CN106119199A - A kind of method of quick obtaining peripheral blood PBMC - Google Patents
A kind of method of quick obtaining peripheral blood PBMC Download PDFInfo
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- CN106119199A CN106119199A CN201610483091.8A CN201610483091A CN106119199A CN 106119199 A CN106119199 A CN 106119199A CN 201610483091 A CN201610483091 A CN 201610483091A CN 106119199 A CN106119199 A CN 106119199A
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- centrifuge tube
- pbmc
- peripheral blood
- blood
- quick obtaining
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
A kind of method that the invention discloses quick obtaining peripheral blood PBMC, comprise the steps: that the plastic blood bag that will be equipped with blood sample is put in centrifuge tube adapter, centrifugal after trim, after removing upper plasma, middle sallow layer is taken out, in the sallow layer taken out, adds normal saline dilution；Being sent in the centrifuge tube containing lymphocyte separation medium by the product obtained, the wherein said product obtained is positioned at lymphocyte separation medium upper strata；Centrifugation, draws middle tunica albuginea layer, washing, is centrifuged and abandons supernatant, obtain peripheral blood PBMC.The present invention need not cut off the blood vessel of plastic blood bag during processing whole blood use, blood sample is put into the operation carrying out being correlated with in centrifuge tube again, easy and simple to handle, a large amount of human peripheral PBMC are separated and not only can reduce operating procedure, and saved operating time, reagent consumptive material and the separation of reagent dosage, beneficially PBMC and the motility rate of cell.
Description
Technical field
The present invention relates to peripheral blood PBMC separation technology field, a kind of method particularly relating to quick obtaining peripheral blood PBMC
Background technology
Autoimmune T cell technology, is to isolate T cell from the peripheral blood of patient, by In vitro culture, expands, makes
Agent, feeds back in the patient, and induction human body self produces immunne response.The acquisition of T cell at present is mainly derived from human peripheral
In PBMC.Main separation method is by direct for human peripheral and PBS mixed in equal amounts, is subsequently adding in lymph separation liquid and carries out ladder
Degree separates, and finally obtains PBMC.For the sample that this method is few to blood volume the most operable, but separate substantial amounts of blood volume time
(> 100ML), complex operation step, the longest, it is unfavorable for the separation of PBMC and the motility rate of cell；The most comparatively large number of blood sampling
(> 100ml) use Plastic Blood Bags, need first to cut short the blood vessel of plastic blood bag during use, blood sample is proceeded to
Centrifuge tube carries out follow-up PBMC operation, complex operation step again.
Summary of the invention
The present invention proposes a kind of method of quick obtaining peripheral blood PBMC, need not cut short plastic blood bag during use
Blood vessel, blood sample is put into the operation carrying out being correlated with in centrifuge tube again.The method separates not only for a large amount of human peripheral PBMC
Operating procedure can be reduced, shorten the operating time, it is ensured that Cell viability, and save experiment consumptive material and reagent dosage, favorably
Cost control in PBMC.
The method of a kind of quick obtaining peripheral blood PBMC that the present invention proposes, comprises the steps:
S1, the plastic blood bag that will be equipped with blood sample are put in centrifuge tube adapter, centrifugal after trim；
After S2, removing upper plasma, middle sallow layer is taken out, in the sallow layer taken out, add normal saline dilute
Release；
S3, the product obtained by S2 are sent in the centrifuge tube containing lymphocyte separation medium, the product position that wherein S2 obtains
In lymphocyte separation medium upper strata；
S4, centrifugation, draw middle tunica albuginea layer, washing, be centrifuged and abandon supernatant, obtain peripheral blood PBMC.
Preferably, in S1, centrifugal speed is 1200-1800rpm, and centrifugation time is 10-20min.
Preferably, in S1, will be equipped with in the adapter that the plastic blood bag of blood sample puts into adaptive 250ml centrifuge tube.
Preferably, in S2, in absorption sallow layer to centrifuge tube, add normal saline dilution to centrifuge tube cumulative volume
70-80%.
Preferably, in S3, draw the product that 20-30ml S2 obtains, join containing 15-30ml separation of lymphocytes
In the centrifuge tube of liquid.
Preferably, in S3, draw the product that obtains of 25ml S2, join containing 20ml lymphocyte separation medium from
In heart pipe.
Preferably, in S4, centrifugal speed is 1500-1800rpm, and centrifugation time is 10-30min.
The present invention can T cell after quick obtaining a large amount of blood PBMC, method is easy, it is easy to operation, the present invention directly will adopt
Blood bag after blood is put in the centrifuge of adaptive centrifuge tube and is directly centrifuged, and after artificial extrusion blood plasma, extrudes the sallow of centre gently
Layer carries out follow-up operation in centrifuge tube, thus has saved time, reagent, consumptive material.
Detailed description of the invention
Below, by specific embodiment, technical scheme is described in detail.
Embodiment 1
A kind of method of quick obtaining peripheral blood PBMC, comprises the steps:
S1, the plastic blood bag that will be equipped with blood sample are put in centrifuge tube adapter, centrifugal after trim；
After S2, removing upper plasma, middle sallow layer is taken out, in the sallow layer taken out, add normal saline dilute
Release；
S3, the product obtained by S2 are sent in the centrifuge tube containing lymphocyte separation medium, the product position that wherein S2 obtains
In lymphocyte separation medium upper strata；
S4, centrifugation, draw middle tunica albuginea layer, washing, be centrifuged and abandon supernatant, obtain peripheral blood PBMC.
Embodiment 2
A kind of method of quick obtaining peripheral blood PBMC, comprises the steps:
S1,30ml is put in centrifuge tube adapter equipped with the plastic blood bag of blood sample, centrifugal after trim, centrifugal speed
For 1800rpm, centrifugation time is 10min；
After S2, removing upper plasma, middle sallow layer is taken out, in the sallow layer taken out, add normal saline dilute
Release；
S3, the product obtained by S2 are sent in the centrifuge tube containing lymphocyte separation medium, the product position that wherein S2 obtains
In lymphocyte separation medium upper strata；
S4, centrifugation, draw middle tunica albuginea layer, washing, be centrifuged and abandon supernatant, obtain peripheral blood PBMC.
Embodiment 3
A kind of method of quick obtaining peripheral blood PBMC, comprises the steps:
S1,150ml is put in the adapter of adaptive 250ml centrifuge tube equipped with the plastic blood bag of blood sample, after trim
Centrifugal, centrifugal speed is 1200rpm, and centrifugation time is 20min；
After S2, removing upper plasma, middle sallow layer is taken out, in the sallow layer taken out, add normal saline dilute
Release；
S3, the product obtained by S2 are sent in the centrifuge tube containing lymphocyte separation medium, the product position that wherein S2 obtains
In lymphocyte separation medium upper strata；
S4, centrifugation, draw middle tunica albuginea layer, washing, be centrifuged and abandon supernatant, obtain peripheral blood PBMC.
Embodiment 4
A kind of method of quick obtaining peripheral blood PBMC, comprises the steps:
S1,40ml is sent in adaptive 50ml centrifuge tube adapter equipped with the plastic blood bag of blood sample, centrifugal after trim,
Centrifugal speed is 1500rpm, and centrifugation time is 16min；
After S2, extrusion upper plasma, in extrusion 10ml sallow layer to 1 50ml centrifuge tube, add normal saline dilution extremely
The 70% of centrifuge tube volume, residue sallow layer is processed by above-mentioned steps dilution；
S3, the product obtained by 30ml S2 are sent in the centrifuge tube containing 15ml lymphocyte separation medium, and wherein S2 obtains
Product be positioned at lymphocyte separation medium upper strata；
S4, centrifugation, centrifugal speed is 1500rpm, and centrifugation time is 30min, and the product that residue S2 obtains is by above-mentioned
Step centrifugal treating；Tunica albuginea layer in the middle of drawing, washing, it is centrifuged and abandons supernatant, obtain peripheral blood PBMC.
Embodiment 5
A kind of method of quick obtaining peripheral blood PBMC, comprises the steps:
S1,40ml is sent in adaptive 50ml centrifuge tube adapter equipped with the plastic blood bag of blood sample, centrifugal after trim,
Centrifugal speed is 1600rpm, and centrifugation time is 14min；
After S2, extrusion upper plasma, draw 17.5ml sallow layer in 50ml centrifuge tube, add normal saline dilution to from
The 80% of heart pipe volume, residue sallow layer is processed by above-mentioned steps dilution；
S3, the product obtained by 20ml S2 are sent in the centrifuge tube containing 30ml lymphocyte separation medium, and wherein S2 obtains
Product be positioned at lymphocyte separation medium upper strata；
S4, centrifugation, centrifugal speed is 1800rpm, and centrifugation time is 10min, and the product that residue S2 obtains is by above-mentioned
Step centrifugal treating；Tunica albuginea layer in the middle of drawing, washing, it is centrifuged and abandons supernatant, obtain peripheral blood PBMC.
Embodiment 6
A kind of method of quick obtaining peripheral blood PBMC, comprises the steps:
S1,120ml is sent in the adapter of adaptive 250ml centrifuge tube equipped with the plastic blood bag of blood sample, after trim
Centrifugal, centrifugal speed is 1500rpm, and centrifugation time is 15min；
After S2, extrusion upper plasma, in absorption 15ml sallow layer to 50ml centrifuge tube, add normal saline dilution to centrifugal
The 75% of pipe cumulative volume, residue sallow layer is processed by above-mentioned steps dilution；
S3, the product obtained by 25ml S2 are sent in the centrifuge tube containing 20ml lymphocyte separation medium, and wherein S2 obtains
Product be positioned at lymphocyte separation medium upper strata；
S4, centrifugation, centrifugal speed is 1600rpm, and centrifugation time is 20min, and the product that residue S2 obtains is by above-mentioned
Step centrifugal treating；In the middle of drawing, tunica albuginea layer is in 50ml centrifuge tube, washing, centrifugal abandons supernatant, obtains peripheral blood PBMC.
The method using embodiment 6 being contrasted with traditional method, wherein traditional method is for mix human peripheral and PBS equivalent
Close, be subsequently adding lymph separation liquid and carry out gradient separations, finally obtain PBMC.
The usage amount of lymph separation liquid, pipet quantity, the centrifuge tube quantity that both are used, that the operating time is carried out is right
Ratio, test result is as shown in table 1 below:
Table 1 the inventive method contrasts table with traditional method test result
The present invention can save substantial amounts of lymph separation liquid, has only to the lymph separation liquid of 60ml by this method, and traditional
Method but needs nearly 280mL lymph separation liquid；Only about need the consumptive material of traditional method 1/10, such as centrifuge tube, pipet etc.；Phase
The substantial amounts of time is saved than traditional method.
The above, the only present invention preferably detailed description of the invention, but protection scope of the present invention is not limited thereto,
Any those familiar with the art in the technical scope that the invention discloses, according to technical scheme and
Inventive concept equivalent or change in addition, all should contain within protection scope of the present invention.
Claims (7)
1. the method for a quick obtaining peripheral blood PBMC, it is characterised in that comprise the steps:
S1, the plastic blood bag that will be equipped with blood sample are put in centrifuge tube adapter, centrifugal after trim；
After S2, removing upper plasma, middle sallow layer is taken out, in the sallow layer taken out, add normal saline dilution；
S3, the product obtained by S2 are sent in the centrifuge tube containing lymphocyte separation medium, and the product that wherein S2 obtains is positioned at pouring
Bar cell separation liquid upper strata；
S4, centrifugation, draw in the middle of tunica albuginea layer, washing, centrifugal abandon supernatant and obtain peripheral blood PBMC.
The method of quick obtaining peripheral blood PBMC the most according to claim 1, it is characterised in that in S1, centrifugal speed
For 1200-1800rpm, centrifugation time is 10-20min.
The method of quick obtaining peripheral blood PBMC the most according to claim 1, it is characterised in that in S1, will be equipped with blood
The plastic blood bag of liquid sample is put in the adapter of adaptive 250ml centrifuge tube.
The method of quick obtaining peripheral blood PBMC the most according to claim 1, it is characterised in that in S2, draws sallow
Layer in centrifuge tube, adds normal saline dilution to the 70-80% of centrifuge tube cumulative volume.
The method of quick obtaining peripheral blood PBMC the most according to claim 1, it is characterised in that in S3, draws 20-
The product that 30ml S2 obtains, joins in the centrifuge tube containing 15-30ml lymphocyte separation medium.
The method of quick obtaining peripheral blood PBMC the most according to claim 4, it is characterised in that in S3, draws 25ml
The product that S2 obtains, joins in the centrifuge tube containing 20ml lymphocyte separation medium.
The method of quick obtaining peripheral blood PBMC the most according to claim 1, it is characterised in that in S4, centrifugal speed
For 1500-1800rpm, centrifugation time is 10-30min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610483091.8A CN106119199A (en) | 2016-06-24 | 2016-06-24 | A kind of method of quick obtaining peripheral blood PBMC |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610483091.8A CN106119199A (en) | 2016-06-24 | 2016-06-24 | A kind of method of quick obtaining peripheral blood PBMC |
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| CN106119199A true CN106119199A (en) | 2016-11-16 |
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| CN201610483091.8A Pending CN106119199A (en) | 2016-06-24 | 2016-06-24 | A kind of method of quick obtaining peripheral blood PBMC |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106085954A (en) * | 2016-06-24 | 2016-11-09 | 安徽未名细胞治疗有限公司 | A kind of a large amount of human peripheral PBMC fast separating process |
| CN114410580A (en) * | 2022-02-07 | 2022-04-29 | 无锡观合医学检验所有限公司 | Extraction method of PBMC (peripheral blood mononuclear cell) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101914497A (en) * | 2010-07-19 | 2010-12-15 | 济南迪博生物技术有限公司 | Clinical N-CIK cell culture and quality control and identification kit and application |
| CN105112373A (en) * | 2015-09-15 | 2015-12-02 | 上海麦禾生物科技有限公司 | Method for separating peripheral blood monouclear cells of hemolytic anemia patient |
-
2016
- 2016-06-24 CN CN201610483091.8A patent/CN106119199A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101914497A (en) * | 2010-07-19 | 2010-12-15 | 济南迪博生物技术有限公司 | Clinical N-CIK cell culture and quality control and identification kit and application |
| CN105112373A (en) * | 2015-09-15 | 2015-12-02 | 上海麦禾生物科技有限公司 | Method for separating peripheral blood monouclear cells of hemolytic anemia patient |
Non-Patent Citations (2)
| Title |
|---|
| 王维等: "穿心莲内酯对人PBMC/PBM表达IL-18相关细胞因子的影响", 《中国免疫学杂志》 * |
| 罗玉敏等: "《脑血管病实验方法学》", 30 April 2014 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106085954A (en) * | 2016-06-24 | 2016-11-09 | 安徽未名细胞治疗有限公司 | A kind of a large amount of human peripheral PBMC fast separating process |
| CN114410580A (en) * | 2022-02-07 | 2022-04-29 | 无锡观合医学检验所有限公司 | Extraction method of PBMC (peripheral blood mononuclear cell) |
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Application publication date: 20161116 |
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