CN109439625A - A kind of preparation method of the controllable scale of NK cell exosomes - Google Patents
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Abstract
The present invention relates to a kind of preparation methods of the controllable scale of NK cell exosomes, comprising the following steps: (1) collects NK cell culture medium supernatant, and be diluted with phosphate buffer, obtain dilution；(2) dilution is successively centrifuged under the slow-speed of revolution and high revolving speed, and takes supernatant to abandon precipitating after centrifugation every time；(3) centrifuged supernatant is placed in super filter tube and is centrifuged, take supernatant, obtained concentrated supernatant carries out ultracentrifugation, takes precipitating after ultracentrifugation；(4) precipitating obtained after step (3) ultracentrifugation is placed in phosphate buffer and is transferred in bag filter and dialysed to get the NK cell exosomes.Compared with prior art, particle diameter distribution of the present invention is uniform, and surface carries significant PROTEIN C D9, CD81, CD63, overcome existing technology of preparing there are purity low, low output, the deficiencies such as characterization expressing quantity is low, and it is used directly for subsequent detection, safety coefficient is high.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of preparation side of the controllable scale of NK cell exosomes
Method.
Background technique
Exosomes is " saucer shape " phospholipid bilayer vesica of the 30-150nm secreted by many cell types, density
For 1.13-1.19g/mL, multiple proteins, lipid and nucleic acid are carried, function is complicated, participates in immunological regulation, the outer base of regulating cell
Matter reconstruct, signal path of active cell etc..In cancer research, previous studies majority is to explore cancer cell to discharge
Exosomes, but seldom understand the function of the exosomes of immunocyte release.NK cell is to metastatic and hematologic malignancies
With natural tachysynthesis effect, the antitumor properties of NK cell are in clinical trial.However living cells treatment can band
Carry out intrinsic risk: as Vascular diseases cause pulmonary embolism and dead, transplanted cells be converted into undesirable cell type or cancer,
Immunological rejection, arrhythmia cordis, ossification and/or calcification etc..With exosomes replacement cell therapy can to avoid these problems,
Such as due to exosomes small size can to avoid the generation of vascular occlusion and undesirable cell type, have at present research (such as:
Zhu L et al.Theranostics, 2017,7 (10): 2732-2745) show that NK cell exosomes has tumour cell
There is cytotoxicity, but the treatment means that further research is developed as cancer are worth without influence on normal cell.To incite somebody to action
The high-purity large-scale production of NK cell exosomes development and application, NK cell exosomes is a unavoidable ring.Tradition
The obtained exosomes purity of ultracentrifugation it is fine, but its yield is not high, and according to the design of exosomes dimensioning principles
Ultrafiltration, the methods of dialysis cannot remove and the consistent foreign protein of exosomes size, and polyethylene glycol well again
(polyethylene glycol, the PEG) precipitation method and immunomagnetic beads method all introduce to some extent is not suitable for the outer of clinical treatment
Source property substance.Although also having been reported that the method for purification of extensive NK cell exosomes (such as: Jong A Y, et al. at present
[J].Journal of Extracellular Vesicles,2017,6(1):1294368).But NK cell exosomes's is pure
Degree and production problems are not well solved simultaneously, therefore high-purity, high yield, Gao Sheng can be extracted by lacking one at present
The antihunt means of the clinical rank NK cell exosomes of object activity.
Summary of the invention
It is an object of the present invention to overcome the above-mentioned drawbacks of the prior art and provide a kind of clinics of purity is high
The preparation method of the grade controllable scale of NK cell exosomes.
The purpose of the present invention can be achieved through the following technical solutions: a kind of controllable scale of NK cell exosomes
Preparation method, comprising the following steps:
(1) NK cell culture medium supernatant is collected, and is diluted with phosphate buffer, dilution is obtained；
(2) dilution is successively centrifuged under the slow-speed of revolution and high revolving speed, and takes supernatant to abandon precipitating after centrifugation every time, in height
Centrifuged supernatant is obtained after being centrifuged under revolving speed；
(3) centrifuged supernatant is placed in super filter tube and is centrifuged, take supernatant, obtained concentrated supernatant carries out superelevation
Speed is centrifuged, and takes precipitating after ultracentrifugation；
(4) precipitating obtained after step (3) ultracentrifugation is placed in be resuspended in phosphate buffer and dilutes and is transferred to
It dialyses in analysis bag, extra small molecular impurity is dialysed away to get the NK cell exosomes of the purifying.
It preferably, include KCl, NaCl, KH in the phosphate buffer2PO4And Na2HPO4, wherein the KCl,
NaCl、KH2PO4And Na2HPO4Mass ratio be (0.1~0.3): (6~10): (0.2~0.3): (1~2), wherein the phosphorus
The total mass concentration of salt is 8.88~9.88g/L in phthalate buffer.
Preferably, the volume ratio of the phosphate buffer and NK cell culture medium supernatant is (1~40): 1.
Preferably, the slow-speed of revolution be 2000~3000rpm, under the slow-speed of revolution centrifugation time be 5~15min, this from
Under heart rate, cell can be removed.
Preferably, the high revolving speed is 8000~11000rpm, and centrifugation time is 10~35min under high revolving speed；At this
It is centrifuged under revolving speed, cell fragment and impurity can be removed.
Preferably, the specification of the super filter tube is 100~150kDa, centrifugal rotational speed in super filter tube is 3000~
4000rpm, centrifugation time are 10~30min, while can removing small molecular weight impurity using super filter tube centrifugation on concentrating cells
Clear liquid reduces the workload and cost of next ultracentrifugation.
Preferably, the revolving speed of the ultracentrifugation is 8000~100000g, time of ultracentrifugation is 60~
90min, the precipitating left and taken herein are excretion body particle.
Preferably, the aperture of the bag filter is 30~35nm, and dialysis time is 3~9h, the phosphate that when dialysis is added
The mass ratio of the precipitating obtained after buffer and step (3) ultracentrifugation is (200~5000): 1, dialysis can remove small point
Sub- impurity.
Preferably, in whole preparation process, temperature is 4~37 DEG C, to keep the bioactivity of NK cell exosomes.
Compared with prior art, the beneficial effects of the present invention are embodied in following several respects:
(1) present invention can obtain a large amount of high-purities stable NK cell exosomes, average-size 30-150nm, completely
Saucer exosomes spline structure, particle diameter distribution is uniform, and surface carries significant PROTEIN C D9, CD81, CD63, overcomes
Existing technology of preparing there are purity low, low output, the deficiencies such as characterization expressing quantity is low；
(2) present invention is used directly for subsequent detection, and safety coefficient is high, will not cause lacking for exosomes function
It loses, scientific research value with higher.
Detailed description of the invention
Fig. 1 is transmission electron microscope (TEM) photo of 1 gained NK cell exosomes of embodiment；
Fig. 2 is the Western Blot testing result of 1 gained NK cell exosomes of embodiment；
Fig. 3 is the granularmetric analysis result of 1 gained NK cell exosomes of embodiment.
Specific embodiment
It elaborates below to the embodiment of the present invention, the present embodiment carries out under the premise of the technical scheme of the present invention
Implement, the detailed implementation method and specific operation process are given, but protection scope of the present invention is not limited to following implementation
Example.
1, phosphate-buffered formula of liquid: KCl 200mg/L；NaCl 8g/L；KH2PO4240mg/L；Na2HPO4 1.44g/
L。
2, it the acquisition of the culture supernatant containing NK cell exosomes: first by the anticoagulant dilution of volunteer's peripheral blood, uses
Lymphocyte separation medium separating peripheral blood mononuclear cells, 4*107A mononuclearcell is resuspended in the complete activation medium of 50ml
(IL-12 of concentration 800-1,000U/mL, the IL-15 of 800-1,000U/ml, 800-1, the DMEM of the IFN-γ of 000U/mL
Culture medium) in, 37 DEG C, 5%CO2It is cultivated in incubator 3-5 days plus (dense without exosomes NK cell amplification culture medium 1,000mL
Degree is 800-1, the IL-2 of 000U/mL, 800-1, and the IL-15's of 000U/mL removes exosomes RPMI-1640 culture medium), after
8-10 days acquisition NK cell culture supernatants of continuous culture
Embodiment 1
(1) it using being diluted under the conditions of 4 DEG C using NK cell culture supernatant of the phosphate buffer to collection, is centrifuged
3,000rpm, 8min, are precipitated as cell, and removal precipitating leaves and takes supernatant；
(2) supernatant for obtaining step (1) is centrifuged under the conditions of 4 DEG C with the revolving speed 15min of 9,000rpm, leaves and takes supernatant
Liquid abandons precipitating；
(3) supernatant obtained in phosphate buffer dilution step (2) under the conditions of 4 DEG C, continues super with 110kDa
Chimney filter is centrifuged 5min with the speed of 3,500rpm, and concentrating cells supernatant while removing small molecular weight impurity is reduced next
The workload and cost of ultracentrifugation；
(4) concentrated supernatant for obtaining step (3) is centrifuged 60min, centrifugation knot with the revolving speed of 100,000g under the conditions of 4 DEG C
Shu Hou abandons supernatant, keeps precipitating here；
(5) the precipitating phosphate buffer resuspension that step (4) obtains is transferred in bag filter, dialysis 8h can be obtained
The a large amount of exosomes of target high-purity, loss late are extremely low；
The present invention is tracked using transmission electron microscope (TEM), immunoblotting (Western Blot), nano particle
Analysis (Nanoparticle Tracking Analysis) characterizes the NK cell exosomes of acquisition, embodiments knot
Fruit difference is as follows:
Fig. 1: transmission electron microscope (TEM) photo: the characterization method of exosomes purity is mainly TEM Electronic Speculum, if
Many impurity are had if exosomes purity is inadequate, do not see exosomes.The NK cell that the present embodiment is obtained
Exosomes shoots TEM picture, as shown in Figure 1, its within sweep of the eye, NK cell exosomes complete display, this explanation is logical
It crosses this method and obtains NK cell exosomes purity is high, and exosomes structural integrity.
Fig. 2: Western Blot detection: the main characterization method of NK cell exosomes bioactivity that the present embodiment obtains
Be Western Blot detection, from Fig. 2 it is known that NK cell exosomes expression exosomes marker protein CD9,
CD81、CD63。
Fig. 3: NTA detection exosomes granularmetric analysis: the present embodiment, which obtains NK cell exosomes cell quantity characterization, is
NTA, the exosomes that as can be seen from Figure 3 present invention obtains, average grain diameter are 113nm ± 20nm, every milliliter of exosomes
Quantity is up to 6*107。
Embodiment 2
(1) it using being diluted under the conditions of 4 DEG C using NK cell culture supernatant of the phosphate buffer to collection, is centrifuged
3,000rpm, 9min are precipitated as cell, and removal precipitating leaves and takes supernatant,
(2) supernatant for obtaining step (1) is centrifuged under the conditions of 4 DEG C with the revolving speed 20min of 10,000rpm, is left and taken
Clear liquid abandons precipitating；
(3) supernatant obtained in phosphate buffer dilution step (2) under the conditions of 4 DEG C, continues super with 110kDa
Chimney filter is centrifuged 5min with the speed of 3,500rpm, and concentrating cells supernatant while removing small molecular weight impurity is reduced next
The workload and cost of ultracentrifugation.
(4) concentrated supernatant for obtaining step (3) is centrifuged 60min, centrifugation knot with the revolving speed of 100,000g under the conditions of 4 DEG C
Shu Hou abandons supernatant, keeps precipitating here.
(5) the precipitating phosphate buffer resuspension that step (4) obtains is transferred in bag filter, dialysis 7h can be obtained
The a large amount of exosomes of target high-purity, through detecting, the loss late of exosomes is extremely low.
Embodiment 3
(1) it using being diluted under the conditions of 4 DEG C using NK cell culture supernatant of the phosphate buffer to collection, is centrifuged
3,000rpm, 8min are precipitated as cell, and removal precipitating leaves and takes supernatant,
(2) supernatant for obtaining step (1) is centrifuged under the conditions of 4 DEG C with the revolving speed 10min of 10,000rpm, is left and taken
Clear liquid abandons precipitating；
(3) supernatant obtained in phosphate buffer dilution step (2) under the conditions of 4 DEG C, continues super with 110kDa
Chimney filter is centrifuged 10min with the speed of 3,000rpm, and concentrating cells supernatant while removing small molecular weight impurity is reduced next
The workload and cost of ultracentrifugation.
(4) concentrated supernatant for obtaining step (3) is centrifuged 60min, centrifugation knot with the revolving speed of 100,000g under the conditions of 4 DEG C
Shu Hou abandons supernatant, keeps precipitating here.
(5) the precipitating phosphate buffer resuspension that step (4) obtains is transferred in bag filter, dialysis 6h can be obtained
The a large amount of exosomes of target high-purity, through detecting, the loss late of exosomes is extremely low.
Embodiment 4
(1) it using being diluted under the conditions of 4 DEG C using NK cell culture supernatant of the phosphate buffer to collection, is centrifuged
4,000rpm, 10min are precipitated as cell, and removal precipitating leaves and takes supernatant,
(2) supernatant for obtaining step (1) is centrifuged under the conditions of 4 DEG C with the revolving speed 20min of 10,000rpm, is left and taken
Clear liquid abandons precipitating；
(3) supernatant obtained in phosphate buffer dilution step (2) under the conditions of 4 DEG C, continues super with 110kDa
Chimney filter is centrifuged 5min with the speed of 3,500rpm, and concentrating cells supernatant while removing small molecular weight impurity is reduced next
The workload and cost of ultracentrifugation.
(4) concentrated supernatant for obtaining step (3) is centrifuged 60min, centrifugation knot with the revolving speed of 100,000g under the conditions of 4 DEG C
Shu Hou abandons supernatant, keeps precipitating here.
(5) the precipitating phosphate buffer resuspension that step (4) obtains is transferred in bag filter, dialysis 5h can be obtained
The a large amount of exosomes of target high-purity, through detecting, the loss late of exosomes is extremely low.
Embodiment 5
(1) it using being diluted under the conditions of 4 DEG C using NK cell culture supernatant of the phosphate buffer to collection, is centrifuged
2500rpm, 15min are precipitated as cell, and removal precipitating leaves and takes supernatant,
(2) supernatant for obtaining step (1) is centrifuged under the conditions of 4 DEG C with the revolving speed 15min of 10,000rpm, is left and taken
Clear liquid abandons precipitating；
(3) supernatant obtained in phosphate buffer dilution step (2) under the conditions of 4 DEG C, continues super with 100kDa
Chimney filter is centrifuged 10min with the speed of 3500rpm, and concentrating cells supernatant while removing small molecular weight impurity is reduced next
The workload and cost of ultracentrifugation.
(4) concentrated supernatant for obtaining step (3) is centrifuged 60min, centrifugation knot with the revolving speed of 100,000g under the conditions of 4 DEG C
Shu Hou abandons supernatant, keeps precipitating here.
(5) the precipitating phosphate buffer resuspension that step (4) obtains is transferred in bag filter, dialysis 4h can be obtained
The a large amount of exosomes of target high-purity, through detecting, the loss late of exosomes is extremely low.
Embodiment 6
(1) it using being diluted under the conditions of 4 DEG C using NK cell culture supernatant of the phosphate buffer to collection, is centrifuged
3,000rpm, 8min, are precipitated as cell, and removal precipitating leaves and takes supernatant；
(2) supernatant for obtaining step (1) is centrifuged under the conditions of 4 DEG C with the revolving speed 30min of 9,000rpm, leaves and takes supernatant
Liquid abandons precipitating；
(3) supernatant obtained in phosphate buffer dilution step (2) under the conditions of 4 DEG C, continues super with 110kDa
Chimney filter is centrifuged 5min with the speed of 3,500rpm, and concentrating cells supernatant while removing small molecular weight impurity is reduced next
The workload and cost of ultracentrifugation；
(4) concentrated supernatant for obtaining step (3) is centrifuged 60min, centrifugation knot with the revolving speed of 100,000g under the conditions of 4 DEG C
Shu Hou abandons supernatant, keeps precipitating here；
(5) the precipitating phosphate buffer resuspension that step (4) obtains is transferred in bag filter, dialysis 8h can be obtained
The a large amount of exosomes of target high-purity, through detecting, the loss late of exosomes is extremely low.
Embodiment 7
(1) it using being diluted under the conditions of 4 DEG C using NK cell culture supernatant of the phosphate buffer to collection, is centrifuged
3,000rpm, 8min, are precipitated as cell, and removal precipitating leaves and takes supernatant；
(2) supernatant for obtaining step (1) is centrifuged under the conditions of 4 DEG C with the revolving speed 15min of 10,000rpm, is left and taken
Clear liquid abandons precipitating；
(3) supernatant obtained in phosphate buffer dilution step (2) under the conditions of 4 DEG C, continues super with 110kDa
Chimney filter is centrifuged 5min with the speed of 3,500rpm, and concentrating cells supernatant while removing small molecular weight impurity is reduced next
The workload and cost of ultracentrifugation；
(4) concentrated supernatant for obtaining step (3) is centrifuged 60min, centrifugation knot with the revolving speed of 100,000g under the conditions of 4 DEG C
Shu Hou abandons supernatant, keeps precipitating here；
(5) the precipitating phosphate buffer resuspension that step (4) obtains is transferred in bag filter, dialysis 8h can be obtained
The a large amount of exosomes of target high-purity, through detecting, the loss late of exosomes is extremely low.
Embodiment 8
Using preparation step same as Example 1, the difference is that:
(1) NaCl, 0.317g/L of KCl, 6.343g/L comprising 0.106g/L in the phosphate buffer used
KH2PO4With the Na of 2.114g/L2HPO4；I.e. total concentration is 8.88g/L, KCl, NaCl, KH2PO4And Na2HPO4Mass ratio be
0.1:6:0.3:2；
(2) volume ratio of phosphate buffer and NK cell culture medium supernatant is 1:1；
(3) slow-speed of revolution is 2000rpm, and the centrifugation time under the slow-speed of revolution is 15min；
(4) high revolving speed is 8000rpm, and the centrifugation time under high revolving speed is 35min；
(5) specification of super filter tube is 100kDa, and the centrifugal rotational speed in super filter tube is 3000rpm, and centrifugation time is
30min；
(6) ultracentrifugal revolving speed is 8000g, and the ultracentrifugal time is 90min；
(7) aperture of bag filter is 30nm, dialysis time 9h, the phosphate buffer and step (3) that when dialysis is added
The mass ratio of the precipitating obtained after ultracentrifugation is 1:200.
The present embodiment finally obtains a large amount of exosomes of target high-purity, and through detecting, the loss late of exosomes is extremely low.
Embodiment 9
Using preparation step same as Example 1, the difference is that:
(1) NaCl, 0.172g/L of KCl, 8.591g/L comprising 0.258g/L in the phosphate buffer used
KH2PO4With the Na of 0.859g/L2HPO4；I.e. total concentration is 9.88g/L, KCl, NaCl, KH2PO4And Na2HPO4Mass ratio be
0.3:10:0.2:1；
(2) volume ratio of phosphate buffer and NK cell culture medium supernatant is 40:1；
(3) slow-speed of revolution is 3000rpm, and the centrifugation time under the slow-speed of revolution is 5min；
(4) high revolving speed is 11000rpm, and the centrifugation time under high revolving speed is 10min；
(5) specification of super filter tube is 150kDa, and the centrifugal rotational speed in super filter tube is 4000rpm, and centrifugation time is
10min；
(6) ultracentrifugal revolving speed is 100000g, and the ultracentrifugal time is 60min；
(7) aperture of bag filter is 350nm, dialysis time 3h, the phosphate buffer and step (3) that when dialysis is added
The mass ratio of the precipitating obtained after ultracentrifugation is 1:5000.
The present embodiment finally obtains a large amount of exosomes of target high-purity, and through detecting, the loss late of exosomes is extremely low.
Claims (9)
1. a kind of preparation method of the controllable scale of NK cell exosomes, which comprises the following steps:
(1) NK cell culture medium supernatant is collected, and is diluted with phosphate buffer, dilution is obtained；
(2) dilution is successively centrifuged under the slow-speed of revolution and high revolving speed, and takes supernatant to abandon precipitating after centrifugation every time, in high revolving speed
Centrifuged supernatant is obtained after lower centrifugation；
(3) centrifuged supernatant is placed in super filter tube and is centrifuged, take supernatant, obtained concentrated supernatant carry out ultrahigh speed from
The heart takes precipitating after ultracentrifugation；
(4) precipitating obtained after step (3) ultracentrifugation is placed in phosphate buffer and is transferred in bag filter and carried out
Dialysis is to get the NK cell exosomes.
2. a kind of preparation method of the controllable scale of NK cell exosomes according to claim 1, which is characterized in that institute
It include KCl, NaCl, KH in the phosphate buffer stated2PO4And Na2HPO4, wherein described KCl, NaCl, KH2PO4And Na2HPO4
Mass ratio be (0.1~0.3): (6~10): (0.2~0.3): (1~2), wherein salt is total in the phosphate buffer
Mass concentration is 8.88-9.88g/L.
3. a kind of preparation method of the controllable scale of NK cell exosomes according to claim 1, which is characterized in that institute
The volume ratio of the phosphate buffer and NK cell culture medium supernatant stated is (1~40): 1.
4. a kind of preparation method of the controllable scale of NK cell exosomes according to claim 1, which is characterized in that institute
The slow-speed of revolution stated is 2000~3000rpm, and centrifugation time is 5~15min under the slow-speed of revolution.
5. a kind of preparation method of the controllable scale of NK cell exosomes according to claim 1, which is characterized in that institute
The high revolving speed stated is 8000~11000rpm, and centrifugation time is 10~35min under high revolving speed.
6. a kind of preparation method of the controllable scale of NK cell exosomes according to claim 1, which is characterized in that institute
The specification for stating super filter tube is 100~150kDa, and the centrifugal rotational speed in super filter tube is 3000~4000rpm, centrifugation time 10
~30min.
7. a kind of preparation method of the controllable scale of NK cell exosomes according to claim 1, which is characterized in that institute
The revolving speed for stating ultracentrifugation is 8000~100000g, and the time of ultracentrifugation is 60~90min.
8. a kind of preparation method of the controllable scale of NK cell exosomes according to claim 1, which is characterized in that institute
The aperture for stating bag filter is 30~35nm, and dialysis time is 3~9h, and the phosphate buffer and step (3) that when dialysis is added are super
The mass ratio of the precipitating obtained after high speed centrifugation is (200~5000): 1.
9. the preparation method of any controllable scale of a kind of NK cell exosomes according to claim 1~8, feature
It is, in whole preparation process, temperature is 4~37 DEG C.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115109742A (en) * | 2022-05-22 | 2022-09-27 | 上海交通大学 | Clinical grade high-purity exosome separation and purification kit in blood or urine |
| CN115197908A (en) * | 2022-06-27 | 2022-10-18 | 上海交通大学 | Extraction and modification method of natural killer cell-derived exosome and application thereof |
| CN115521914A (en) * | 2022-10-12 | 2022-12-27 | 西北工业大学 | Human primary natural killer cell in-vitro amplification system and method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6812023B1 (en) * | 2000-04-27 | 2004-11-02 | Anosys, Inc. | Methods of producing membrane vesicles |
| US20150125950A1 (en) * | 2012-05-18 | 2015-05-07 | Agency For Science, Technology And Research (A*Sta (A*Star) | Umbilical cord mesenchymal stem cell exosomes |
| CN106929467A (en) * | 2017-02-16 | 2017-07-07 | 上海交通大学 | The method and kit of a kind of separating high-purity urine excretion body |
-
2018
- 2018-10-31 CN CN201811290297.4A patent/CN109439625A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6812023B1 (en) * | 2000-04-27 | 2004-11-02 | Anosys, Inc. | Methods of producing membrane vesicles |
| US20150125950A1 (en) * | 2012-05-18 | 2015-05-07 | Agency For Science, Technology And Research (A*Sta (A*Star) | Umbilical cord mesenchymal stem cell exosomes |
| CN106929467A (en) * | 2017-02-16 | 2017-07-07 | 上海交通大学 | The method and kit of a kind of separating high-purity urine excretion body |
Non-Patent Citations (2)
| Title |
|---|
| 殷永健: "干扰素α 2b通过降低MDSC抑制小鼠肾癌生长的研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 潘柏申: "《2018检验医学进展》", 31 August 2018 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115109742A (en) * | 2022-05-22 | 2022-09-27 | 上海交通大学 | Clinical grade high-purity exosome separation and purification kit in blood or urine |
| CN115197908A (en) * | 2022-06-27 | 2022-10-18 | 上海交通大学 | Extraction and modification method of natural killer cell-derived exosome and application thereof |
| CN115521914A (en) * | 2022-10-12 | 2022-12-27 | 西北工业大学 | Human primary natural killer cell in-vitro amplification system and method |
| CN115521914B (en) * | 2022-10-12 | 2024-04-19 | 西北工业大学 | In-vitro amplification system and method for human primary natural killer cells |
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