CN112251406A

CN112251406A - Exosome sorting method for NK cell activation stage

Info

Publication number: CN112251406A
Application number: CN202011196500.9A
Authority: CN
Inventors: 曹韪凡; 滕春波; 于泽; 曹峰林; 陈京京; 曹涵
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-10-30
Filing date: 2020-10-30
Publication date: 2021-01-22
Anticipated expiration: 2040-10-30
Also published as: CN112251406B

Abstract

An exosome sorting method for an NK cell activation stage, which relates to the field of biology. The invention aims to solve the problem that no technology for directionally sorting the specific exosomes of the umbilical cord blood exists at present. The invention adopts the extracellular exosome separation of the umbilical cord blood NK cell period after the specific stimulation and activation, utilizes IL-2 to proliferate the NK cells, IL-12, IL-15, GM-SCF and Fit3-L can promote the activation of the NK cells and can induce CD34⁺The stem cells are differentiated into NK cells. And sorting and collecting the NK cells at about 10d of in vitro culture to obtain a larger amount of exosomes secreted by the NK cells. The invention utilizes the characteristics of innovatively optimizing the culture method and the feeding method of the NK cell factors to extract the activated NK cell exosomes in a large scale and achieve the purpose of activating the NK cells. The invention is applied to the field of exosomes.

Description

Exosome sorting method for NK cell activation stage

Technical Field

The invention relates to the field of biology, and particularly relates to an exosome sorting method for an NK cell activation stage.

Background

The pathogenesis of viral infection or tumorigenesis is mainly due to the inability of the body's immune capacity to overcome pathogenic microorganisms or to eliminate tumor cells. Human natural killer cells (NK cells) are natural killer immune cells possessed by the body itself, and are the first line of defense against viral infection and removal of senescent diseased cells, and have definite actions in the treatment of influenza, HIV and other major viral infectious diseases and in the process of anti-tumorigenesis, and the major immunological features are production of cytokines, mediation of cytotoxic activity by secretion of cytokines IFN- γ and the like, and mediation of killing of target cells by release of perforin and granzyme, (viieret al, 2008). NK cells are the core cells in the innate immune system, account for about 15% of the leukocytes in the blood, are critical to both innate and adaptive immunity, and are essential in defending against viral infections.

NK cells have many own immunological properties and advantages. First, lymphocytes that can cause nonspecific killing of tumor cells and virus-infected cells without prior sensitization are obtained. Secondly, NK cells are not limited by Major Histocompatibility Complex (MHC) to open the killing effect of target cells, mainly through surface cytotoxic receptors, the release of perforin and granzyme is triggered, and cytokines such as cytotoxic factors and TNF-alpha are generated, but cytokines such as IL-2 are not generated, so that severe cytokine storm can be avoided. Meanwhile, NK cells can also induce antibody-dependent cell-mediated cytotoxicity (ADCC) by binding surface antigens to specific antibody Fc fragments. Therefore, the intervention of the virus infection and the tumor diseases by using NK cells and related technologies has innovative and existing biological treatment modes and means.

However, the number of NK cells and the ability to resist viruses in immunocompromised patients also decrease. NK cells can now exert graft-versus-leukemia cell capacity during clinical hematopoietic stem cell transplantation without causing graft-versus-host disease (Ruggeri et al, 2002). In addition, NK cell infusion can provide remission in patients with hematological tumors (Miller et al, 2005). Therefore, NK cells are currently very promising immune cells for hematological tumor therapy. However, there are still some unsolved problems in clinical application due to the limitation of NK cell application. For example, the expansion of primary NK cells or the differentiation and expansion of stem cells to obtain a sufficient number of NK cells with stronger killing property is a prerequisite for promoting the clinical application of NK cells and improving the curative effect of treatment (Cheng et al, 2013; Luevano et al, 2012). Secondly, the problems of non-specific cytotoxicity, poor biological safety, low delivery efficiency and the like exist, and the cell preparation is rapidly cleared in vivo due to the action of the immune system in vivo after being infused. The influence of immune reaction is completely eliminated (the influence of blood brain barrier, tissue physiological structure and immune microenvironment of a human body causes that immune cells cannot act on target cells and the cells cannot be contacted with each other, and meanwhile, the targeting of cellular immunotherapy is not strong), and the only method is to use an endogenous nano-scale carrier.

Exosomes (Exosomes) are membrane vesicles which are secreted by a series of biological mechanisms such as endocytosis, fusion and efflux through various active cell secretion modes, have the diameter of about 30-200nm and the buoyancy density of 1.10-1.21g/mL in a sucrose gradient and mainly have a phospholipid bilayer structure. Exosomes occur naturally in body fluids, including blood, saliva, urine, cerebrospinal fluid, and breast milk, among others. The exosome is relatively uniform in size, is in a cup shape or a double-concave dish shape, and is spherical in human body fluid.

Exosomes contain some important bioactive molecules, such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), messenger ribonucleic acid (mRNA) microrna (mirna), and the like. Due to the specific mode of production of exosomes, exosomes contain more protein components such as cytoplasmic membranes and endosomes than the relevant protein components such as golgi, nucleus, mitochondria. Exosomes also comprise MHC molecules; the four-transmembrane protein superfamily, such as CD81, CD82, CD63 and CD9, has lipid bilayer, and the biomolecules such as nucleic acid and protein wrapped in the exosome can keep stable activity and play multiple biological roles after being taken by receptor cells. Among them, extracellular secretion can carry various bioactive substances such as RNA, protein and lipid, and plays an important role in biological processes such as cell communication, immune response and antigen presentation. The exosome of the NK has the function of regulating NK cells and has better histocyte compatibility. The traditional Chinese medicine composition has the advantages of easiness in passing through a blood brain barrier, stable performance, easiness in storage, no tumorigenicity and the like, so that the comprehensive activation of NK cells is very critical to the effect of the NK cells on the immune function (Long et al, 2013). The study shows that the cells obtained from human peripheral blood have different cell uniformity and unstable functional activity due to individual difference. And the immortalized tool cell line derived from tumor cells, such as NK-92 cell line, has poor biological safety and is not suitable for subsequent exosome application. Meanwhile, the peripheral blood cell proliferation effect can influence the scale purification and application of exosomes.

There are two main ways of expanding NK cells: the first mode of expansion is by feeder cell co-culture. Feeder cell lines are commonly used, such as K562 cells, but since K562 cells are tumor cell lines, there is a biosafety risk. Yet another expansion approach is to use cytokine culture treatments, but NK cell depletion may result due to their cost, as well as associated molecular regulatory disturbances. Therefore, how to obtain NK cells with safe sources, stable and uniform and activated states is a key technology which needs to be broken through.

The umbilical cord blood is an important source of NK cells required by people at this time, and the advantages of the umbilical cord blood are mainly safety, convenience and strong amplification. Researches show that the GMP-grade allogeneic umbilical cord blood can be amplified to reach the clinical required quantity without the stimulation of feeder cells, and the NK cell product obtained in the way can reach the standard in the aspects of activity, cell phenotype, purity, sterility and the like. The phenotype characteristics of the cord blood NK cells and the peripheral blood NK cells are different: in addition to the similar expression levels of CD56, NCRs and NKG2D, the expression levels of CD16, adhesion molecules (CD2, CD11a, CD18 and CD62L), KIRs, DNAM-1, NKG2C, IL-2R and CD57 of NK cells in cord blood are lower, and the higher expression level of an inhibitory receptor NKG2A causes slightly lower cytotoxicity. The cord blood NK cells are easy to obtain, low in cost and small in individual difference, and can be standardized and prepared into universal NK cell products.

Stem cells (Stem cells) are insufficiently differentiated and immature cells, have potential functions of regenerating various tissues, organs and human bodies, and are called "universal cells" in the medical field. Among them, hematopoietic stem cells are the only source of various blood cells in the body, and are mainly present in bone marrow, peripheral blood, and umbilical cord blood. Transplantation of hematopoietic stem cells is the most effective method for treating hematological diseases, congenital genetic diseases, and multiple metastatic neoplastic diseases. Compared with bone marrow transplantation and peripheral blood stem cell transplantation, the umbilical cord blood stem cell transplantation has the advantages of no source limitation, low requirement on HLA match and difficult pollution by viruses or diseases.

The development, proliferation and activation of NK cells are regulated by a variety of cytokines. Resting NK cells are generally not effective in killing target cells and can significantly promote NK cytotoxic activity and cytokine secretion capacity by cytokine treatment. Currently, IL-2, IL-12, IL-15, GM-SCF, etc., which alone or in combination play an important role in promoting the differentiation, maturation, activation, proliferation and cytotoxic activity of NK cells, are more commonly reported and have been applied to clinical disease treatment (Fehniger et al, 2003; Long, 2007; Walzer et al, 2005; Nandagopal et al, 2014). IL-2 is a multifunctional inflammatory cytokine, can activate and induce NK cell proliferation, is the most common cytokine in an in vitro expansion NK cell system, and IL-2 activates NK cells before activating other lymphocytes, enhances the cytotoxic effect of the NK cells, generates lymphocyte activated killer cells, induces CD34⁺The stem cells are differentiated into NK cells. (Malek, 2008; Sadlack et al, 1993; Suzuki et al, 1995; Willerford et al, 1995). IL-12 is able to induce high levels of IFN- γ secretion by NK cells (Jalah et al, 2013; Kobayashi et al, 1989). IL-12 activates the cellular tyrosine kinase JAK2, leading to phosphorylation of STATs, ultimately promoting IFN-. gamma.production and other biological responses. Can induce the production of cytokines such as IFN-gamma, improve the cytotoxic effect of peripheral blood NK cells, mediate the mature and differentiation of human in vitro NK cells and promote the in vitroNK cell expansion can be enhanced by combining with IL-2. (Hunter, 2005; Kaplan et al, 1996; McIntyre et al, 1996; Trinchieri et al, 2003). NK cells under IL-15 sensitization will enhance IL-12-induced IFN- γ secretion. Can induce the differentiation of NK-like cells and promote the proliferation thereof. Improve the cytotoxic effect and increase the production of cytokines. Meanwhile, pre-stimulation of IL-15 may make NK cells more sensitive to subsequent stimulation, thus causing a higher level of immune cell response (Fehniger et al, 1999; Lucas et al, 2007; Ma et al, 2006).

Disclosure of Invention

The invention aims to solve the problem that no technology exists for directionally sorting specific exosomes in umbilical cord blood at present, and provides an exosome sorting method in an NK cell activation stage.

The invention relates to an exosome sorting method in an NK cell activation stage, which is carried out according to the following steps:

step one, taking umbilical cord blood, and centrifugally collecting umbilical cord blood cells;

step two, separating plasma and cells: centrifuging for 15min at 700g, sucking the upper layer of yellowish plasma into a 50mL centrifuge tube, adding 1-fold volume of physiological saline into the lower layer of blood cells, and diluting to separate mononuclear cells in the umbilical cord blood cells; the separation of the mononuclear cells in the umbilical cord blood cells is carried out by adopting a density gradient centrifugation method;

step three, incubating the mononuclear cells separated in the last step by immunomagnetic beads and separating by a magnetized cell separator to obtain the cord blood-derived CD56⁺NK cells and cord blood CD34⁺Stem cells;

step four, separating the obtained cord blood-derived CD56⁺NK cell and cord blood CD34⁺Stem cell combined in-vitro expanding culture:

1) the cell concentration was 1.0X 10⁶CD56 containing cord blood source⁺NK cells and cord blood CD34⁺Adding NK cell in-vitro culture synergist into NK cell serum-free culture medium of stem cells, placing in incubator, and culturing at 37 deg.C with 5% CO₂Culturing the cells for 1-14 days under the condition(ii) a Wherein the volume ratio of the NK cell in-vitro culture synergist to cell fluid in a serum-free culture medium is 1: 10;

wherein the CD56 derived from cord blood in serum-free medium⁺NK cell and cord blood CD34⁺The number ratio of the stem cells is 4-5: 1; the NK cell in-vitro culture synergist consists of 100ng/mL IL-2, 100ng/mL IL-12, 100ng/mL IL-15, 50ng/mLGM-SCF granulocyte-macrophage colony stimulating factor, 50 ng/mLfms-like tyrosine kinase 3 ligand, 10% human serum albumin and 3% folic acid;

2) supplementing the NK cell serum-free culture medium and the NK cell in-vitro culture synergist in the culture 3 d;

3) in the 5 th and 6 th days of culture, NK cell serum-free culture medium is respectively supplemented, and the cell concentration is adjusted to 1 x 10⁸Per mL;

4) and 7d, performing sterility detection, supplementing an NK cell serum-free medium, and counting cells if the total number of the cells is 6-12 multiplied by 10⁷Then the NK cell in-vitro culture synergist is supplemented;

5) replenishing an NK cell serum-free culture medium in the 9 th, 10 th and 11 th days of culture respectively, and counting cells;

6) replenishing the NK cell serum-free culture medium in the culture 12d, and performing quality control inspection;

7) at 14d of culture, cell counting was performed;

and (3) centrifugally collecting the culture medium containing the cell mixed solution for 7-14 days, washing the culture medium with normal saline for 1 time, adding an NK cell in-vitro culture synergist to resuspend NK cells, and obtaining the exosome by adopting an anion exchange chromatography column method.

Further, the method for obtaining the exosome by using the anion exchange chromatography column is carried out by the following steps: and (3) removing large particles and cell fragments from the resuspended NK cells by two-step centrifugation, filtering the cells through a 0.22-micron filter membrane, concentrating and washing the cells by TFF if the sample amount is 50-200 mL, loading the cells onto a column, and eluting the cells to obtain exosomes.

Further, the step of loading the column and eluting to obtain exosomes refers to the following steps: loading a column by using a GE Sepharose Fast Flow ion exchange filler of 4mLGE company, balancing by using 8mL of an equilibrium buffer solution, washing by using 40mL of a washing solution to remove proteome impurities and continuously using 1mL of an elution buffer solution for 6 times for each 150mL of supernatant, collecting to obtain an exosome, and temporarily storing PBS at 4 ℃.

Further, the NK cell serum-free medium is an X-VIVO 15 medium.

Further, the density gradient centrifugation method is adopted to separate the mononuclear cells in the cord blood in the step two: diluting with PBS, diluting the solution diluted in the centrifuge tube of 50mL in the previous step to 30mL, and adding lymphocyte separation solution to obtain cell suspension; adding Ficoll human lymphocyte separating medium into the cell suspension according to the volume ratio of 2:1, 600g, centrifuging at room temperature for 15min, and stopping rotation without braking; sucking the middle leucocyte layer by using a rubber-tipped dropper, adding the sucked middle leucocyte layer into another new 50mL centrifuge tube, resuspending the cell sediment in each umbilical cord blood specimen by using 20mL PBS without calcium and magnesium ions, and centrifuging for 15min at room temperature at 800 g; sucking the middle white atomized cell layer, washing with PBS buffer solution for 2 times, suspending with culture medium, counting cells under microscope, and separating to obtain mononuclear cells in cord blood.

Further, the cell suspension was spread on the surface of the Ficoll human lymphocyte separation medium along the tube wall.

Further, the mononuclear cells separated in the last step are separated by a magnetized cell separator, specifically:

labeling the separated stem cell and NK cell mixture in a water bath: incubating cells with anti-CD34 and CD56 monoclonal antibodies and monoclonal antibodies for resisting surface antigens for 10min, washing, adding magnetic bead sorting buffer, incubating for 10min, washing, adding a cell plate coated with a CD34 antibody, reacting for 10min, washing, adding biotin-labeled magnetic particles, and reacting for 8 min; washing with 10 times volume of DPBS during culture, centrifuging at 3000rpm/min for 8min to obtain cord blood NK cells and cord blood CD34⁺Stem cells; the volume ratio of the anti-CD34 monoclonal antibody to the biotin-labeled magnetic particles is 1:1, the volume ratio of the magnetic beads to the anti-CD34 monoclonal antibody is 3 mL: 100 μ L.

Further, the washing was followed by the addition of a cell plate coated with antibodies, magnetic bead sorting antibody from CD 34.

Further, the following operations are carried out before the exosome is obtained by adopting an anion exchange chromatography column method:

1mL of the resuspended NK cells are taken for continuous culture for 3d, and the cells are counted; adding the mixture into a test hole containing 5-100 IU of X-VIVO 15 solution, and culturing for 1-6 h in an incubator; adjusting the cell suspension concentration to 1X 10⁷A liquid transfer gun is used for sucking 200 mu L of cell suspension into a 24-well plate for culture, and liquid is changed for cells every 48 hours; counting and dividing cells every 7 days to obtain a period, observing the growth condition of NK cells in cord blood by a microscope when the cultured cells grow into a logarithmic phase, and enriching and extracting exosomes when the cell density reaches 60-70%.

The invention has the following beneficial effects:

the present invention selects umbilical cord blood NK cells as compared with peripheral blood NK cells:

the NK cells from the cord blood have better cell activity, cell state and exosome consistency and homogeneity obtained in the later stage than the NK cells from the peripheral blood. The optimization of the material source is better than the biological safety of NK tool cell line (tumor source). Meanwhile, CD34 is adopted in the culture of cord blood NK cells⁺And (3) improving a stem cell feeding culture method, and constructing a cord blood NK cell culture system. The biosafety and the functionality of the exosome can be conveniently acquired at the later stage. Although the killing activity of the cord blood NK cells on target cells is slightly inferior to that of peripheral blood NK cells, the cord blood NK cells are easy to obtain, low in cost and small in individual difference, and can be easily standardized to prepare universal NK cell products.

Because NK cells are not easy to be amplified and cultured in vitro and need to be mixed into a tumor cell line for co-culture, the reinfusion has the problem of biological safety, or the traditional cell reinfusion has the problems of ethics, biological safety and the like and is limited by condition application, the invention avoids directly reinfusion of the cells, and the invention utilizes exosomes generated in the cell activation (state under cytokine stimulation activation treatment) stage as the screening and extracting target. Meanwhile, the invention is prepared in a large scale by combining the stimulating factors.

Because of the high amount of non-NK cells contained in cord blood. Therefore, the obtained exosome has low purity, and the exosome with relatively high purity is obtained by the conventional operation such as column purification, but the method is complex and has high components. The invention selects the cord blood NK cells and the cord blood CD34⁺Stem cell co-culture using CD34⁺The stem cell characteristics of the stem cells assist the culture of cord blood NK cells, and the purpose of obtaining exosomes with less impurities is achieved.

Because cells in different periods can secrete different exosomes, because the content in the exosomes contains a plurality of biological mediators and impurities, the directional sorting is difficult, and no technology exists at present for directionally sorting specific exosomes, the invention adopts the separation of the extracellular exosomes in the cell period after specific stimulation and activation, utilizes IL-2 to proliferate NK cells, and utilizes IL-12, IL-15, GM-SCF and Fit3-L to promote the activation of the NK cells and induce CD34⁺The stem cells are differentiated into NK cells. And sorting and collecting the NK cells at about 10d of in vitro culture to obtain a larger amount of exosomes secreted by the NK cells.

Compared with an ultracentrifugation mode, the exosome obtaining method disclosed by the invention well maintains the exosome integrity and the particle size; the recovery rate is about 80 percent; the sample purity, the EV Markers and the target cell intake the exosomes, which are all comparable to the ultracentrifugation mode; time saving (small volume 85 min; large scale 150 min); the repeatability is good; can be amplified.

The invention utilizes the characteristics of innovatively optimizing the culture method and the feeding method of the NK cell factors to extract the activated NK cell exosomes in a large scale and achieve the purpose of activating the NK cells.

Drawings

FIG. 1 is a graph showing the results of primary sorting of cord blood-derived NK cells;

FIG. 2 is a graph showing the growth of NK-92 cells cultured in vitro;

FIG. 3 is a diagram of NK cell activation state; the left graph is 100 μm, and the right graph is 1000 μm;

FIG. 4 is a graph showing the effect of different concentrations of X-VIVO 15 solution culture on the number of amplified NK cells;wherein,

is 0IU of X-VIVO 15 solution,

is 25IU of X-VIVO 15 solution,

is a 50IU X-VIVO 15 solution,

is 75IU of X-VIVO 15 solution,

is 100IU X-VIVO 15 solution;

FIG. 5 is an extracted exosome electroscope map;

FIG. 6 is a western blot of exosome detection of CD63 protein;

FIG. 7 is a graph showing NK cell activation detection of the extracted exosomes;

FIG. 8 is a schematic diagram of scale extraction of exosomes;

FIG. 9 is a functional assay of exosome incubated cells in vitro; wherein, A is a diagram of the influence of the function of NK cells of an exosome-added group and an exosome-not-added group, and B is a diagram of a mechanism path influenced by differential gene expression;

FIG. 10 is a graph showing the results of cluster analysis of different genes.

Detailed Description

It will be understood by those of ordinary skill in the art that the foregoing embodiments are specific examples for carrying out the invention, and that various changes in form and details may be made therein without departing from the spirit and scope of the invention in practice.

To make the objects, aspects and advantages of the embodiments of the present invention more apparent, the following detailed description clearly illustrates the spirit of the disclosure, and any person skilled in the art, after understanding the embodiments of the disclosure, may make changes and modifications to the technology taught by the disclosure without departing from the spirit and scope of the disclosure.

The exemplary embodiments of the present invention and the description thereof are provided to explain the present invention and not to limit the present invention.

The invention is not limited to the above embodiments, and one or a combination of several embodiments may also achieve the object of the invention.

The exosome sorting method in the NK cell activation stage of the present embodiment is performed according to the following steps:

firstly, collecting 20mL-40 mL/part of fresh anticoagulated cord blood, and centrifugally collecting cord blood mononuclear cells;

II, a cord blood mononuclear cell separation method: centrifuging at low speed of 700g for 15min, sucking the upper layer of yellowish plasma into a 50mL centrifuge tube, adding 1-fold volume of physiological saline into the lower layer of blood cells, and diluting to separate mononuclear cells;

separating mononuclear cells in cord blood by adopting a density gradient centrifugation method: diluting the solution diluted in a 50mL centrifuge tube in the previous step by PBS, fixing the volume to 30mL, adding the diluted cell fluid to the lymphocyte separation fluid in the previous step to ensure that a liquid interface is not damaged, then adding about 20mL of Ficoll human lymphocyte separation fluid into the cell fluid at a ratio of 2:1, carefully paving the cell suspension on the surface of the Ficoll human lymphocyte separation fluid (the Ficoll human lymphocyte separation fluid is purchased from Tianjin Shanghai biological products science and technology Limited) along the tube wall, slowly increasing the centrifugal force, and centrifuging 15min from rest to 600g at the lowest rising speed level at room temperature for 15min without braking to stop; sucking the middle leucocyte layer by using a rubber-tipped dropper, adding the sucked middle leucocyte layer into another new 50mL centrifuge tube, resuspending the cell sediment in each umbilical cord blood specimen by using 20mL PBS without calcium and magnesium ions, and centrifuging for 15min at room temperature at 800 g; sucking the middle white atomized cell layer, washing for 2 times by using PBS buffer solution, suspending by using a culture medium, counting cells under a microscope, namely separating mononuclear cells in the cord blood for later use;

thirdly, separating CD34 incubated by magnetic beads by magnetic field⁺Stem cells and CD56⁺NK cells: isolated stem cellsAnd NK cells are labeled in a water bath, the cells are firstly incubated with monoclonal antibodies resisting surface antigens for 10min, the cells are washed, then 3mL of magnetic bead sorting buffer is added for incubation for 10min, after washing, a cell plate coated with antibodies (CD56 and CD34 Meitian whirlwind magnetic bead sorting antibodies) is added, reaction is carried out for 10min, washing is carried out, and after washing, biotin-labeled magnetic particles (100 uL of magnetic particles added for a monoclonal antibody resisting CD56 and 100uL of magnetic particles resisting CD 34) are added for reaction for 8 min; washing with 10 times volume of DPBS during culture, centrifuging at 3000r/min for 8min to obtain cord blood NK cells and cord blood CD34⁺Stem cells;

fourthly, the separated cord blood NK cells and cord blood CD34⁺Stem cell combined in-vitro expanding culture:

1) the cell concentration was 1.0X 10⁷The cell fluid containing NK cell serum-free medium is placed in a culture bottle and is subjected to 5% CO at 37 DEG C₂Culturing the conventional cells in an incubator; then the added NK cell in vitro culture potentiators (100ng/mL IL-2, 100ng/mL IL-12, 100ng/mL IL-15, 50ng/mL granulocyte-macrophage colony stimulating factor (GM-SCF), 50 ng/mLfms-like tyrosine kinase 3 ligand (Fit3-L), 10% human serum albumin, and 3% folic acid) were placed in an incubator at 37 deg.C with 5% CO₂Culturing under the condition; wherein the volume ratio of the NK cell in-vitro culture synergist to the cell liquid containing the NK cell serum-free culture medium is 1: 10; cord blood NK cell and cord blood CD34 in cell liquid⁺The number ratio of the stem cells is 4-5: 1;

2) supplementing the NK cell serum-free culture solution in the culture period of 3 d;

3) in the 5 th and 6 th culture period, NK cell serum-free culture solution is respectively supplemented until the cell concentration is 1 x 10⁸/mL or not more than 1 time of the volume of the serum-free culture solution of the existing NK cells;

4) and 7d, performing sterility detection, supplementing NK cell serum-free culture solution, and counting cells if the total number of cells is 6-12 multiplied by 10⁷Then the NK cell in-vitro culture synergist is supplemented;

5) at 8d, the culture medium was supplemented with NK cell serum-free medium to a cell concentration of 1X 10⁹Transferring the cells into a cell culture bag, and discharging air bubbles;

6) replenishing the NK cell serum-free culture solution in the 9 th, 10 th and 11 th days of culture respectively, and counting cells;

7) replenishing the NK cell serum-free culture solution in the culture 12d, and performing quality control inspection;

8) at 14d of culture, cell counting was performed;

centrifugally collecting the culture medium containing the cell mixed solution for 7-14 days, washing the culture medium for 1 time by using normal saline, and then adding an NK cell in-vitro culture synergist to resuspend NK cells; the exosome is obtained by adopting an anion exchange chromatography column method.

And the NK cell is cultured in a serum-free culture solution X-VIVO 15 culture medium.

The beneficial effects of the invention are verified by the following experiments:

2) main apparatus and equipment

Instrument and equipment specific information, see table 1.

TABLE 1 Main instrumentation

3) Main experimental reagent and consumable

Consumable name and manufacturer name:

(1) gun tips, Eppendorf tubes, were purchased from Axygen, USA;

(2)5mL pipettes (Cat:4487), 10mL pipettes (Cat:4488), 25mL pipettes (Cat:4489) were purchased from Corning, USA;

(3) pipette gun was purchased from Thermo Fisher Scientific, usa;

(4) t25 flask T75 flask T175 flask T225 flask was purchased from Corning, usa;

(5)35mm petri dishes (Cat:430165), 60mm petri dishes (Cat:430166), 100mm petri dishes (Cat:430167) were purchased from Corning, USA;

(6) 6-well plates (Cat:3513), 12-well plates (Cat:3516), 24-well plates (Cat:3517), 96-well plates (Cat:3519) were purchased from Corning, USA;

(7) cryovial (Cat:430659) was purchased from Corning, USA;

(8) cell counting plates were purchased from Shanghai silent instruments and meters;

(9) flow tubes (Cat:352054) were purchased from Becton Dickinson, USA;

(10)50mL centrifuge tubes (Cat:430829), 15mL centrifuge tubes (Cat:430791) were purchased from Corning, USA;

(11) cell culture bags were purchased from Life Technology, usa;

reagent name and company name:

(1) the human lymphocyte separation solution and the serum-free lymphocyte culture solution are purchased from the science and technology limited company of the tertiary ocean biological products in Tianjin;

(2) IFN-gamma, IL-2, IL-12, IL-15, GM-CSF antibodies were purchased from BioLegend, USA;

(3) fetal Bovine Serum (FBS) was purchased from GIBCO;

(4) CD34, CD56 sorting/activating magnetic beads (cat # 11141D) were purchased from Life technologies

(5) The CBA kit was purchased from Becton Dickinson, USA;

(6) Anti-CD56-APC, Anti-CD56-PE, Anti-CD56-FITC, Anti-CD34-PE, Anti-CD34-FITC, Anti-CD34-APC, Anti-CD16-PE from Becton Dickinson/BioLegend, USA;

(7) fluorescently labeled mouse anti-human CD34, CD56 monoclonal antibodies, purchased from Becton Dickinson, usa;

(8) goat anti-mouse IgG/HRP Santa cruz;

(9)1kb DNA marker, 6 × Loading buffer, dNTPs, RNase inhibitor and DNase were purchased from NEB;

(10) tween-20, methylene bisacrylamide, acrylamide, TMED, DMSO, beta-mercaptoethanol (beta-ME), puromycin, and the like were purchased from Sigma;

(11) DMEM high-sugar (25mM) medium, 0.25% trypsin, Trizol, Sodium Dodecyl Sulfate (SDS), streptomycin, glycine, Tris-HCl buffer (containing 0.05% Tween 20, pH 7.4), PBS, polyvinylidene fluoride (PVDF) membrane, CELL COUNTING KIT-8 detection KIT (Cat: NC9864731) purchased from Thermo Fisher and Invitrogen;

(12) anti-human CD63 antibody was purchased from Abcam;

(13) anti-human β -actin antibody (Cat: A5316) was purchased from Sigma;

PolyA polymerase, RNase H enzyme (Cat:18-021-014), SuperScript TM First-Strand Synthesis System reverse transcription kit (Cat:11904018), transfection reagent Lipofectamine 2000(Cat:11668500) were purchased from Invitrogen;

(14) rapid DNA gel recovery kit (Cat:28704) was purchased from Qiagen;

(15) CD34, CD56 monoclonal antibody, Bio Legend, USA;

(16) non-radioactive cytotoxicity kit CytoTox

Promega corporation;

(17) X-VIVO 15 Medium, AIM-V Medium #087-

(18) RetroNectin was purchased from TaKaRa;

(19) the small-scale plasmid extraction kit and the large-scale plasmid extraction kit are purchased from Axygen company;

experimental methods

Method for separating mononuclear cells from cord blood

Collecting cord blood: collecting 20mL-40mL of fresh anticoagulated cord blood per part.

Separating plasma and cells: centrifuging at low speed of 700g for 15min, sucking the upper layer of yellowish plasma into a 50mL centrifuge tube, and adding 1 volume of physiological saline into the rest part for diluting cells to conveniently separate mononuclear cells. Inactivation of plasma: the plasma was inactivated in a 56 ℃ water bath for 30 minutes and then centrifuged at 400g low speed for 10min at room temperature. The upper plasma layer was transferred to a new 50mL centrifuge tube and stored in a refrigerator at 4 ℃ with the plasma remaining for use. Low speed centrifugation is required to remove the pellet before each use of the stored plasma. Separating mononuclear cells in cord blood by adopting a density gradient centrifugation method (Ficoll): the supernatant pale yellow plasma in the previous 50mL centrifuge tube was diluted with PBS and then made up to 30 mL. The cell fluid diluted in the previous step is added to the lymphocyte separation fluid, taking care not to disrupt the fluid interface. Then adding the mixture into about 20mL of Ficoll human lymphocyte separation solution in a ratio of 2:1, finally carefully paving the cell suspension on the surface of the Ficoll separation solution along the tube wall, slowly increasing the centrifugal force, and centrifuging for 15min at normal temperature from rest to 600g at the lowest rising speed. After the centrifugation is finished, the middle leucocyte layer is sucked by using a rubber-tipped dropper, and the sucked middle leucocyte layer is added into another new 50mL centrifuge tube. After 20mL of DPBS without calcium and magnesium ions is used for resuspending each cord blood sample, 500g of DPBS is used for centrifuging at room temperature for 10min so as to precipitate cells; the middle leukocyte layer was aspirated, washed 2 times with DPBS buffer, suspended in X-VIVO 15 medium and counted.

Collecting mononuclear cells separated from cord blood, transferring the mononuclear cells into a 50mL centrifuge tube, carrying out centrifugal collection at 4 ℃, and discarding supernatant; resuspending PBS, centrifugally collecting, and discarding supernatant; resuspend with MACS buffer, 40. mu.L/10, depending on the number of cells⁷Adding 10. mu.L/10 of each cell⁷NK cell biotin-antibody cocktail, 4 ℃, 10 min; add MACS buffer 30. mu.L/10⁷One cell, 20. mu.L/10⁷NK cell microbead cocktail, 4 ℃, 15 min; adding 10-20 mL of MACS buffer for resuspension, centrifuging and collecting; resuspend with 1mL MACS buffer, perform the corresponding MACS protocol, centrifuge the flow down cells, and collect (results are shown in FIG. 1).

Cord blood NK cell and feeder cell CD34⁺Stem cell isolation method

The magnetic bead NKcell Isolation Kit is selected by adopting German and American whirlpool cells, and the NK cell selection Kit is an indirect magnetic labeling system and is used for selecting NK cells which are not combined with antibodies from umbilical cord blood. High-purity NK cells are obtained by removing non-NK cells. non-NK cells (i.e., B cells, T cells, DCs, macrophages, granulocytes and erythrocytes) were incubated with a series of biotinylated CD19, CD4(L3T4), CD8a (Ly-2), CD5(Ly-1), Gr-1(Ly-6G/C) and Ter-119 antibody mixtures, followed by indirect magnetic labeling with avidin microbeads to remove magnetically labeled cells, thereby separating NK cells.

The NK cell sorting kit comprises a series of biotinylated antibody mixtures that label non-NK cells and anti-biotin microbeads. MACS magnetic beads are superparamagnetic particles coupled to highly specific monoclonal antibodies for magnetic labeling of cells of interest or removal of cells. The beads are about 50nm in diameter, many times 200 times smaller than the cells, one part per million in volume, and are not visible under an optical microscope. The magnetic beads are composed of polysaccharide and ferric oxide, have no toxicity, no damage to cells, and can be biodegraded. The MACS beads are compatible with a flow cytometer, and the light scattering property of cells cannot be influenced; the magnetic label only occupies 20-30% of the binding sites, and does not affect the fluorescent antibody label of the cells. Furthermore, MACS magnetic beads can avoid cell activation to the maximum extent; the magnetic beads do not need to be dissociated, and the subsequent experiment can be directly carried out: such as flow cytometry analysis or sorting, cell culture, molecular biology research, and the like.

Separating mononuclear cells from the sample, removing mononuclear cells by adhesion method, removing B cells by nylon column method, and removing the remaining CD34⁺Stem cells and NK cells were separated by a magnetized cell separator (MACS).

The classical analysis methods include two methods: negative selection method, adding antibody in the reaction, making the cell be specifically combined with it, then making it form magnetic separation column to remove labeled cell, washing the unlabeled cell; for example, in the positive selection method, anti-CD16 and anti-CD56 are added to the reaction, so that NK cells are specifically bound to form a magnetic immune complex, the magnetic immune complex is retained in the column, after unlabeled cells are washed away, the labeled NK cells are eluted with a washing solution under gentle pressure. The specific operation method comprises the following steps:

1. the mononuclear cells were adjusted to 1X 10 with 10% serum-containing X-VIVO 15 medium⁷Perml, added to sterile plastic dishes at 37 ℃ with 5% CO₂After 2h of culture, monocytes and B cells adhering to the plate were removed.

2. Pre-incubating non-adherent cells with 10% serum X-VIVO 15 solution for 1 hr, passing through a nylon cotton column, adhering B cells and residual mononuclear cells to the nylon cotton column, washing the column with culture solution, and collecting washed CD34⁺Stem cellsAnd NK cells; magnetic separation of CD34⁺Stem cells and NK cells.

3. The newly separated cells are marked in a water bath, the cells are firstly incubated with the monoclonal antibody of the epitope for 10min, the cells are washed and then incubated with 100uL goat anti-mouse antiserum marked with biotin for 10min, 25uL of FITC marked streptavidin is added after washing for reaction for 8min, and the biotin marked magnetic particles are added after washing for reaction for 8 min.

4. After each reaction, the reaction was stopped by washing with 10-fold volume of PBS and centrifuging at 3000r/min for 8 min.

5. Immunomagnetic separation using a magnetized cell separator (MACS). Resuspending the magnetic complex-labeled cell suspension in 2-5mL of PBS to adjust the cell concentration to 5X 10⁷～1×10⁸cells/mL.

6. Pre-incubating the separation column with PBS for 30min, precooling at 4 ℃, placing in a magnetic field of a permanent magnet, and adding 80-100mL of labeled cell suspension.

7. The column was removed from the magnetic field, the labeled cells were eluted under gentle pressure with 50mL of wash solution under aseptic conditions using a syringe, the cells were pelleted by centrifugation, the viability was analyzed using a fluorescence microscope, and the cells were stored in X-VIVO 15.

Note that: the NK cell is CD16⁺、CD56⁺A cell. If anti-CD3 monoclonal antibody is added in the reaction, the eluted unlabeled cells are NK cells; anti-CD16 and anti-CD56 monoclonal antibodies are added in the reaction, and the labeled cells remained in the column are NK cells. The former method is a negative selection method, and the latter method is a positive selection method.

In-vitro amplification culture of cord blood NK cells

The first day of culture: about 1.0X 10⁷Adding 50mL of NK cell serum-free culture solution (NK cell culture solution: lymphocyte serum-free culture solution + lymphocyte culture solution additive, preparation concentration is 5% according to initial culture solution, and the rest culture solution is added according to 1-2%) to be prepared in the kit, adding NK cell in-vitro culture synergist into a T175 culture bottle, culturing in an incubator (37 ℃, 5% CO, 5%) (₂Concentration). The NK cell in-vitro culture synergist is the core test in the kitAgents (100ng/mL IL-2, 100ng/mL IL-12, 100ng/mL IL-15, 50ng/mL GM-SCF, 50ng/mL Fit3-L, 10% human serum albumin and 3% folic acid). The number of cells to be cultured is 1X 10 per cell⁷50mL of cells were required. Before use, the extract is taken out of a liquid nitrogen or a refrigerator at the temperature of-80 ℃, immediately placed in a water bath tank at the temperature of 37-40 ℃ for instant dissolution, then centrifuged at 1200rpm for 5min, the supernatant is discarded, the precipitate is washed for 2 times by using normal saline, and then 3mLNK cell culture solution is used for light suspension for standby. Can be selected according to the total number of NK cells required by actual conditions.

And (3) fluid infusion on day 3: approximately 50mL of the prepared NK cell culture medium was supplemented.

And (5) fluid infusion: about 50mL of NK cell culture solution is supplemented, the amount of the supplemented culture solution is not more than 1 time of the existing volume, or the cell concentration is adjusted to 1X 10⁷/mL。

And (6) fluid infusion: NK cell culture broth was supplemented at about 50 mL.

And (3) supplementing liquid and supplementing the NK cell in-vitro culture synergist on the 7 th day, and simultaneously carrying out sterile detection: supplementing about 50mL of NK cell culture solution, counting cells, and when the total number of cells is more than 1X 10⁸Then adding the NK cell in-vitro culture synergist; if the total number of cells is 6-12X 10⁷In this case, the addition was extended to day 8. A first sterility test is performed.

And (3) supplementing liquid and performing bag transfer on the 8 th day: NK cell culture broth was supplemented at about 200 mL. Transferring into cell culture bag.

Fluid infusion on days 9, 10 and 11: NK cell culture was supplemented daily with about 150 mL.

And (3) fluid infusion and quality control inspection on day 12: and 350mL of NK cell culture solution is supplemented.

Quality control inspection: comprehensive quality control inspection, and continuous culture of qualified indexes.

NK cell quality control criteria at day 12 were as follows:

cells were collected on day 14: the cells were counted, and the required amount of NK cells was collected by centrifugation and washed 1 time with physiological saline. Using 300mL of raw materialSaline solution (cell concentration not exceeding 2X 10)⁹mL, further 10% human albumin).

Reserving a sample and carrying out quality control inspection: after cell collection, 2 samples were taken, one for product release detection and the other as a retained sample for half a year at 4 ℃ for follow-up testing.

NK cells released the assay at day 14. (the culture results are shown in FIG. 2)

In-vitro activation culture and activity state stage exosome extraction of cord blood NK cells

1mL of fresh complete medium was taken to resuspend the pellet, and culture was continued for 1d, 2d, 3d (as shown in FIG. 3), and the cells were counted; selecting proper concentration from the X-VIVO 15 solution (as shown in figure 4, the optimal concentration is under the condition of 100IU of the X-VIVO 15 solution), adding the solution into a culture dish, and respectively culturing 1d, 3d, 4d, 5d and 10d … … in an incubator to examine the optimal culture time, as shown in figure 4, the culture effect is better after 4 d.

1) Adjusting the cell suspension concentration to 1X 10⁷Per mL, pipette 200uL of cell suspension into a 24-well plate;

3) changing the X-VIVO 15 solution for the cells every 48 hours;

4) every 7 days for one cycle, cells were counted to re-stimulate cells;

5) according to the actual condition of cell proliferation, the culture system is divided into pores or expanded correspondingly.

6) Cells were grown into log phase and cells were collected for subsequent experiments.

Observing the growth condition of NK cells in cord blood by a microscope, and carrying out exosome enrichment extraction (the extraction thumbnail is shown in figure 8) after the cell density reaches 60-70%.

The exosome is obtained by adopting an anion exchange chromatography column method through enrichment extraction in the following way: and (3) removing large particles and cell fragments from the resuspended NK cells by two-step centrifugation, filtering the cells through a 0.22-micron filter membrane, concentrating and washing the cells by TFF if the sample amount is 50-200 mL, loading the cells onto a column, and eluting the cells to obtain exosomes (shown in figure 5). And the exosome detection CD63 protein western detection is shown in figure 6.

And loading the obtained exosome on a column, eluting, loading the obtained exosome into the column by using a GE Q-Sepharose Fast Flow ion exchange filler of 4mLGE company, balancing by using GE Health Care Life Science and 8mL of balance buffer solution, washing with 40mL of washing solution to remove proteome impurities and continuously using 1mL of elution buffer solution for 6 times per 150mL of supernatant, collecting to obtain the exosome, and temporarily storing PBS at 4 ℃.

Function verification and analysis of extracted exosome

First, an NK cell in-vitro validation set is set, and the number of cells is 1 multiplied by 10⁴The exosomes collected and separated were added to NK cell basal medium at different concentrations (0IU, 25IU, 75IU), respectively, and expression of NK cell surface active receptor NKG2D was detected after 7 days of co-incubation (as shown in fig. 7, the effect of co-incubation of exosomes at different concentrations with NK cells was found, and NK cell active receptor was activated). High concentrations of exosome were found to be more active. The results are shown in FIGS. 9 and 10.

Claims

1. A method for sorting exosomes in an NK cell activation stage is characterized by comprising the following steps of:

1) the cell concentration was 1.0×10⁶CD56 containing cord blood source⁺NK cells and cord blood CD34⁺Adding NK cell in-vitro culture synergist into NK cell serum-free culture medium of stem cells, placing in incubator, and culturing at 37 deg.C with 5% CO₂Culturing the cells for 1-14 days under the condition; wherein the volume ratio of the NK cell in-vitro culture synergist to cell fluid in a serum-free culture medium is 1: 10;

7) at 14d of culture, cell counting was performed;

2. The method for sorting exosomes in NK cell activation stage according to claim 1, characterized in that the exosomes are obtained by the method using anion exchange chromatography column by: and (3) removing large particles and cell fragments from the resuspended NK cells by two-step centrifugation, filtering the cells through a 0.22-micron filter membrane, concentrating and washing and filtering the cells if the sample amount is 50-200 mL, and then loading the cells onto a column to obtain exosomes after elution.

3. The method for sorting exosomes in NK cell activation stage according to claim 2, wherein the step of loading the column and obtaining exosomes after elution is: loading with 4mL of GE Sepharose Fast Flow ion exchange filler, equilibrating with 8mL of equilibration buffer, washing with 40mL of washing solution per 150mL of supernatant to remove proteome impurities and eluting with 1mL of elution buffer for 6 times, collecting exosomes, and storing in PBS at 4 deg.C.

4. The method according to claim 1, wherein the NK cell serum-free medium is X-VIVO 15 medium.

5. The method for sorting exosomes in NK cell activation stage according to claim 1, wherein the mononuclear cells in cord blood are separated by density gradient centrifugation in step two: diluting with PBS, diluting the solution diluted in the centrifuge tube of 50mL in the previous step to 30mL, and adding lymphocyte separation solution to obtain cell suspension; then adding Ficoll human lymphocyte separation liquid into the cell suspension according to the volume ratio of 2:1, centrifuging for 15min at 600g room temperature, and stopping rotation without braking; sucking the middle leucocyte layer by using a rubber-tipped dropper, adding the sucked middle leucocyte layer into another new 50mL centrifuge tube, resuspending the cell sediment in each umbilical cord blood specimen by using 20mL PBS without calcium and magnesium ions, and centrifuging for 15min at the room temperature of 800 g; sucking the middle white atomized cell layer, washing with PBS buffer solution for 2 times, suspending with culture medium, counting cells under microscope, and separating to obtain mononuclear cells in cord blood.

6. The method of claim 5 wherein the cell suspension is applied to the surface of the Ficoll human lymphocyte separation medium along the vessel wall.

7. The method for sorting exosomes in NK cell activation stage according to claim 1, wherein the mononuclear cells separated in the previous step are separated by a magnetized cell separator, specifically:

labeling the separated stem cell and NK cell mixture in a water bath: incubating cells with anti-CD34 and CD56 monoclonal antibodies and monoclonal antibodies of surface antigens for 10min, washing, adding magnetic bead sorting buffer, incubating for 10min, washing, adding a cell plate coated with a CD34 antibody, reacting for 10min, washing, adding biotin-labeled magnetic particles, and reacting for 8 min; washing with 10 times volume of DPBS during culture, centrifuging at 3000r/min for 8min to obtain cord blood NK cells and cord blood CD34⁺Stem cells; the volume ratio of the anti-CD34 monoclonal antibody to the biotin-labeled magnetic particles is 1:1, the volume ratio of the magnetic beads to the anti-CD34 monoclonal antibody is 3 mL: 100 μ L.

8. The method of claim 7 wherein said washing is followed by the addition of a cell plate coated with antibodies selected from the group consisting of CD34 and CD56 magnetic bead sorting antibodies from Meitian and whirlpool.

9. The method for sorting exosomes in NK cell activation stage according to claim 1, characterized in that the following operations are carried out before the exosomes are obtained by adopting an anion exchange chromatography column method:

1mL of the resuspended NK cells are taken for continuous culture for 3d, and the cells are counted; adding the mixture into a culture bag or a culture dish containing 5-100 IU of X-VIVO 15 solution, and culturing for 1-6 h in an incubator; adjusting the cell suspension concentration to 1X 10⁷A liquid transfer gun is used for sucking 200 mu L of cell suspension into a 24-well plate for culture, and liquid is changed for cells every 48 hours; counting and dividing cells every 2 days to obtain cells, observing the growth of NK cells in cord blood under microscope, and allowing cultured cells to grow into logarithmic phaseIn potential, exosome enrichment extraction is performed.