CN106854661A - A kind of preparation method of the CAR T cell preparations for treating prostate cancer - Google Patents
A kind of preparation method of the CAR T cell preparations for treating prostate cancer Download PDFInfo
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- CN106854661A CN106854661A CN201611220895.5A CN201611220895A CN106854661A CN 106854661 A CN106854661 A CN 106854661A CN 201611220895 A CN201611220895 A CN 201611220895A CN 106854661 A CN106854661 A CN 106854661A
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Abstract
The invention discloses a kind of preparation method of the CAR T cell preparations for treating prostate cancer, comprise the following steps：S1, to in culture dish and sequentially add 100 500mg/L L glutamine, the vitamin C of 15 150mg/L, the insulin of 10 20mU/L and 5 255 μ g/L VEGF, pH value is adjusted by NaOH, and adds hyclone and filtration sterilization, obtain new culture medium；S2, gathers the blood of patient itself, therefrom isolates T cell, and T cell is put into centrifuge tube；S3, by the centrifuge tube in physiological saline addition S2, by the 6min of solution centrifugal 4 in centrifuge tube, centrifugal force is 150 250g；S4, the T cell after centrifugation is transferred on culture medium, and culture medium is put into incubator, and the temperature in incubator is 35 39 DEG C, contains 4 8% CO2, amplification cultivation.The present invention is simple for production, and by CAR T cell preparation for treating prostate cancers, significantly, human serum albumin can maintain the activity of CAR T cells to therapeutic effect.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of preparation of the CAR-T cell preparations for treating prostate cancer
Method.
Background technology
Chimeric antigen receptor is the artificial receptors for simulating φt cell receptor function, and by antigen recognizing domain, (part is single-stranded
Antibody) and a series of signal domain of T cell be connected in sequence.To can be matched somebody with somebody using Chimeric antigen receptor modification T cell
The specific recognition effect of body or antibody is joined together with the effector function of T cell, carries out cancer target immunization therapy.This therapy is
It is used to treat after transforming human body self T-cell, relative to putting and chemotherapy, its toxic and side effect is weaker.Due to chimeric antigen
Then the T cell that acceptor is repaiied starts lethal effect, therefore its by its antigen recognizing domain tumor cell surface antigen
Treatment has targeting.Because this kind of T cell passes through its Chimeric antigen receptor Direct Recognition TCSA, Ran Houtong
Cross its signal domain and identification signal is directly passed into speed to intracellular, activate the killing activity of T cell, therefore can get around conventional thin
The major histocompatibility complex (MHC) that born of the same parents are immunized is restricted, thus effectively overcome tumour cell MHC expression quantity it is low cause exempt from
Epidemic disease is escaped, it is possible to target nonprotein tumour antigen.
Prostate cancer (Prostate Cancer, PCa) is common male urinary system malignant tumour, and its death rate has surpassed
Lung cancer is crossed, the second largest malignant tumour of men's health is threatened as serious.Although early stage, PCa was easily treated, productivity ratio is high, three
Phase, the fourth phase PCa death rates are high, and Bone tumour easily occur.PCa current therapeutic modality mainly has operation, radiation and chemotherapy.PCa
Resection operation wound is big, the incidence of the postoperative urinary incontinence and erectile dysfunction is high, and operative mortality rate is also high；And radiotherapy
It is big with the side effect that chemotherapy is brought, the quality of life of patient can be had a strong impact on, it is that this treats prostate cancer we have proposed one kind
CAR-T cell preparations preparation method.
The content of the invention
Based on the technical problem that background technology is present, the present invention proposes a kind of CAR-T cell systems for treating prostate cancer
The preparation method of agent.
A kind of preparation method of CAR-T cell preparations for treating prostate cancer proposed by the present invention, comprises the following steps：
S1, in culture dish and sequentially add the Glu of 100-500mg/L, the vitamin C of 15-150mg/L,
The insulin of 10-20mU/L and the VEGF of 5-255 μ g/L, adjust pH value, and add tire by NaOH
Cow's serum and filtration sterilization, obtain new culture medium；
S2, gathers the blood of patient itself, therefrom isolates T cell, and T cell is put into centrifuge tube；
S3, by the centrifuge tube in physiological saline addition S2, by the solution centrifugal 4-6min in centrifuge tube, centrifugal force is
150-250g；
S4, the T cell after centrifugation is transferred on culture medium, and culture medium is put into incubator, the temperature in incubator
It is 35-39 DEG C, the CO containing 4-8%2, amplification cultivation；
S5, slow virus to the T cell of S4 transfect and amplification cultivation, obtains CAR-T cells；
S6, by human serum albumin, trypsase with 1：The ratio of 1-3 is mixed, and adds CAR-T cells, is mixed,
Can be prepared by treating the CAR-T cell preparations of prostate cancer.
Preferably, in the S1, in culture dish and sequentially add the Glu of 300mg/L, 75mg/L dimension give birth to
The VEGF of the insulin of plain C, 15mU/L and 150 μ g/L, adjusts pH value, and add tire ox by NaOH
Serum and filtration sterilization, obtain new culture medium.
Preferably, in the S1, the pH value 7-7.4 of culture medium.
Preferably, in the S3, by physiological saline add S2 in centrifuge tube in, by the solution centrifugal in centrifuge tube
5min, centrifugal force is 200g, after washing 2 times after cell precipitation after centrifugation.
Preferably, in the S4, the number of days of amplification cultivation is 7-15 days.
Preferably, in the S3, centrifugation rate is 2000r/min.
The present invention first configures special culture medium, then gathers the blood of patient itself, therefrom isolates T cell, and T is thin
Born of the same parents are put into centrifuge tube, by physiological saline addition centrifuge tube, the solution centrifugal in centrifuge tube are turned the T cell after centrifugation
Move on culture medium, and culture medium be put into incubator amplification cultivation, slow virus to T cell transfect and amplification cultivation,
CAR-T cells are obtained, then human serum albumin, trypsase are mixed, add CAR-T cells, mixed, you can, this
Invention is simple for production, and prostate cancer is treated by CAR-T cell preparations, and significantly, human serum albumin can be maintained therapeutic effect
The activity of CAR-T cells.
Specific embodiment
The present invention is made with reference to specific embodiment further explain.
Embodiment one
A kind of preparation method of CAR-T cell preparations for treating prostate cancer proposed by the present invention, comprises the following steps：
S1, in culture dish and sequentially adding the pancreas of the Glu of 100mg/L, the vitamin C of 15mg/L, 10mU/L
The VEGF of island element and 5 μ g/L, pH value is adjusted by NaOH, and adds hyclone and filtration sterilization,
Obtain new culture medium；
S2, gathers the blood of patient itself, therefrom isolates T cell, and T cell is put into centrifuge tube；
S3, by the centrifuge tube in physiological saline addition S2, by the solution centrifugal 4min in centrifuge tube, centrifugal force is
150g；
S4, the T cell after centrifugation is transferred on culture medium, and culture medium is put into incubator, the temperature in incubator
It is 35 DEG C, contains 4% CO2, amplification cultivation；
S5, slow virus to the T cell of S4 transfect and amplification cultivation, obtains CAR-T cells；
S6, by human serum albumin, trypsase with 1：1 ratio is mixed, and adds CAR-T cells, is mixed, i.e.,
The CAR-T cell preparations for the treatment of prostate cancer can be obtained.
Embodiment two
A kind of preparation method of CAR-T cell preparations for treating prostate cancer proposed by the present invention, comprises the following steps：
S1, in culture dish and sequentially adding the pancreas of the Glu of 200mg/L, the vitamin C of 40mg/L, 12mU/L
The VEGF of island element and 50 μ g/L, pH value is adjusted by NaOH, and adds hyclone and filtration sterilization,
Obtain new culture medium；
S2, gathers the blood of patient itself, therefrom isolates T cell, and T cell is put into centrifuge tube；
S3, by the centrifuge tube in physiological saline addition S2, by the solution centrifugal 4.5min in centrifuge tube, centrifugal force is
175g；
S4, the T cell after centrifugation is transferred on culture medium, and culture medium is put into incubator, the temperature in incubator
It is 36 DEG C, contains 5% CO2, amplification cultivation；
S5, slow virus to the T cell of S4 transfect and amplification cultivation, obtains CAR-T cells；
S6, by human serum albumin, trypsase with 1：1.5 ratio is mixed, and adds CAR-T cells, is mixed,
Can be prepared by treating the CAR-T cell preparations of prostate cancer.
Embodiment three
A kind of preparation method of CAR-T cell preparations for treating prostate cancer proposed by the present invention, comprises the following steps：
S1, in culture dish and sequentially adding the pancreas of the Glu of 300mg/L, the vitamin C of 70mg/L, 17mU/L
The VEGF of island element and 100 μ g/L, adjusts pH value, and add hyclone and filter excessively by NaOH
Bacterium, obtains new culture medium；
S2, gathers the blood of patient itself, therefrom isolates T cell, and T cell is put into centrifuge tube；
S3, by the centrifuge tube in physiological saline addition S2, by the solution centrifugal 5min in centrifuge tube, centrifugal force is
200g；
S4, the T cell after centrifugation is transferred on culture medium, and culture medium is put into incubator, the temperature in incubator
It is 37 DEG C, contains 6% CO2, amplification cultivation；
S5, slow virus to the T cell of S4 transfect and amplification cultivation, obtains CAR-T cells；
S6, by human serum albumin, trypsase with 1：2 ratio is mixed, and adds CAR-T cells, is mixed, i.e.,
The CAR-T cell preparations for the treatment of prostate cancer can be obtained.
Example IV
A kind of preparation method of CAR-T cell preparations for treating prostate cancer proposed by the present invention, comprises the following steps：
S1, in culture dish and sequentially adding the Glu of 300mg/L, the vitamin C of 120mg/L, 19mU/L
The VEGF of insulin and 200 μ g/L, adjusts pH value, and add hyclone and filter excessively by NaOH
Bacterium, obtains new culture medium；
S2, gathers the blood of patient itself, therefrom isolates T cell, and T cell is put into centrifuge tube；
S3, by the centrifuge tube in physiological saline addition S2, by the solution centrifugal 5.5min in centrifuge tube, centrifugal force is
225g；
S4, the T cell after centrifugation is transferred on culture medium, and culture medium is put into incubator, the temperature in incubator
It is 38 DEG C, contains 7% CO2, amplification cultivation；
S5, slow virus to the T cell of S4 transfect and amplification cultivation, obtains CAR-T cells；
S6, by human serum albumin, trypsase with 1：2.5 ratio is mixed, and adds CAR-T cells, is mixed,
Can be prepared by treating the CAR-T cell preparations of prostate cancer.
Embodiment five
A kind of preparation method of CAR-T cell preparations for treating prostate cancer proposed by the present invention, comprises the following steps：
S1, in culture dish and sequentially adding the Glu of 500mg/L, the vitamin C of 15-150mg/L, 20mU/L
Insulin and 255 μ g/L VEGF, pH value is adjusted by NaOH, and add hyclone and filter
It is degerming, obtain new culture medium；
S2, gathers the blood of patient itself, therefrom isolates T cell, and T cell is put into centrifuge tube；
S3, by the centrifuge tube in physiological saline addition S2, by the solution centrifugal 6min in centrifuge tube, centrifugal force is
250g；
S4, the T cell after centrifugation is transferred on culture medium, and culture medium is put into incubator, the temperature in incubator
It is 39 DEG C, contains 8% CO2, amplification cultivation；
S5, slow virus to the T cell of S4 transfect and amplification cultivation, obtains CAR-T cells；
S6, by human serum albumin, trypsase with 1：3 ratio is mixed, and adds CAR-T cells, is mixed, i.e.,
The CAR-T cell preparations for the treatment of prostate cancer can be obtained.
The present invention is simple for production, and prostate cancer is treated by CAR-T cell preparations, and therapeutic effect is notable, human serum albumin
The activity of CAR-T cells can be maintained.
The above, the only present invention preferably specific embodiment, but protection scope of the present invention is not limited thereto,
Any one skilled in the art the invention discloses technical scope in, technology according to the present invention scheme and its
Inventive concept is subject to equivalent or change, should all be included within the scope of the present invention.
Claims (6)
1. a kind of preparation method of the CAR-T cell preparations for treating prostate cancer, it is characterised in that comprise the following steps：
S1, in culture dish and sequentially adding the Glu of 100-500mg/L, the vitamin C of 15-150mg/L, 10-
The insulin of 20mU/L and the VEGF of 5-255 μ g/L, adjust pH value, and add tire ox blood by NaOH
Clear and filtration sterilization, obtains new culture medium；
S2, gathers the blood of patient itself, therefrom isolates T cell, and T cell is put into centrifuge tube；
S3, by the centrifuge tube in physiological saline addition S2, by the solution centrifugal 4-6min in centrifuge tube, centrifugal force is 150-
250g；
S4, the T cell after centrifugation is transferred on culture medium, and culture medium is put into incubator, and the temperature in incubator is 35-
39 DEG C, the CO containing 4-8%2, amplification cultivation；
S5, slow virus to the T cell of S4 transfect and amplification cultivation, obtains CAR-T cells；
S6, by human serum albumin, trypsase with 1：The ratio of 1-3 is mixed, and adds CAR-T cells, is mixed, you can
The CAR-T cell preparations for the treatment of prostate cancer are obtained.
2. a kind of preparation method of CAR-T cell preparations for treating prostate cancer according to claim 1, its feature exists
In in the S1, in culture dish and sequentially adding the Glu of 300mg/L, the vitamin C of 75mg/L, 15mU/L
The VEGF of insulin and 150 μ g/L, adjusts pH value, and add hyclone and filter excessively by NaOH
Bacterium, obtains new culture medium.
3. a kind of preparation method of CAR-T cell preparations for treating prostate cancer according to claim 1, its feature exists
In, in the S1, the pH value 7-7.4 of culture medium.
4. a kind of preparation method of CAR-T cell preparations for treating prostate cancer according to claim 1, its feature exists
In in the S3, by the centrifuge tube in physiological saline addition S2, by the solution centrifugal 5min in centrifuge tube, centrifugal force is
200g, after washing 2 times after cell precipitation after centrifugation.
5. a kind of preparation method of CAR-T cell preparations for treating prostate cancer according to claim 1, its feature exists
In in the S4, the number of days of amplification cultivation is 7-15 days.
6. a kind of preparation method of CAR-T cell preparations for treating prostate cancer according to claim 1, its feature exists
In in the S3, centrifugation rate is 2000r/min.
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| CN107523550A (en) * | 2017-08-08 | 2017-12-29 | 安徽惠恩生物科技股份有限公司 | A kind of Car T cell preparation methods for ED-SCLC |
| CN107557335A (en) * | 2017-08-08 | 2018-01-09 | 安徽惠恩生物科技股份有限公司 | A kind of preparation method of Car NK cells for ED-SCLC |
| CN107686831A (en) * | 2017-08-23 | 2018-02-13 | 安徽惠恩生物科技股份有限公司 | A kind of Car T cell preparation methods for cancer of pancreas |
| CN108707579A (en) * | 2018-05-28 | 2018-10-26 | 北京美迪阿姆科技发展有限公司 | The serum free medium and preparation method and cultural method of a kind of human T lymphocyte's culture |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107523550A (en) * | 2017-08-08 | 2017-12-29 | 安徽惠恩生物科技股份有限公司 | A kind of Car T cell preparation methods for ED-SCLC |
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| CN108707579A (en) * | 2018-05-28 | 2018-10-26 | 北京美迪阿姆科技发展有限公司 | The serum free medium and preparation method and cultural method of a kind of human T lymphocyte's culture |
| CN108707579B (en) * | 2018-05-28 | 2022-08-09 | 北京美迪阿姆科技发展有限公司 | Serum-free medium for human T lymphocyte culture, preparation method and culture method |
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