CN1275618A - Method for cultivating T-lymphocytes - Google Patents
Method for cultivating T-lymphocytes Download PDFInfo
- Publication number
- CN1275618A CN1275618A CN 00109663 CN00109663A CN1275618A CN 1275618 A CN1275618 A CN 1275618A CN 00109663 CN00109663 CN 00109663 CN 00109663 A CN00109663 A CN 00109663A CN 1275618 A CN1275618 A CN 1275618A
- Authority
- CN
- China
- Prior art keywords
- lymphocyte
- nutrient solution
- cultural method
- culture fluid
- lymphocytes
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000001744 T-lymphocyte Anatomy 0.000 title claims abstract description 16
- 238000000034 method Methods 0.000 title claims description 19
- 210000004369 blood Anatomy 0.000 claims abstract description 9
- 239000008280 blood Substances 0.000 claims abstract description 9
- 235000015097 nutrients Nutrition 0.000 claims description 9
- 239000012531 culture fluid Substances 0.000 claims description 7
- 230000008014 freezing Effects 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 210000004698 lymphocyte Anatomy 0.000 abstract description 4
- 238000012136 culture method Methods 0.000 abstract 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- 230000000527 lymphocytic effect Effects 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 244000309466 calf Species 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 229920000669 heparin Polymers 0.000 description 2
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 description 2
- 229960001008 heparin sodium Drugs 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 238000005498 polishing Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- QTENRWWVYAAPBI-YCRXJPFRSA-N streptomycin sulfate Chemical compound OS(O)(=O)=O.OS(O)(=O)=O.OS(O)(=O)=O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O QTENRWWVYAAPBI-YCRXJPFRSA-N 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- IHPYMWDTONKSCO-UHFFFAOYSA-N 2,2'-piperazine-1,4-diylbisethanesulfonic acid Chemical compound OS(=O)(=O)CCN1CCN(CCS(O)(=O)=O)CC1 IHPYMWDTONKSCO-UHFFFAOYSA-N 0.000 description 1
- OTMSDBZUPAUEDD-UHFFFAOYSA-N Ethane Chemical compound CC OTMSDBZUPAUEDD-UHFFFAOYSA-N 0.000 description 1
- 241000594182 Sarcophaga sigma Species 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 108010086652 phytohemagglutinin-P Proteins 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000005204 segregation Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention relates to a culture method for T-lymphocytes. It has no need of separating lymphocyte, and features little blood demand and simple operation.
Description
The present invention relates to a kind of cultural method of cell, particularly a kind of lymphocytic cultural method.
The lymphocytic cultural method of T-in the prior art need carry out cellular segregation, complicated operating process.
T-lymphocyte cultural method of the present invention has overcome the deficiencies in the prior art, and is simple to operate, and blood using amount is few.
T-lymphocyte cultural method of the present invention, be applicable to the T-lymphocyte cultivation of using homemade T-lymphocyte culture fluid to carry out, the composition of relevant self-control T-lymphocyte culture fluid, prescription and preparation method thereof, apply for patent of invention separately, the moiety of described self-control T-lymphocyte culture fluid and prepare 1000 the gram nutrient solutions consumptions be:
N-(2-hydroxyethyl) piperazine-N '-2 ethane sulfonic aicd (Hepes) 3-6g
Sodium bicarbonate 1-3g
RPMI 1640 substratum (in the dry powder amount) 5-10g
L-glutaminate (LG) 0.1-0.3g
Heparin sodium (150u/mg) 0.1-0.3g
Phytohaemagglutinin (PHA) 5-20mg
Foetal calf serum 100-300ml
Penicillin (800,000 u/2ml) 0.1-0.5ml
Streptomycin sulphate (1,000,000 u/2ml) 0.1-0.5ml
Surplus distilled water polishing.
The pH value of this nutrient solution is 6.5-7.5.
T-lymphocyte cultural method of the present invention and the steps include:
(1) gets above-mentioned freezing T-lymphocyte culture fluid, at room temperature thaw;
(2) venous blood samples injects in the container of containing nutrient solution, and immediately with both mixings, the volume ratio of blood volume and nutrient solution is 1: 8-12;
(3) said vesse is placed 37 ℃ ± 0.5 ℃ incubator, promptly finish cell cultures after 48-96 hour.
In implementing process of the present invention, employed vessel and other apparatus all need sterilization to handle, and all operations all should meet aseptic technique, and method for disinfection and sterilization and aseptic technique method all belong to the known technology of this area.
T-lymphocyte cultural method of the present invention compared with prior art has following advantage:
(1) blood using amount is few, only need the 0.4-0.5ml blood sample, and prior art generally needs the 5-10 milliliter;
(2) method easy, save time, easy to operate, equipment is simple, separate and need to do lymphocyte in the prior art, and need expensive CO2gas incubator with lymphocyte separation medium, complex operation, time-consuming, instrument is expensive.
Further specify the present invention below in conjunction with embodiment:
Step 1, get moiety and prepare 1000 the gram nutrient solutions the following T-lymphocyte culture fluid of consumption:
Hepes 4.76g
Sodium bicarbonate 1.6g
RPMI 1640 substratum 8.32g
L-glutaminate (LG) 0.3g
Heparin sodium (150u/mg) 0.1g
The PHA-P 10mg that U.S. sigma produces
Penicillin (800,000 u/2ml) 0.35ml
Streptomycin sulphate (1,000,000 u/2ml) 0.3ml
Foetal calf serum 200ml
Surplus distilled water polishing.
The pH value of this nutrient solution is 7.0-7.2.
Step 2, carry out the lymphocytic cultivation of T-according to following method and step:
(1) lymphocyte culture fluid that above-mentioned 4ml is freezing is thawed under the room temperature;
(2) venous blood samples 0.4-0.5ml injects in the container of containing nutrient solution, immediately with both mixings;
(3) said vesse is placed 37 ℃ ± 0.5 ℃ incubator, promptly finish cell cultures after 72 hours.
Using such method, the T-lymphocyte transformation rate reaches more than 80%, and karyon rises greatly by the kernel division.
Claims (1)
1. T-lymphocyte cultural method is characterized in that it realizes as follows:
(1) under the room temperature freezing T-lymphocyte culture fluid is thawed;
(2) venous blood samples injects in the container of containing nutrient solution, and immediately with both mixings, the volume ratio of blood and nutrient solution is 1: 8-12;
(3) said vesse is placed 37 ℃ ± 0.5 ℃ incubator 48-96 hour.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00109663 CN1275618A (en) | 2000-06-21 | 2000-06-21 | Method for cultivating T-lymphocytes |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00109663 CN1275618A (en) | 2000-06-21 | 2000-06-21 | Method for cultivating T-lymphocytes |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1275618A true CN1275618A (en) | 2000-12-06 |
Family
ID=4579774
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 00109663 Pending CN1275618A (en) | 2000-06-21 | 2000-06-21 | Method for cultivating T-lymphocytes |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1275618A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1306027C (en) * | 2002-12-12 | 2007-03-21 | 中国医学科学院基础医学研究所 | External amplification process of gamma delta T-lymphocyte |
| CN100410368C (en) * | 2005-07-19 | 2008-08-13 | 翁炳焕 | Pretreating method for chromosome nuclear type analysis lymphocyte culture specimen |
| CN101129405B (en) * | 2007-07-31 | 2011-05-11 | 崔澂 | Biological formulation product for treating habitual abortion |
| CN103184193A (en) * | 2013-04-12 | 2013-07-03 | 阳小卫 | Rapid proliferation method of T cells |
| CN103898192A (en) * | 2014-04-23 | 2014-07-02 | 重庆庞通医疗器械有限公司 | Blood culture method |
| CN110669730A (en) * | 2019-10-23 | 2020-01-10 | 杭州宝荣科技有限公司 | Human peripheral blood lymphocyte culture medium |
-
2000
- 2000-06-21 CN CN 00109663 patent/CN1275618A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1306027C (en) * | 2002-12-12 | 2007-03-21 | 中国医学科学院基础医学研究所 | External amplification process of gamma delta T-lymphocyte |
| CN100410368C (en) * | 2005-07-19 | 2008-08-13 | 翁炳焕 | Pretreating method for chromosome nuclear type analysis lymphocyte culture specimen |
| CN101129405B (en) * | 2007-07-31 | 2011-05-11 | 崔澂 | Biological formulation product for treating habitual abortion |
| CN103184193A (en) * | 2013-04-12 | 2013-07-03 | 阳小卫 | Rapid proliferation method of T cells |
| CN103898192A (en) * | 2014-04-23 | 2014-07-02 | 重庆庞通医疗器械有限公司 | Blood culture method |
| CN103898192B (en) * | 2014-04-23 | 2015-01-28 | 重庆庞通医疗器械有限公司 | Blood culture method |
| CN110669730A (en) * | 2019-10-23 | 2020-01-10 | 杭州宝荣科技有限公司 | Human peripheral blood lymphocyte culture medium |
| CN110669730B (en) * | 2019-10-23 | 2022-03-29 | 杭州杰毅麦特医疗器械有限公司 | Human peripheral blood lymphocyte culture medium |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| ASS | Succession or assignment of patent right |
Owner name: BEIJING JIANERKANG MEDICINE EQUIPMENT CO., LTD. Free format text: FORMER OWNER: ZHANG GUOQING Effective date: 20010912 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20010912 Address after: 100088 Building No. 7, West Lane, Zhichun Road, Haidian District, Beijing, Luozhuang Applicant after: Beijing jianerkang Medical Equipment Co., Ltd. Address before: 100080 Beijing city Haidian District Xiyuan No. 15 Haidian Sports Institute in the playground Applicant before: Zhang Guoqing |
|
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |