CN110317786B

CN110317786B - Immune cell capable of preventing tumorigenesis and preparation method and application thereof

Info

Publication number: CN110317786B
Application number: CN201910630204.6A
Authority: CN
Inventors: 古松海; 吴茂友; 蔡晓晴; 康国媛
Original assignee: Sidt Biotechnology Development Co ltd
Current assignee: Saidete biopharmaceutical Co.,Ltd.
Priority date: 2019-07-12
Filing date: 2019-07-12
Publication date: 2020-09-29
Anticipated expiration: 2039-07-12
Also published as: CN110317786A

Abstract

The invention provides an immune cell capable of preventing tumorigenesis and a preparation method and application thereof, wherein the preparation method of the immune cell comprises the following steps: separating and purifying peripheral blood mononuclear cells in vitro; activating peripheral blood mononuclear cells by using a cell activating factor I containing IL-2, IFN-gamma, GM-CSF and TNF-alpha; expanding the activated cells by using a cell activating factor II containing IL-2, IFN-gamma and IL-12; and (5) harvesting the immune cells. The immune cells can identify tumor precursor cells expressing variant proteins caused by susceptible factors in a body and micro tumors which are invisible in imaging; tumor precursor cells expressing variant proteins can be eliminated after recognition by APC; preventing clonal proliferation of transformed cells, and eliminating microscopic tumor invisible in imaging, thereby preventing tumor occurrence and recurrence, and preventing early development of tumor.

Description

Immune cell capable of preventing tumorigenesis and preparation method and application thereof

Technical Field

The invention relates to an immune cell capable of preventing tumorigenesis, a preparation method and application thereof, in particular to an immune cell TPKT (tumor prediction Killer T cells) prepared by activating and amplifying peripheral blood mononuclear cells, a preparation method thereof and application thereof in preparing a preparation for preventing tumorigenesis and preventing early tumor development, belonging to the field of biotechnology.

Background

Under the action of various tumorigenic factors, cells of local tissues lose normal regulation and control of growth at the gene level to cause clonal abnormal hyperplasia, and the tumor is often expressed as a local lump. Tumors are common diseases and frequently encountered diseases, wherein malignant tumors are the most serious diseases which endanger human health at present. Malignant tumors are a multifactorial, multistep complex process involving very complex environmental-gene interactions that involve not only single gene but multiple gene combinations. Tumors are formed by the progressive proliferation of a transformed cell, and the natural growth history of a typical malignant tumor can be divided into several stages: malignant transformation of one cell → clonal proliferation of transformed cells → local infiltration → distant metastasis. The tumor has the characteristic of immortal property when occurring. While many normal cells have a limited proliferation cycle, tumor cells can proliferate indefinitely, providing an inexhaustible population of precursor cells.

The primary treatment means of early and middle stage tumors is surgical resection, and although the surgical resection method is thorough in treatment, the postoperative trauma is large, and if cancer cells invade and spread to adjacent tissues or metastasize at a far end, the effect of surgical resection is greatly limited. This method is also not suitable for the treatment of microscopic lesions, and the tumor recurrence or metastasis rate after surgery is high for tumor patients. The chemotherapy mode can have certain treatment effect on primary focus and metastatic focus, but can kill normal cells of human body while killing tumor cells, and has larger toxic and side effect. The radiation therapy can generate better treatment effect on the regional sensitive tumor, but the radiation therapy is more limited by the tumor part and easily causes damage to the normal tissue of the human body.

In summary, the current technology cannot accurately diagnose the tumor precursor cells expressing variant proteins and invisible micro-tumors in vivo caused by unknown susceptibility factors, which causes great difficulty in preventing and treating early cancer.

Tumor immunotherapy is a therapeutic method for restoring the normal anti-tumor immune response of the body by restarting and maintaining the recognition and killing of tumor cells by the immune system, thereby controlling and eliminating tumors. This therapy has attracted attention in recent years because it is effective and avoids damage to the body. One of the major directions in tumor immunotherapy is the activation of immune cells. Many techniques for activating immune cells have been reported.

CN106754704A discloses a method for in vitro induced expansion of immune cells, CN106591233B discloses a method for in vitro induced expansion and cryopreservation of immune cells, and CN106701681B discloses a method for in vitro induced expansion, cryopreservation and resuscitation of immune cells, wherein the in vitro induced expansion of immune cells in these documents is substantially consistent, and the method comprises: coating the culture container with the CD16 antibody to obtain a coated culture container; placing the single nuclear cells into a coated culture container by using an immune cell activation culture medium to carry out the immune cell activation culture medium, so as to obtain immune cells which are subjected to primary induced amplification; performing second induced amplification culture on the immune cells subjected to the primary induced amplification by using an immune cell amplification culture medium to obtain differentiated immune cells; and performing third induced amplification culture on the differentiated immune cells by using the large-scale amplification culture of the immune cells to obtain the large-scale function-activated immune cells. Wherein the immune cell activation medium is a serum-free lymphocyte culture medium added with plasma, interleukin-2 and saperin; the immune cell amplification culture medium is a serum-free lymphocyte culture medium added with interleukin-2 and saperin; the large-scale amplification culture of the immune cells is a serum-free lymphocyte culture medium added with interleukin-2. The method has the advantages of high induction efficiency, high amplification speed, high safety, low cost and the like, and the cryopreserved large-scale immune cells have long effective preservation time and high cell recovery rate.

CN105505870B discloses an immune cell culture method and the use of artificial feeder cells in immune cell culture, wherein feeder cells expressing cell growth factors and co-stimulatory molecules are added in the immune cell culture process, and the cell growth factors expressed by the feeder cells are IL-21 cell growth factors; the costimulatory molecule expressed by the trophoblast is 4-1BBL costimulatory molecule, and the immune cell is continuously stimulated by utilizing the immune cell growth factor and costimulatory signal on the surface of the trophoblast, so that the effect of promoting the amplification of the immune cell is achieved. Because the trophoblast is directly added, rather than the immune cell growth factor and the cell co-stimulatory molecule signal protein are independently added, the action time of the cell growth factor and the co-stimulatory signal is prolonged, the production cost of the immune cell is reduced, and the cultured immune cell also has the advantages of good proliferation effect, high ratio of T cells with specific antigen, high specificity, strong function of killing tumor cells and the like.

CN105219708A discloses an immune cell culture kit and an immune cell culture method, wherein the kit comprises a culture solution A, the culture solution A is prepared by adding rhIFN-gamma, rhIL-2, rhIL-15 and human transferrin into serum-free lymphocyte culture solution, the number of cultured immune cells can reach about 100 hundred million cells, and besides CTL cell groups, the immune cells also contain more NK cells and NKT cell groups.

Disclosure of Invention

An object of the present invention is to provide an immune cell which can prevent tumorigenesis and prevent the early development of tumors.

Another object of the present invention is to provide a method for producing the above immune cell.

The invention also aims to provide application of the immune cell.

In order to achieve the purpose, the invention provides a preparation method of an immune cell for preventing tumorigenesis, so that the immune cell is obtained, the prepared immune cell is called TPKT (TPKT) which can recognize invisible infection antigens through Antigen-presenting Cells (APC) so as to remove the antigens, tumor precursor Cells expressing variant proteins caused by unknown susceptibility factors in a body are removed, residual cancer Cells and microscopic focuses invisible in imaging after operation, radiotherapy and chemotherapy are removed, and the tumor metastasis and recurrence are reduced, so that the early development of tumors can be prevented, and the tumorigenesis and recurrence are prevented.

Specifically, in one aspect, the present invention provides a method for preparing an immune cell, the method comprising:

peripheral Blood Mononuclear Cells (PBMCs) were isolated and purified in vitro;

activating peripheral blood mononuclear cells (activated cells become TPKT cells) by using cell activating factor I (also called TPKT cell activating factor I in the invention); wherein, the TPKT cell activating factor I contains IL-2, IFN-gamma, GM-CSF, TNF-alpha, and is prepared by normal saline;

expanding the activated cell, namely the TPKT cell, by using a cell activating factor II (also called TPKT cell activating factor II in the invention); wherein, the TPKT cell activating factor II contains IL-2, IFN-gamma and IL-12, and is prepared by normal saline;

immune cells (TPKT cells) were harvested.

According to a specific embodiment of the present invention, the process for preparing immune cells according to the present invention, wherein the process for in vitro isolation and purification of peripheral blood mononuclear cells comprises:

diluting blood with RPMI1640 culture solution, placing on lymphocyte separation solution, centrifuging, collecting upper layer lymphocyte layer, adding RPMI1640 culture medium, centrifuging, and discarding supernatant;

adding RPMI1640 culture medium to suspend cells, centrifuging, and discarding the supernatant;

complete medium was added to suspend the cells.

According to some embodiments of the present invention, in the above method for preparing immune cells, the centrifugation is 1800rpm for 5-15 min.

According to a specific embodiment of the present invention, in the method for preparing an immune cell of the present invention, the complete medium is prepared according to the following method:

centrifuging whole blood, removing supernatant, standing overnight at 4 deg.C, centrifuging again, and collecting supernatant as plasma added into RPMI1640 culture medium; in some embodiments of the invention, wherein the centrifugation conditions are 1500g centrifugation for 15 to 20 minutes;

to the RPMI1640 medium was added plasma and gentamicin at a concentration of 100U/ml to prepare a complete medium containing 10v/v% of autologous plasma.

According to the specific embodiment of the invention, in the preparation method of the immune cell of the invention, the cell activator I comprises IL-2 with concentration of 100-5000IU/ml, IFN-gamma with concentration of 200-10000IU/ml, GM-CSF with concentration of 150-1500IU/ml and TNF-alpha with concentration of 0-500IU/ml, and is prepared by normal saline.

According to the specific embodiment of the present invention, in the method for preparing the immune cell of the present invention, the cytokine II comprises IL-2 concentration of 300-.

In some specific embodiments of the invention, the cell activator I comprises IL-2 concentration 2500-; the cell activator II comprises 6500IU/mL of IL-2 concentration of 5000-.

According to a specific embodiment of the present invention, the process of activating peripheral blood mononuclear cells with TPKT cell activating factor i in the method for preparing immune cells of the present invention comprises:

adding TPKT cell activating factor I0.5 ml into 20ml cell suspension, and heating at 38.5 deg.C and 6.5% CO₂The culture was carried out in a constant temperature incubator for 72 hours.

According to a specific embodiment of the present invention, the method for preparing an immune cell of the present invention, the process of amplifying TPKT cells using TPKT cell activating factor ii comprises:

after inoculating the cells for 72h, transferring the culture into a centrifuge tube, centrifuging (in some embodiments, the centrifugation conditions are 1800rpm and 5-15 min), discarding the supernatant, suspending the cells in RPMI1640 medium, and adjusting the cell density to 0.5 × 10⁶-1×10⁶Adding TPKT cell activating factor II into the mixture per ml; the cell suspension was added to an uncoated cell culture flask at 38.5 deg.C with 6.5% CO₂The incubation was continued for 72 hours.

According to a specific embodiment of the present invention, in the method for preparing an immune cell of the present invention, the process of harvesting TPKT cells comprises:

centrifuging the cell culture product (in some embodiments, centrifuging at 1800rpm for 5-15 min), discarding the supernatant, suspending the cells with normal saline containing albumin or RPMI1640 medium, and freezing.

According to a specific embodiment of the present invention, in the method for preparing an immune cell of the present invention, the cryopreservation step of the TPKT cell comprises:

centrifuging the cell culture product (in some specific embodiments, the centrifugation condition is 1800rpm and 5-15 min), discarding the supernatant, adding the precooled cryopreservation liquid A for resuspension, fully and uniformly mixing, placing on ice, slowly adding the precooled cryopreservation liquid B, fully and uniformly mixing, and immediately transferring into a cryopreservation tube for preservation at-80 ℃;

in some embodiments of the invention, the cryopreservation solution a is: adding 100mL PBS buffer solution with pH7.0 into every 100mL of human serum albumin solution with the concentration of 200 g/L; mixing, and storing at-20 deg.C; the frozen stock solution B comprises: PBS buffer ph7.0 was mixed with dimethyl sulfoxide according to 4: 1 volume ratio and mixing evenly.

Wherein the cryopreservation step of the TPKT cell is as follows: the cells were centrifuged (at 1800rpm for 5-15 min in some embodiments) and resuspended in 4ml of freezing solution A, mixed well, placed on ice, and 4ml of pre-cooled freezing solution B was added slowly. Mixing, subpackaging in 6 freezing tubes, storing in liquid nitrogen or a low temperature refrigerator of-80 deg.C, and storing in liquid nitrogen after 24 hr. Note that: freezing solution A and freezing solution B, wherein the freezing solution A is as follows: adding 100mL PBS buffer solution with pH7.0 into 100mL human serum albumin solution with concentration of 200 g/L; mixing, and storing at-20 deg.C; the frozen stock solution B comprises: PBS buffer ph7.0 was mixed with dimethyl sulfoxide according to 4: 1 volume ratio and mixing evenly. Pre-preparing in advance, pre-cooling in a 4 ℃ refrigerator, or subpackaging after preparation and freezing in a-20 ℃ refrigerator for later use. The frozen stock solution is shaken up and down and mixed evenly when being taken out. And placing the freezing tube, the freezing tube frame and the foam box into a refrigerator with the temperature of-20 ℃ in advance for precooling for about 30 min.

In another aspect, the present invention provides an immune cell, which is detected by a flow cytometer as a leukocyte differentiation antigen on the surface of the immune cell, and has the following properties:

CD3-FITC/CD4-PE/CD8-PECy5 identification: the cell content of CD3+ is more than or equal to 90%, the cell content of CD3+/CD4+ is more than or equal to 40%, and the cell content of CD3+/CD8+ is more than or equal to 40%;

CD3-FITC/CD16-PE/CD56-PECy5 identification: the cell ratio of CD3+/CD56+ is more than or equal to 10 percent, and the cell ratio of CD3-/CD56+ and CD3-/CD16+ is more than or equal to 5 percent;

Lin-FITC/CD83-PE/CD1a-PECy5 identification: Lin-/CD83+/CD1a + cells are more than or equal to 1%;

FoxP3-FITC/CD25-PE/-CD4-PECy5 identified: FoxP3+/CD25 +/CD4+ cells are less than or equal to 1%;

CD3-FITC/CD14-PE/CD19-PECy5 identification: CD3-/CD14+ cells are less than or equal to 1%, and CD3-/CD19+ cells are less than or equal to 1%.

According to a particular embodiment of the invention, the immune cell of the invention, which is prepared according to the method of the invention.

The immune cells of the invention have other quality identification and qualification standards: the final product of cell culture is required to be tested according to the aseptic test procedure of biological products in Chinese biological products, and the final product of cell culture is negative through the culture results of gram-positive and gram-negative bacteria; the result is negative through the identification of fungus culture; the result is negative through mycoplasma detection; the result of syphilis detection is negative; the result of hepatitis B antigen detection is negative; the endotoxin detection result was negative.

On the other hand, the invention also provides the application of the immune cell in preparing a preparation for preventing tumorigenesis and preventing early tumor development.

According to some embodiments of the present invention, the agent for preventing tumorigenesis, preventing the development of early stage tumor is administered by intradermal injection, intravenous injection, intramuscular injection, or administration in or around the tumor at a dose of 50 to 3000 ten thousand immune cells per time.

According to some embodiments of the invention, the agent for preventing tumorigenesis, preventing the early development of tumors is an agent that eliminates antigens by recognizing stealth infection antigens by antigen presenting cells, eliminates tumor precursor cells expressing variant proteins, and/or prevents clonal proliferation of transformed cells and eliminates imagewise invisible micro-tumors.

According to a specific embodiment of the present invention, the immune cells are revived before being returned for specific applications. The recovery steps of TPKT cells are as follows: thawing a cell freezing tube to be resuscitated in a constant-temperature water bath at 37 ℃, quickly transferring the cell freezing tube into a centrifugal tube preset with 10ml of cell resuscitating solution, centrifuging (the centrifugation conditions in some specific embodiments are 1800rpm and 5-15 min), resuspending the cell freezing tube with 10ml of physiological saline, and then centrifuging. Resuspend with 2ml of cell resuscitating solution and prepare for reinfusion. Sampling, carrying out trypan blue staining, counting and calculating the survival rate. The preparation method of the resuscitation liquid comprises the following steps: adding 5ml of 200g/L human serum albumin solution into 95ml of sterile physiological saline, mixing uniformly, and filtering with a 0.22 μm filter. Wherein the feedback steps of TPKT cells are as follows: the recovered TPKT cells are administered by intradermal injection, intravenous injection, intramuscular injection or administration in or around the tumor at a dose of 50-3000 ten thousand cells per injection. Bacterial identification and other quality inspection reports must be obtained before the feedback to prevent infection caused by the culture process.

The invention has the beneficial effects that:

the specific factor combination of IL-2, IFN-gamma, GMCSF and TNF-alpha is used in the in vitro activation stage of the cell, and under the combined action of all the components, the peripheral blood mononuclear cell can be quickly activated into TPKT cell, so that the TPKT cell has the capacity of identifying tumor precursor cells expressing variant protein caused by recessive infection factors and imaging invisible micro tumor through the presentation of APC.

And (II) a cytokine combination of IL-2, IFN-gamma and IL-12 is used in the cell expansion stage, so that TPKT cells are greatly expanded, the number of memory T killer cells in a cell population is increased, and the tumor specific killing activity of a final product is ensured.

And thirdly, the TPKT cells obtained by the amplification of the method can eliminate tumor precursor cells expressing variant proteins.

And (IV) the TPKT cells obtained by the amplification of the method can clear the microscopic tumors which cannot be seen in imaging.

Drawings

FIG. 1 is a graph showing the proliferation of TPKT cells in example 2.

FIG. 2 is a graph showing the results of TPKT cytotoxicity in vitro testing for the US survival time of intervertebral discs in example 5.

FIG. 3 is a graph showing the results of tumor size in the TPKT cytotoxicity in vitro assay in example 5.

Detailed Description

The following examples are intended to illustrate the practice and advantageous effects of the present invention, but are not to be construed as limiting the scope of the present invention. The procedures and operating conditions not specified in the examples were carried out according to the usual techniques in the art or according to the recommended procedures of the manufacturer of the apparatus, using commercially available starting materials.

Example 1 preparation of TPKT cells

1. Isolation of peripheral blood lymphocytes

(1) 150ml of blood is taken at one time, heparin anticoagulation is carried out, and 1.5-2 × 10 can be obtained under the normal condition of 150ml of whole blood⁸One cell can be planted by 75cm²A culture bottle; and (3) subpackaging the peripheral blood subjected to heparin anticoagulation treatment into centrifuge tubes, centrifuging at 1800rpm for 15min, and discarding the supernatant.

(2) Diluting blood: the RPMI1640 medium diluted blood in equal times.

(3)25ml of the diluted blood was carefully placed on 12.5ml of lymphocyte separation medium, centrifuged at 1800rpm for 15min, and the rise and fall speed was adjusted to the minimum.

(4) Taking the upper plasma layer for use, and storing in a refrigerator at 4 deg.C.

(5) The lymphocyte layer was collected and placed in a 50ml centrifuge tube, and RPMI1640 medium was added to the tube to a total volume of 50ml, and centrifuged at 1800rpm for 5 min.

(6) The supernatant was discarded, a certain amount of RPMI1640 medium was added, the cells were suspended, and a small amount was counted.

(7) Centrifuge at 1800rpm for 5 min.

(8) Discarding the supernatant to obtain peripheral blood mononuclear cells; complete medium was added to suspend the cells.

2. Preparation method of reagent and solution prepared culture medium

Centrifuging whole blood at 1500g for 20 min, discarding supernatant, standing overnight at 4 deg.C, centrifuging at 1500g for 15min, and collecting supernatant as plasma added into culture medium; to the RPMI1640 medium was added plasma and gentamicin at a concentration of 100U/ml to prepare a complete medium containing 10v/v% of autologous plasma.

3. Cell culture flask treatment

Sucking out the coating liquid, adding 30ml of normal saline into each bottle, slightly shaking, and sucking out the normal saline.

TPKT cell activation

The TPKT cell activation method through the TPKT cell activation factor I culture comprises the following steps: after the cell culture flask is treated, 20ml of cell suspension is immediately added, 0.5ml of TPKT cell activating factor I is added, and the temperature is kept at 38.5 ℃ and 6.5% CO₂The culture was carried out in a constant temperature incubator for 72 hours.

Wherein TPKT cell activating factor I comprises IL-2(3500IU/ml), IFN-gamma (3000IU/ml), GM-CSF (550IU/ml) and TNF-alpha (320IU/ml), and is prepared by normal saline.

Amplification of TPKT cells

(1) After 72h of cell inoculation, the culture is transferred into a 50ml centrifuge tube, when partial adherent cells cannot be blown down, 15ml of normal saline can be added, and the bottle is flapped or scraped to separate from the bottom of the bottle and is combined with the original cell suspension. Centrifuge at 1800rpm for 5 min.

(2) The supernatant was discarded, and cells were suspended in RPMI1640 medium and counted.

(3) Adding RPMI1640 culture medium, and adjusting cell density to 0.5 × 10⁶-1×10⁶Adding 0.5ml of TPKT cell activating factor II into the mixture per ml.

(4) Adding the cell suspension to the new 75cm²Cell culture flasks (uncoated), 50ml or so per flask.

(5) The culture flask was maintained at 38.5 ℃ and 6.5% CO₂The incubation was continued for 72 hours.

Wherein TPKT cell activating factor II comprises IL-2(5500IU/ml), IFN-gamma (3000IU/ml) and IL-12(5800IU/ml), and is prepared with physiological saline.

TPKT cell harvesting and cryopreservation

The cell culture product was centrifuged at 1800rpm for 5min, the supernatant was discarded, and the cells were suspended in albumin-containing physiological saline or RPMI1640 medium and counted. According to the cell number, the freezing quantity is determined, and simultaneously, a sample is taken and sent to quality control detection.

Wherein the submission step is as follows:

the leukocyte differentiation antigen on the surface of finished cells is detected by flow cytometry, and the antibodies usually used for fluorescent staining comprise the following groups:

(1) CD3-FITC/CD4-PE/CD8-PECy5, and the qualification standard for identifying the final product of the cultured cells is as follows: the cell content of CD3+ is more than or equal to 90%, the cell content of CD3+/CD4+ is more than or equal to 40%, and the cell content of CD3+/CD8+ is more than or equal to 40%;

(2) CD3-FITC/CD16-PE/CD56-PECy5, and the qualification standard for identifying the final product of the cultured cells is as follows: the cell ratio of CD3+/CD56+ is more than or equal to 10 percent, and the cell ratio of CD3-/CD56+ and CD3-/CD16+ is more than or equal to 5 percent;

(3) Lin-FITC/CD83-PE/CD1a-PECy5 identifies the final product qualification standard of cultured cells as follows: Lin-/CD83+/CD1a + cells are more than or equal to 1%;

(4) FoxP3-FITC/CD25-PE/-CD4-PECy5 identifies cultured cells as the final product qualification standard: FoxP3+/CD25 +/CD4+ cells are less than or equal to 1%;

(5) CD3-FITC/CD14-PE/CD19-PECy5 identifies the final product qualification standard of cultured cells as follows: CD3-/CD14+ cells are less than or equal to 1%, and CD3-/CD19+ cells are less than or equal to 1%.

Other quality identification and qualification standards:

the final product of cell culture is required to be tested according to the aseptic test procedure of biological products in the Chinese biological product protocol, and the final product of cell culture is negative through the culture results of gram-positive and gram-negative bacteria; the result is negative through the identification of fungus culture; the result is negative through mycoplasma detection; the result of syphilis detection is negative; the result of hepatitis B antigen detection is negative; the endotoxin detection result was negative.

Through detection, the finished product cell of the embodiment meets the requirement of the quality inspection qualification standard.

Wherein the cryopreservation step of the TPKT cell is as follows:

the cells were centrifuged and resuspended in 4ml of freezing solution A, mixed well, placed on ice and 4ml of pre-cooled freezing solution B was added slowly. Mixing, subpackaging in 6 freezing tubes, storing in liquid nitrogen or a low temperature refrigerator of-80 deg.C, and storing in liquid nitrogen after 24 hr.

Note that: freezing solution A and freezing solution B, wherein the freezing solution A is as follows: adding 100mL PBS buffer solution with pH7.0 into 100mL human serum albumin solution with concentration of 200 g/L; mixing, and storing at-20 deg.C; the frozen stock solution B comprises: PBS buffer ph7.0 was mixed with dimethyl sulfoxide according to 4: 1 volume ratio and mixing evenly. Pre-preparing in advance, pre-cooling in a 4 ℃ refrigerator, or subpackaging after preparation and freezing in a-20 ℃ refrigerator for later use. The frozen stock solution is shaken up and down and mixed evenly when being taken out. And placing the freezing tube, the freezing tube frame and the foam box into a refrigerator with the temperature of-20 ℃ in advance for precooling for about 30 min.

Resuscitation of TPKT cells

Thawing a cell freezing tube to be resuscitated in a constant-temperature water bath at 37 ℃, transferring the cell freezing tube into a centrifuge tube preset with 10ml of cell resuscitating solution, centrifuging, re-suspending with 10ml of physiological saline, re-centrifuging, and repeating the steps of re-suspending and centrifuging for 3 times. Samples were taken, stained with trypan blue and counted. Resuspend with 2ml cell resuscitating solution, prepare to return CAPRI cell, used for tumor patient postoperative treatment.

The preparation method of the resuscitation liquid comprises the following steps: adding 5ml of 200g/L human serum albumin solution into 95ml of sterile physiological saline, mixing uniformly, and filtering with a 0.22 μm filter.

Wherein the feedback steps of TPKT cells are as follows: the recovered TPKT cells are administered by intradermal injection, intravenous injection, intramuscular injection or administration in or around the tumor at a dose of 50-3000 ten thousand cells per injection. Bacterial identification and other quality inspection reports must be obtained before the feedback to prevent infection caused by the culture process.

Example 2 detection of the amplification Capacity of TPKT cells

TPKT cells were cultured in 3 cases in the same manner as in example 1, and the growth of TPKT cells was examined at 0, 3, 5, 7, 9 and 12 days of culture, as shown in FIG. 1.

As can be seen from FIG. 1, TPKT cells cultured by the method of the present invention have good proliferation.

Example 3 assay of TPKT cell viability

The cells were cultured by the method of example 1, 100. mu.l of the cells cultured for 12 days were taken, 100. mu.l of 0.4% trypan blue staining solution was added, viable cells were not stained, dead cells were stained blue, and 500 cells were counted per sample to calculate cell viability. As in table 1. As can be seen from Table 1, the cell activity of the TPKT prepared by the method is more than 95%, and the activity of the cultured TPKT is better.

TABLE 1

	Sample 1	Sample 2	Sample 3
				Percentage of viable cells	96.4％	97.6％	95.8％

Example 4 TPKT cell flow phenotyping

The method of example 1 is adopted for culture, 100 mul of cells cultured for 0, 4, 7 and 10 days are respectively taken, the cells with the concentration of about 106/ml are respectively added with mouse anti-human CD3-PerCP/CD4-FITC/CD8-PE, CD3-PerCP/CD19-FITC/CD14-PE, CD3-PerCP/CD16-FITC/CD56-PE, CD4-PerCP/CD25-FITC/FOX-P3-PE antibody combination for detection, each antibody is 10 mul, and the cells are incubated in a dark environment for 30min at room temperature. Flow cytometric assays were then performed with two washes in PBS as shown in table 2.

TABLE 2

Detecting content	Example 1	Example 2	Example 3
				CD3+/CD19-/CD14- (T-cell)	94.7％	93.5％	92.5％
CD3+/CD8+/CD4- (T killer cell)	67.7％	70.8％	62.1％
				CD3+/CD4+/CD8- (T helper cell)	28.0％	23.5％	30.2％
CD3+/CD56+ or CD16+ (NKT cells)	21.4％	16.8％	20.2％
				CD3-/CD56+/CD16+ (NK cells)	4.7％	5.8％	6.3％
CD4+/CD25+/FoxP3+ (Treg cells)	0.6％	0.3％	0.8％

As can be seen from Table 2, TPKT cells cultured by the method of the present invention contain more CD8+ T killer cells, less CD4+ T helper cells and Treg cells with immunoregulatory function. Specifically, the cells cultured by the method have the advantages that CD3+/CD8+/CD4- (T killer cells) are more than 62.1 percent, and the performance of TPKT cells is 50.4 +/-6.9 percent; the content of CD3+/CD4+/CD8- (T helper cell) is below 30.2 percent, and the performance of TPKT cell is 46.6 percent +/-4.3 percent; the content of CD4+/CD25+/FoxP3+ (Treg cells) is below 0.8 percent, and the performance of TPKT cells is 11.3 percent +/-4.1 percent.

Example 5 in vitro test results for TPKT cell toxin

TPKT cells were cultured as described in example 1 using peripheral blood mononuclear cells of lung patients, each as containing 1 × 10⁶0.1ml of physiological saline per cell was injected into 6-8 weeks old nude mice (6 mice per group) in tail vein every day, and the cells were continuously administered for one week, after TPKT cells were administered for one week, autologous tumor cells 1 × 10 of lung patients were administered⁵One of the mice in the control group was injected with autologous peripheral blood lymphocytes 1 × 10 of the patient before tumor inoculation by subcutaneous injection into nude mice⁶One/only. Mice survival time was recorded (21 days total observation), mice were sacrificed after 21 days with statistical analysis and tumor volume was recorded or measured at the time of death of the mice. FIG. 2 is a comparison of survival time of TPKT cell preventive immunoprotection group control autologous peripheral blood lymphocyte protection group after inoculation to nude mouse autologous lung cancer tumor cell line.

As shown in FIG. 2, the median survival time TPKT was 19.5 days, and the peripheral blood lymphocyte cell count was 11.00 days, which are statistically significant (X)²5.335, P0.0209 < 0.05), i.e. the TPKT group survived longer than the peripheral blood lymphocytes group, and had antitumor effect.

FIG. 3 comparison of tumor volumes after inoculation of autologous lung cancer tumor cell lines in nude mice with TPKT cell preventive immunoprotection group control autologous peripheral blood lymphocyte protection group.

As can be seen from FIG. 3, the mean volume of TPKT group tumors was 1.867+/-1.111cm³The mean tumor volume of the control group was 3.358+/-0.465cm³And has statistical significance (T ═ T ═3.034, P ═ 0.0126 < 0.05), i.e., TPKT group kills tumors better than peripheral blood lymphocytes.

Claims

1. A method for preparing immune cells capable of preventing tumorigenesis and preventing the development of tumors in an early stage, wherein the immune cells capable of preventing tumorigenesis and preventing the development of tumors in an early stage have the following properties:

the killing rate of CD3+/CD8+/CD 4-T cells is more than 62.1%; the content of CD3+/CD4+/CD 8-T helper cells is below 30.2%; the content of CD4+/CD25+/FoxP3+ Treg cells is below 0.8%;

the method comprises the following steps:

separating and purifying peripheral blood mononuclear cells in vitro;

activating peripheral blood mononuclear cells with cell activating factor I; wherein, the cell activating factor I consists of IL-2, IFN-gamma, GM-CSF, TNF-alpha, and is prepared by normal saline; wherein the concentration of IL-2 is 3500IU/ml, the concentration of IFN-gamma is 3000IU/ml, the concentration of GM-CSF is 550IU/ml, and the concentration of TNF-alpha is 320 IU/ml;

amplifying the activated cells by using a cell activating factor II; wherein, the cell activating factor II consists of IL-2, IFN-gamma and IL-12 and is prepared by normal saline; wherein the concentration of IL-2 is 5500IU/ml, the concentration of IFN-gamma is 3000IU/ml, and the concentration of IL-12 is 5800 IU/ml;

and (5) harvesting the immune cells.

2. The preparation method of claim 1, wherein the process of isolating and purifying peripheral blood mononuclear cells in vitro comprises:

complete medium was added to suspend the cells.

3. The production method according to claim 2, wherein the complete medium is produced by:

centrifuging whole blood, removing supernatant, standing overnight at 4 deg.C, centrifuging again, and collecting supernatant as plasma added into RPMI1640 culture medium;

4. The method of claim 1, wherein the process of activating peripheral blood mononuclear cells with cytokine I comprises:

adding 0.5ml of cell activator I to 20ml of cell suspension at 38.5 deg.C and 6.5% CO₂Culturing for 72 hours in a constant-temperature incubator;

the process of expanding the activated cells by using the cell activating factor II comprises the following steps:

72 hours after cell inoculation, the culture was transferred to a centrifuge tube, centrifuged, the supernatant was discarded, the cells were suspended in RPMI1640 medium, and the cell density was adjusted to 0.5 × 10⁶-1×10⁶Adding cell activating factor II into the mixture per ml; the cell suspension was added to an uncoated cell culture flask at 38.5 deg.C with 6.5% CO₂The incubation was continued for 72 hours.

5. The preparation method according to claim 1, wherein the process of harvesting immune cells comprises:

the cell culture product was centrifuged, the supernatant was discarded, and the cells were suspended in albumin-containing physiological saline or RPMI1640 medium and frozen.

6. The method of claim 1, wherein the step of cryopreserving the immune cells comprises:

centrifuging the cell culture product, discarding the supernatant, adding the precooled freezing solution A, resuspending, fully mixing, placing on ice, slowly adding the precooled freezing solution B, fully mixing, immediately transferring into a freezing tube for preservation at-80 ℃;

the frozen stock solution A comprises: adding 100mL PBS buffer solution with pH7.0 into every 100mL of human serum albumin solution with the concentration of 200 g/L; mixing, and storing at-20 deg.C;

the frozen stock solution B comprises: PBS buffer ph7.0 was mixed with dimethyl sulfoxide according to 4: 1 volume ratio and mixing evenly.

7. An immune cell capable of preventing tumorigenesis and preventing tumor development in an early stage, which is prepared by the method according to any one of claims 1 to 6, and which has the following properties by detecting a leukocyte differentiation antigen on the surface of the immune cell by flow cytometry:

CD3-FITC/CD4-PE/CD8-PECy5 identification: the cell of CD3+ is more than or equal to 90%, the cell of CD3+/CD4+ is less than 40%, and the cell of CD3+/CD8+ is more than or equal to 40%;

CD3-FITC/CD14-PE/CD19-PECy5 identification: CD3-/CD14+ cells are less than or equal to 1%, and CD3-/CD19+ cells are less than or equal to 1%;

and, the immune cell which can prevent tumorigenesis and prevent the development of a tumor at an early stage has the following properties:

the killing rate of CD3+/CD8+/CD 4-T cells is more than 62.1%; the content of CD3+/CD4+/CD 8-T helper cells is below 30.2%; the content of CD4+/CD25+/FoxP3+ Treg cells is below 0.8%.

8. Use of an immune cell according to claim 7 for the preparation of a formulation for preventing tumorigenesis, preventing the development of tumors at an early stage, wherein the tumor is a lung tumor.

9. The use according to claim 8, wherein the agent for preventing tumorigenesis, preventing the development of early stage tumor is administered by intradermal injection, intravenous injection, intramuscular injection, or administration in or around the tumor at a dose of 50-3000 ten thousand immune cells per time.