CN114672457A - T lymphocyte derived from tumor tissue and having tumor specific killing effect, preparation method thereof and cell preparation - Google Patents

T lymphocyte derived from tumor tissue and having tumor specific killing effect, preparation method thereof and cell preparation Download PDF

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CN114672457A
CN114672457A CN202210595579.5A CN202210595579A CN114672457A CN 114672457 A CN114672457 A CN 114672457A CN 202210595579 A CN202210595579 A CN 202210595579A CN 114672457 A CN114672457 A CN 114672457A
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冯春玲
陈晓波
韩颢
韩洪起
吴学涛
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Yousai Medical Technology Tianjin Co ltd
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Abstract

The invention discloses a T lymphocyte with tumor specific killing effect from tumor tissue, a preparation method and a cell preparation thereof. The invention can obtain the cell number meeting the preparation requirement in a shorter culture period and prepare the cell suspension by digesting and separating the tumor tissue to obtain the seed cells, giving continuous stimulation to the seed cells and using a specific culture medium for amplification culture. The invention adopts an amplification mode of continuous stimulation and directional culture, and stimulation is given at the initial stage of cell inoculation, compared with the traditional culture method of firstly amplifying the number of seed cells and then stimulating, the invention can greatly improve the cell amplification efficiency, shorten the culture period and ensure that the cells maintain high-level biological activity.

Description

T lymphocyte derived from tumor tissue and having tumor specific killing effect, preparation method thereof and cell preparation
Technical Field
The invention relates to the field of T lymphocyte preparations, in particular to a T lymphocyte with a tumor specific killing effect and a preparation method thereof and a cell preparation.
Background
In recent years, with the rapid development of the field of biological medicine, immunotherapy is becoming the fourth kind of tumor treatment means after surgery, radiotherapy and chemotherapy, wherein adoptive cell therapy, immune checkpoint blockade therapy, tumor vaccines, bispecific antibodies and other treatment methods all achieve certain clinical effects. However, during the development of solid tumors, the physical barrier of solid tumors and their complex tumor microenvironment limit the efficacy of nonspecific immunotherapy.
Tumor tissue infiltrating lymphocytes from the interior of a tumor or the vicinity of the tumor tissue have specific recognition capability for the tumor cells, and are first mentioned in 1922, Mac carty speculates that the lymphocytes into the tumor tissue are a phenomenon that an in vivo immune system resists the tumor, and proposes that the tumor tissue infiltrating lymphocytes are high-efficiency anti-cancer immune effector cells. Rosenberg et al discovered and isolated tumor tissue infiltrating lymphocytes in tumor-bearing mice in 1986, and the isolated cells were cultured by in vitro amplification and then returned to the tumor-bearing mice, and the tumor tissue was found to have obvious regression, thus proving that the tumor tissue infiltrating lymphocytes have the anti-tumor effect.
The conventional culture method for the tumor specific killing T lymphocytes adopts a two-step method for culture, the number of cells is firstly amplified by using high-concentration IL-2, then the cells are stimulated by using a CD3 monoclonal antibody, a CD28 monoclonal antibody or irradiated human peripheral blood PBMC and the like to improve the proportion of effector cells, the culture period is usually 20-25 days, and research culture is carried out for 36 days.
Disclosure of Invention
In order to solve the technical problems, the invention provides a T lymphocyte which is derived from tumor tissues and has a tumor specific killing effect, a preparation method thereof and a cell preparation.
In a first aspect, the present invention provides a method for preparing a T lymphocyte having a tumor-specific killing effect, which is derived from a tumor tissue, according to the following technical scheme.
A method for preparing T lymphocyte with tumor specificity killing effect from tumor tissue comprises the following steps:
s1, cleaning and dividing tumor tissues, and putting the tumor tissues into digestive juice to perform shake digestion for 30-40 min;
s2, filtering, washing and centrifuging after tissue digestion, adding the suspension sediment after the complete culture basis weight of the T lymphocytes into a culture bottle coated in advance, supplementing IFN-gamma to the final concentration of 1000-1500IU/ml, and carrying out cell culture; the method for coating the culture flask comprises the following steps: the culture flask is coated with complete culture medium containing CD3 and CD28 monoclonal antibody for T lymphocyte and is placed at 37 deg.C and 5.0% CO2Under the condition of 0.5-1 h;
s3, observing the cell state every day, sampling and counting, supplementing a complete T lymphocyte culture medium according to the counting result, and adjusting the cell concentration to be 0.5-0.8 multiplied by 109L, continuing cell culture;
s4, continuously observing the cell state every day, sampling and counting when the culture system reaches 10-15ml, transferring the cells into a culture bottle coated in advance, wherein the coating method is the same as the step S2; adding complete culture medium of T lymphocyte according to counting result, adjusting cell concentration to 0.5-0.8 × 109Supplementing IFN-gamma to the final concentration of 1000-1500IU/ml, and continuing cell culture;
s5, continuously observing the cell state every day, sampling and counting, when the culture system reaches 25-45ml, transferring the cells into another culture bottle, supplementing a complete culture medium of the T lymphocytes according to counting results, and adjusting the cell concentration to be 0.5-0.8 multiplied by 109L, continuing cell culture;
s6, continuously observing the cell state every day, sampling and counting, and when the culture system reaches 180-20%Transferring the cells into cell culture bag at 0ml, supplementing CD3 and CD28 monoclonal antibody into the culture bag according to 20-50ng/ml, supplementing complete culture medium for T lymphocyte according to counting result, and adjusting cell concentration to 0.5-0.8 × 109L, continuing cell culture;
s7, continuously observing the cell state every day, sampling and counting, when the culture system reaches 1600-9At the above time, all cells were harvested.
By adopting the technical scheme, the tumor tissue is digested and separated, the obtained seed cells are continuously stimulated and are subjected to amplification culture by using a specific culture medium, the number of the cells meeting the preparation requirement is obtained in a short culture period, and the cells are prepared into a cell suspension. The invention adopts an amplification mode of continuous stimulation and directional culture, and stimulation is given at the initial stage of cell inoculation, compared with the traditional culture method of firstly amplifying the number of seed cells and then stimulating, the invention can greatly improve the cell amplification efficiency, shorten the culture period and ensure that the cells maintain high-level biological activity.
Further, in step S1, 10-15ml of digestive juice is added per cubic centimeter of tissue mass.
Further, in step S1, the digestion solution is 1640 medium containing 0.075-0.15g/ml collagenase type I.
Further, the complete T lymphocyte culture medium is X-VIVO 15 medium containing 10-20 v% fetal bovine serum, 2 v% 50X essential amino acids and 4000-.
Further, in step S2, the content of the two monoclonal antibodies CD3 and CD28 is 20-50ng/cm2。
In a second aspect, the present application provides a T lymphocyte derived from a tumor tissue and having a tumor-specific killing effect, which is achieved by the following technical means.
T lymphocyte with tumor specific killing effect derived from tumor tissue and prepared by the preparation method.
In a third aspect, the present application provides a T lymphocyte preparation with a tumor-specific killing effect, which is achieved by the following technical scheme.
A T lymphocyte preparation with tumor-specific killing effect comprises the above T lymphocytes with tumor-specific killing effect derived from tumor tissue.
The present application has the following advantageous effects.
The application separates and obtains seed cells from tumor tissues containing a large number of immune cells with specificity recognition tumor antigens and surrounding tissues thereof, and can greatly improve the proportion of the cells with specificity recognition tumor antigens through in vitro directional culture. The invention adopts an amplification mode of continuous stimulation and directional culture, and stimulation is given at the initial stage of cell inoculation, compared with the traditional culture method of firstly amplifying the number of seed cells and then stimulating, the invention can greatly improve the cell amplification efficiency, shorten the culture period, maintain the high-level biological activity of the cells and reduce the proportion of regulatory T lymphocytes in the final preparation.
Drawings
FIG. 1 is a diagram showing the state of cell expansion observed under the microscope in example 1D 3 of the present invention (4-fold microscope);
FIG. 2 is a graph showing the flow pattern of D16 cultured in example 1 of the present invention;
FIG. 3 is a graph showing cell expansion curves in example 1 of the present invention;
FIG. 4 is a diagram showing the state of cell expansion observed under the microscope of D3 in example 2 of the present invention (4-fold microscope);
FIG. 5 is a graph showing the flow pattern of D18 cultured in example 2 of the present invention;
FIG. 6 is a graph showing cell expansion in example 2 of the present invention.
Detailed Description
The patent application is further described below with reference to the drawings and examples.
A method for preparing T lymphocyte with tumor specificity killing effect from tumor tissue comprises the following steps:
(1) extraction of seed cells: tumor tissue obtained by surgery has a volume of not less than 1cm × 1cm × 1 cm. Make itAfter washing once with 1640 medium, the tissue was placed in a sterile petri dish and cut into several 2-3mm sections with sterile scalpel and forceps3Putting the small blocks into a centrifuge tube, supplementing digestive juice, mixing uniformly, and putting into a constant temperature oscillator at 37 ℃ for oscillation digestion for 30-40 min. Supplementing 10-15ml of digestive juice per cubic centimeter of tissue block, wherein the components of the digestive juice are as follows: 1640 medium contains 0.075-0.15g/ml collagenase type I.
(2) T lymphocyte culture: the T-25 flask was previously coated with 1ml of complete T lymphocyte culture medium containing CD3 and CD28 mAbs and placed at 37 ℃ in a 5.0% CO atmosphere2Under the condition of 0.5-1h for standby, the content of the two monoclonal antibodies is 20-50ng/cm2. After tissue digestion, filtering the mixture by a 200-micron pore size filter screen, supplementing 20-30ml 1640 medium and 500g of 300-25K culture medium, centrifuging the mixture for 8-10min, then removing the supernatant, suspending the precipitate by using 4-6ml of T lymphocyte complete culture medium, and adding the suspended precipitate into a coated T-25 culture bottle, wherein the complete culture medium is X-VIVO 15 culture medium (LONZA/04-418Q) comprising 10-20% of fetal bovine serum by volume fraction, 2% of 50X essential amino acids (BI/01-325-1B) by volume fraction and 4000-6000IU/ml IL-2. IFN-gamma is supplemented to a final concentration of 1000-1500IU/ml in culture flasks, placed at 37 ℃ and 5% C02The incubator is used for culture.
(3) Observing cell state every day, sampling, counting, supplementing complete culture medium for T lymphocyte according to counting result, and adjusting cell concentration to 0.5-0.8 × 109/L, mixing, adding 5% C0 at 37 deg.C2The incubator continues to cultivate.
(4) Observing cell state every day, sampling and counting when culture system reaches 10-15ml, coating T-75 culture flask with 3ml T lymphocyte complete culture medium containing CD3 and CD28 monoclonal antibody for 0.5-1h, and keeping the two monoclonal antibodies at 20-50ng/cm2. Transferring the cells to coated T-75 culture flask, supplementing complete culture medium for T lymphocytes according to counting result, and adjusting cell concentration to 0.5-0.8 × 109L, supplementing IFN-gamma to the final concentration of 1000-1500IU/ml, mixing uniformly, placing at 37 ℃, 5% C02The incubator continues to culture.
(5) Continuously observing cell state every day, sampling and counting, and transferring cells into the culture system when the culture system reaches 25-45mlAdding complete culture medium for T lymphocytes according to counting result, and adjusting cell concentration to 0.5-0.8 × 109/L, mixing, adding 5% C0 at 37 deg.C2The incubator continues to culture.
(6) Continuously observing the cell state every day, sampling and counting, transferring the cells into a new cell culture bag when the culture system reaches 180-9L, supplementing CD3 and CD28 monoclonal antibody into the culture bag according to 20-50ng/ml, mixing uniformly, placing at 37 deg.C and 5% C02The incubator continues to cultivate.
(7) The cell status was observed daily and the samples were counted when the culture system reached 1600-2000ml and the total number of cells was 4X 109In the above process, all cell suspensions are harvested to 4 500ml centrifuge cups, centrifugation is carried out for 10-20min by using 500g of 300-plus-one solution, then centrifugation supernatant is discarded, after the cells are resuspended by adding sodium chloride injection containing 0.2-0.4% of human serum albumin, centrifugation is carried out for 10-20min by using 500g of 300-plus-one solution, the cells are washed for 2-3 times, and the cells are resuspended by using sodium chloride injection containing 1-3% of human serum albumin, so that the T lymphocyte preparation with the tumor specific killing effect is prepared.
Example 1
Sample information: male, 60 years old, after liver cancer surgery, was collected from the fifth medical center of the general hospital of liberty military.
Preparation of T lymphocyte preparation with tumor specific killing effect:
(1) extraction of seed cells: tumor tissue obtained by surgery, 1.2cm × 0.9cm × 1 cm. After washing once with 10ml 1640 medium, the tissue was placed on a sterile petri dish and cut into 2-3mm sections with a scalpel and forceps3The tissue mass of (1) was put into 150 ml centrifuge tube, supplemented with 15ml of a digestion solution (digestion solution components: 13.5ml 1640 medium and 1.5ml of type I collagenase stock solution (1640 medium contains 1g/ml type I collagenase)), mixed well and digested for 30min with shaking at 37 ℃ in a constant temperature shaker.
(2) T lymphocyte culture: coating 1T-25 culture flask with 1ml of coating solution of CD3 and CD28 monoclonal antibody for 0.5h for use (coating solution component: T gonorrhea containing CD3 and CD28 monoclonal antibody at concentration of 1ug/mlComplete culture medium of blast cells), the content of the two monoclonal antibodies is 40ng/cm2. Filtering the digested tissue by a 200-micron filter screen into 1 new 50ml centrifuge tube, supplementing 20ml 1640 culture medium, centrifuging for 10min at 500g, discarding the supernatant, suspending and precipitating by using 4ml T lymphocyte complete culture medium, adding into the coated T-25 culture bottle, supplementing IFN-gamma to the final concentration of 1000IU/ml, mixing uniformly, placing at 37 ℃, and adding 5% C02The incubator is used for culture. The complete culture medium of T lymphocyte is X-VIVO 15 culture medium (LONZA/04-418Q) comprising 10% volume fraction of fetal calf serum, 2% volume fraction of 50X essential amino acids (BI/01-325-1B), and 4000IU/ml IL-2.
(3) Observing the cell state every day, observing a small amount of cell masses under a microscope after culturing D3 days (see figure 1), blowing and mixing the cells, sampling and counting, and counting the results: WBC 0.3X 109And L. Placing at 37 deg.C with 5% C02The incubator continues to culture.
(4) Culturing for D4 days, sampling and counting the cells uniformly mixed by blowing, and counting the results: WBC 1.0X 109L, adding 5ml of complete T lymphocyte culture medium, mixing, adding 5% C0 at 37 deg.C2The incubator continues to culture.
(5) Culturing for D5 days, sampling and counting the cells uniformly mixed by blowing, and counting the results: WBC 1.0X 109L, coating 1T-75 culture flask with 3ml of coating solution of CD3 and CD28 monoclonal antibodies for 0.5h for later use, wherein the content of the two monoclonal antibodies is 40ng/cm2. Transferring the cells to a coated T-75 culture flask, supplementing 7ml of T lymphocyte complete culture medium according to counting results, and adjusting the cell concentration to 0.5 multiplied by 109and/L, adding IFN-gamma to a final concentration of 1000 IU/ml. Mixing, adding 5% C0 at 37 deg.C2The incubator continues to culture.
(6) Culturing for D6 days, sampling and counting the cells uniformly mixed by blowing, and counting the results: WBC 0.8X 109L, adding 12ml of complete T lymphocyte culture medium according to counting results, and adjusting the cell concentration to 0.5X 109L, mixing, adding 5% C0 at 37 deg.C2The incubator continues to culture.
(7) Culturing for D7 days, blowing, mixing, sampling and counting the cells, and counting the results: WBC 1.0X 109L, transferring the cells into a new T-175 flask,adding 32ml of T lymphocyte complete culture medium according to counting results, and adjusting the cell concentration to 0.5 multiplied by 109/L, mixing, adding 5% C0 at 37 deg.C2The incubator continues to culture.
(8) Culturing for D8 days, sampling and counting the cells uniformly mixed by blowing, and counting the results: WBC 1.0X 109L, adding 64ml of complete T lymphocyte culture medium according to counting results, and adjusting the cell concentration to 0.5X 109/L, mixing, adding 5% C0 at 37 deg.C2The incubator continues to culture.
(9) Culturing for D9 days, sampling and counting the cells uniformly mixed by blowing, and counting the results: WBC 1.1X 109L, transferring the cells into a new cell culture bag, supplementing 148.36ml of T lymphocyte complete culture medium according to counting results, and adjusting the cell concentration to 0.5 multiplied by 109Adding 5.64ml of coating solution of CD3 and CD28 monoclonal antibodies into the culture bag according to the ratio of 20ng/ml of CD3 and CD28 monoclonal antibodies to the culture bag, uniformly mixing, placing at 37 ℃, and 5% C02The incubator continues to culture.
(10) Culturing for D10 days, sampling and counting the cells uniformly mixed by blowing, and counting the results: WBC 0.9X 109Per L, 226ml of complete T lymphocyte culture medium is added according to counting results, and the cell concentration is adjusted to 0.5X 109/L, mixing, adding 5% C0 at 37 deg.C2The incubator continues to culture.
(11) Culturing for D11 days, sampling and counting the cells uniformly mixed by blowing, and counting the results: WBC 0.9X 109Adding 688ml of T lymphocyte complete culture medium according to counting results, and adjusting the cell concentration to 0.5X 109/L, mixing, adding 5% CO at 37 deg.C2The incubator continues to culture.
(12) Culturing for D12 days, sampling and counting the cells uniformly mixed by blowing, and counting the results: WBC 1.0X 109Adding 915ml of complete T lymphocyte culture medium according to counting results, and adjusting cell concentration to 0.5 × 109L, mixing, adding 5% C0 at 37 deg.C2The incubator continues to culture. The total liquid volume reached 1830ml at this point.
(13) Culturing for D13 days, sampling and counting the cells uniformly mixed by blowing, and counting the results: WBC 1.2X 109/L, placing at 37 ℃ with 5% C02The incubator continues to culture.
(14) Culturing for D14 days, sampling and counting the cells uniformly mixed by blowing, and counting the results: WBC 1.5X 109/L, placing at 37 ℃ with 5% C02The incubator continues to cultivate.
(15) Culturing for D15 days, sampling and counting the cells uniformly mixed by blowing, and counting the results: WBC 1.8X 109/L, placing at 37 ℃ with 5% C02The incubator continues to culture.
(16) Culturing for D16 days, blowing and mixing the cells uniformly, sampling, counting the survival rate, and counting the result: WBC 2.2X 109L, total number of cells: 4.026X 109. 2.3ml of cell suspension (5X 10) was taken6Individual cells) were subjected to surface marker detection (see figure 2 for results). The rest cell suspension was harvested into 4 500ml centrifuge cups, the supernatant was discarded after balancing 500g and centrifuging for 10min, and the cells were resuspended in 2 500ml centrifuge cups using two bottles of wash solution (500 ml sodium chloride injection per bottle with 1ml human serum albumin, human serum albumin concentration 0.2%). After the cells are resuspended, the cells are balanced, the cells are washed for 3 times by 500g centrifugation for 10min, 200ml of preservation solution is used for resuspending the cells (the preservation solution is 194ml of sodium chloride injection and 6ml of human serum albumin, the concentration of the human serum albumin is 3 percent), and the T lymphocyte preparation with the tumor specific killing effect is prepared. The amplification curve is shown in FIG. 3 (3 a: cell amplification concentration curve; 3 b: cell amplification amount curve), and the cell viability is shown in Table 1.
Table 1: d16 day cell viability
Figure DEST_PATH_IMAGE001
Example 2
Sample information: male, age 63, post hepatoma surgery, from the fifth medical center of the general hospital for the liberty military.
Preparation of T lymphocyte preparation with tumor-specific killing effect:
(1) extraction of seed cells: tumor tissue obtained by surgery, 1.1cm × 0.7cm × 1.0 cm. After washing once with 10ml 1640 medium, the tissue was placed on a sterile petri dish and cut into 2-3mm sections with a scalpel and forceps3The tissue blocks of (1) are put into 50ml bagsThe heart tube was supplemented with 15ml of a digestion solution (digestion solution components: 13.5ml of 1640 medium and 1.5ml of type I collagenase stock solution (1 g/ml of type I collagenase contained in the 1640 medium)), mixed and digested at 37 ℃ for 30min with shaking in a constant temperature shaker.
(2) T lymphocyte culture: coating 1T-25 culture flask with 1ml of coating solution containing CD3 and CD28 monoclonal antibodies for 0.5h (coating solution component: T lymphocyte complete culture medium containing CD3 and CD28 monoclonal antibodies at concentration of 1 ug/ml), and the content of the two monoclonal antibodies is 40ng/cm2. Filtering the digested tissue by a 200-micron filter screen into 1 new 50ml centrifuge tube, supplementing 30ml 1640 culture medium, centrifuging for 10min, discarding the supernatant, suspending and precipitating with 4ml T lymphocyte complete culture medium, adding into the coated T-25 culture bottle, supplementing IFN-gamma to the final concentration of 1500IU/ml, mixing, adding into 37 deg.C, and 5% C02The incubator is used for cultivation. The complete culture medium of T lymphocytes is X-VIVO 15 medium (LONZA/04-418Q) comprising 10% volume fraction of fetal bovine serum, 2% volume fraction of MEM-Eagle Amino Acids Solution (BI/01-325-1B), and 4000IU/ml of IL-2.
(3) The cell state is observed every day, a small amount of cell masses can be seen under a D3 day lens of culture (see figure 4), the cells are uniformly blown and sampled for counting, and the counting result is as follows: WBC 0.2X 109L is the ratio of the total weight of the composition to the total weight of the composition. Placing at 37 deg.C with 5% C02The incubator continues to cultivate.
(4) Culturing for D4 days, sampling and counting the cells uniformly mixed by blowing, and counting the results: WBC 0.4X 109/L, placing at 37 ℃ with 5% C02The incubator continues to culture.
(5) Culturing for D5 days, sampling and counting the cells uniformly mixed by blowing, and counting the results: WBC 1.1X 109L, adding 6ml of complete T lymphocyte culture medium according to counting results, and adjusting the cell concentration to 0.5X 109L, mixing, adding 5% C0 at 37 deg.C2The incubator continues to culture.
(6) Culturing for D6 days, sampling and counting the cells uniformly mixed by blowing, and counting the results: WBC 1.2X 109L, coating 1T-75 culture flask with 3ml of coating solution of CD3 and CD28 monoclonal antibodies for 0.5h for later use, wherein the content of the two monoclonal antibodies is 40ng/cm2. Transferring the cells to coated T-75 culture flasks, and countingAdding 12ml of complete culture medium of T lymphocytes, and adjusting the cell concentration to 0.5X 109and/L, adding IFN-gamma to a final concentration of 1500 IU/ml. Mixing, adding 5% C0 at 37 deg.C2The incubator continues to culture.
(7) Culturing for D7 days, sampling and counting the cells uniformly mixed by blowing, and counting the results: WBC 0.8X 109L, transferring the cells into a new T-175 culture flask, supplementing 16ml of T lymphocyte complete culture medium according to counting results, and adjusting the cell concentration to 0.5X 109/L, mixing, adding 5% C0 at 37 deg.C2The incubator continues to culture.
(8) Culturing for D8 days, sampling and counting the cells uniformly mixed by blowing, and counting the results: WBC 1.0X 109L, adding 42ml of T lymphocyte complete culture medium according to counting results, and adjusting the cell concentration to 0.5X 109/L, mixing, adding 5% C0 at 37 deg.C2The incubator continues to culture.
(9) Culturing for D9 days, sampling and counting the cells uniformly mixed by blowing, and counting the results: WBC 1.1X 109Per L, according to 20ng/cm2Supplementing 3.7ml of CD3 and CD28 monoclonal antibody coating solution into the culture bag, transferring the cells into a new cell culture bag, supplementing 97.3ml of T lymphocyte complete culture medium according to counting results, and adjusting the cell concentration to 0.5X 109/L, mixing, adding 5% C0 at 37 deg.C2The incubator continues to culture.
(10) Culturing for D10 days, sampling and counting the cells uniformly mixed by blowing, and counting the results: WBC 1.2X 109L, adding 259ml of complete T lymphocyte culture medium according to counting results, and adjusting cell concentration to 0.5X 109L, mixing, adding 5% C0 at 37 deg.C2The incubator continues to culture.
(11) Culturing for D11 days, sampling and counting the cells uniformly mixed by blowing, and counting the results: WBC 1.0X 109L, supplementing 444ml of T lymphocyte complete culture medium according to counting results, and adjusting the cell concentration to 0.5X 109L, mixing, adding 5% C0 at 37 deg.C2The incubator continues to culture.
(12) Culturing for D12 days, sampling and counting the cells uniformly mixed by blowing, and counting the results: WBC 0.9X 109L, adding 712ml of T lymphocytes to complete culture according to counting resultsCulturing, adjusting cell concentration to 0.5 × 109/L, mixing, adding 5% C0 at 37 deg.C2The incubator continues to culture. The total volume of liquid has reached 1600 ml.
(13) Culturing for D13 days, sampling and counting the cells uniformly mixed by blowing, and counting the results: WBC 1.1X 109/L, placing at 37 ℃ with 5% C02The incubator continues to cultivate.
(14) Culturing for D14 days, sampling and counting the cells uniformly mixed by blowing, and counting the results: WBC 1.3X 109/L, placing at 37 ℃ with 5% C02The incubator continues to culture.
(15) Culturing for D15 days, sampling and counting the cells uniformly mixed by blowing, and counting the results: WBC 1.5X 109/L, placing at 37 ℃ with 5% C02The incubator continues to culture.
(16) Culturing for D16 days, sampling and counting the cells uniformly mixed by blowing, and counting the results: WBC 1.8X 109/L, placing at 37 ℃ with 5% C02The incubator continues to culture.
(17) Culturing for D17 days, sampling and counting the cells uniformly mixed by blowing, and counting the results: WBC 2.1X 109/L, placing at 37 ℃ with 5% C02The incubator continues to culture.
(18) Culturing for D18 days, blowing and mixing the cells uniformly, sampling, counting the survival rate, and counting the result: WBC 2.7X 109L, total number of cells: 4.31X 109. 1.9ml of cell suspension (5X 10) was taken6Individual cells) were tested for surface markers (see figure 5 for results), the remaining cell suspension was harvested into 4 500ml centrifuge cups, the 500g was trimmed and centrifuged for 10min and the supernatant discarded, and the cells were resuspended in 2 500ml centrifuge cups using two bottles of wash solution (500 ml sodium chloride injection plus 1ml human albumin per bottle of wash solution, human albumin content 0.2%). After the cells are resuspended, the cells are balanced, the cells are washed for 3 times by 500g centrifugation for 10min, 200ml of preservation solution is used for resuspending the cells (the preservation solution is 194ml of sodium chloride injection and 6ml of human serum albumin, the content of the human serum albumin is 3 percent), and the T lymphocyte preparation with the tumor specific killing effect is prepared. The amplification curve is shown in FIG. 6 (6 a: cell amplification concentration curve; 6 b: cell amplification number curve), and the cell viability is shown in Table 2.
Table 2: d18 day cell viability
Figure 154263DEST_PATH_IMAGE002
The embodiments of the present invention are preferred embodiments of the present invention, and the scope of the present invention is not limited by these embodiments, so: all equivalent changes made according to the structure, shape and principle of the invention are covered by the protection scope of the invention.

Claims (7)

1. A method for preparing T lymphocytes with tumor specific killing effect from tumor tissues, which is characterized by comprising the following steps: the method comprises the following steps:
s1, cleaning and dividing tumor tissues, and putting the tumor tissues into digestive juice to perform shake digestion for 30-40 min;
s2, filtering, washing and centrifuging after tissue digestion, adding the suspension sediment after the complete culture basis weight of the T lymphocytes into a culture bottle coated in advance, supplementing IFN-gamma to the final concentration of 1000-1500IU/ml, and carrying out cell culture; the method for coating the culture flask comprises the following steps: the culture flask is coated with complete culture medium containing CD3 and CD28 monoclonal antibody for T lymphocyte and is placed at 37 deg.C and 5.0% CO2Under the condition of 0.5-1 h;
s3, observing cell state every day, sampling and counting, supplementing a complete T lymphocyte culture medium according to counting results, and adjusting the cell concentration to be 0.5-0.8 multiplied by 109L, continuing cell culture;
s4, continuously observing the cell state every day, sampling and counting when the culture system reaches 10-15ml, transferring the cells into a culture bottle coated in advance, wherein the coating method is the same as the step S2; adding complete culture medium of T lymphocyte according to counting result, adjusting cell concentration to 0.5-0.8 × 109L, adding IFN-gamma to the final concentration of 1000-1500IU/ml, and continuing cell culture;
s5, continuously observing the cell state every day, sampling and counting, when the culture system reaches 25-45ml, transferring the cells into another culture bottle, supplementing a complete culture medium of the T lymphocytes according to counting results, and adjusting the cell concentration to be 0.5-0.8 multiplied by 109L, continuing cell culture;
s6, continuously observing the cell state every day, sampling and counting, when the culture system reaches 180-200ml, transferring the cells into a cell culture bag, supplementing CD3 and CD28 monoclonal antibodies into the culture bag according to 20-50ng/ml, supplementing a complete culture medium of the T lymphocytes according to the counting result, and adjusting the cell concentration to be 0.5-0.8 multiplied by 109L, continuing cell culture;
s7, continuously observing the cell state every day, sampling and counting, when the culture system reaches 1600-9At the above time, all cells were harvested.
2. The method according to claim 1, wherein the T lymphocytes having a tumor-specific killing effect are derived from tumor tissue, and the method comprises: in step S1, 10-15ml of digestive juice is added to each cubic centimeter of tissue mass.
3. The method according to claim 1, wherein the T lymphocytes having a tumor-specific killing effect are derived from tumor tissue, and the method comprises: in step S1, the digestion solution is 1640 medium containing 0.075-0.15g/ml collagenase type I.
4. The method according to claim 1, wherein the T lymphocytes having a tumor-specific killing effect are derived from tumor tissue, and the method comprises: the complete culture medium of T lymphocytes is X-VIVO 15 medium containing 10-20 v% fetal bovine serum, 2 v% 50X essential amino acids and 4000-6000IU/ml IL-2.
5. The method according to claim 1, wherein the T lymphocytes having a tumor-specific killing effect are derived from tumor tissue, and the method comprises: in step S2, the content of the two monoclonal antibodies CD3 and CD28 is 20-50ng/cm2。
6. T lymphocytes having a tumor-specific killing effect, which are derived from tumor tissues, and which are obtained by the production method according to any one of claims 1 to 5.
7. A T lymphocyte preparation having a tumor-specific killing effect, comprising: comprising the tumor-tissue-derived T lymphocyte with a tumor-specific killing effect according to claim 6.
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