CN110872575A

CN110872575A - In-vitro amplification method of iNKT cells

Info

Publication number: CN110872575A
Application number: CN201911001417.9A
Authority: CN
Inventors: 吴勇君; 江良旗; 王俊
Original assignee: Zhongguan Sail Biotechnology Beijing Co ltd
Current assignee: Zhongguan Sail Biotechnology Beijing Co ltd
Priority date: 2019-10-21
Filing date: 2019-10-21
Publication date: 2020-03-10

Abstract

The embodiment of the invention discloses an in-vitro amplification method of iNKT cells, which comprises the following steps: mixing the DC cells and the iNKT cells to obtain mixed cells; and inoculating and culturing the mixed cells. The amplification method adopts a mixed culture mode of dendritic cells (which are professional antigen presenting cells with the strongest organism functions) and iNKT cells, can improve the amplification multiple of the iNKT cells and the capability of the iNKT cells for releasing granzyme, perforin, IFN-gamma, IL-2, IL-4 and TNF, repairs the immune system in a patient body while killing mutant cells in the body, and further improves the killing capability of the iNKT cells.

Description

In-vitro amplification method of iNKT cells

Technical Field

The embodiment of the invention relates to the technical field of cell amplification, in particular to an in-vitro amplification method of iNKT cells.

Background

With the continuous and deep research on the mechanism of tumor development and the mechanism of immune tolerance, the autoimmune system is gradually recognized to play a very important role in preventing the occurrence and treatment of tumors. The immune system of the human body can play the immune monitoring function to identify and eliminate the mutant cells generated in the body and remove the mutant cells in time, thereby avoiding the occurrence of body tumors.

Immunotherapy is also emerging as the fourth largest modality for treating tumors following radiation therapy, chemotherapy, and surgical resection, with eosin for combating malignant tumors. Human constant natural killer T cells (iNKT) are connected with innate immunity and specific immunity, play an important bridge role between two immunization modes, play an important role in anti-tumor immunity and immune regulation through a specific immune response mechanism, show huge potential in the immunotherapy of cancers, and play a remarkable role in inhibiting the occurrence, development and diffusion of various tumors.

The number and the cell killing capacity of the iNKT cells are directly related to the anti-tumor effect, the expansion growth times of the traditional used interleukin 2 combined α -galactosyl ceramide are limited (average 100 times), the purity is low (20% -30%), the killing function of the expanded iNKT cells is low, the clinical requirement cannot be met, and some K562 cells (engineering cells) which are transfected with CD8 α -interleukin 21, CD14, CD19, CD86, CD163 and CD137L proteins are used for expanding the iNKT cells, the number can meet the clinical treatment requirement, but the ethical risk of preparing the human constant killing T cells by adopting the engineering cells exists.

Disclosure of Invention

Therefore, the embodiment of the invention provides an in vitro amplification method of iNKT cells, which aims to solve the problems of low amplification multiple, purity and killing function and ethical risk in the existing amplification method.

In order to achieve the above object, the embodiments of the present invention provide the following technical solutions:

according to a first aspect of embodiments of the present invention, there is provided a method for in vitro expansion of iNKT cells, comprising the steps of:

mixing the DC cells and the iNKT cells to obtain mixed cells;

and inoculating and culturing the mixed cells.

The amplification method adopts a mixed culture mode of dendritic cells (which are professional antigen presenting cells with the strongest organism functions) and iNKT cells, can improve the amplification multiple of the iNKT cells and the capability of the iNKT cells for releasing granzyme, perforin, IFN-gamma, IL-2, IL-4 and TNF, repairs the immune system in a patient body while killing mutant cells in the body, and further improves the killing capability of the iNKT cells.

Further, the number ratio of DC cells to iNKT cells in the mixed cells is 1: (18-22).

Further, the inoculation density is (1-2) multiplied by 10⁶One per ml.

Further, the culture medium used for the culture was Corning 88-551.

Further, in the culture process, the culture medium is supplemented at intervals of 2-3 days, and the supplement amount is the same as the initial culture volume.

Further, the culture conditions are: the temperature is 37 ℃; CO 2₂The concentration is 5%; humidity is 40-60%; the culture time is 12-16 days.

The present invention can improve the amplification factor of iNKT cells and the antigen killing ability more effectively by the above-mentioned specific limitations.

Further, the DC cells are prepared by the following method:

(a) extracting mononuclear cells from peripheral blood;

(b) adding a blank culture medium into the mononuclear cells, suspending, inoculating and culturing for a period of time, then sucking the supernatant into a blank centrifuge tube, washing the culture bottle with the supernatant by adopting a PBS buffer solution, transferring the washing solution into the blank centrifuge tube filled with the supernatant, centrifuging and washing with water, wherein the centrifugation speed is 1500rpm, and the time is 10 min;

(c) adding 10ml Corning88-551 blank culture medium into the centrifuge tube after washing, adding 250ng GM-CSF and 200ng IL-4, culturing, and adding 250ng GM-CSF and 200ng IL-4 into the centrifuge tube respectively when culturing is carried out for 2 days and 4 days;

(d) and (3) adding 250ng of GM-CSF, 200ng of IL-4, 250ng of TNF- α and K562 cell lysis supernatant with the final protein concentration of 5 mu g/mL into a centrifuge tube until the culture reaches 6 days, continuing to culture, and collecting cells to obtain the DC cells.

Further, the hollow white medium in the step (b) is Corning88-551 serum-free medium.

Further, the iNKT cells are prepared by the following method:

(a) extracting mononuclear cells from peripheral blood, adding a blank culture medium into the mononuclear cells, suspending, inoculating and culturing for a period of time, then sucking a supernatant, centrifuging, and collecting precipitated cells;

(b) suspending the precipitated cells by using a blank culture medium, uniformly mixing, and transferring into a culture flask coated with a CD3 monoclonal antibody and a CD16 monoclonal antibody;

(c) adding IL-15, IL-2, IL-21 and α -galactosylceramide into the culture flask for culture;

(d) culturing to the 3 rd day, transferring the cells in the culture bottle to another culture bottle, adding IL-15, IL-2, IL-21 and a blank culture medium, supplementing liquid at intervals of 2 days, and culturing to 7-8 days to obtain the iNKT cells.

According to the invention, the iNKT cell is activated by combining the CD3 monoclonal antibody and the CD16 monoclonal antibody, the ethical risk is avoided, and the amplification multiple and the killing performance of the iNKT cell can be better improved by adding the cell factor.

Further, the blank medium in the step (a) is Corning88-551 serum-free medium.

Further, the hollow white medium in the steps (b) and (d) is Corning88-581 serum-free medium.

Further, in the step (c), the addition amounts of IL-15, IL-2, IL-21 and α -galactosylceramide are 10ng/ml, 1000U/ml, 20ng/ml and 5ug/ml respectively, and the culture system in the culture flask is 50 ml.

Further, in the step (d), the addition amounts of IL-15, IL-2, IL-21 and blank medium are respectively 10ng/ml, 1000U/ml, 20ng/ml and 50 ml; the amount of the supplement solution was 50ml, and the supplement solution was corning88-551 medium containing IL-2.

In the present invention, the extraction method for extracting mononuclear cells from peripheral blood comprises the following steps:

(a) centrifuging peripheral blood to collect serum, and then carrying out water bath and centrifugation on the serum to collect supernatant and blood cells;

(b) adding normal saline into blood cells to full blood volume, adding hydroxyethyl starch into blood cell solution according to the volume ratio of 1: 1.8-2.2, mixing, precipitating for 25-30min, collecting supernatant, centrifuging, collecting precipitate, and resuspending;

(c) and (b) adding the resuspended cell suspension into the supernatant obtained in the step (a), centrifuging, collecting middle leucocyte layer cells, adding physiological saline into the collected middle leucocyte layer cells, and centrifuging to obtain the mononuclear cells.

The embodiment of the invention has the following advantages:

(1) the amplification method adopts a mixed culture mode of dendritic cells (which are professional antigen presenting cells with the strongest organism functions) and iNKT cells, can improve the amplification multiple of the iNKT cells and the capability of the iNKT cells for releasing granzyme, perforin, IFN-gamma, IL-2, IL-4 and TNF, repairs the immune system in a patient body while killing mutant cells in the body, and further improves the killing capability of the iNKT cells.

(2) According to the invention, the iNKT cell is activated by combining the CD3 monoclonal antibody and the CD16 monoclonal antibody, the ethical risk is avoided, and the iNKT cell expansion fold and the killing performance can be better improved by adding the specific cell factor.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

FIG. 1 is a graph showing the activity of cells after expansion in example 5 of the present invention;

FIG. 2 is a graph showing the CD3 positive peak of cells after expansion in example 5 of the present invention;

FIG. 3 is a diagram showing the flow analysis of iNKT cells of the cells after expansion in example 5 of the present invention.

Detailed Description

The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

This embodiment is a method for extracting mononuclear cells from human peripheral blood, comprising the steps of:

(a) taking a sterile centrifuge tube, adding peripheral blood into the centrifuge tube according to the amount of 30ml per tube, centrifuging at 2800rpm for 10min, taking the upper serum into the centrifuge tube, then putting the centrifuge tube into a 56 ℃ water bath for 30min, centrifuging at 3500rpm for 20min, collecting the supernatant into a clean centrifuge tube, preserving at 4 ℃ for later use, and collecting the lower blood cells;

(b) adding normal saline into blood cells to full blood volume, adding hydroxyethyl starch into blood cell solution according to the volume ratio of 1: 2, mixing uniformly, precipitating for 30min, taking supernatant into a centrifuge tube, centrifuging at 2000rpm for 10min, collecting precipitate, and suspending in 20ml normal saline;

(c) slowly adding the resuspended cell suspension into the supernatant obtained in the step (a) along the wall of the centrifugal tube, centrifuging at 2000rpm for 20min, carefully sucking the middle leucocyte (cells 1cm above and below the leucocyte) into the centrifugal tube by using a pipette, adding physiological saline into the collected middle leucocyte to a 45ml scale position, centrifuging at 2000rpm for 10min, discarding the supernatant, centrifuging at 1200rpm for 10min, and discarding the supernatant to obtain the mononuclear cells.

Example 2

This example 2 is a method for preparing DC cells, which was prepared by the following method:

(a) mononuclear cells were obtained according to the method of example 1;

(b) corning88-551 Medium was added to the mononuclear cells, and the mixture was suspended and seeded at a density of 3X 10⁶Seed/ml, inoculate 10ml of cell suspension into a 90mm petri dish at 37 ℃ with 5% CO₂The incubator was incubated under the conditions for 4 hours,then, sucking the supernatant into a blank centrifuge tube, washing the culture bottle with the supernatant by adopting PBS buffer solution, transferring the washing solution into the blank centrifuge tube filled with the supernatant, centrifuging and washing with light force in the washing process so as to avoid washing the mononuclear cells for 2 times by adopting physiological saline, wherein the centrifugal rotating speed is 1500rpm, the time is 10 min;

(d) and (2) adding 250ng of GM-CSF, 200ng of IL-4, 250ng of TNF- α and K562 cell lysis supernatant with the final protein concentration of 50 mu g into a centrifuge tube until the culture is carried out for 6 days (K562 cell strains are purchased, and the culture is carried out by repeated freeze thawing of-196 ℃, recovery in water bath at 37 ℃, freezing again at-196 ℃ after dissolution, recovery in water bath at 37 ℃ and repeated freeze thawing for four times), and then continuously culturing and collecting cells to obtain the DC cells.

Example 3

This example is a method for producing iNKT cells, which were produced by the following method:

(a) mononuclear cells were obtained in the same manner as in example 1, and Corning88-551 serum-free medium was added to the mononuclear cells to suspend the cells, thereby adjusting the seeding density to 3X 10⁶Seed/ml, inoculate 10ml of cell suspension into a 90mm petri dish at 37 ℃ with 5% CO₂Incubating the culture box for 4 hours under the condition, then slightly shaking the culture dish, sucking supernatant, putting the supernatant into a 50ml centrifuge tube, centrifuging for 10min at 2000prm, and collecting precipitated cells;

(b) resuspending the precipitated cells in Corning88-581 serum-free medium, and mixing to obtain a suspension with a cell concentration of 2 × 10⁶Individual/ml, followed by transfer of the cell suspension to 5ml T-75 flasks coated with CD3(1ug/ml) and CD16(1ug/ml) monoclonal antibodies;

(c) adding IL-15, IL-2, IL-21 and α -galactosylceramide into a culture flask for culture, wherein the concentration of each component after the addition is IL-1510 ng/ml, IL-21000 u/ml, IL-2120 ng/ml and α -galactosylceramide 10ng/ml, and the culture system in the culture flask is 50 ml;

(d) culturing to day 3, transferring the cells in the T-75 culture flask to another T-75 culture flask, adding IL-15, IL-2, IL-21 and Corning88-581 serum-free culture medium, supplementing liquid at intervals of 2 days, and culturing for 7-8 days to obtain iNKT cells, wherein the addition amounts of the IL-15, IL-2, IL-21 and Corning581 serum-free culture medium are 10ng/ml, 1000U/ml, 20ng/ml and 50ml respectively; the amount of the supplement solution was 50ml, and the supplement solution was corning88-551 medium containing IL-2.

Example 4

This example is a method for in vitro expansion of iNKT cells, comprising the steps of:

mixing the DC cells and the iNKT cells according to the number ratio of the DC cells to the iNKT cells of 1: 18 to obtain mixed cells;

mixing the cells according to 1 × 10⁶Inoculating into Corning88-551 medium at an inoculation density of one/ml, and culturing at 37 deg.C and CO₂Culturing at 5% concentration and 50% humidity, adding culture medium at an interval of 3 days, culturing for 16 days, and collecting co-cultured cells.

Example 5

mixing the DC cells and the iNKT cells according to the number ratio of the DC cells to the iNKT cells of 1: 20 to obtain mixed cells;

mixing the cells according to 2X 10⁶Inoculating into Corning88-551 medium at an inoculation density of one/ml, and culturing at 37 deg.C and CO₂Culturing at 5% concentration and 50% humidity, adding culture medium at an interval of 2 days in the culture process, culturing for 12 days, and collecting co-cultured cells.

Performing flow cytometry analysis on the cells obtained by the co-culture, and analyzing the results as shown in FIGS. 1 to 3;

as can be seen from fig. 1-3, CD3+ was 75.6%; iNKT CD3+ CD16+ CD56+ was 90.9%.

Experimental example 1

The killing activity of the iNKT cells prepared in example 3 and the co-cultures of DC cells and iNKT cells obtained in example 5 on K562 cells (human leukemia cells) and HepG2 cells (human liver cancer cells), respectively, was measured by MTT chromogenic assay;

using 96-well culture plates (pre-UV irradiation for 20 min), effector cells (2X 10) were added to each well of the experimental groups⁶4X 10 pieces/ml⁶8 × 10 pieces/ml⁶One/ml) and target cells (2X 10)⁵Each 100ul of suspension is mixed evenly, namely the effective target ratio is 10:1, 20:1 and 40:1, three holes are added; simultaneously setting effector cell and target cell controls, setting three wells, respectively taking 100ul, adding RPMI-1640 (containing 10% calf serum) culture solution 100ul into all control wells, respectively, 37 deg.C, 5% CO₂Co-cultivating for 24 hours under the condition;

after 24 hours, the supernatant was aspirated, 10ul of MTT stock solution (5mg/ml in 0.02M, pH7.4 in 0.05% glucose in PBS) was added, 37 ℃, 5% CO₂Under the condition, co-culturing for 2-4 hours;

carefully absorbing and discarding the supernatant, and adding dimethyl sulfoxide to dissolve and reduce the formazan particles for 20 minutes;

measuring OD490nm value on a microplate reader;

the killer cell activity is expressed as the killer cytotoxic activity, and is calculated according to the following formula:

% cytotoxic activity ═ 1- (experimental well OD value-effector cell alone OD value)/target cell alone OD value } × 100%; the calculation results are shown in tables 1 and 2:

TABLE 1 killing of K562

Effective target ratio	10:1	20:1	40:1
				iNKT	39.6％	57.3％	63.2％
DC-iNKT	43.2％	60.5％	75.8％

TABLE 2 killing of HepG2 cells

Effective target ratio	10:1	20:1	40:1
				iNKT	29.4％	63.8％	76.9％
DC-iNKT	36.7％	69.2％	83.7％

As is clear from tables 1 and 2, iNKT cells obtained by amplification in the examples of the present invention have higher killing activity against K562 cells (human leukemia cells) and HepG2 cells (human hepatoma cells).

Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. An in vitro amplification method of iNKT cells, comprising the steps of:

mixing the DC cells and the iNKT cells to obtain mixed cells;

and inoculating and culturing the mixed cells.

2. The method of claim 1, wherein the ratio of the number of DC cells to iNKT cells in the mixed cells is 1: 18-22.

3. The amplification method of claim 1, wherein the seeding density is (1-2) x 10⁶One per ml.

4. The amplification method according to claim 1, wherein the culture medium used for the culture is Corning 88-551.

5. The amplification method according to claim 1, wherein the culture medium is supplemented at intervals of 2 to 3 days in the course of the culture in the same amount as the initial culture volume.

6. The amplification method of claim 1, wherein the culture conditions are: the temperature is 37 ℃; CO 2₂The concentration is 5%; humidity is 40-60%; the culture time is 12-16 days.

7. The amplification method according to claim 1, wherein the DC cells are prepared by:

(a) extracting mononuclear cells from peripheral blood;

(b) adding a blank culture medium into the mononuclear cells, suspending, inoculating and culturing for a period of time, then sucking the supernatant into a blank centrifuge tube, washing the culture bottle with the supernatant by adopting a PBS (phosphate buffer solution), transferring the washing liquid into the blank centrifuge tube filled with the supernatant, centrifuging and washing with water;

(c) adding 10ml Corning88-551 blank culture medium into the centrifuge tube after washing, adding 250ng GM-CSF and 200ng IL-4, culturing, and adding 250ng GM-CSF and 200ng IL-4 into the centrifuge tube respectively when culturing is carried out for 2 and 4 days;

8. The method according to claim 1, wherein the iNKT cells are produced by the method comprising:

9. The amplification method according to claim 8, wherein in step (c), the amounts of IL-15, IL-2, IL-21 and α -galactosylceramide added are 10ng/ml, 1000U/ml, 20ng/ml and 5ug/ml, respectively, and the culture system in the culture flask is 50 ml.

10. The amplification method according to claim 8, wherein in step (d), the IL-15, IL-2, IL-21 and blank medium are added in amounts of 10ng/ml, 1000U/ml, 20ng/ml and 50ml, respectively; the amount of the supplement solution was 50ml, and the supplement solution was corning88-551 medium containing IL-2.