CN110607275B

CN110607275B - Culture method of enhanced natural killer cells

Info

Publication number: CN110607275B
Application number: CN201910769177.0A
Authority: CN
Inventors: 魏彩华; 梁丽香
Original assignee: Beijing Zhiren Biotechnology Co ltd
Current assignee: Beijing Zhiren Biotechnology Co ltd
Priority date: 2019-08-20
Filing date: 2019-08-20
Publication date: 2021-06-15
Anticipated expiration: 2039-08-20
Also published as: CN110607275A

Abstract

The invention discloses a culture method of enhanced natural killer cells, which comprises the following steps: s1, separating to obtain neutrophilic granulocyte and PBMC; s2, loading zoledronic acid on the neutrophils; s3, and adding the zoledronic acid-loaded neutrophils and PBMCs into NK cells for culture. By adopting the culture method of the enhanced natural killer cells, the purity of the NK cells is improved, the safety of clinical application of the NK cells is greatly improved, and the cost is saved.

Description

Culture method of enhanced natural killer cells

Technical Field

The invention relates to the technical field of biomedicine, in particular to a culture method of enhanced natural killer cells.

Background

In recent years, the incidence of cancer has been on the rise. According to (2012, the annual report of Chinese tumor registration), the incidence of cancers in China is 285.91/10 ten thousand, and the incidence of cancers in cities and rural areas is 303.91/10 ten thousand and 249.98/10 ten thousand respectively, it is estimated that the incidence of cancers in China can reach the level of 400/10 ten thousand by 2049 years.

At present, although the treatment measures such as surgery, chemotherapy, radiotherapy and the like are continuously developed, the prognosis of patients with advanced malignant tumors is still poor, and the 5-year survival rate is less than 15%.

With the development of tumor biology, molecular biology and immunology, immunotherapy has become the fourth tumor treatment mode after traditional tumor treatment methods (surgery, chemotherapy, radiotherapy). A large number of researches show that the adoptive cellular immunotherapy has incomparable superiority to other treatment modes for patients with advanced malignant tumors, is one of effective treatment means, and has wide clinical application prospect.

The traditional tumor treatment means has poor targeting property and large side effect, is easy to generate stronger drug resistance phenomenon, also has the risk of metastasis and relapse, and is difficult to eradicate tumor cells. Adoptive cellular immunotherapy is the reinfusion of cultured, activated, genetically modified immune cells in vitro into patients with the aim of eliminating tumor cells and preventing tumor recurrence, improving the survival rate and quality of life of the patients. It is suitable for patients with low cellular immune function, such as patients with high dose chemotherapy, radiotherapy, bone marrow transplantation, and virus infection and damage to the number and function of immune cells.

With the continuous development of adoptive cellular immunotherapy, several major breakthroughs have been made in this field. The most studied immune cell types are tumor-infiltrating lymphocytes (TILs), LAK, cytokine-induced killer Cells (CIK), Donor Lymphocyte Infusion (DLI), NK-92, EBV-specific CTL cell lines, T Cell Receptors (TCRs) or CAR gene-modified T cells.

NK cells are core cells in the immune system of a human body, have the strongest anticancer activity and are the first barrier and the first line of defense against cancer of the human body. NK cells kill virus-infected or altered target cells mainly through four pathways: directly killing target cells by releasing granzyme and perforin; tumor cell apoptosis mediated by death receptor ligands (such as TRAIL or Fasl); killing the target cell by antibody-dependent cytokine-mediated cytotoxicity (ADCC); cytokine mediated killing. In addition, NK cells do not kill normal cells because the combination of MHC-class I molecules on the surface of normal cells with inhibitory receptors on the surface of NK cells can prevent NK cell attack.

How to amplify and purify NK cells in vitro in large quantities and how to enhance the antitumor activity of NK cells are the key points and difficulties of the adoptive immunotherapy nowadays.

The existing NK cell in vitro amplification technology mainly comprises 2 technologies: factor method and trophoblast method. The factor method is to activate and amplify NK cells under the action of various cytokines, the purity and the activity of the NK cells often do not meet the clinical requirements, and the required factor combinations comprise CD16 monoclonal antibody, OK432, IL15 and the like, and the factors are very expensive and complex to operate. The trophoblast method is a method of activating and amplifying NK cells in vitro by using trophoblast cells (often tumor cell lines), and the method causes risks such as DNA residue of the trophoblast cells, genome integration and the like, and is prohibited in advanced cell therapy countries such as Japan.

Disclosure of Invention

The invention aims to provide a culture method of enhanced natural killer cells, which improves the purity of NK cells, greatly improves the safety of clinical application of the NK cells and saves the cost.

In order to achieve the above object, the present invention provides a method for culturing enhanced natural killer cells, comprising the steps of:

s1, separating to obtain neutrophilic granulocyte and PBMC;

s2, loading zoledronic acid on the neutrophils;

s3, and adding the zoledronic acid-loaded neutrophils and PBMCs into NK cells for culture.

Preferably, the isolation of neutrophils and PBMCs is performed as follows:

s1, extracting 20-100ml of donor peripheral blood,

s2, preparing a lymphocyte separation solution:

a. preparing Percoll stock solution by adding 1 part of normal saline into 9 parts of Percoll;

b. diluting Percoll stock solution with physiological saline to obtain solution A with 70% Percoll concentration and solution B with 50% Percoll concentration;

s3, adding the solution A into a centrifuge tube, then slowly adding the solution B into the upper layer of the solution A, balancing at room temperature, then slowly adding peripheral blood, and obtaining PBMC and neutrophilic granulocyte through density gradient centrifugation.

Preferably, the loading of the neutrophils with zoledronic acid is performed as follows:

s1, washing the PBMC and the neutrophils with physiological saline respectively, then resuspending the PBMC and the neutrophils respectively with RPMI-1640 culture medium, culturing the PBMC and the neutrophils for 2 hours, and counting the PBMC and the neutrophils;

s2, after the PBMC is cultured for 2 hours, eluting nonadherent cells in the PBMC, resuspending and centrifuging the PBMC in a PBMC culture bottle by using physiological saline, culturing the obtained PBMC by using a first culture solution, and calling the PBMC cultured by the first culture solution as cells C;

s3, adding the neutrophils into a PMBC adherent cell culture bottle, adding a second culture solution for culture, and obtaining cells cultured by the second culture solution, namely cells D;

s4, after 24 hours, washing the cell D3 times with physiological saline, and then pouring the cell C into the cell D together with the culture medium;

s5, double volume of the medium was added every 3 days to the flask in step S4, and the culture was continued for 14-21 days.

Preferably, the composition of the first culture medium comprises GT-T551H3, 10% autologous serum and 2000IU IL 2.

Preferably, the composition of the second culture medium comprises RPMI-1640, 10% autologous serum and 5uM zoledronic acid.

Preferably, C, D cells are added to a medium in which NK cells are cultured and then analyzed for NK cell phenotype by flow cytometry.

Preferably, the killing effect of the obtained NK cells in vitro is evaluated by A549, K562 and Hep G2.

Therefore, the culture method of the enhanced natural killer cells has the following technical effects:

1) the zoledronic acid can be loaded by C, D cells, and the C, D cells loaded with the zoledronic acid can activate NK cells, so that allogeneic trophoblasts are not needed, and the joint stimulation of various factors is not needed, thereby greatly improving the safety of clinical application of the NK cells and saving the cost;

2) although zoledronic acid can also activate gamma delta T cells, neutrophils inhibit the proliferation of the gamma delta T cells by combining the zoledronic acid, so that the purity of the NK cells is higher than that of the traditional trophoblast method;

3) the neutrophil granulocytes and the PD1 antibody can obviously enhance the antitumor activity of the NK cells, and the antitumor effect of the neutrophil granulocytes and the PD1 antibody is more than 2 times that of the traditional NK cells.

The technical solution of the present invention is further described in detail by the accompanying drawings and embodiments.

Drawings

FIG. 1 is a schematic diagram of the stratification of peripheral blood in a test tube in a method of culturing enhanced natural killer cells according to the present invention;

FIG. 2 is a diagram showing the content of neutrophils separated by FACS detection in the enhanced natural killer cell culturing method of the present invention;

FIG. 3 is a schematic diagram of a method for culturing enhanced natural killer cells according to the present invention, wherein the neutrophils are loaded with zoledronic acid;

FIG. 4 is a schematic diagram of FACS detection of NK cell purity for 14 days of culture of enhanced natural killer cells according to the present invention;

FIG. 5 is a diagram showing the classical method for 14-day FACS detection of NK cell purity;

FIG. 6 is a graph comparing the lethality of NK cells cultured according to the present invention and the prior art to different tumor cells.

Detailed Description

Embodiments of the present invention will be further described with reference to the accompanying drawings.

Example one

The procedure for neutrophil and PBMC isolation was as follows:

1. 20ml of peripheral blood of the donor is extracted;

2. preparing a lymphocyte separation solution:

3. injecting 3ml of the solution A into a 15ml centrifuge tube, slowly adding 2ml of the solution B to the upper layer of the solution A, and balancing at room temperature for 1 hour;

4. slowly injecting 5ml of peripheral blood into the upper layer of the solution B, rotating for 1800 turns, and centrifuging for 20 minutes;

5. the liquid is clearly visible in 6 layers as shown in fig. 1: the layer a is serum, the layer B is PBMC, the layer C is B fluid, the layer d is neutrophilic granulocyte, the layer e is A fluid, and the layer f is erythrocyte;

6. the a, b and d layers were aspirated separately for use, and the content of neutrophils in the d layer cells was examined by FACS, as shown in FIG. 2, whereby neutrophils of 99% purity could be obtained.

Example two

The steps for loading zoledronic acid on neutrophils are as follows:

1. 20ml of peripheral blood of the donor is extracted, and the neutrophil is obtained according to the method of the first example;

2. by 1 x 10⁷Adding the cell amount per well into a six-well plate, and adding 2ml of RPMI-1640 culture medium into each well plate for culture;

3. adding 5uM zoledronic acid into each of the six-well plates, and loading the antigen for 24 hours;

4. after 24 hours, FACS is used for measuring the antigen loading condition of the neutrophils, as shown in figure 3, the left graph is a blank test, namely no zoledronic acid is added, the right graph is a test group, namely zoledronic acid is added, and the result shows that the neutrophils can efficiently load the zoledronic acid.

EXAMPLE III

NK cell culture

1. 20ml of blood from the donor was collected and layer b PBMC and layer a serum were obtained as in example one;

2. sucking the serum by a pipette into a new 50ml centrifuge tube, covering the centrifuge tube, and incubating in a 56 ℃ water bath for 30 min;

3. after the incubation is finished, carrying out ice bath on the centrifuge tube at 4 ℃ for 20min, then centrifuging at the room temperature of 2000g for 10min, transferring the supernatant to a new 50ml centrifuge tube, and storing at 4 ℃ for later use;

4. sucking the PBMC of the layer b to a 50ml centrifuge tube by using a Pasteur pipette, and centrifuging for 5min by using 1800rpm of normal saline to wash for 2 times;

5. resuspending the cell pellet in RPMI-1640 medium and counting, 2-3 x 10⁶Inoculating the seeds in a 24-pore plate at a density of/ml, and placing the plates in an incubator to adhere to the wall for 2 hours;

5. after 2 hours, the nonadherent cells were aspirated, centrifuged at 1500rpm for 5 minutes, counted and inoculated into a T75 flask, 40ml of GT-T551H3 medium, 10% of autologous serum, 2000ng/ml of IL2, equal cell number of zoledronic acid-loaded neutrophils prepared in example two, and incubated at 5% CO₂The culture was carried out at 37 ℃.

6. On days 3-4, adding GT-T551 culture medium containing 10% heat-inactivated plasma and 1000ng/ml IL-2 in an amount of 1-1.5 times the cell growth state, and culturing at 37 deg.C in a 5% carbon dioxide incubator; then, fluid infusion was performed every 1 day until day 14.

7. On day 14, cells were harvested, counted by a hemocytometer, and purity of NK cells was checked by FACS, as shown in FIG. 4, and on day 14 of the culture method of the present invention, purity of NK cells was 88% and the number of cells was 5.76X 10⁹。

8. 20ml of peripheral blood from the same donor was drawn and NK cells were cultured using the current optimal protocol:

(1) constructing K562 engineering cells stably expressing IL21, CD137L, CD86, CD64 and CD 28;

(2) separating PBMC with lymphocyte separating liquid;

(3) adjusting the concentration of PBMC and the concentration of the engineering cells to 1: 10, and culturing in a culture solution of IL 21000 ng/ml, 10% autologous serum and GT-T-H3.

(4) After 14 days, the cells were harvested, counted by a hemocytometer, and the purity of NK cells was examined by FACS, as shown in FIG. 5, the purity of NK cells was 82% and the number of cells was 3.84X 10 in the classical method at 14 days⁹. It can be seen that the cell purity obtained by the culture method of the present invention is not inferior to that of the classical method, and the cell amplification amount of the present invention is slightly superior to that of the classical method.

Example four

Killing experiment of different tumor cell lines with NK cells prepared by the invention in example III and NK prepared by the classical method in control group

1. Pretreatment

(1) Preparing target cells: centrifuging at 1000rpm/min for 5min for target cells (A549, K562, Hep G2) with good growth state, resuspending in RPMI-1640 complete culture medium, counting phenol blue to detect cell viability, and adjusting cell number to 2 × 10⁵Ml, 2ml (50 ul/well, 21 wells total);

(2) treating effector cells, re-suspending NK cells, centrifuging at 1500rpm/min for 5min 2 times, counting the survival rate of Taiwanese, re-suspending in complete culture medium, and regulating cell number to 4 x 10⁶Perml (maximum effective target ratio of 20: 1, 50 ul/well, 6 wells) for use.

Effector cell treatment (according to target cell 10)⁴Hole)

20: 1 treatment: adjusting 1ml of NK cells, mixing uniformly, 50 ul/hole, 6 holes.

10: 1 treatment: taking 500ul NK cells, adding 500ul RPMI-1640, mixing evenly, 50 ul/hole, 6 holes.

5: 1 treatment: taking 500ul NK cells, adding 500ul RPMI-1640, mixing evenly, 50 ul/hole, 6 holes.

2: 1 treatment: taking NK cells 400ul, adding RPMI-1640 600ul, mixing evenly, 50 ul/hole, 6 holes.

1: 1 treatment: taking 500ul NK cells, adding 500ul RPMI-1640, mixing evenly, 50 ul/hole, 6 holes.

0.5: 1 treatment: taking 500ul NK cells, adding 500ul RPMI-1640, mixing evenly, 50 ul/hole, 6 holes.

2. NK cytotoxicity assay

And (3) mixing the treated TIL-TCM with target cells according to different effective target ratios E: adding T of 8: 1, 4: 1, 2: 1, 1: 1 and 0.5: 1 into a 96-well plate respectively, adding RPMI-1640 complete culture medium to 100ul of the 96-well plate with less than 100ul of the volume; and (3) taking target cells with corresponding cell numbers and TIL-TCM as a control, taking the culture medium with the same volume as a blank control, and adding PBS with the same volume to the peripheral holes of the test holes for overnight culture (16-24 h).

The next day, 10% CCK-8 by volume was added to each well, and the mixture was placed at 37 ℃ and 5% CO₂After incubation for 2-4h in the incubator, the OD was measured at a wavelength of 450nm, while 630nm was set as the reference wavelength.

And (4) calculating a result:

the killing rate (%) - (control target cell OD value + corresponding control CTL OD value-experimental OD value-blank medium OD value)/(control target cell OD value-blank medium OD value) × 100%

As shown in FIG. 6, according to the line graph of the killing rate results, the broken line is the NK cultured by the prior optimal method, the solid line is the NK of the invention, and the killing capacity of the NK of the invention on different tumor cells is more than 1 time of that of the prior scheme.

Therefore, the method for culturing the enhanced natural killer cells improves the purity of the NK cells, greatly improves the safety of clinical application of the NK cells and saves the cost.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting the same, and although the present invention is described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that: modifications and equivalents may be made to the invention without departing from the spirit and scope of the invention.

Claims

1. A method for culturing enhanced natural killer cells, which is characterized by comprising the following steps:

s1, separating to obtain neutrophilic granulocyte and PBMC;

(1) 20-100ml of peripheral blood of the donor is extracted,

(2) preparing a lymphocyte separation solution:

(3) adding the solution A into a centrifuge tube, slowly adding the solution B into the upper layer of the solution A, balancing at room temperature, slowly adding peripheral blood, and respectively obtaining PBMC and neutrophilic granulocyte through density gradient centrifugation;

s2, loading zoledronic acid on the neutrophils;

(1) washing PBMC and neutrophils with physiological saline, respectively, then resuspending the PBMC and the neutrophils with an RPMI-1640 culture medium, culturing the PBMC and the neutrophils for 2 hours, and counting the PBMC and the neutrophils;

(2) after culturing the PBMC for 2 hours, eluting nonadherent cells in the PBMC, carrying out resuspension centrifugation in a PBMC culture flask by using physiological saline, culturing the obtained PBMC by using a first culture solution, and calling the PBMC cultured by using the first culture solution as cells C;

the composition of the first culture solution comprises GT-T551H3, 10% autologous serum and 2000IU IL 2;

(3) adding the neutrophils into a PMBC adherent cell culture bottle, adding a second culture solution for culture, and obtaining cells cultured by the second culture solution as cells D;

the composition of the second culture solution comprises RPMI-1640, 10% autologous serum and 5 mu M zoledronic acid;

(4) after 24 hours, the cells D were washed 3 times with physiological saline, and then the cells C were poured into the cells D together with the first culture solution;

(5) supplementing double volume of RPMI-1640 culture medium into the culture flask in step (4) every 3 days, and continuously culturing for 14-21 days;

2. The method of claim 1, wherein the natural killer cell culture is enhanced by: adding the cells C and the cells D into a culture medium for culturing the NK cells, and analyzing the NK cell phenotype by a flow cytometer.

3. The method of claim 1, wherein the natural killer cell culture is enhanced by: the killing effect of the obtained NK cells in vitro is evaluated by A549, K562 and Hep G2.