CN108148805B - Human Tsccm cell and preparation method and application thereof - Google Patents
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Abstract
The invention belongs to the field of biological immunology, and particularly relates to a human Tsccm cell and a preparation method and application thereof, wherein the preparation method comprises the following steps: (1) collecting human umbilical cord blood, and obtaining human umbilical cord blood mononuclear cells by adopting a density gradient centrifugation method; (2) sorting the CD8+ CD45RA + CCR7+ primary T cell subpopulation using a flow cytometric sorter; (3) adding the sorted primary T cell subsets to a culture medium containing stimulators CD3/CD28beads, cytokines IL-2 and metformin (metformin) for culture; (4) and changing the solution every 2d and supplementing the cytokine IL-2, and expanding to 14d to obtain the CD8+ T cells with the Tsccm phenotype. The method of the invention can obtain a large amount of T cells with memory property and low differentiation state for treating tumors.
Description
Technical Field
The invention belongs to the field of biological immunology, and particularly relates to a human Tsccm cell and a preparation method and application thereof.
Background
With the continuous and deep research in the biomedical field and the changing day by day of the theoretical technology of immunology, immunotherapy has become a new hope for overcoming tumors in human beings. Adoptive immunotherapy, which is the infusion of immunocompetent cells, as an important component of immunotherapy, refers to the infusion of in vitro activated immune effector cells into a patient to kill tumor cells in the patient. But this therapy also faces significant challenges while meeting the expectations: the immune cells infused into the patient by adoptive cellular immunotherapy are in a terminal differentiation state, have short survival time in vivo and limited anti-tumor effect. How to obtain more young, durable and stable anti-tumor cells is a hot spot of current immunotherapy research.
CD8+ memory T cells play an important role in anti-tumor responses and are divided into different subgroups, stem cell-like T cells (Tscm), central memory T cells (Tcm) and effector memory T cells (Tem). Of these, the Tsccm subset is of great interest because of its good self-renewal capacity and pluripotent potential and its greater ability to kill tumor cells.
At present, the method for culturing and expanding Tsccm cells at home and abroad cannot meet the actual clinical requirements, on one hand, most of blood sources come from patients or the peripheral blood of relatives of the patients, so that the acquisition of initial cells is greatly limited; on the other hand, the Tsccm cells are not suitable for stable maintenance in vitro culture and are easy to be further differentiated, so that the number of the Tsccm cells obtained by culture is small. Therefore, how to solve the problem of obtaining initial cells and maintaining the Tsccm cells is the biggest bottleneck of the in vitro culture of the Tsccm at present.
Metformin (metformin) is a safe and efficient biguanide hypoglycemic agent, and is used as an insulin sensitizer, and metformin can effectively reduce the blood insulin level by reducing hepatic gluconeogenesis, increasing the sensitivity of peripheral tissues to insulin, inhibiting glucose absorption by intestinal tract cells to play a role in reducing blood glucose. Meanwhile, in the research of cell energy metabolism, metformin is found to be an agonist of an energy regulation signal molecule adenylate activated protein kinase (AMPK), and activates the AMPK to negatively regulate a downstream mammalian rapamycin target (mTOR) pathway.
Disclosure of Invention
The invention mainly provides a human Tsccm cell and a preparation method and application thereof, and a large number of T cells with memory characteristics and low differentiation state can be obtained for treating tumors. The technical scheme is as follows:
a method for preparing human Tsccm cells, comprising the steps of:
(1) collecting human umbilical cord blood, and obtaining human umbilical cord blood mononuclear cells by adopting a density gradient centrifugation method;
(2) sorting the CD8+ CD45RA + CCR7+ primary T cell subpopulation using a flow cytometric sorter;
(3) adding the sorted primary T cell subset into a culture medium containing stimulators CD3/CD28beads, cytokines IL-2 and metformin for culture;
(4) and changing the solution every 2d and supplementing the cytokine IL-2, and expanding to 14d to obtain the CD8+ T cells with the Tsccm phenotype.
Preferably, the specific method for obtaining the human umbilical cord blood mononuclear cells in the step (1) is to collect human umbilical cord blood under the aseptic condition, firstly centrifuge the human umbilical cord blood under the conditions of 1500rpm and 10min, transfer serum into a sterile centrifuge tube to inactivate at 56 ℃ for 25min, then centrifuge the serum at 4000rpm and 20min, dilute the precipitated blood cells with a buffer solution, slowly add the blood cell diluent to the lymph separation solution, centrifuge the blood cell diluent under the conditions of 2500rpm and 25min, finally suck a leucocyte layer, wash the leucocyte layer with the buffer solution, and centrifuge the human umbilical cord blood mononuclear cells at 1500rpm and 10min to obtain the human umbilical cord blood mononuclear cells.
Preferably, the buffer solution is PBS buffer solution, when the precipitated blood cells are diluted by the buffer solution, the PBS buffer solution with the blood cell amount of 1/2-1/4 is used for dilution, and the volume amount of the lymph separation liquid is 1/2-1/4 of the volume amount of the blood cell dilution liquid.
Preferably, the step (2) of sorting the initial T cell subpopulation using a flow cytometric sorter is performed in a manner of every 2X 1061-3 μ L of antibodies, CD3-FITC, CD8-APC-cy-7, CD45RA-APC and CCR7-Percp, were added to individual monocytes in the dark and flow sorted for CD8+ CD45RA + CCR7+ naive T cells.
Preferably, the volume of the stimulant CD3/CD28beads in step (3) is 1/3 of the initial T cell volume, the concentration of the cytokine IL-2 is 500-1000IU/mL, and the concentration of metformin is 50-150 μ M.
Preferably, the medium used in step (3) is Takara GT-551 medium, and the culture conditions are 37 ℃ and 5% CO2And (4) environment.
Preferably, the culture medium in step (3) further comprises 5% volume of autologous serum, penicillin and streptomycin, and the density of T cells in the culture medium is (1-3). times.106/mL。
Preferably, the expansion of CD8+ T cells to 14d is up to 180-220 fold.
A human Tscm cell obtained by the above method.
The human Tsccm cells can be applied to preparations for treating tumor diseases.
The method for preparing the human Tsccm cells has the following advantages:
compared with the traditional method for obtaining the initial immune cells from the peripheral blood, the method has the following unique advantages: the umbilical cord blood has rich sources, unique biological and immunological characteristics and contains a large amount of initial T cells, so that the stem cell-like T cells are more easily induced, and the difficulty in obtaining the initial cells is perfectly solved;
in an in vitro experiment, the first research shows that metformin can activate an AMPK signal path of a T cell to maintain the number of Tsccm cells and inhibit the further differentiation of the Tsccm cells, so that the in vitro culture and amplification efficiency of the Tsccm cells is greatly improved, and the high-efficiency anti-tumor capacity of the Tsccm cells is maintained while the self-renewal capacity and the pluripotent potential of the group of cells are maintained.
Obtaining initial T cells from umbilical cord blood, adding CD3/CD28beads and IL-2 to induce activation, and using metfromin to maintain the proportion of Tsccm cells, thereby obtaining a large amount of T cells with memory characteristics and low differentiation state, and further obtaining T cells with high antitumor activity and long in-vivo survival time for clinical adoptive cellular immunotherapy.
The method can realize the in vitro mass culture and amplification of the Tsccm cells, can effectively promote the further development of cellular immunotherapy, solves the technical bottleneck of the current clinical culture, prolongs the survival time of the adoptive immune cells in the body of a patient, enhances the lasting and efficient anti-tumor capability of the adoptive immune cells, and lays a solid foundation for the further development of large-scale clinical therapy of the cellular immunotherapy.
Drawings
FIG. 1 is a diagram showing a ratio of cord blood mononuclear cells Tn cells;
FIG. 2 is a diagram showing a Tn cell ratio of adult peripheral blood mononuclear cells;
FIG. 3 is a statistical chart of Tn cells of cord blood mononuclear cells and peripheral blood mononuclear cells;
FIG. 4 is a diagram showing a cell ratio of Tsccm prepared in comparative example 1;
FIG. 5 is a diagram showing a cell ratio of Tsccm prepared in example 1;
FIG. 6 is a statistical plot of the proportion of Tsccm cells prepared in comparative example 1 and example 1;
FIG. 7 is a graph showing the expression of the killing marker CD107a in the method of comparative example 1 by flow assay;
FIG. 8 is a graph showing the expression of the killing marker CD107a in the method of example 1;
FIG. 9 is a statistical graph of the killing ratio of Tsccm cells prepared in comparative example 1 and example 1.
Detailed Description
Example 1
Collecting 50mL of cord blood under aseptic condition, and obtaining human cord blood mononuclear cells CBMCs by adopting a density gradient centrifugation method, which comprises the following steps: centrifuging at 1500rpm for 10min, with the acceleration of acceleration and deceleration being 9m/s2Transferring the serum into a sterile 50mL centrifuge tube, inactivating the serum at 56 ℃ for 25min, centrifuging the serum at 4000rpm for 20min, wherein the acceleration of the acceleration and the deceleration is 9m/s2. The precipitated blood cells were diluted with PBS buffer at a ratio of 1:3 to the amount of blood cells, and then the blood cell dilution was slowly added to the lymph separation medium in an amount 1/3 by volume based on the volume of the blood cell dilution medium. Finally, centrifuging at 2500rpm for 25min, wherein the acceleration of acceleration and deceleration is 5m/s2Carefully sucking the leucoderma layer, washing twice with PBS buffer solution, centrifuging at 1500rpm for 10min, and accelerating and decelerating at 9m/s2Obtaining the cord blood mononuclear cells.
Counting the separated CBMCs at each 2X 106And adding 2 mu L of flow antibodies which are CD3-FITC, CD8-APC-cy-7, CD45RA-APC and CCR7-Percp into the cells in a dark place, and carrying out flow sorting on CD8+ CD45RA + CCR7+ T cells.
The sorted initial T cell subsets were placed in GT551 medium containing 1/3T cell subset volumetric amounts of the stimulants CD3/CD28beads and 1000IU/mL IL-2, with 5% autologous serum, cyanStreptomycin and streptomycin, and adjusting cell density to 2 × 106and/mL. At 37 ℃ 5% CO2Culturing in incubator, adding 100 μ M metformin after 24 hr, observing cell growth state every 2d, half-changing liquid and supplementing full amount of cytokine IL-2. When the cells are cultured to 7d, 90 percent of CD8+ T cells with the Tscm phenotype can be obtained, and the amplification factor reaches 220 times when the cells are expanded to 14 d.
Among the reagents used above, the lymphocyte separation medium was selected from the third-class Biotechnology, Inc., available in Tianjin, GT-551 medium was selected from Takara, IL-2 was produced by Beijing Shuanglu pharmaceutical Co., Ltd, metformin was obtained from sigma, CD3/CD28beads was produced by Life technology, and the flow antibody was obtained from biologid.
Example 2
Collecting 50mL of cord blood under aseptic condition, and obtaining human cord blood mononuclear cells CBMCs by adopting a density gradient centrifugation method, which comprises the following steps: centrifuging at 1500rpm for 10min, with the acceleration of acceleration and deceleration being 9m/s2Transferring the serum into a sterile 50mL centrifuge tube, inactivating the serum at 56 ℃ for 25min, centrifuging the serum at 4000rpm for 20min, wherein the acceleration of the acceleration and the deceleration is 9m/s2. The precipitated blood cells were diluted with PBS buffer at a ratio of 1:2 to the amount of blood cells, and then the blood cell dilution was slowly added to the lymph separation medium in an amount 1/4 by volume based on the volume of the blood cell dilution medium. Finally, centrifuging at 2500rpm for 25min, wherein the acceleration of acceleration and deceleration is 5m/s2Carefully sucking the leucoderma layer, washing twice with PBS buffer solution, centrifuging at 1500rpm for 10min, and accelerating and decelerating at 9m/s2Obtaining the cord blood mononuclear cells.
Counting the separated CBMCs at each 2X 106mu.L of antibodies, CD3-FITC, CD8-APC-cy-7, CD45RA-APC and CCR7-Percp, were added to the cells in the dark, and flow sorted for CD8+ CD45RA + CCR7+ T cells.
The sorted initial T cell subsets were placed in GT551 medium containing 1/2T cell subset volumetric amounts of stimulants CD3/CD28beads and 900IU/mL IL-2, along with 5% autologous serum, penicillinAnd streptomycin, adjusting cell density to 3 × 106and/mL. At 37 ℃ 5% CO2Culturing in incubator, adding 50 μ M metformin after 24 hr, observing cell growth state every 2d, half-changing liquid and supplementing full amount of cytokine IL-2. When the cells are cultured to the 7 th day, 80% of CD8+ T cells with the Tsccm phenotype can be obtained, and the amplification times reach 180 times when the cells are expanded to the 14 th day.
Among the reagents used above, the lymphocyte separation medium was selected from the third-class Biotechnology, Inc., available in Tianjin, GT-551 medium was selected from Takara, IL-2 was produced by Beijing Shuanglu pharmaceutical Co., Ltd, metformin was obtained from sigma, CD3/CD28beads was produced by Life technology, and the flow antibody was obtained from biologid.
Example 3
Collecting 50mL of cord blood under aseptic condition, and obtaining human cord blood mononuclear cells CBMCs by adopting a density gradient centrifugation method, which comprises the following steps: centrifuging at 1500rpm for 10min, with the acceleration of acceleration and deceleration being 9m/s2Transferring the serum into a sterile 50mL centrifuge tube, inactivating the serum at 56 ℃ for 25min, centrifuging the serum at 4000rpm for 20min, wherein the acceleration of the acceleration and the deceleration is 9m/s2. The precipitated blood cells were diluted with PBS buffer at a ratio of 1:4 to the amount of blood cells, and then the blood cell dilution was slowly added to the lymph separation medium in an amount 1/2 by volume based on the volume of the blood cell dilution medium. Finally, centrifuging at 2500rpm for 25min, wherein the acceleration of acceleration and deceleration is 5m/s2Carefully sucking the leucoderma layer, washing twice with PBS buffer solution, centrifuging at 1500rpm for 10min, and accelerating and decelerating at 9m/s2Obtaining the cord blood mononuclear cells.
Counting the separated CBMCs at each 2X 1063 μ L of antibodies, CD3-FITC, CD8-APC-cy-7, CD45RA-APC and CCR7-Percp, were added to the cells in the dark, and flow sorted for CD8+ CD45RA + CCR7+ T cells.
The sorted initial T cell subsets were placed in GT551 medium containing 1/4T cell subset volumetric amounts of the stimulants CD3/CD28beads and 500IU/mL IL-2, along with 5% autologous serum, penicillin and streptomycesAdjusting cell density to 1 × 106and/mL. At 37 ℃ 5% CO2Culturing in incubator, adding 150 μ M metformin after 24 hr, observing cell growth state every 2d, half-changing liquid and supplementing full amount of cytokine IL-2. When the cells are cultured to 7d, 85% of CD8+ T cells with the Tscm phenotype can be obtained, and the amplification times of the cells are up to 200 times when the cells are expanded to 14 d.
Among the reagents used above, the lymphocyte separation medium was selected from the third-class Biotechnology, Inc., available in Tianjin, GT-551 medium was selected from Takara, IL-2 was produced by Beijing Shuanglu pharmaceutical Co., Ltd, metformin was obtained from sigma, CD3/CD28beads was produced by Life technology, and the flow antibody was obtained from biologid.
Comparative example 1
This example differs from example 1 in that metformin was not added and other culture conditions were consistent.
Detection and results
The phenotype, activity and metformin of the Tsccm cells were examined for the maintenance and functional enhancement of Tsccm using the sample from example 1. The specific operation steps are as follows:
1. the cord blood mononuclear cells obtained in example 1 were taken and compared with adult peripheral blood for Tn cell ratio, and the results are shown in FIGS. 1 to 3. Wherein, FIG. 1 is the ratio of cord blood mononuclear cells Tn cells detected by flow, FIG. 2 is the ratio of adult peripheral blood mononuclear cells Tn cells detected by flow, and FIG. 3 is the statistical chart of cord blood mononuclear cells and peripheral blood mononuclear cells Tn cells. As shown in FIGS. 1-3, the Tn cells of umbilical cord blood mononuclear cells are lower in content than those of peripheral blood mononuclear cells, so that the induction activation of the Tn cells of umbilical cord blood mononuclear cells is required to obtain a large amount of T cells with memory characteristics and low differentiation state.
Tsccm cell morphology and activity assays: observing the cell state under a microscope, culturing until the 3 rd cell is aggregated and rapidly grows, and culturing until the 7 th cell is subjected to flow cytometry to detect the apoptosis condition of the cell. Collecting 1X 106Resuspending the cells in 200. mu.L Annexin-V binding buffer, adding 2. mu.L Annexin-V-APC antibody in dark place, incubating in dark at 4 ℃ for 15min, and addingAdding PI before the machine.
Tsccm cell phenotype and function assay: collecting 1X 10 cells cultured to 7d and 14d6Each was resuspended in 200 μ L PBS containing 1% FBS (fetal bovine serum) and flow antibody was added in the dark: CD8-APC-cy-7, CD45RA-APC, CCR7-Percp and CD95-PE-cy-7, incubated at 4 ℃ for 15min in the absence of light, and the phenotype of Tsccm is detected by flow. The phenotype results of the cells obtained by analyzing and culturing by using Diva software show that the proportion of Tsccm reaches 95 percent and is higher than that of the Tsccm cells obtained by inducing and culturing peripheral blood by more than 30 percent.
4. The ratio of the Tscm cells prepared in example 1 to the Tscm cells prepared in comparative example 1 was measured by flow assay and the results are shown in fig. 4-6, where fig. 4 is the ratio of the Tscm cells prepared in comparative example 1, fig. 5 is the ratio of the Tscm cells prepared in example 1, and fig. 6 is a statistical graph of the ratio of the Tscm cells prepared in comparative example 1 and example 1. From the results, it was found that the method of example 1 produces Tsccm cells at a high ratio, and facilitates induction and activation of the Tsccm cells.
5. The Tscm cells prepared in example 1 and the Tscm cells prepared in comparative example 1 were incubated with the lung cancer tumor cell line a549 and the esophageal cancer tumor cell line TE7 respectively, and the expression of the Tscm cell killing marker CD107a was detected, with the results shown in fig. 7-9, wherein fig. 7 is a flow detection of the expression of the method killing marker CD107a in comparative example 1, fig. 8 is a flow detection of the expression of the method killing marker CD107a in example 1, fig. 9 is a statistical graph of the killing ratio of the Tscm cells prepared in comparative example 1 and example 1, and fig. 7-9 show that the Tscm cell killing function prepared in the present invention is 5-10 times higher than the Tscm cell killing function induced by the method in example 1, and has a strong anti-tumor effect.
The results of the Tsccm cells prepared in examples 2-3 after the above-described measurement are similar to those of example 1, and are not described herein again.
Various other modifications and changes may be made by those skilled in the art based on the above-described technical solutions and concepts, and all such modifications and changes should fall within the scope of the claims of the present invention.
Claims (6)
1. A method for producing human stem cell-like T cells, comprising: the method comprises the following steps:
(1) collecting human umbilical cord blood, and obtaining human umbilical cord blood mononuclear cells by adopting a density gradient centrifugation method;
(2) sorting the CD8+ CD45RA + CCR7+ primary T cell subpopulation using a flow cytometric sorter;
(3) adding the sorted primary T cell subsets to a culture medium containing stimulators CD3/CD28beads, cytokines IL-2 and metformin for culture;
(4) changing the solution every 2 days, supplementing a cytokine IL-2, and amplifying to 14 days to obtain CD8+ T cells with the phenotype of stem cell-like T cells;
the specific method for sorting the initial T cell subsets in step (2) by using a flow cytometric sorter is that the T cell subsets are sorted every 2 x 106Adding 1-3 mu L of antibodies which are CD3-FITC, CD8-APC-cy-7, CD45RA-APC and CCR7-Percp into each mononuclear cell in a dark place, and carrying out flow sorting on CD8+ CD45RA + CCR7+ initial T cells;
the volume amount of the stimulants CD3/CD28beads in the step (3) is 1/2-1/4 of the volume amount of the initial T cells, the concentration of the cytokine IL-2 is 500-1000IU/mL, and the concentration of the metformin is 50-150 mu M.
2. The method for producing human stem cell-like T cells according to claim 1, wherein: the specific method for obtaining the human umbilical cord blood mononuclear cells in the step (1) comprises the steps of collecting human umbilical cord blood under the aseptic condition, centrifuging at 1500rpm for 10min, transferring serum into an aseptic centrifuge tube, inactivating the serum at 55-57 ℃ for 20-30min, centrifuging the serum at 4000rpm for 20min, diluting precipitated blood cells with a buffer solution, slowly adding a blood cell diluent to a lymph separation solution, centrifuging at 2500rpm for 25min, sucking a leucocyte layer, cleaning with the buffer solution, and centrifuging at 1500rpm for 10min to obtain the human umbilical cord blood mononuclear cells.
3. The method for producing human stem cell-like T cells according to claim 2, wherein: the buffer solution is PBS buffer solution, when the precipitated blood cells are diluted by the buffer solution, the PBS buffer solution with the blood cell amount of 1/2-1/4 is adopted for dilution, and the volume amount of the lymph separation liquid is 1/2-1/4 of the volume amount of the blood cell dilution liquid.
4. The method for producing human stem cell-like T cells according to claim 1, wherein: the culture medium in the step (3) is Takara GT-551 culture medium, and the culture conditions are 37 ℃ and 5% CO2And (4) environment.
5. The method for producing human stem cell-like T cells according to claim 1, wherein: the culture medium in the step (3) also comprises 5% volume of autologous serum, penicillin and streptomycin, and the density of T cells in the culture medium is (1-3) multiplied by 106/mL。
6. The method for producing human stem cell-like T cells according to claim 1, wherein: the expansion time of CD8+ T cells reaches 180-220 times when the cells are expanded to 14 d.
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