CN107686831A - A kind of Car T cell preparation methods for cancer of pancreas - Google Patents
A kind of Car T cell preparation methods for cancer of pancreas Download PDFInfo
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Abstract
The invention discloses a kind of Car T cell preparation methods for cancer of pancreas, this method comprises the following steps：Peripheral blood in patients is gathered, extracts PMNC；Nucleus is washed with PBS, abandoning supernatant, take and GT551 resuspensions are added in cell precipitation；By nucleus with 1x106Individual/hole is inoculated in 24 orifice plates, and nutrient solution and 60IU/mL IL 2 are added in every hole, cultivates 24 36h, adds 20 40 μ L PersonGen in every orifice plate afterwardsTMBiomembrane；Divide the cell of culture to hole once every 16 18h, the supplement 2mL of nutrient solution 1 per hole, every the μ L of 36 48h supplement PersonGenTM biomembranes 10 15, persistently cultivate 16 18d.The invention discloses a kind of preparation method of the Car T cells for cancer of pancreas, obtained Car T cells can be injected by cell preparation to be entered in Pancreas cancer patients body, a kind of effective treatment of pancreatic cancer method is this method provide, and effectively reduces ill recurrence probability.
Description
Technical field
The invention belongs to biomedical sector, more particularly to a kind of Car-T cell preparation methods for cancer of pancreas.
Background technology
Cancer of pancreas is that a kind of grade malignancy is very high, diagnoses and treat all highly difficult malignant tumor of digestive tract, about 90% is
Duct adenocarcinoma originating from glandular tube epithelium.Its morbidity and mortality substantially rises in recent years.5 years survival rates<1%, it is prognosis
One of worst malignant tumour.The diagnosis rate of cancer of pancreas early stage is not high, and operative mortality rate is higher, and cure rate is very low.This disease is sent out
Sick rate male is 1.5~2 higher than the ratio between women, men and women:1, male patient is common far beyond premenopausal women, postmenopausal women's
The incidence of disease is similar with male.The cause of disease of cancer of pancreas is not still fully aware of.Its generation and smoking, drink, higher fatty acid and high protein drink
Food, excessive coffee for drinking, environmental pollution and inherent cause are relevant；Survey report in recent years finds cancer of pancreas in diabetic population
The incidence of disease apparently higher than general population；Also someone notices that the morbidity of chronic pancreatitis patient and cancer of pancreas has certain close
System, it is found that the ratio of chronic pancreatitis patient generation cancer of pancreas substantially increases；There are many factors sick to have with this in addition
Certain relation, such as occupation, environment, geography.
Cancer of pancreas clinical manifestation depend on the position of cancer, the course of disease sooner or later, whether there is transfer and situation that adjacent organs involves.
Its clinical characters is that the whole course of disease is short, disease development is fast and rapid deterioration.Most common is the glutted uncomfortable, pain of upper abdomen.Though
So have and feel pain, but not all patient has tenderness, if tenderness is then consistent with the position of conscious pain.
The content of the invention
It is an object of the invention to provide a kind of Car-T cell preparation methods for cancer of pancreas, one this method provide
The effective treatment of pancreatic cancer method of kind, and effectively reduce ill recurrence probability.
The present invention is achieved by the following technical solutions：
A kind of Car-T cell preparation methods for cancer of pancreas, this method comprise the following steps：
S1, collection peripheral blood in patients, extract PMNC；
S2, the PMNC obtained in S1 washed with PBS, abandoning supernatant, take in cell precipitation and add
GT551 is resuspended；
S3, by the PMNC obtained in S2 with 1x106Individual/hole is inoculated in 24 orifice plates, in every Kong Zhongjia
Enter nutrient solution and 60IU/mL IL-2, cultivate 24-36h, add 20-40 μ L PersonGen in every orifice plate afterwardsTMBiomembrane；
S4, every 16-18h by the cell point hole cultivated in S3 once, per hole supplement nutrient solution 1-2mL, every 36-48h
PersonGenTM biomembrane 10-15 μ L are supplemented, persistently cultivate 16-18d；
S5, slow virus to the T cell in S4 transfect to simultaneously amplification cultivation, obtain CAR-T cells.
Further, concretely comprising the following steps in the S1, collection patient peripheral are enriched with blood, slowly moved into after filtrating leukocytes
In centrifuge tube equipped with lymphocyte separation medium, centrifuged with density-gradient centrifugation method, draw tunica albuginea layer, obtain the single core of peripheral blood
Cell.
Further, nutrient solution composition described in S2 is to add 10%FBS in RPMI1640.
Further, it is 2-2.5x10 that cell concentration is adjusted after being resuspended in S26。
The invention has the advantages that：
The invention discloses a kind of preparation method of the Car-T cells for cancer of pancreas, this method is by gathering cancer of pancreas
Peripheral blood in patients obtains mononuclearcell, and nucleus is inoculated in into addition has PersonGenTMIn 24 orifice plates of biomembrane, lead to
Cross in vitro culture and obtain T cell, and CAR-T cells are obtained with the method for virus infection, obtained Car-T cells can be by thin
The injection of born of the same parents' preparation enters in Pancreas cancer patients body, this method provide a kind of effective treatment of pancreatic cancer method, and have
Effect reduces ill recurrence probability.
Certainly, any product for implementing the present invention it is not absolutely required to reach all the above advantage simultaneously.
Embodiment
Technical scheme in the embodiment of the present invention is clearly and completely described, it is clear that described embodiment is only
Part of the embodiment of the present invention, rather than whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art
The all other embodiment obtained under the premise of creative work is not made, belongs to the scope of protection of the invention.
The present invention is a kind of Car-T cell preparation methods for cancer of pancreas, and this method comprises the following steps that：
Embodiment 1
S1, collection patient peripheral are enriched with blood, and the centrifuge tube equipped with lymphocyte separation medium is slowly moved into after filtrating leukocytes
In, centrifuged with density-gradient centrifugation method, wherein parameter of noncentricity is 2000r/min, 20 DEG C, 15min, draws tunica albuginea layer, is obtained outer
All blood mononuclear cells；
S2, the PMNC obtained in S1 washed with PBS, abandoning supernatant, take in cell precipitation and add
GT551 is resuspended, and adjustment cell concentration is 2x106；
S3, by the PMNC obtained in S2 with 1x106Individual/hole is inoculated in 24 orifice plates, in every Kong Zhongjia
Enter nutrient solution and 60IU/mL IL-2, be placed in 37 DEG C, 5%CO224h is cultivated in being cultivated in incubator, is added afterwards in every orifice plate
20μL PersonGenTMBiomembrane；
Wherein, the nutrient solution composition is to add 10%FBS in RPMI1640；
S4, every 16h by the cell point hole cultivated in S3 once, per hole supplement nutrient solution 1-2mL, every 36h supplement
The μ L of PersonGenTM biomembranes 10, persistently cultivate 16d；
S5, slow virus to the T cell in S4 transfect to simultaneously amplification cultivation, obtain CAR-T cells.
Embodiment 2
S1, collection patient peripheral are enriched with blood, and the centrifuge tube equipped with lymphocyte separation medium is slowly moved into after filtrating leukocytes
In, centrifuged with density-gradient centrifugation method, wherein parameter of noncentricity is 2200r/min, 25 DEG C, 20min, draws tunica albuginea layer, is obtained outer
All blood mononuclear cells；
S2, the PMNC obtained in S1 washed with PBS, abandoning supernatant, take in cell precipitation and add
GT551 is resuspended, and adjustment cell concentration is 2.5x106；
S3, by the PMNC obtained in S2 with 1x106Individual/hole is inoculated in 24 orifice plates, in every Kong Zhongjia
Enter nutrient solution and 60IU/mL IL-2, be placed in 37 DEG C, 5%CO236h is cultivated in being cultivated in incubator, is added afterwards in every orifice plate
40μL PersonGenTMBiomembrane；
Wherein, the nutrient solution composition is to add 10%FBS in RPMI1640；
S4, every 18h by the cell point hole cultivated in S3 once, per hole supplement nutrient solution 2mL, every 48h supplement
The μ L of PersonGenTM biomembranes 15, persistently cultivate 16-18d；
S5, slow virus to the T cell in S4 transfect to simultaneously amplification cultivation, obtain CAR-T cells.
Embodiment 3
S1, collection patient peripheral are enriched with blood, and the centrifuge tube equipped with lymphocyte separation medium is slowly moved into after filtrating leukocytes
In, centrifuged with density-gradient centrifugation method, wherein parameter of noncentricity is 2100r/min, 22 DEG C, 18min, draws tunica albuginea layer, is obtained outer
All blood mononuclear cells；
S2, the PMNC obtained in S1 washed with PBS, abandoning supernatant, take in cell precipitation and add
GT551 is resuspended, and adjustment cell concentration is 2.3x106；
S3, by the PMNC obtained in S2 with 1x106Individual/hole is inoculated in 24 orifice plates, in every Kong Zhongjia
Enter nutrient solution and 60IU/mL IL-2, be placed in 37 DEG C, 5%CO230h is cultivated in being cultivated in incubator, is added afterwards in every orifice plate
30μL PersonGenTMBiomembrane；
Wherein, the nutrient solution composition is to add 10%FBS in RPMI1640；
S4, every 17h by the cell point hole cultivated in S3 once, per hole supplement nutrient solution 1.5mL, every 40h supplement
The μ L of PersonGenTM biomembranes 12, persistently cultivate 16-18d；
S5, slow virus to the T cell in S4 transfect to simultaneously amplification cultivation, obtain CAR-T cells.
Above content is only citing made for the present invention and explanation, and affiliated those skilled in the art are to being retouched
The specific embodiment stated is made various modifications or supplement or substituted using similar mode, without departing from invention or super
More scope defined in the claims, protection scope of the present invention all should be belonged to.
Claims (4)
1. a kind of Car-T cell preparation methods for cancer of pancreas, it is characterised in that this method comprises the following steps：
S1, collection peripheral blood in patients, extract PMNC；
S2, the PMNC obtained in S1 washed with PBS, abandoning supernatant, take and GT551 is added in cell precipitation
It is resuspended；
S3, by the PMNC obtained in S2 with 1x106Individual/hole is inoculated in 24 orifice plates, and culture is added in every hole
Liquid and 60IU/mL IL-2,24-36h is cultivated, add 20-40 μ L PersonGen in every orifice plate afterwardsTMBiomembrane；
S4, every 16-18h by the cell point hole cultivated in S3 once, per hole supplement nutrient solution 1-2mL, every 36-48h supplement
PersonGenTM biomembrane 10-15 μ L, persistently cultivate 16-18d；
S5, slow virus to the T cell in S4 transfect to simultaneously amplification cultivation, obtain CAR-T cells.
A kind of 2. Car-T cell preparation methods for cancer of pancreas according to claim 3, it is characterised in that：The S1
In concretely comprise the following steps, collection patient peripheral is enriched with blood, slowly move into after filtrating leukocytes equipped with lymphocyte separation medium from
In heart pipe, centrifuged with density-gradient centrifugation method, draw tunica albuginea layer, obtain PMNC.
A kind of 3. Car-T cell preparation methods for cancer of pancreas according to claim 3, it is characterised in that：Institute in S2
Nutrient solution composition is stated to add 10%FBS in RPMI1640.
A kind of 4. Car-T cell preparation methods for cancer of pancreas according to claim 3, it is characterised in that：Weight in S2
It is 2-2.5x10 that cell concentration is adjusted after outstanding6。
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710728773.5A CN107686831A (en) | 2017-08-23 | 2017-08-23 | A kind of Car T cell preparation methods for cancer of pancreas |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710728773.5A CN107686831A (en) | 2017-08-23 | 2017-08-23 | A kind of Car T cell preparation methods for cancer of pancreas |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108441481A (en) * | 2018-05-15 | 2018-08-24 | 河南省肿瘤医院 | A kind of Chimeric antigen receptor T cell and its cultural method |
| CN108641953A (en) * | 2018-05-16 | 2018-10-12 | 余春 | Totally enclosed type intelligent biology production system and production method |
| WO2020000533A1 (en) * | 2018-06-26 | 2020-01-02 | 深圳市北科生物科技有限公司 | High-adaptive full-automatic cell culture system and method therefor |
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| CN106511379A (en) * | 2016-12-26 | 2017-03-22 | 武汉波睿达生物科技有限公司 | Preparation method of CAR-T cell preparation for treating breast cancer |
| CN106854661A (en) * | 2016-12-26 | 2017-06-16 | 武汉波睿达生物科技有限公司 | A kind of preparation method of the CAR T cell preparations for treating prostate cancer |
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2017
- 2017-08-23 CN CN201710728773.5A patent/CN107686831A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106511379A (en) * | 2016-12-26 | 2017-03-22 | 武汉波睿达生物科技有限公司 | Preparation method of CAR-T cell preparation for treating breast cancer |
| CN106854661A (en) * | 2016-12-26 | 2017-06-16 | 武汉波睿达生物科技有限公司 | A kind of preparation method of the CAR T cell preparations for treating prostate cancer |
Non-Patent Citations (1)
| Title |
|---|
| 周如苑 等: "PersonGenTM 技术和CIK 细胞培养方法对T 细胞扩增与活化效果的比较", 《中国肿瘤生物治疗杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108441481A (en) * | 2018-05-15 | 2018-08-24 | 河南省肿瘤医院 | A kind of Chimeric antigen receptor T cell and its cultural method |
| CN108641953A (en) * | 2018-05-16 | 2018-10-12 | 余春 | Totally enclosed type intelligent biology production system and production method |
| WO2020000533A1 (en) * | 2018-06-26 | 2020-01-02 | 深圳市北科生物科技有限公司 | High-adaptive full-automatic cell culture system and method therefor |
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