CN113817676A - Peripheral blood T cell culture method - Google Patents

Peripheral blood T cell culture method Download PDF

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CN113817676A
CN113817676A CN202111309326.9A CN202111309326A CN113817676A CN 113817676 A CN113817676 A CN 113817676A CN 202111309326 A CN202111309326 A CN 202111309326A CN 113817676 A CN113817676 A CN 113817676A
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peripheral blood
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马永光
黄国喜
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Jilin Yikang Medical Technology Co.,Ltd.
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Abstract

The invention discloses a method for culturing peripheral blood T cells, which comprises the following steps: (1) collecting peripheral blood, and separating by adopting a Ficoll density gradient centrifugation method to obtain peripheral blood mononuclear cells; (2) adding the coating solution into the culture bottle for coating overnight, and removing the coating solution to serve as an induction culture bottle for later use; (3) resuspending the peripheral blood mononuclear cells in the step (1) by adopting an induction culture medium, and culturing in the culture bottle in the step (2) for 2-3 days to finish induction culture; (4) adding coating liquid into the culture bottle for coating overnight, and discarding the culture liquid to serve as a proliferation culture bottle for later use; (5) and (4) adding the cells subjected to the induction culture in the step (3) into the culture bottle in the step (4), and culturing by adopting a proliferation culture medium for 12 days continuously to complete the culture. The culture method provided by the invention comprises the steps of carrying out induction culture on peripheral blood mononuclear cells by using an induction culture medium, and then carrying out amplification on the cells by using a proliferation culture medium, so that the obtained T cells have good activity and large quantity.

Description

Peripheral blood T cell culture method
Technical Field
The invention relates to the field of cell culture, in particular to a method for culturing peripheral blood T cells.
Background
Cancer has been a great problem that afflicts mankind. Although treatment modalities such as surgery, chemotherapy, and radiotherapy can greatly improve the survival rate of cancer patients, these therapies are far from sufficient for saving millions of cancer patients all over the world. With the rapid development of biotechnology, tumor immune cell therapy has become the fourth major therapy in the field of cancer therapy, which can make up for the disadvantages of conventional therapies and is considered as the most promising therapeutic means in the 21 st century tumor comprehensive treatment mode.
The cellular immunotherapy is a novel biological treatment technology with obvious curative effect, and the novel treatment method for improving autoimmunity and resisting cancer mainly utilizes the biological technology and biological agents to carry out in-vitro culture and improvement on self immune cells collected by a patient and then return the self immune cells to the patient, so as to stimulate, identify and guide the immune cells to kill and kill tumor cells, thereby enhancing the autoimmunity of the body and achieving the effect of treating tumors. In the field of genetically modified immune cell therapy, CAR-T therapy (chimeric antigen receptor T cell immunotherapy), TCR-T therapy (T cell receptor modified T cell therapy) have become hot spots for research in recent years.
T cells are derived from bone marrow pluripotent stem cells. In human embryonic and primary stages, a part of pluripotent stem cells or pre-T cells in bone marrow migrate into the thymus and differentiate and mature under the induction of thymic hormone to become T cells with immunocompetence. Mature T cells distribute to thymus dependent area of peripheral immune organ through blood stream to colonize, and can be recycled through lymphatic vessel, peripheral blood and tissue fluid, etc., to exert functions of cellular immunity and immunoregulation, etc.
T cells in the existing cellular immunotherapy are mainly used for tumor therapy by separating lymphocytes in blood, obtaining a certain amount of cells through in vitro culture and various cytokine stimulation induced amplification culture, and then returning the cells to a human body. The existing culture medium for the T cells mainly comprises a basic culture medium, autologous plasma (or animal serum) and various factors, the proliferation times of the cultured T cells are limited, the number of the cultured T cells is small, the T cells cannot be widely used for treatment, and the application of the T cells in immune cell treatment is limited.
Therefore, it is necessary to provide a culture method capable of efficiently culturing a large amount of T lymphocytes in a short period while maintaining the tumor killing ability.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for culturing peripheral blood T cells, which does not add autologous plasma and animal serum, has safe components and effectively improves the in-vitro proliferation efficiency of the T cells.
The purpose of the invention is realized by adopting the following technical scheme:
a method for culturing peripheral blood T cells, comprising the steps of:
(1) collecting peripheral blood, and separating by adopting a Ficoll density gradient centrifugation method to obtain peripheral blood mononuclear cells;
(2) adding physiological saline containing dibutyryladenosine cyclophosphate and human CD3 monoclonal antibody as coating solution into culture flask, and culturing at 37 deg.C and 5% CO2Coating overnight under the condition, and removing a coating solution to serve as an induction culture bottle for later use;
(3) resuspending the peripheral blood mononuclear cells in the step (1) by adopting an induction culture medium, and culturing the peripheral blood mononuclear cells in the culture bottle in the step (2) for 2-3 days to finish induction culture, wherein the induction culture medium consists of a basic culture medium and spirulina polysaccharide, allantoin, sodium pyruvate, IFN-gamma, interleukin-2 and interleukin-12 which are added in the basic culture medium;
(4) adding physiological saline containing ciclopirox and human CD3 monoclonal antibody as coating solution into culture flask, and culturing at 37 deg.C with 5% CO2Coating overnight under the condition, discarding the culture solution, and using as a proliferation culture bottle for later use;
(5) adding the cells subjected to the induction culture in the step (3) into the culture bottle in the step (4), culturing by adopting a proliferation culture medium for 12 days, and completing the continuous culture, wherein the proliferation culture medium comprises the following components: a basal medium, wherein the following components are added in the basal medium: bismuth potassium citrate, chitin, naringin, interleukin-2 and transforming growth factor-alpha.
Preferably, the concentration of dibutyryl cyclic adenosine monophosphate and the human CD3 monoclonal antibody in the physiological saline in the step (2) is as follows: 1.5-2 mu g/mL of dibutyryl cyclic adenosine monophosphate and 0.5-1 mu g/mL of human CD3 monoclonal antibody.
Preferably, the basic culture medium of the induction culture medium in the step (3) is X-VIVO culture medium, and the concentration of each component in the culture medium is 10-15ng/mL of spirulina polysaccharide, 5-10ng/mL of allantoin, 3-5 ng/mL of sodium pyruvate, 20-25 ng/mL of IFN-gamma, 230-35 ng/mL of interleukin and 1210-15 ng/mL of interleukin.
Preferably, the concentration of each component in the culture medium is 12ng/mL of spirulina polysaccharide, 8ng/mL of allantoin, 4ng/mL of sodium pyruvate, 22 ng/mL of IFN-gamma, 231 ng/mL of interleukin and 1213 ng/mL of interleukin.
Preferably, the concentration of ciclopirox and the human CD3 monoclonal antibody in the physiological saline in the step (4) is as follows: dibutyryl cyclic adenosine monophosphate 3.5-6.5ng/mL, human CD3 monoclonal antibody 3-5 μ g/mL.
Preferably, the basic culture medium of the proliferation culture medium in the step (5) is X-VIVO culture medium, and the concentration of each component in the culture medium is as follows: 1-5ng/mL of bismuth potassium citrate, 8-12ng/mL of chitin, 5-10ng/mL of naringin, 220-30 ng/mL of interleukin and 10-20 ng/mL of transforming growth factor-alpha.
Preferably, the concentrations in the respective component media are: 3ng/mL of bismuth potassium citrate, 10ng/mL of chitin, 8ng/mL of naringin, 225 ng/mL of interleukin and 15ng/mL of transforming growth factor-alpha.
Preferably, step (5) is performed by changing the medium every 2 to 3 days during the culture.
Compared with the prior art, the invention has the beneficial effects that: the invention provides a method for culturing peripheral blood T cells, which comprises the steps of respectively coating a culture bottle with physiological saline containing dibutyryl cyclic adenosine monophosphate and a human CD3 monoclonal antibody and physiological saline containing ciclopirox and a human CD3 monoclonal antibody before induction culture and proliferation culture, then performing induction culture on peripheral blood mononuclear cells by using an induction culture medium, and adding spirulina polysaccharide, allantoin, sodium pyruvate and other components into the induction culture medium to improve the cell induction culture efficiency. Finally, the proliferation culture medium is adopted to amplify the cells, the components of bismuth potassium citrate, chitin and the like in the proliferation culture medium effectively improve the proliferation speed of the cells and the proliferation efficiency, and the obtained T cells have good activity and large quantity.
Detailed Description
The present invention is further described below with reference to specific embodiments, and it should be noted that, without conflict, any combination between the embodiments or technical features described below may form a new embodiment.
Example 1
A method for culturing peripheral blood T cells, comprising the steps of:
(1) adding the collected peripheral blood into a centrifugal tube, then uniformly mixing the collected peripheral blood with PBS (phosphate buffer solution) with the same volume, adding the peripheral blood into the centrifugal tube added with the Ficoll separating medium, absorbing the middle leucocyte after centrifugation, washing the cell with the PBS, and separating to obtain peripheral blood mononuclear cells;
(2) adding physiological saline containing dibutyryladenosine cyclophosphate and human CD3 monoclonal antibody as coating solution into T75 culture flask, and culturing at 37 deg.C and 5% CO2Coating overnight under the condition, and removing a coating solution to serve as an induction culture bottle for later use; the concentration of dibutyryl cyclic adenosine monophosphate and the human CD3 monoclonal antibody in the physiological saline is as follows: 1.8 mu g/mL of dibutyryl cyclic adenosine monophosphate and 0.6 mu g/mL of human CD3 monoclonal antibody;
(3) resuspending the peripheral blood mononuclear cells obtained in the step (1) by using an induction medium, adding the resuspended peripheral blood mononuclear cells into the culture flask obtained in the step (2) and enabling the cell density to be 2 x 106one/mL, 5% CO at 37 ℃2The culture is carried out for 2 days in the incubator, and a culture medium is supplemented in time during the culture process to complete the induction culture; the basic culture medium of the induction culture medium is an X-VIVO culture medium, and the concentrations of the added components in the culture medium are 12ng/mL of spirulina polysaccharide, 8ng/mL of allantoin, 4ng/mL of sodium pyruvate, 22 ng/mL of IFN-gamma, 231 ng/mL of interleukin and 1213 ng/mL of interleukin;
(4) adding physiological saline containing ciclopirox and human CD3 monoclonal antibody as coating solution into T75 culture flask, and culturing at 37 deg.C with 5% CO2Coating overnight under the condition, discarding the culture solution, and using as a proliferation culture bottle for later use; the concentrations of ciclopirox and human CD3 monoclonal antibody in saline were: 5ng/mL of dibutyryl cyclic adenosine monophosphate and 4 mu g/mL of human CD3 monoclonal antibody;
(5) adding the cells subjected to the induction culture in the step (3) into the culture bottle in the step (4), and adopting a proliferation culture medium at 37 ℃ and 5% CO2The culture box continuously cultures for 12 days, the culture solution is changed every 3 days, and the cell density in the culture medium is 1 multiplied by 106Per mL; the proliferation culture medium comprises the following components: the basic culture medium X-VIVO culture medium is added with the following components in the basic culture medium: 3ng/mL of bismuth potassium citrate, 10ng/mL of chitin, 8ng/mL of naringin, 225 ng/mL of interleukin and 15ng/mL of transforming growth factor-alpha.
Example 2
A method for culturing peripheral blood T cells, comprising the steps of:
(1) adding the collected peripheral blood into a centrifugal tube, then uniformly mixing the collected peripheral blood with PBS (phosphate buffer solution) with the same volume, adding the peripheral blood into the centrifugal tube added with the Ficoll separating medium, absorbing the middle leucocyte after centrifugation, washing the cell with the PBS, and separating to obtain peripheral blood mononuclear cells;
(2) adding physiological saline containing dibutyryladenosine cyclophosphate and human CD3 monoclonal antibody as coating solution into T75 culture flask, and culturing at 37 deg.C and 5% CO2Coating overnight under the condition, and removing a coating solution to serve as an induction culture bottle for later use; the concentration of dibutyryl cyclic adenosine monophosphate and the human CD3 monoclonal antibody in the physiological saline is as follows: 1.5 mu g/mL of dibutyryl cyclic adenosine monophosphate and 0.5 mu g/mL of human CD3 monoclonal antibody;
(3) resuspending the peripheral blood mononuclear cells obtained in the step (1) by using an induction medium, adding the resuspended peripheral blood mononuclear cells into the culture flask obtained in the step (2) and enabling the cell density to be 2 x 106one/mL, 5% CO at 37 ℃2Culturing for 3 days in the incubator, and supplementing a culture medium in time during the culture process to finish the induction culture; the basic culture medium of the induction culture medium is an X-VIVO culture medium, and the concentrations of the added components in the culture medium are 10ng/mL of spirulina polysaccharide, 5ng/mL of allantoin, 3ng/mL of sodium pyruvate, 20ng/mL of IFN-gamma, 230 ng/mL of interleukin and 1210 ng/mL of interleukin;
(4) adding physiological saline containing ciclopirox and human CD3 monoclonal antibody as coating solution into T75 culture flask, and culturing at 37 deg.C with 5% CO2Coating overnight under the condition, discarding the culture solution, and using as a proliferation culture bottle for later use; the concentrations of ciclopirox and human CD3 monoclonal antibody in saline were: dibutyryl cyclic adenosine monophosphate (DAP) 3.5ng/mL, human CD3 monoclonal antibody 3 mug/mL;
(5) adding the cells subjected to the induction culture in the step (3) into the culture bottle in the step (4), and adopting a proliferation culture medium at 37 ℃ and 5% CO2The culture box continuously cultures for 12 days, the culture solution is changed every 2 days, and the cell density in the culture medium is 1 multiplied by 106Per mL, the composition of the proliferation medium is as follows: the basic culture medium X-VIVO culture medium is added with the following components in the basic culture medium: 1ng/mL of bismuth potassium citrate, 8ng/mL of chitin, 5ng/mL of naringin, 220 ng/mL of interleukin and 10ng/mL of transforming growth factor-alpha.
Example 3
A method for culturing peripheral blood T cells, comprising the steps of:
(1) adding the collected peripheral blood into a centrifugal tube, then uniformly mixing the collected peripheral blood with PBS (phosphate buffer solution) with the same volume, adding the peripheral blood into the centrifugal tube added with the Ficoll separating medium, absorbing the middle leucocyte after centrifugation, and washing the cell with the PBS to obtain peripheral blood mononuclear cells;
(2) adding physiological saline containing dibutyryladenosine cyclophosphate and human CD3 monoclonal antibody as coating solution into T75 culture flask, and culturing at 37 deg.C and 5% CO2Coating overnight under the condition, and removing a coating solution to serve as an induction culture bottle for later use; the concentration of dibutyryl cyclic adenosine monophosphate and the human CD3 monoclonal antibody in the physiological saline is as follows: dibutyryl cyclic adenosine monophosphate 2 μ g/mL, human CD3 monoclonal antibody 1 μ g/mL;
(3) resuspending the peripheral blood mononuclear cells obtained in the step (1) by using an induction medium, adding the resuspended peripheral blood mononuclear cells into the culture flask obtained in the step (2) and enabling the cell density to be 2 x 106one/mL, 5% CO at 37 ℃2The culture is carried out for 2 days in the incubator, and a culture medium is supplemented in time during the culture process to complete the induction culture; the basic culture medium of the induction culture medium is an X-VIVO culture medium, and the concentrations of the added components in the culture medium are 15ng/mL of spirulina polysaccharide, 10ng/mL of allantoin, 5ng/mL of sodium pyruvate, 25 ng/mL of IFN-gamma, 235 ng/mL of interleukin and 1215 ng/mL of interleukin;
(4) adding physiological saline containing ciclopirox and human CD3 monoclonal antibody as coating solution into T75 culture flask, and culturing at 37 deg.C with 5% CO2Coating overnight under the condition, discarding the culture solution, and using as a proliferation culture bottle for later use; the concentrations of ciclopirox and human CD3 monoclonal antibody in saline were: 6.5ng/mL of dibutyryl cyclic adenosine monophosphate and 5 mu g/mL of human CD3 monoclonal antibody;
(5) adding the cells subjected to the induction culture in the step (3) into the culture bottle in the step (4), and adopting a proliferation culture medium at 37 ℃ and 5% CO2The culture box continuously cultures for 12 days, the culture solution is changed every 3 days, and the cell density in the culture medium is 1 multiplied by 106complete/mL, the composition of the multiplication medium being: the basic culture medium X-VIVO culture medium is added with the following components in the basic culture medium: 5ng/mL bismuth potassium citrate, 12ng/mL chitin, 10ng/mL naringin, 230 ng/mL interleukin, transforming growth factorSub-. alpha.20 ng/mL.
Comparative example 1
Comparative example 1 provides a method for culturing peripheral blood T cells, which is different from example 1 in that: the amount of the allantoin was adjusted to 20ng/mL, except that the spirulina polysaccharide was omitted, and the same procedure as in example 1 was repeated.
Comparative example 2
Comparative example 2 provides a method for culturing peripheral blood T cells, which is different from example 1 in that: allantoin was omitted, and the amount of spirulina polysaccharide was adjusted to 20ng/mL, and the procedure was repeated as in example 1.
Comparative example 3
Comparative example 3 provides a method for culturing peripheral blood T cells, which is different from example 1 in that: the amount of bismuth potassium citrate was omitted, and the amount of chitin was adjusted to 13ng/mL, the rest being the same as in example 1.
Comparative example 4
Comparative example 4 provides a method for culturing peripheral blood T cells, which is different from example 1 in that: the chitin was omitted and the amount of bismuth potassium citrate was adjusted to 13ng/mL, the remainder being the same as in example 1.
Comparative example 5
Comparative example 5 provides a method for culturing peripheral blood T cells, which is different from example 1 in that: the procedure of example 1 was repeated except that sodium citrate was used instead of bismuth potassium citrate.
Comparative example 6
Comparative example 6 provides a method for culturing peripheral blood T cells, which is different from example 1 in that: the dibutyryl cyclic adenosine monophosphate in the step (2) is omitted and the same as in example 1 is applied.
Comparative example 7
Comparative example 7 provides a method for culturing peripheral blood T cells, which is different from example 1 in that: ciclopirox in step (4) was omitted and the same procedure as in example 1 was repeated.
Comparative example 8
Comparative example 8 provides a method for culturing peripheral blood T cells, which is different from example 1 in that: the same as in example 1 was repeated except that the dibutyryladenosine cyclophosphate in step (2) was replaced with ciclopirox, and the ciclopirox in step (4) was replaced with dibutyryladenosine cyclophosphate.
And (3) phenotype detection: the CD3 in the cells was determined by staining the day 12 cultured T cells of example 1 and comparative examples 1 to 8 with flow antibody+The results are shown in Table 1.
TABLE 1
Group of CD3+Percentage of cells (%)
Example 1 94.21
Comparative example 1 82.76
Comparative example 2 84.33
Comparative example 3 91.06
Comparative example 4 90.57
Comparative example 5 91.55
Comparative example 6 83.64
Comparative example 7 91.89
Comparative example 8 86.47
It can be seen from Table 1 that the proportion of T cells was the highest in example 1, and the proportion of T cells was decreased to various degrees after adjusting the composition of the coating solution, the induction medium and the proliferation medium during the culture in comparative examples 1 to 8. Thus, the purity of the cells obtained by the culture method of the present invention was higher.
The total cell numbers of the cells of example 1 and comparative examples 1 to 8 after proliferation culture for 12 days were counted by trypan blue staining method, respectively, and the proliferation fold was calculated as compared with the cell numbers inoculated before proliferation culture, and the results are shown in table 2.
TABLE 2
Group of Fold of proliferation
Example 1 119
Comparative example 1 91
Comparative example 2 95
Comparative example 3 81
Comparative example 4 86
Comparative example 5 72
Comparative example 6 102
Comparative example 7 98
Comparative example 8 93
As can be seen from Table 2, the T cells in example 1 had better proliferative activity and the number of cells was the greatest after proliferation culture. The cell numbers in comparative examples 1 to 8 were decreased to different degrees from those in example 1, indicating that the culture method of the present invention is useful for increasing the proliferation activity of T cells.
Detecting the killing activity of T cells on K562 cells: t cells cultured for 12 days in example 1 and comparative examples 1 to 8 were collected, and prepared into cell suspensions using serum-free X-VIVO medium at a cell concentration of 1X 10 as effector cells6The cells/mL were seeded in 96-well plates at an effective target ratio of 20:1 with T cells and K562 cells at 37 ℃ with 5% CO2The culture box is used for culturing for 5 hours, an effector cell control group and a blank control group are arranged at the same time, a target cell control group is arranged in each group, 3 holes are formed in each group, the absorbance value (A) of each group at 570nm is measured, and the killing activity is calculated, wherein the result is shown in table 3, and the killing activity calculation formula is as follows:
killing activity (%) = [ target cell control well a value- (experimental well a value-effector cell control well a) ]/target cell control well a value × 100%
TABLE 3
Group of Killing activity (%)
Example 1 82.37
Comparative example 1 68.49
Comparative example 2 70.21
Comparative example 3 74.59
Comparative example 4 72.15
Comparative example 5 75.69
Comparative example 6 69.88
Comparative example 7 76.57
Comparative example 8 73.34
It can be seen from Table 3 that the cell killing activity of example 1 was high. Comparative examples 1 to 8 all had different degrees of reduction. Thus, the activity of the T cells obtained by the culture method of the present invention was better.
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.

Claims (8)

1. A method for culturing peripheral blood T cells, comprising the steps of:
(1) collecting peripheral blood, and separating by adopting a Ficoll density gradient centrifugation method to obtain peripheral blood mononuclear cells;
(2) adding physiological saline containing dibutyryladenosine cyclophosphate and human CD3 monoclonal antibody as coating solution into culture flask, and culturing at 37 deg.C and 5% CO2Coating overnight under the condition, and removing a coating solution to serve as an induction culture bottle for later use;
(3) resuspending the peripheral blood mononuclear cells in the step (1) by adopting an induction culture medium, and culturing the peripheral blood mononuclear cells in the culture bottle in the step (2) for 2-3 days to finish induction culture, wherein the induction culture medium consists of a basic culture medium and spirulina polysaccharide, allantoin, sodium pyruvate, IFN-gamma, interleukin-2 and interleukin-12 which are added in the basic culture medium;
(4) adding physiological saline containing ciclopirox and human CD3 monoclonal antibody as coating solution into culture flask, and culturing at 37 deg.C with 5% CO2Coating overnight under the condition, discarding the culture solution, and using as a proliferation culture bottle for later use;
(5) adding the cells subjected to the induction culture in the step (3) into the culture bottle in the step (4), culturing by adopting a proliferation culture medium for 12 days, and completing the continuous culture, wherein the proliferation culture medium comprises the following components: a basal medium, wherein the following components are added in the basal medium: bismuth potassium citrate, chitin, naringin, interleukin-2 and transforming growth factor-alpha.
2. The method for culturing peripheral blood T cells according to claim 1, wherein the concentration of dibutyryl cyclic adenosine monophosphate (DAP) and the human CD3 monoclonal antibody in the physiological saline in the step (2) is: 1.5-2 mu g/mL of dibutyryl cyclic adenosine monophosphate and 0.5-1 mu g/mL of human CD3 monoclonal antibody.
3. The method for culturing peripheral blood T cells according to claim 1, wherein the basic medium of the induction medium in the step (3) is X-VIVO medium, and the concentration of each component in the medium is 10-15ng/mL of spirulina polysaccharide, 5-10ng/mL of allantoin, 3-5 ng/mL of sodium pyruvate, 20-25 ng/mL of IFN-. gamma., 230-35 ng/mL of interleukin, or 1210-15 ng/mL of interleukin.
4. The method for culturing peripheral blood T cells according to claim 3, wherein the concentration of each component in the culture medium is 12ng/mL of spirulina polysaccharide, 8ng/mL of allantoin, 4ng/mL of sodium pyruvate, 22 ng/mL of IFN-. gamma., -231 ng/mL of interleukin, or-1213 ng/mL of interleukin.
5. The method for culturing peripheral blood T cells according to claim 1, wherein the concentration of ciclopirox and human CD3 monoclonal antibody in the physiological saline of step (3) is: dibutyryl cyclic adenosine monophosphate 3.5-6.5ng/mL, human CD3 monoclonal antibody 3-5 μ g/mL.
6. The method for culturing peripheral blood T cells according to claim 1, wherein the basic medium of the proliferation medium in the step (5) is X-VIVO medium, and the concentrations of the components in the medium are: 1-5ng/mL of bismuth potassium citrate, 8-12ng/mL of chitin, 5-10ng/mL of naringin, 220-30 ng/mL of interleukin and 10-20 ng/mL of transforming growth factor-alpha.
7. The method for culturing peripheral blood T cells according to claim 6, wherein the concentrations in the respective component media are: 3ng/mL of bismuth potassium citrate, 10ng/mL of chitin, 8ng/mL of naringin, 225 ng/mL of interleukin and 15ng/mL of transforming growth factor-alpha.
8. The method for culturing peripheral blood T cells according to claim 1, wherein the step (5) comprises changing the culture medium every 2 to 3 days during the culture.
CN202111309326.9A 2021-11-06 2021-11-06 Peripheral blood T cell culture method Pending CN113817676A (en)

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