CN113444685A - Culture method for culturing immune cells by autologous peripheral blood - Google Patents
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Abstract
The invention discloses a culture method for culturing immune cells by autologous peripheral blood, belongs to the technical field of cell culture, and aims to solve the problem that the activity expression of cryopreserved cells is easily influenced by pollution due to the influence of operation of technicians, environment and performance of cryopreserving liquid in the process of cryopreservation. According to the invention, the cryopreservation step is improved on the basis of the existing culture mode, the cryopreservation stabilizer is added, the main components of the cryopreservation stabilizer comprise proline, sucrose, polyvinylpyrrolidone, ethylene glycol, glycerol, sodium citrate, ectoin and Al ys-Basa l basal cell culture solution, and no exogenous animal-derived protein is introduced due to the serum-free cell cryopreservation solution which does not contain animal serum, dimethyl sulfoxide and animal-derived components, so that the possibility of animal-derived pollution is reduced, the probability of infecting fungi and bacteria is reduced, the glycerol has a cryoprotective effect, the damage of low-temperature cryopreservation on cells is reduced, and the preservation effect is improved.
Description
Technical Field
The invention belongs to the technical field of cell culture, and relates to a culture method for culturing immune cells by autologous peripheral blood.
Background
The autologous cell immunotherapy is to extract immature immune cells in a tumor patient body by blood collection, activate and culture the immature immune cells in a laboratory so that the immature immune cells have the capacity of efficiently recognizing and killing the tumor cells, and then return the immature immune cells to the tumor patient body.
Currently, the method of culturing Cytokine-induced killer (CIK) immune cells in vitro is one of autologous cell immunotherapy methods, and is used to treat a broad spectrum of tumor patients. The technical principle of CIK therapy is as follows: singulating peripheral blood of humanA heterogeneous population of cells obtained by co-culturing nuclear cells in vitro with a variety of cytokines (e.g., anti-CD 3 monoclonal antibodies, IL-2, IFN- γ, etc.) for a period of time; a group of heterogeneous cells exist in the group, and the cells express CD3 simultaneously+And CD56+Cells of two membrane protein molecules, NK cell-like T lymphocytes (NKT cells), are the main effector cells with killing effect on tumor cells. The CIK cells have the characteristics of high proliferation speed, no toxic effect on normal cells, wide tumor killing spectrum, high safety and the like. Therefore, the use of CIK cells is considered to be one of the important approaches for anti-tumor adoptive cellular immunotherapy.
In general, a culture method for culturing immune cells in autologous peripheral blood includes static amplification and intermittent liquid exchange, and then cryopreserving the cultured cells, but in the cryopreservation process, the cryopreserved cells are contaminated due to the operation of technicians, the environment and the influence of the performance of the cryopreserving liquid, so that the activity expression of the cryopreserved cells is influenced, and therefore, a safer culture method for culturing immune cells in autologous peripheral blood is required to be developed.
Disclosure of Invention
The invention aims to provide a culture method for culturing immune cells by autologous peripheral blood.
The problems to be solved by the invention are as follows: during the process of cryopreservation, due to the influence of the operation, environment and cryopreservation liquid performance of technicians, cryopreserved cells are polluted to influence the activity expression of the cryopreserved cells.
The purpose of the invention can be realized by the following technical scheme:
a method for culturing immune cells in autologous peripheral blood comprises the following steps:
step A1, collecting peripheral blood, performing centrifugal separation to obtain peripheral blood mononuclear cells and upper plasma, and performing inactivation, freeze thawing, high-speed centrifugation and filtration on the upper plasma to obtain autologous serum;
step A2, adding the autologous serum prepared in the step A1, IL-4 and GM-CSF to an immune cell culture medium to obtain a culture medium A;
step A3, adding 7DW8-5 into an immune cell culture medium to obtain a culture medium B;
step A4, adding IFN-gamma, IL-2 and CD3 monoclonal antibodies into an immune cell culture medium to obtain a culture medium C;
step A5, adding IL-2 into an immune cell culture medium to obtain a culture medium D;
step A6, take (5-10) x 107Inoculating peripheral blood mononuclear cells into a culture bottle, adding an immune cell culture medium, autologous serum and a culture medium A, culturing for 20-24h, then adding the culture medium A, culturing for 40-48h, blowing and amplifying the cells, then adding the immune cell culture medium, the culture medium A and the autologous serum, culturing for 20-24h, adding a culture medium B and a culture medium C, blowing and amplifying the cells, adding the immune cell culture medium, the culture medium D and the autologous serum, and culturing for 40-48h to obtain target cells;
and step A7, adding a freezing stabilizer into the target cells to form a cell suspension, subpackaging the cell suspension into freezing tubes, placing the freezing tubes into a programmed cooling box, firstly placing the freezing tubes into a temperature-80 ℃ freezing box for 12 hours, and then transferring the freezing tubes into liquid nitrogen for long-term freezing.
Further, the immune cell culture medium in the step A2 is a TexMACS immune cell culture medium, the volume fraction of the autologous serum is 6-10%, the concentration of IL-4 is 80-150IU/mL, and the concentration of GM-CSF is 800-1400 IU/mL.
Further, the concentration of 7DW8-5 in the step A3 is 1 ng/. mu.L.
Further, in step A4, the concentration of IFN-gamma is 100U/. mu.L, the concentration of IL-2 is 200U/. mu.L, and the concentration of CD3 monoclonal antibody is 5 ng/. mu.L.
Further, the concentration of IL-2 in step A5 was 100U/. mu.L.
Further, the cryopreservation stabilizer in the step A7 is prepared by mixing the following components in parts by mass: 1-2% of proline, 5-6% of sucrose, 3-6% of polyvinylpyrrolidone, 5-8% of ethylene glycol, 5-8% of glycerol, 2-4% of sodium citrate, 2-5% of ectoin and the balance of aly-basic cell culture solution.
The invention has the beneficial effects that: the invention aims to provide a method for culturing immune cells by using autologous peripheral blood, which improves the freezing step on the basis of the existing mature culture mode, adds a freezing stabilizer, and mainly comprises proline, sucrose, polyvinylpyrrolidone, glycol, glycerol, sodium citrate, ectoin and Alys-Basal cell culture solution, reduces the possibility of animal source pollution and the probability of infecting fungi and bacteria due to the fact that the serum-free cell freezing solution does not contain animal serum, dimethyl sulfoxide and animal source components and does not introduce exogenous animal source protein, avoids adverse reaction and toxic and side effects when the cells are returned to patients after recovery, does not produce harm to human cell immunotherapy, ensures the safety of clinical application, can be directly used for clinical cell immunotherapy, is safe and effective, and the polyvinylpyrrolidone is secondly, The damage of ethylene glycol to cytotoxicity is lower than that of dimethyl sulfoxide, the cell protection effect is better, and in addition, the glycerol has the freezing protection effect, so that the damage of low-temperature freezing to cells can be reduced, and the preservation effect is improved.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A method for culturing immune cells in autologous peripheral blood comprises the following steps:
step A1, collecting peripheral blood, performing centrifugal separation to obtain peripheral blood mononuclear cells and upper plasma, and performing inactivation, freeze thawing, high-speed centrifugation and filtration on the upper plasma to obtain autologous serum;
step A2, adding the autologous serum prepared in the step A1, IL-4 and GM-CSF into a TexMACS immune cell culture medium to obtain a culture medium A, wherein the volume fraction of the autologous serum is 6%, the concentration of the IL-4 is 80IU/mL, and the concentration of the GM-CSF is 800 IU/mL;
step A3, adding 7DW8-5 into a TexMACS immune cell culture medium to obtain a culture medium B, wherein the concentration of 7DW8-5 is 1 ng/. mu.L;
step A4, adding IFN-gamma, IL-2 and CD3 monoclonal antibodies into a TexMACS immune cell culture medium to obtain a culture medium C, wherein the concentration of the IFN-gamma is 100U/mu L, the concentration of the IL-2 is 200U/mu L, and the concentration of the CD3 monoclonal antibodies is 5 ng/mu L;
step A5, adding IL-2 into the TexMACS immune cell culture medium to obtain a culture medium D, wherein the concentration of the IL-2 is 100U/. mu.L;
step A6, choose 5X 107Inoculating peripheral blood mononuclear cells into a culture bottle, adding a TexMACS immune cell culture medium, autologous serum and a culture medium A, culturing for 20h, adding the culture medium A, culturing for 40h, blowing and expanding the cells, adding the TexMACS immune cell culture medium, the culture medium A and the autologous serum, culturing for 20h, adding a culture medium B and a culture medium C, blowing and expanding the cells, adding the TexMACS immune cell culture medium, a culture medium D and the autologous serum, and culturing for 40h to obtain target cells;
step A7, adding a freezing stabilizer into target cells to form a cell suspension, subpackaging the cell suspension in freezing tubes, placing the freezing tubes in a programmed cooling box, firstly placing the freezing tubes in a temperature-80 ℃ freezing box for 12 hours, and then transferring the freezing tubes into liquid nitrogen for long-term freezing, wherein the freezing stabilizer is formed by mixing the following components in percentage by mass and comprises the following components: 1% of proline, 5% of sucrose, 3% of polyvinylpyrrolidone, 5% of ethylene glycol, 5% of glycerol, 2% of sodium citrate, 2% of ectoin and 77% of an aly-basic cell culture solution.
Example 2
A method for culturing immune cells in autologous peripheral blood comprises the following steps:
step A1, collecting peripheral blood, performing centrifugal separation to obtain peripheral blood mononuclear cells and upper plasma, and performing inactivation, freeze thawing, high-speed centrifugation and filtration on the upper plasma to obtain autologous serum;
step A2, adding the autologous serum prepared in the step A1, IL-4 and GM-CSF into a TexMACS immune cell culture medium to obtain a culture medium A, wherein the volume fraction of the autologous serum is 8%, the concentration of the IL-4 is 120IU/mL, and the concentration of the GM-CSF is 1100 IU/mL;
step A3, adding 7DW8-5 into a TexMACS immune cell culture medium to obtain a culture medium B, wherein the concentration of 7DW8-5 is 1 ng/. mu.L;
step A4, adding IFN-gamma, IL-2 and CD3 monoclonal antibodies into a TexMACS immune cell culture medium to obtain a culture medium C, wherein the concentration of the IFN-gamma is 100U/mu L, the concentration of the IL-2 is 200U/mu L, and the concentration of the CD3 monoclonal antibodies is 5 ng/mu L;
step A5, adding IL-2 into the TexMACS immune cell culture medium to obtain a culture medium D, wherein the concentration of the IL-2 is 100U/. mu.L;
step A6, get 7X 107Inoculating peripheral blood mononuclear cells into a culture bottle, adding a TexMACS immune cell culture medium, autologous serum and a culture medium A, culturing for 22h, adding the culture medium A, culturing for 44h, blowing and expanding the cells, adding the TexMACS immune cell culture medium, the culture medium A and the autologous serum, culturing for 22h, adding a culture medium B and a culture medium C, blowing and expanding the cells, adding the TexMACS immune cell culture medium, a culture medium D and the autologous serum, and culturing for 44h to obtain target cells;
step A7, adding a freezing stabilizer into target cells to form a cell suspension, subpackaging the cell suspension in freezing tubes, placing the freezing tubes in a programmed cooling box, firstly placing the freezing tubes in a temperature-80 ℃ freezing box for 12 hours, and then transferring the freezing tubes into liquid nitrogen for long-term freezing, wherein the freezing stabilizer is formed by mixing the following components in percentage by mass and comprises the following components: proline 2%, sucrose 5%, polyvinylpyrrolidone 4%, ethylene glycol 6%, glycerol 7%, sodium citrate 3%, ectoin 3%, and aly-basic cell culture fluid 70%.
Example 3
A method for culturing immune cells in autologous peripheral blood comprises the following steps:
step A1, collecting peripheral blood, performing centrifugal separation to obtain peripheral blood mononuclear cells and upper plasma, and performing inactivation, freeze thawing, high-speed centrifugation and filtration on the upper plasma to obtain autologous serum;
step A2, adding the autologous serum prepared in the step A1, IL-4 and GM-CSF into a TexMACS immune cell culture medium to obtain a culture medium A, wherein the volume fraction of the autologous serum is 10%, the concentration of the IL-4 is 150IU/mL, and the concentration of the GM-CSF is 1400 IU/mL;
step A3, adding 7DW8-5 into a TexMACS immune cell culture medium to obtain a culture medium B, wherein the concentration of 7DW8-5 is 1 ng/. mu.L;
step A4, adding IFN-gamma, IL-2 and CD3 monoclonal antibodies into a TexMACS immune cell culture medium to obtain a culture medium C, wherein the concentration of the IFN-gamma is 100U/mu L, the concentration of the IL-2 is 200U/mu L, and the concentration of the CD3 monoclonal antibodies is 5 ng/mu L;
step A5, adding IL-2 into the TexMACS immune cell culture medium to obtain a culture medium D, wherein the concentration of the IL-2 is 100U/. mu.L;
step A6, get 10 × 107Inoculating peripheral blood mononuclear cells into a culture bottle, adding a TexMACS immune cell culture medium, autologous serum and a culture medium A, culturing for 24h, adding the culture medium A, culturing for 48h, blowing and expanding the cells, adding the TexMACS immune cell culture medium, the culture medium A and the autologous serum, culturing for 24h, adding a culture medium B and a culture medium C, blowing and expanding the cells, adding the TexMACS immune cell culture medium, a culture medium D and the autologous serum, and culturing for 48h to obtain target cells;
step A7, adding a freezing stabilizer into target cells to form a cell suspension, subpackaging the cell suspension in freezing tubes, placing the freezing tubes in a programmed cooling box, firstly placing the freezing tubes in a temperature-80 ℃ freezing box for 12 hours, and then transferring the freezing tubes into liquid nitrogen for long-term freezing, wherein the freezing stabilizer is formed by mixing the following components in percentage by mass and comprises the following components: proline 2%, sucrose 6%, polyvinylpyrrolidone 6%, ethylene glycol 8%, glycerol 8%, sodium citrate 4%, ectoin 5%, and aly-basic cell culture solution 61%.
Comparative example 1
The method for culturing immune cells in autologous peripheral blood according to comparative example 1 is different from that of example 1 in that a cryopreservative agent is not added.
Fungi and bacteria were detected in examples 1 to 3 and comparative example 1, and the cultured immune cells were inoculated into a thioglycolate fluid culture medium and a trypticase soytone fluid culture medium, incubated at 35 ℃ for 6 days, and observed, and the results were determined: if the sample detection tube grows in a clear and sterile manner within 6 days, judging that the sample meets the specification, namely the sample is qualified; if any tube in the sample detection tubes is turbid and the bacteria growth is confirmed, the sample is judged not to meet the specification, namely the sample is not qualified, and the test data is shown in the table 1:
TABLE 1
As is clear from Table 1, the fungi and bacteria tests carried out in examples 1 to 3 showed negative results, and the samples were judged to be in accordance with the specifications, whereas the comparative examples were judged to be in accordance with the specifications because no cryopreservation stabilizer was added and infection with mycoplasma, bacteria and fungi occurred.
The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.
Claims (6)
1. A method for culturing immune cells in autologous peripheral blood, which comprises the following steps:
step A1, collecting peripheral blood, performing centrifugal separation to obtain peripheral blood mononuclear cells and upper plasma, and performing inactivation, freeze thawing, high-speed centrifugation and filtration on the upper plasma to obtain autologous serum;
step A2, adding the autologous serum prepared in the step A1, IL-4 and GM-CSF to an immune cell culture medium to obtain a culture medium A;
step A3, adding 7DW8-5 into an immune cell culture medium to obtain a culture medium B;
step A4, adding IFN-gamma, IL-2 and CD3 monoclonal antibodies into an immune cell culture medium to obtain a culture medium C;
step A5, adding IL-2 into an immune cell culture medium to obtain a culture medium D;
step A6, choose 5X 107-10×107Inoculating peripheral blood mononuclear cells into a culture bottle, adding an immune cell culture medium, autologous serum and a culture medium A, culturing for 20-24h, then adding the culture medium A, culturing for 40-48h, blowing and amplifying the cells, then adding the immune cell culture medium, the culture medium A and the autologous serum, culturing for 20-24h, adding a culture medium B and a culture medium C, blowing and amplifying the cells, adding the immune cell culture medium, the culture medium D and the autologous serum, and culturing for 40-48h to obtain target cells;
and step A7, adding a freezing stabilizer into the target cells to form a cell suspension, subpackaging the cell suspension into freezing tubes, placing the freezing tubes into a programmed cooling box, firstly placing the freezing tubes into a temperature-80 ℃ freezing box for 12 hours, and then transferring the freezing tubes into liquid nitrogen for long-term freezing.
2. The method according to claim 1, wherein the immune cells are cultured in autologous peripheral blood, and the culture method comprises: the immune cell culture medium in the step A2 is a TexMACS immune cell culture medium, the volume fraction of the autologous serum is 6-10%, the concentration of IL-4 is 80-150IU/mL, and the concentration of GM-CSF is 800-1400 IU/mL.
3. The method according to claim 1, wherein the immune cells are cultured in autologous peripheral blood, and the culture method comprises: the concentration of 7DW8-5 in step A3 was 1 ng/. mu.L.
4. The method according to claim 1, wherein the immune cells are cultured in autologous peripheral blood, and the culture method comprises: the concentration of IFN-gamma in the step A4 is 100U/mu L, the concentration of IL-2 is 200U/mu L, and the concentration of CD3 monoclonal antibody is 5 ng/mu L.
5. The method according to claim 1, wherein the immune cells are cultured in autologous peripheral blood, and the culture method comprises: the IL-2 concentration in step A5 was 100U/. mu.L.
6. The method according to claim 1, wherein the immune cells are cultured in autologous peripheral blood, and the culture method comprises: the freezing storage stabilizer in the step A7 is prepared by mixing the following components in percentage by mass: 1-2% of proline, 5-6% of sucrose, 3-6% of polyvinylpyrrolidone, 5-8% of ethylene glycol, 5-8% of glycerol, 2-4% of sodium citrate, 2-5% of ectoin and the balance of aly-basic cell culture solution.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114667999A (en) * | 2022-05-27 | 2022-06-28 | 广东先康达生物科技有限公司 | Immune cell cryopreservation stabilizer, cryopreservation liquid and cryopreservation method |
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