CN106701548A - Lymphocyte extraction device and extraction method - Google Patents
Lymphocyte extraction device and extraction method Download PDFInfo
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- CN106701548A CN106701548A CN201710072789.5A CN201710072789A CN106701548A CN 106701548 A CN106701548 A CN 106701548A CN 201710072789 A CN201710072789 A CN 201710072789A CN 106701548 A CN106701548 A CN 106701548A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/04—Cell isolation or sorting
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- 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
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- 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
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Abstract
The invention relates to a lymphocyte extraction device and an extraction method. The lymphocyte extraction device comprises an injector, a separating medium displacer and a filter device, wherein the injector consists of an outer tube body, a pull handle, a piston and a needle head; the connection section of the separating medium displacer is detachably connected with the outer tube body; the filter device comprises a connection tube, a liquid storage tube and a mixed cellulose filter membrane placed in the connection tube; a liquid inlet is formed in the bottom of the liquid storage tube and is connected with a liquid suction pipe, and a liquid outlet is formed in the side wall of the liquid storage tube and is connected with a liquid discharging pipe; check valves are arranged at the liquid inlet and the liquid outlet; and the upper end of the connection tube is detachably connected with the outer tube body. According to the extraction method implemented through the device, cells to be extracted can be always retained in the original container, and unnecessary cells and plasma can be easily and conveniently removed, so that time and labor for cell separation and extraction are greatly saved, the working efficiency is high, more cells with higher purity are extracted, and the possibility of pollution is lower.
Description
Technical field
The present invention relates to a kind of lymphocyte extraction element and extracting method.
Background technology
It is a kind of preliminary cellular method of purification commonly used in clinical examination and scientific research, base that separation of lymphocytes is extracted
Present principles are all density-gradient centrifugation methods, and conventional method is that initial sample is added on lymphocyte separation medium to be centrifuged, after centrifugation
Mononuclearcell superposed layer is drawn, physiological saline is eventually adding and is centrifuged repeatedly washing again.Operated in accordance with conventional methods is comparatively laborious time-consuming,
And cell needs repeatedly to be shifted between different utensils, more than time centrifugation, process easily causes that cell loss is damaged and pollution.
Because complex operation is cumbersome, the extracted amount that there is cell is few, the low weak point of operating efficiency of extraction.
The content of the invention
The technical problem to be solved in the present invention is：A kind of lymphocyte extraction element and extracting method, extraction dress are provided
Put and extracting method can allow and need the cell for extracting to remain in the container of script, without cell and blood plasma etc.
Removal that can be easier, make cell separation extract it is more time saving save trouble, operating efficiency cell extraction is more purer, and the probability of pollution is more
It is small.
Solving the technical scheme of above-mentioned technical problem is：A kind of lymphocyte extraction element, including syringe, separating liquid are put
Parallel operation and diafiltration device, described syringe are made up of outer cylinder body, pull handle, piston and syringe needle, between pull handle and piston, outer barrel
Can be connected by removably between body and syringe needle, described separating liquid displacer is that lower end is closed, upper end open cylinder
Structure, separating liquid displacer is made up of reservoir and linkage section, can be led between the linkage section and outer cylinder body of separating liquid displacer
Removably connection is crossed, described diafiltration device includes connecting cylinder, the liquid storage cylinder for being arranged on connecting cylinder lower end and the company of being placed on
The cellulose mixture filter membrane in cylinder is connect, the bottom of liquid storage cylinder is provided with liquid inlet and is connected with pipette, on the side wall of liquid storage cylinder
Be provided with liquid outlet and be connected with drain pipe, be provided with check-valves on liquid inlet and liquid outlet, the upper end of connecting cylinder with
Can be connected by removably between outer cylinder body.
External screw thread, the upper end of syringe needle, the upper end open of separating liquid displacer and connecting cylinder are provided with described outer cylinder body
It is provided with the internal thread coordinated with the external screw thread on outer cylinder body.
It is to be connected through a screw thread between described pull handle and piston.
The aperture of described cellulose mixture filter membrane is 0.5~2 μm.
Lymphocyte separation medium, the upper end of separating liquid displacer have also been filled in the reservoir of described separating liquid displacer
Opening is sealed by sealed membrane.
Another technical scheme of the invention is：A kind of lymphocyte extracting method, using lymphocyte of the present invention
Extraction element, concrete operation step is as follows：
（1）Sample peripheral blood is gathered using syringe, by sample collection to outer cylinder body；
（2）The pull handle and syringe needle of syringe are removed, lymphocyte separation medium is added in separating liquid displacer and liquid storage is filled it up with
Groove, the addition of lymphocyte separation medium is 1 with the volume ratio of sample：0.9~1, then separating liquid displacer is installed to outer barrel
On body, then it is centrifuged, the liquid in outer barrel is layered as serum, mononuclearcell layer and lymph successively from top to bottom
Cell separation liquid, liquid is followed successively by lymphocyte separation medium and red blood cell from top to bottom in separating liquid displacer,
（3）Separating liquid displacer is removed, pull handle is connected with piston, then diafiltration device is installed on outer cylinder body, then up
Pull pull handle to suck physiological saline from the pipette of diafiltration device to wash the liquid in outer cylinder body, pull down pull handle
Serum is discharged from the discharging tube of diafiltration device, mononuclearcell liquid is deposited in outer cylinder body, complete once washing process；So
Repeated multiple times afterwards to take out crowded physiological saline the liquid in outer cylinder body is washed, the liquid for finally being obtained in outer cylinder body is to be washed
Wash cell suspension.
It is 2-3 times that crowded physiological saline is taken out to the number of times that the liquid in outer cylinder body is washed.
Separation of lymphocytes its it is liquid-tight degree between mononuclearcell and neutrophil leucocyte and red blood cell, by centrifugation make
Different densities cell is layered, and takes fresh anticoagulation and is gently added on the liquid level of cell separation liquid, up to descends cell to divide after centrifugation
Four layers：Ground floor is plasma layer（Serum）；The second layer is ring-type milky lymphocyte（Mononuclearcell layer）；Third layer is
Bright lymphocyte separation medium layer；4th layer is red blood cell layer.This programme is dispensed lymphocyte separation medium based on the principle in advance
In in a container, sample tube and container are centrifuged by connecting, and red blood cell and apocyte are sink to the container after centrifugation, by container
By the tangible Impurity removal of the overwhelming majority in original sample by removing, filter wash then is carried out to the cell liquid in sample tube again
Removal soluble impurity.
The present invention is the Optimal Experimental process in the principle for keeping density-gradient centrifugation method, can be by holding after layering is centrifuged
Device disassembles the removal for realizing unwanted contributions, then is washed to extracting cell by the way that diafiltration method is quick.Using lymph of the present invention
Cell extraction device and extracting method, can allow need extract cell remain in the container of script, and other cell and
Blood plasma etc. can be easier removal.I.e. can be always held at cell in the outer cylinder body of syringe by the present invention, can avoid thin
Born of the same parents and extraneous excessive contact, also cannot be easily caused the sample wrong number and cell contamination caused by transfer, sample cell separation and
The method of washing is more time-consuming than conventional method, and reduces the use of some medical disposable materials.
Brief description of the drawings
Fig. 1：The lymphocyte extraction element structural representation of the embodiment of the present invention 1.
Fig. 2：Syringe decomposition texture schematic diagram of the present invention.
Fig. 3：The separating liquid displacer schematic diagram of the embodiment of the present invention 2.
Fig. 4：Lymphocyte extraction process front half section flow chart of the present invention.Wherein,（A）Represent step（1）Operation,（B）Table
Show step（2）The operation of syringe pull handle and syringe needle is removed,（C）Represent step（2）Separating liquid displacer is installed to outer cylinder body
On operation,（D）Represent step（2）The operation of centrifugation.
Fig. 5：Lymphocyte extraction process second half section flow chart of the present invention.Wherein,（D）Represent step（2）Centrifugation
Operation,（E）Represent step（3）Separating liquid displacer is removed, pull handle is connected with piston, then diafiltration device is installed to outer cylinder body
On operation,（F）Represent step（3）The operation that suction physiological saline is washed to the liquid in outer cylinder body first,（G）Represent
Step（3）The operation that repeatedly suction physiological saline is washed to the liquid in outer cylinder body.
In figure：1- syringes, 11- pull handles, 12- outer cylinder bodies, 13- pistons, 14- syringe needles, 2- separating liquid displacers, 21- storages
Liquid bath, 22- linkage sections, 3- diafiltration devices, 31- connecting cylinders, 32- cellulose mixture filter membranes, 33- liquid storage cylinders, 34- pipette, 35-
Drain pipe, 36- check-valves, 4- sealed membranes.
P1 represents peripheral blood, and P2 represents lymphocyte separation medium, and P3 represents serum, and P4 represents mononuclearcell liquid, P5 tables
Show red blood cell, P6 represents physiological saline, and P7 represents washed cell suspension.
Specific embodiment
Embodiment 1：A kind of lymphocyte extraction element, as Figure 1-Figure 2, including syringe 1, separating liquid displacer 2
With diafiltration device 3, described syringe 1 is made up of outer cylinder body 12, pull handle 11, piston 13 and syringe needle 14, pull handle and piston it
Between, can be connected by removably between outer cylinder body and syringe needle, described separating liquid displacer 2 is that lower end is closed, upper end is opened
Mouthful tube structure, separating liquid displacer 2 is made up of reservoir 21 and linkage section 22, the linkage section 22 of separating liquid displacer with
Can be connected by removably between outer cylinder body, described diafiltration device 3 includes connecting cylinder 31, is arranged on connecting cylinder lower end
Liquid storage cylinder 33 and the cellulose mixture filter membrane being placed in connecting cylinder（MCE）32,0.5-2 μm of aperture, the bottom of liquid storage cylinder is provided with
Liquid inlet is simultaneously connected with pipette 34, is provided with liquid outlet on the side wall of liquid storage cylinder and is connected with drain pipe 35, connecting cylinder
Can be connected by removably between upper end and outer cylinder body, check-valves 36 is provided with liquid inlet and liquid outlet, only
It is check valve to return valve, and direction of the valve only towards liquid flowing is opened, and can prevent liquid from flowing back.
In the present embodiment, external screw thread, syringe needle, the upper end open of separating liquid displacer and company are provided with described outer cylinder body
The upper end for connecing cylinder is provided with the internal thread coordinated with the external screw thread on outer cylinder body.It is by spiral shell between described pull handle and piston
Line is connected.Converted as one kind, described syringe needle, between the upper end open of separating liquid displacer and the upper end of connecting cylinder, pull handle
Can also be connected using buckle or other removablies and piston between.
Embodiment 2：A kind of lymphocyte extraction element, as shown in figure 3, including syringe 1, separating liquid displacer 2 and filter
Cleaning device 3, its basic structure is same as Example 1, and difference is：In the reservoir 21 of described separating liquid displacer 2 also
Lymphocyte separation medium P2 is filled, the upper end open of separating liquid displacer 2 is sealed by sealed membrane 4.When using, directly will envelope
Membrana oralis 4 is opened or discloses that wear can be to use, without adding lymphocyte separation medium P2 inside again.
Embodiment 3：A kind of lymphocyte extracting method, it is as shown in fig. 4-5, thin using lymph as described in Example 1
Born of the same parents' extraction element, concrete operation step is as follows：
（1）Sample peripheral blood P1 is gathered using syringe 1, by sample collection to outer cylinder body 12；
（2）The pull handle 11 and syringe needle 14 of syringe are removed, lymphocyte separation medium P2 is added in separating liquid displacer 2, drenched
The addition of bar cell separation liquid is 1 with the volume ratio of sample：1, then separating liquid displacer is installed on outer cylinder body, Ran Houjin
Row centrifugation, makes the liquid in outer barrel be layered as blood-serum P 3, mononuclearcell layer P4 and lymphocyte point successively from top to bottom
Chaotropic P2, liquid is followed successively by lymphocyte separation medium P2 and red blood cell P5 from top to bottom in separating liquid displacer,
（3）Separating liquid displacer 2 is removed, pull handle 11 is connected with piston 13, then diafiltration device 3 is installed on outer cylinder body 12,
Pull pull handle to suck physiological saline P6 from the pipette 34 of diafiltration device 3 then up to wash the liquid in outer cylinder body
（Traditional conventional wash be centrifugation go supernatant to add again physiological saline is resuspended to be repeated twice, as long as the present embodiment suction physiology twice
Salt solution, without being centrifuged again or mixing）, pull down pull handle and serum is discharged from the discharging tube 36 of diafiltration device, it is single
Nucleus liquid is deposited in outer cylinder body, completes once washing process；Then it is repeated multiple times to smoke crowded physiological saline in outer cylinder body
Liquid is washed, and the liquid for finally being obtained in outer cylinder body is the final washed cell suspension for needing to separate acquisition.
Under normal circumstances, the present invention can just rinse out most serum and remaining point for 3 times using brine
Chaotropic, but if running into jaundice or piarhemia sample can suitably increase washing times, can be according to the life from the outlet discharge of discharging tube 36
The turbidity and color for managing salt solution judge, if the physiological saline of discharge more cleans explanation and rinsed out most impurity.
As one kind conversion of embodiment 3, it would however also be possible to employ lymphocyte extraction element as described in Example 2.Use
When, directly by the sealed membrane 4 of separating liquid displacer 2 open or disclose wear can to use, without again toward in separating liquid displacer 2 plus
Enter lymphocyte separation medium P2.
Claims (7)
1. a kind of lymphocyte extraction element, it is characterised in that：Including syringe（1）, separating liquid displacer（2）With diafiltration device
（3）, described syringe（1）It is by outer cylinder body（12）, pull handle（11）, piston（13）And syringe needle（14）Composition, pull handle and piston
Between, can be connected by removably between outer cylinder body and syringe needle, described separating liquid displacer（2）Be lower end it is closed, on
The tube structure of end opening, separating liquid displacer（2）It is by reservoir（21）And linkage section（22）Constitute, separating liquid displacer
Linkage section（22）Can be connected by removably between outer cylinder body, described diafiltration device（3）Including connecting cylinder（31）, set
Put the liquid storage cylinder in connecting cylinder lower end（33）With the cellulose mixture filter membrane being placed in connecting cylinder（32）, the bottom of liquid storage cylinder is opened
There is liquid inlet and be connected with pipette（34）, it is provided with liquid outlet on the side wall of liquid storage cylinder and is connected with drain pipe（35）, liquid
Check-valves is provided with body entrance and liquid outlet（36）, can be by removably between the upper end of connecting cylinder and outer cylinder body
Connection.
2. lymphocyte extraction element according to claim 1, it is characterised in that：Outer spiral shell is provided with described outer cylinder body
Line, the upper end of syringe needle, the upper end open of separating liquid displacer and connecting cylinder is provided with and the external screw thread cooperation on outer cylinder body
Internal thread.
3. lymphocyte extraction element according to claim 1, it is characterised in that：It is logical between described pull handle and piston
Cross threaded connection.
4. lymphocyte extraction element according to claim 1, it is characterised in that：The hole of described cellulose mixture filter membrane
Footpath is 0.5~2 μm.
5. the lymphocyte extraction element according to claim any one of 1-4, it is characterised in that：Described separating liquid displacement
Device（2）Reservoir（21）Lymphocyte separation medium is inside also filled（P2）, separating liquid displacer（2）Upper end open by envelope
Membrana oralis（4）Sealing.
6. a kind of lymphocyte extracting method, it is characterised in that：Carried using the lymphocyte as described in claim any one of 1-4
Device is taken, concrete operation step is as follows：
（1）Using syringe（1）Collection sample peripheral blood（P1）, by sample collection to outer cylinder body（12）It is interior；
（2）Remove the pull handle of syringe（11）And syringe needle（14）, by lymphocyte separation medium（P2）It is added to separating liquid displacer
（2）In and fill it up with reservoir（21）, the addition of lymphocyte separation medium is 1 with the volume ratio of sample：0.9~1, then will separate
Liquid displacer is installed on outer cylinder body, is then centrifuged, and the liquid in outer barrel is layered as serum successively from top to bottom
（P3）, mononuclearcell layer（P4）And lymphocyte separation medium（P2）, liquid is followed successively by pouring from top to bottom in separating liquid displacer
Bar cell separation liquid（P2）And red blood cell（P5）,
（3）Remove separating liquid displacer（2）, by pull handle（11）With piston（13）Connection, then by diafiltration device（3）It is installed to outer barrel
Body（12）On, pull handle from diafiltration device is pulled then up（3）Pipette（34）Middle suction physiological saline（P6）To outer cylinder body
Interior liquid is washed, and pulls down pull handle by serum from the discharging tube of diafiltration device（36）Middle discharge, mononuclearcell liquid
It is deposited in outer cylinder body, completes once washing process；Then it is repeated multiple times to take out crowded physiological saline the liquid in outer cylinder body is carried out
Washing, the liquid for finally being obtained in outer cylinder body as washed cell suspension.
7. lymphocyte extracting method according to claim 6, it is characterised in that：Crowded physiological saline is smoked in outer cylinder body
The number of times that liquid is washed is 2-3 times.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710072789.5A CN106701548A (en) | 2017-02-10 | 2017-02-10 | Lymphocyte extraction device and extraction method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710072789.5A CN106701548A (en) | 2017-02-10 | 2017-02-10 | Lymphocyte extraction device and extraction method |
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| Publication Number | Publication Date |
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| CN106701548A true CN106701548A (en) | 2017-05-24 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201710072789.5A Withdrawn CN106701548A (en) | 2017-02-10 | 2017-02-10 | Lymphocyte extraction device and extraction method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108554655A (en) * | 2018-01-03 | 2018-09-21 | 赛诺菲生物科技（武汉）有限公司 | Platelet rich plasma separator and separation method |
| CN111870258A (en) * | 2020-07-31 | 2020-11-03 | 武汉轻工大学 | High-efficient separation needle of animal serum |
| CN112375654A (en) * | 2020-11-20 | 2021-02-19 | 广州穗阳生物学研究有限公司 | Adipose-derived stem cell separation device and method |
| CN113832028A (en) * | 2020-06-24 | 2021-12-24 | 上海医药集团生物治疗技术有限公司 | Full-automatic cell culture system and culture method |
| CN117305078A (en) * | 2023-09-25 | 2023-12-29 | 成都赛恩吉诺生物科技有限公司 | Tissue dissociation method |
-
2017
- 2017-02-10 CN CN201710072789.5A patent/CN106701548A/en not_active Withdrawn
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108554655A (en) * | 2018-01-03 | 2018-09-21 | 赛诺菲生物科技（武汉）有限公司 | Platelet rich plasma separator and separation method |
| CN108554655B (en) * | 2018-01-03 | 2019-12-27 | 周勇 | Platelet-rich plasma separation device and separation method |
| CN113832028A (en) * | 2020-06-24 | 2021-12-24 | 上海医药集团生物治疗技术有限公司 | Full-automatic cell culture system and culture method |
| CN111870258A (en) * | 2020-07-31 | 2020-11-03 | 武汉轻工大学 | High-efficient separation needle of animal serum |
| CN111870258B (en) * | 2020-07-31 | 2023-07-21 | 武汉轻工大学 | Animal blood serum high-efficiency separating needle |
| CN112375654A (en) * | 2020-11-20 | 2021-02-19 | 广州穗阳生物学研究有限公司 | Adipose-derived stem cell separation device and method |
| CN112375654B (en) * | 2020-11-20 | 2023-12-05 | 广州穗阳生物学研究有限公司 | Adipose-derived stem cell separation device and separation method |
| CN117305078A (en) * | 2023-09-25 | 2023-12-29 | 成都赛恩吉诺生物科技有限公司 | Tissue dissociation method |
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Application publication date: 20170524 |