, and can be applied and popularized in clinical research.

Description

Treatment method for feeder cell proliferation removing capacity for NK cell culture

Technical Field

The invention belongs to the technical field of cell culture, and particularly relates to a treatment method for the de-proliferation capacity of feeder cells for NK cell culture.

Background

NK cells belong to nonspecific immune cells, and can directly kill some tumor and virus-infected target cells without antigen pre-sensitization, and can secrete various cytokines and chemokines to participate in immune regulation. Therefore, plays an important role in the immune process of resisting tumors and resisting virus or intracellular parasitic bacteria infection in the early stage of the body. NK immune cell therapy has wide application prospects in antiviral and antitumor therapy. In recent years, there have been increasing clinical examples of NK cells for immunotherapy.

At present, feeder cells adopting gene recombination, such as K562 cells (41BB L-mbI L-21-K562 cells) stably expressing 41BBB L and membrane expressing I L-21 factors, are widely used for in vitro NK cell amplification, and a large amount of high-purity NK cells can be obtained.

Disclosure of Invention

The feeder cells obtained by the treatment method stimulate the amplification of the NK cells in PBMC, can obtain a large number of NK cells with high killing activity, have a simple culture process, and can completely meet clinical requirements.

The invention firstly provides a method for treating the proliferation removing capacity of feeder cells for NK cell culture, which is to treat the feeder cells by mitomycin C (MMC);

feeder cells for NK cell culture can be subjected to de-proliferation treatment by adopting the method, wherein the feeder cells provided by the embodiment of the invention are K562 cells;

the method of (1), wherein mitomycin C is dissolved in DMSO;

further, the method is characterized in that the mitomycin C concentration in the feeder cells is 5-40 mu g/ml, and the optimized concentration is 33 mu g/ml.

In still another aspect, the present invention provides a feeder cell for NK cell culture, said feeder cell being treated for its capacity to proliferate using mitomycin C;

the provided feeder cells are used for culturing and proliferating NK cells;

in still another aspect, the present invention provides a method for culturing NK cells using the mitomycin C-treated feeder cells described above.

The invention has the following advantages: (1) the method for operating the deproliferation of the feeder cells has the advantages of low cost, simple operation and convenient popularization and use; (2) the feeder cells treated by the method have intact morphology, can better stimulate the amplification of NK cells, so that the prepared NK cells have high purity and killing activity, and when the NK cells are detected and cultured for 14 days, the purity of the NK cells reaches more than 94 percent, and the amplification times reach more than 720; when the effective target ratio is 5:1, the killing activity of the NK cells on K562 cells is more than 60 percent; (3) the feeder cells treated by the method are almost completely killed by NK cells in the 4 th day of culture, have no later-stage residue, and can be applied and popularized in clinical research.

Drawings

FIG. 1: a proportion of feeder cells surviving during culture;

FIG. 2: cytomorphograms under a feeder cell lens before and after mitomycin C treatment;

FIG. 3: graphs of feeder cell stimulated NK cell expansion for different treatment methods;

FIG. 4: a proportion of feeder cells stimulating NK cells for different treatment methods;

FIG. 5: experimental group NK cell flow diagrams;

FIG. 6: graph of tumor killing activity of NK cells stimulated by feeder cells for different treatment methods.

Detailed Description

In order to make the technical solution and advantages of the present invention more clearly understood, the following examples are further detailed, and the embodiments and descriptions of the present invention are only illustrative and not limiting.

DMSO used in the examples of the invention was purchased from Sigma under code D2650, mitomycin C from Selleck under code A1933, DPBS from Takara under code FU0021 feeder cells are K562 cells expressing 41BB L and membrane expressing factor I L-21.

EXAMPLE 1 mitomycin C treatment of feeder cells

Firstly, adding a proper amount of DMSO to dissolve mitomycin C to make the concentration of the mitomycin C33 mg/ml, preparing a mitomycin C storage solution, subpackaging, storing at-20 ℃ for later use, centrifuging at 1200rpm for 5min to collect 41BB L-mbI L-21-K562 feeder cells, and diluting the feeder cells to 1x10 by using an RPMI1640 culture medium⁷cells/ml cell suspension. Mitomycin C was added to the cell suspension to a final concentration of 33. mu.g/ml, 37 ℃ 5% CO₂After 90min of light-shielding treatment, 1200rpm, 5min, cells were collected and washed 4 times with DPBS to remove the residual mitomycin C as much as possible to avoid toxic effects on the cultured cells. The treated feeder cells can be frozen by using a cell freezing medium for standby, and can also be directly used for NK cell amplification or other experiments.

To verify the proliferative capacity of feeder cells treated according to the method of the invention, the treated feeder cells were diluted with fresh complete medium to a concentration of 1 × 10⁶cell suspension in cells/ml, 37 ℃, 5% CO₂Culturing in an incubator, supplementing culture medium according to the growth condition of the cells, and counting the growth condition of the cells on the 5 th day and the 10 th day.

Table 1: feeder cell proliferation following mitomycin C treatment

As can be seen from Table 1, when feeder cells were cultured after the above-mentioned proliferation-removing treatment, the cells continued to die and no proliferation occurred during 10 days of the culture.

Meanwhile, the morphological change of the cells before and after treatment is compared through under-mirror observation, the treated feeder cells are uniform in size and complete in shape, and no obvious cell fragments exist under the mirror. The morphology of the cells after treatment was essentially identical to that before treatment (FIG. 2).

Example 2NK cell culture after 14 days feeder cell retention

The feeder cells used were K562 cells and the proportion of feeder cells was detected by flow-through using Anti-41BB L PE antibody (R & D cat # FAB 2295P).

1. PBMC separation: collecting 50ml of peripheral blood of a tumor patient, anticoagulating by heparin, centrifuging whole blood, 700g, and 20 min. The upper layer is a plasma layer, the middle layer is a white membrane layer, and the lower layer is red blood cells. Collecting plasma layer, inactivating at 56 deg.C for 30min, standing at 4 deg.C for 15min at 1800rpm/800g, 4 deg.C for 25min, and collecting supernatant as autologous plasma. The middle white membrane layer was collected, added to 20ml PBS, mixed well, and then mixed at a ratio of 4:3, slowly adding the centrifugal tube added with the lymph separation liquid at 1800rpm/800g at room temperature for 20min, and washing PBMC; the white middle PBMC layer after centrifugation was aspirated with a Pasteur pipette, washed with about 30ml PBS in a new 50ml centrifuge tube at 1500rpm for 8min, washed once with medium and counted.

2. NK cell Induction and expansion by 3-group repetition, feeder cells treated in example 1 were added at a ratio of 1:1, and then supplemented with 10% autologous plasma and cytokine I L-2, respectively, to a final concentration of 800U/ml, and PBMC was inoculated at a concentration range of 1.2X10⁶cells/ml，37℃，5％CO₂Culturing in an incubator, (2) culturing until the 4 th day, supplementing half and half of fresh culture medium, wherein the final concentration of I L-2 is 50ng/ml, (3) supplementing fresh culture medium and I L-2 every 2-3 days later, the concentration of I L-2 is 50ng/ml, and maintaining the cell concentration at 5x10⁵cells/ml above.

3. Collecting 200 mu L sample, washing twice with PBS, adding 100 mu L PBS, mixing to prepare cell suspension, adding 10 mu L Anti-41BB L PE, incubating at room temperature in dark place for 30min, 3000rpm, at room temperature for 5min, mixing with 100 mu L PBS to prepare cell suspension, performing flow detection, and adding isotype control antibody as control.

The results are shown in FIG. 1, and it can be seen from FIG. 1 and Table 1 that the feeder cells treated according to the invention have no proliferative capacity, almost all of them are apoptotic at day 4 and almost none are detectable after day 6. The results are combined to show that the feeder cells treated by the method do not cause pollution to the later culture process, and can be widely used for clinical application and research.

Example 3 culturing of NK cells Using the treated cells

1. Isolation of PBMC

Collecting 50ml of peripheral blood of a tumor patient, anticoagulating by heparin, centrifuging whole blood, 700g, and 20 min. The upper layer is a plasma layer, the middle layer is a white membrane layer, and the lower layer is red blood cells. Collecting plasma layer, inactivating at 56 deg.C for 30min, standing at 4 deg.C for 15min at 1800rpm/800g, 4 deg.C for 25min, and collecting supernatant as autologous plasma. Collecting the middle leucocyte layer, adding into 20ml PBS, mixing, slowly adding into a centrifuge tube added with lymph separation liquid at a ratio of 4:3, 1800rpm/800g, room temperature, 20min, no-break, washing PBMC; the white middle PBMC layer after centrifugation was aspirated with a Pasteur pipette, washed with about 30ml PBS in a new 50ml centrifuge tube at 1500rpm for 8min, washed once with medium and counted.

2. Induction and expansion of NK cells

(1) Step 1 Total cell count was 3.7X10⁷cells, divided into six groups, experimental (3 replicates) and control (3 replicates), transferred to two T25cm cells²The feeder cells are added into a cell culture bottle according to the proportion of 1:1, the mitomycin C de-proliferation treated K562 cells in example 1 are added into an experimental group, the gamma-ray inactivated 41BB L-mbI L-21-K562 cells are added into a control group, then 10% autologous plasma and I L-2 with the final concentration of 800U/ml are respectively added into the control group, and the inoculation concentration range of PBMC is 1.2x10⁶cells/ml，37℃，5％CO₂Culturing in an incubator;

(2) culturing to the third day, and supplementing half and half with fresh culture medium, wherein the final concentration of I L-2 is 50 ng/ml;

(3) thereafter, fresh medium and I L-2 at a final concentration of 50ng/ml were added every 2 or 3 days to maintain the cell concentration at 5X10⁵cell/ml, and recording the cell expansion times and NK cell purity during fluid infusion.

3. Detection of NK cell biological characteristics

1) Cell proliferation capacity, cell purity, cell activity assay

Taking 20 mu L of sampled cells, adding 20 mu l of 0.1% trypan blue for counting, calculating the proliferation capacity and cell activity of NK cells, and obtaining the calculation results shown in figures 3 and 4, wherein the average amplification multiple of the cells on the 14 th day of an experimental group is 720, the proportion of live cells is 94%, the average amplification multiple of the cells on the 14 th day of a control group is 680, and the proportion of live cells is 90%₅₆CD₁₆The amplified cells were analyzed by flow analysis using the CD3 antibody, and the percentage of NK cells was counted, with NK cells accounting for about 94% on day 14 (see FIG. 5).

2) NK cytotoxicity assay

Taking day 14 cells to perform NK cell killing activity detection, adding a certain amount of NK cells in each well of a 96-well culture plate as effector cells, adding a certain amount of K562 cells as target cells according to an effect-target ratio of 5:1, 10:1, 20:1 and 40:1, wherein the total volume is 20 mu L (the liquid is RPMI1640 culture solution containing fetal calf serum), the temperature is 37 ℃, and CO is₂Incubating in incubator for 4 hr, adding 20 μ L cell counting reagent into each well, incubating at 37 deg.C under CO₂Measuring an absorbance value (A) in an incubator for 2h at the wavelength of 450nm, and calculating a killing rate, wherein an experimental group is NK cells stimulated and amplified by 41BB L-mbI L-21-K562 cells treated by the method, a control group is NK cells stimulated and amplified by 41BB L-mbI L-21-K562 cells inactivated by gamma rays, and control holes only containing the NK cells and only containing the K562 cells are arranged at the same time.

The killing rate is [ 1- (Ae + t-Ae)/At ] × 100% where Ae is the A value for the simple effector cell well, i.e., NK cell well, At is the A value for the simple target cell well, i.e., K562 cell well, and Ae + t is the A value for the effector plus target cell well.

NK cell killing activity was 70% at day 14 with a 10:1 cellular potency target ratio (FIG. 6).

From the above examples, it can be seen that 33. mu.g/ml mitomycin C treated feeder cells were not proliferative. Compared with the feeder cells treated by gamma rays, the feeder cells treated by de-proliferation treatment of the method of the invention have the advantages of stimulating the amplification multiple of NK cells, improving the purity of the NK cells and improving the killing activity of the NK cells. And the mitomycin C deproliferation treatment method is simple to operate and low in cost, and can be better popularized and applied in clinic.

In conclusion, the method for carrying out the deproliferation treatment on the feeder cells by the mitomycin C has the advantages of low requirements on instruments and equipment, low cost, complete cell morphology and capability of improving the NK cell amplification stimulation efficiency of the feeder cells. Mitomycin C has been used mostly in the treatment of feeder cells for stem cells or as an antitumor drug in clinical studies, and studies on the treatment of feeder cells for stimulating NK cell expansion have been rarely reported. Through comparison of multiple groups of experiments, the feeder cells treated by the method can stimulate PBMC to obtain a large number of NK cells with high killing activity, and can be safely and effectively applied to clinical treatment.

Claims

1. A method of providing feeder cell de-proliferative capacity for NK cell culture, said method comprising treating feeder cells with mitomycin C.

2. The method of claim 1, wherein the feeder cells are 41BB L-mbI L-21-K562 cells.

3. The method of claim 1, wherein mitomycin C is dissolved in DMSO.

4. The method of claim 1, wherein the final mitomycin C concentration in said treated feeder cells is from 5 to 40 μ g/ml.

5. The method of claim 1, wherein the final mitomycin C concentration at which the feeder cells are treated is 33 μ g/ml.

6. A feeder cell for NK cell culture, wherein the feeder cell is subjected to a deproliferation treatment using the method according to any one of claims 1 to 5.

7. Use of the feeder cells of claim 6 for the culture propagation of NK cells.

8. A method for culturing NK cells, which comprises culturing NK cells using the feeder cell according to claim 6.