CN105713875B - The method for separating of NK cell - Google Patents
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Abstract
The invention belongs to cell biologies, disclose a kind of method for separating of NK cell.The present invention provides a kind of method for separating of NK cell, take mononuclearcell that blood platelet extract and ethyl mercaptan phosphate is added, and are then added after magnetic bead liquid mixes and are incubated for；It takes the mixed liquor after being incubated for that blood platelet extract is added, is put into magnetic bead and corresponds in magnetic pole and sorted, supernatant is collected after standing.Method for separating of the present invention can play good buffer protection function to the damaging action in the external world such as magnetic bead, stirring, while the efficiency of separation that will not influence cell and the culture after NK cell sorting by addition blood platelet extract, ethyl mercaptan phosphate.The NK cell that experimental result display is obtained using the method for separating of NK cell of the present invention, Cell viability is high, growth rate is fast, the NK cell that higher purity can be obtained, obtained NK Cell killing efficacy are also apparently higher than conventional method for separating and sort to obtain NK cell.
Description
Technical field
The invention belongs to cell biologies, and in particular to a kind of method for separating of NK cell.
Background technique
Natural killer cells (natural killer cell, NK) is the important immunocyte of body, is not only swollen with anti-
Tumor, viral infection resisting and immunological regulation are related, and participate in the hair of hypersensitivity and autoimmune disease in some cases
It is raw.NK cell is distributed mainly in peripheral blood, accounts for PBMC (mononuclearcell) 5%~10%, also has NK in lymph node and marrow
Activity, but it is horizontal low compared with peripheral blood.Since the killing activity of NK cell is limited without MHC, antibody is not depended on, because referred to herein as killing naturally
Wound activity.There is early, 1 hour in vitro, internal 4 hours i.e. visible killing effects in NK cytosis lethal effect after target cell
It answers.The target cell of NK cell mainly has certain tumour cells (including part cell line), virus infected cell, certain autologous tissues
Cell (such as haemocyte), helminth, therefore NK cell is that body is antitumor, anti-infectious important immune factor, also assists in
The hypersensitivity of II type and graft-versus-host reaction.
NK cell is one of immunocyte, is played a significant role in clinical immune cell therapy, however from blood
NK cell content in the lymphocyte separated in liquid is very low, and only 10% or so, even if conventional NK cell Fiber differentiation,
Concentration also only maintains 30%~40% or so, so cannot play the good spectrum of NK cell in immune cell therapy kills tumor
Feature.
Cell sorting is a kind of a kind of method of not high problem of very good solution NK cell purity.Cell sorting is a kind of
A kind of method that cell is separated from many cells group's sample generallys use antibody labeled cells, while antibody is by fluorescence mark
Note or connection immunomagnetic beads, separate cell using the characteristic of fluorescence and immunomagnetic beads.Generally divide key player on a team and negative choosing, key player on a team is mark
Note needs obtained cell, directly the cell that needs of sorting, and negative choosing is to mark unwanted cells, sub-elect should not cell,
Cell to be needed.
Existing various kinds of cell sorting kit is applicable to the sorting of NK cell, and existing preferably cell sorting method is such as
Under:
1. sample is put into.It will include target cell, the turbid solution of magnetic bead and spuious cell marked to be put into sample cup, sample cup
Far from magnetic field；
2. stirring and evenly mixing: mixing head and be downwardly into sample cup, slowly accelerate rotation, combine magnetic bead sufficiently with target cell；
3. standing sorting.Stop stirring, sample cup is placed in above the magnet in screening installation magnetic field, adjusts sample cup and magnet
Distance, using magnetometer monitor sample bottom of a cup portion magnetic field strength, adjust be suitble to height, stand 5-15 minute, removal supernatant, obtain
To sorting cell.
However in above-mentioned sorting technology, along with larger to cell during the addition of magnetic bead, especially stirring, centrifugation etc.
Mechanical damage, the target cell motility rate obtained after sorting is relatively low and cell activity is substantially reduced, to sorting cell it is direct
Using or follow-up cultivation cause very big influence.
Summary of the invention
In view of this, providing a kind of sorting of NK cell it is an object of the invention in view of the problems of the existing technology
Method.Method for separating of the present invention is by addition blood platelet extract, ethyl mercaptan phosphate, to the external world such as magnetic bead, stirring
Damaging action can play good buffer protection function, while after will not influence the efficiency of separation and the NK cell sorting of cell
Culture.
In order to achieve the object of the present invention, the present invention adopts the following technical scheme:
A kind of method for separating of NK cell, including
Step 1 takes mononuclearcell that blood platelet extract and ethyl mercaptan phosphate is added, after the mixing of magnetic bead liquid is then added
It is incubated for；
Step 2 takes the mixed liquor after being incubated for that blood platelet extract is added, and is put into magnetic bead and corresponds in magnetic pole and is sorted, quiet
Postpone collection supernatant.
Ethyl mercaptan phosphate also known as Amifostine, Amifostine category injection can be used as tumor radiotherapy or cytotoxic chemotherapies
Auxiliary therapeutical agent is mainly used for the adjuvant treatment of various cancers.To lung cancer, oophoroma, breast cancer, nasopharyngeal carcinoma, bone tumour, disappear
Change before the kinds cancers patients such as road tumour, hematological system tumor carry out chemotherapy and applies ethyl mercaptan phosphate, it can substantially reduced chemotherapy
Kidney, marrow caused by drug, heart, ear and nervous system toxicity.
Method for separating of the present invention is outer to magnetic bead, stirring etc. by addition blood platelet extract and ethyl mercaptan phosphate
The damaging action on boundary can play good buffer protection function.
Wherein, in some embodiments, the additional amount of blood platelet extract described in step 1 is by every 1 × 106Single core
0.02mL-0.1mL blood platelet extract is added in cell concentration.
In some embodiments, the additional amount of the ethyl mercaptan phosphate is by every 1 × 106Mononuclearcell amount is added
The ethyl mercaptan phosphate of 0.02mL-0.05mL.
Incubation time described in method for separating step 1 of the present invention is preferably 5min-10min.
In some embodiments, the volume ratio of blood platelet extract described in step 2 and the mixed liquor after incubation is 1:2-
1:3.That is the additional amount of blood platelet extract described in step 2 is the 1/3~1/2 of the volume of the mixed liquor after being incubated for.
Time of repose described in method for separating step 2 of the present invention is preferably 5min-15min.
Mononuclearcell described in method for separating of the present invention can be isolated by peripheral blood or Cord blood.
In some embodiments, the preparation method of the mononuclearcell is that physiological saline is added in peripheral blood or Cord blood
Dilution, is slowly added into the centrifuge tube containing lymphocyte separation medium, is layered diluted blood and lymphocyte separation medium clear
Clear, 400-700g is centrifuged 15-30min, collects intermediate tunica albuginea confluent monolayer cells.
Preferably, the volume ratio of the physiological saline and peripheral blood or Cord blood is 1:1.
Preferably, the volume ratio of the blood of the lymphocyte separation medium and normal saline dilution is 1:2.
Ethyl mercaptan phosphate described in method for separating of the present invention can be commercially available by commercial channel.
Blood platelet extract described in method for separating of the present invention can be mentioned by the blood platelet bought by commercial channel
Take acquisition.
In some embodiments, the phosphate buffer that the preparation method of the blood platelet extract is pH 7.2 is resuspended
After blood platelet, -80 DEG C/37 DEG C multigelation 5 times or more, then at 4 DEG C 8000g centrifugation 30min to remove platelet membrane and its
His cell debris collects supernatant.
In some embodiments, the blood platelet can be isolated from peripheral blood or Cord blood.The blood platelet
Preparation method be specially that normal saline dilution is added in peripheral blood or Cord blood, be slowly added into containing lymphocyte separation medium from
In heart pipe, it is layered diluted blood and lymphocyte separation medium clear, 400-700g is centrifuged 15-30min, collects upper layer blood
Slurry, 200-400g are centrifuged 5min, and supernatant 1200-1500g is taken to be centrifuged, and collect the blood platelet of lower layer's enrichment.
Further, in some embodiments, method for separating of the present invention further includes the incubation step after sorting.
Culture after sorting of the present invention specially sorts obtained NK cell inoculation X-VIVO15 serum free medium,
And add OKT-3, IL-2, IL-15, IL-21 and inactivation filtered plasma culture NKT cell.
In some embodiments, the NK cell inoculation X-VIVO15 sorted described in the culture after the sorting without
The inoculum concentration of blood serum medium is 1 × 106/mL-2×106/mL。
Culture after sorting of the present invention joined some cell factors, including OKT-3, IL-2, IL-15, IL-21.
In some embodiments, the final concentration of 100U/mL- of OKT-3 described in the culture after sorting of the present invention
Final concentration of 100U/mL-1000U/mL, IL-21 of final concentration of 100U/mL-1000U/mL, IL-15 of 1000U/mL, IL-2
Final concentration of 100-1000U/mL.
It also added inactivation filtered plasma in culture after sorting of the present invention.
The inactivation filtered plasma can by commercial channel buy or from peripheral blood or Cord blood it is isolated.
In some embodiments, the preparation method of the inactivation filtered plasma is that physiology salt is added in peripheral blood or Cord blood
Water dilution, is slowly added into the centrifuge tube containing lymphocyte separation medium, is layered diluted blood and lymphocyte separation medium
Clearly, 400-700g is centrifuged 15-30min, collects upper plasma, filters after 56 DEG C of inactivations through 0.22 μm of filter.
In some embodiments, the additive amount of inactivation filtered plasma described in the culture after sorting of the present invention is
5v/v%.
The incubation time of culture after sorting of the present invention is preferably 14 days or more.
Further, in some embodiments, the culture after sorting of the present invention is primary every fluid infusion in 2-3 days.
Wherein, each fluid infusion cell density is 0.5 × 106/mL-2×106/ mL, and add OKT-3, IL-2, IL-15,
IL-21 and inactivation filtered plasma.
Preferably, the final concentration of 100U/mL- of final concentration of 100U/mL-1000U/mL, IL-2 of each fluid infusion OKT-3
The final concentration of 100U/mL-1000U/mL of final concentration of 100U/mL-1000U/mL, IL-21 of 1000U/mL, IL-15, inactivation
The final concentration of 5v/v% of filtered plasma.
As shown from the above technical solution, the present invention provides a kind of method for separating of NK cell, mononuclearcell is taken to be added
Then blood platelet extract and ethyl mercaptan phosphate are added after magnetic bead liquid mixes and are incubated for；Take the mixed liquor after being incubated for that blood is added small
Plate extract, is put into magnetic bead and corresponds in magnetic pole and sorted, and supernatant is collected after standing.Method for separating of the present invention is by adding
Platelet extract, ethyl mercaptan phosphate are healed, good buffer protection can be played to the damaging action in the external world such as magnetic bead, stirring and made
With, while the efficiency of separation that will not influence cell and the culture after NK cell sorting.Experimental result display uses institute of the present invention
The NK cell that the method for separating of NK cell obtains is stated, Cell viability is high, growth rate is fast, and the NK that can obtain higher purity is thin
Born of the same parents, obtained NK Cell killing efficacy are also apparently higher than conventional method for separating and sort to obtain NK cell.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 shows 2 each group growth curve of test example；WhereinShow comparative example 1 sorting obtain NK cell,Show reality
Apply example 3 sorting obtain NK cell,Show embodiment 4 sorting obtain NK cell,It is thin that the sorting of embodiment 5 obtains NK
Born of the same parents；
Fig. 2 shows in test example 2 cell surface marker object CD3 after the sorting of comparative example 1 obtains NK cell culture 14 days+CD56+Table
Up to situation map；
Fig. 3 shows in test example 2 cell surface marker object CD3 after the sorting of embodiment 5 obtains NK cell culture 14 days+CD56+Table
Up to situation map；.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the invention is clearly and completely described,
Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based in the present invention
Embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts, all
Belong to the scope of protection of the invention.
In order to better understand the present invention, the present invention will be described in detail combined with specific embodiments below.It is wherein described
Ethyl mercaptan phosphate (Amifostine) is purchased from Shanghai Chen Xi Biotechnology Co., Ltd.
Embodiment 1: the separation of mononuclearcell
Peripheral blood 20mL is acquired, 10mL normal saline dilution is added, is slowly added into the 15mL's containing lymphocyte separation medium
In centrifuge tube, diluted blood and lymphocyte separation medium is made to be layered clear, 400g-700g centrifugal force, lifting speed 0, centrifugation
15-30min extracts intermediate tunica albuginea confluent monolayer cells to get mononuclearcell, adds physiological saline to clean twice, count, cell concentration 2
×107A -4 × 107It is a.
The preparation of embodiment 2, blood platelet extract
Peripheral blood 20mL is acquired, 10mL normal saline dilution is added, is slowly added into the 15mL's containing lymphocyte separation medium
In centrifuge tube, diluted blood and lymphocyte separation medium is made to be layered clear, 400g-700g centrifugal force, lifting speed 0, centrifugation
15-30min collects upper plasma, and 200-400g is centrifuged 5min, and supernatant 1200-1500g is taken to be centrifuged, and collects the blood of lower layer's enrichment
Platelet.
After blood platelet is resuspended in the phosphate buffer of pH 7.2, -80 DEG C/37 DEG C multigelation 5 times or more, then at 4 DEG C
8000g is centrifuged 30min, and -80 DEG C of supernatant of collection freezes spare.
The method for separating of embodiment 3, NK cell of the present invention
The mononuclearcell (PBMC) 4 × 10 that Example 1 is prepared7It is a, by every 1 × 1060.02mL is added in cell concentration
Then the ethyl mercaptan phosphate of blood platelet extract and 0.02mL is added the magnetic bead of German U.S. day girl, is added and incubates after incubation 5min
The blood platelet extract of 1/3 volume of mixed liquor after educating, the magnetic pole for being put into corresponding German U.S. day girl are sorted, are stood
Supernatant is collected after 15min obtains NK cell.
The method for separating of embodiment 4, NK cell of the present invention
The mononuclearcell (PBMC) 4 × 10 that Example 1 is prepared7It is a, by every 1 × 1060.1mL is added in cell concentration
Then the ethyl mercaptan phosphate of blood platelet extract and 0.05mL is added the magnetic bead of German U.S. day girl, is added and incubates after incubation 10min
The blood platelet extract of 1/2 volume of mixed liquor after educating, the magnetic pole for being put into corresponding German U.S. day girl sort, and stand 5min
Supernatant is collected afterwards obtains NK cell.
The method for separating of embodiment 5, NK cell of the present invention
The mononuclearcell (PBMC) 4 × 10 that Example 1 is prepared7It is a, by every 1 × 1060.05mL is added in cell concentration
Then the ethyl mercaptan phosphate of blood platelet extract and 0.03mL is added the magnetic bead of German U.S. day girl, is added 1/3 after being incubated for 8min
The blood platelet extract of volume, the magnetic pole for being put into corresponding German U.S. day girl sort, and collection supernatant obtains after standing 10min
To NK cell.
Comparative example 1,
Take mononuclearcell (PBMC) 4 × 107It is a, the magnetic bead of German U.S. day girl is added, examination is sorted using German U.S. day girl
Agent box is sorted, and NK cell is obtained.
Test example 1, Cell viability and flow cytometer detection
Mononuclearcell, embodiment 3-5 and the sorting of comparative example 1 that Example 1 is prepared obtain NK cell and carry out carefully
Born of the same parents' motility rate and flow cytometer detection, the results are shown in Table 1.
1 Cell viability of table and flow cytometer detection
| Group | Cell viability | CD3-CD56+ expression rate |
| PBMC (embodiment 1) | 99.8% | 8.6% |
| The NK cell that embodiment 3 obtains | 97.9% | 98.9% |
| The NK cell that embodiment 4 obtains | 97.8% | 98.8% |
| The NK cell that embodiment 5 obtains | 97.2% | 98.8% |
| The NK cell that comparative example 1 obtains | 90.7% | 98.8% |
The results show that NK cell content is 8.6% in the PBMC of initially-separate, after sorted, NK cell proportion all reaches
99% or so；The PBMC Cell viability of initially-separate is up to 99% or more, after sorted, the NK cell that comparative example 1 obtains
Motility rate reduces obviously, and 90% or so, and the Cell viability for the NK cell that embodiment 3-5 is obtained still has 97% or more, hence it is evident that
Better than comparative example 1.
The Fiber differentiation experiment of NK cell after test example 2, sorting
Are obtained by NK cell and carries out Fiber differentiation respectively by embodiment 3-5 and the sorting of comparative example 1.
Wherein, the method for Fiber differentiation specifically: sorting obtains NK cell and the dilution of 1640 culture mediums, 400g centrifugation is added
5min adds X-VIVO15 serum free medium to be resuspended, and according to cell concentration, controlling inoculum density is 1 × 106/mL-2×106/ mL,
And add OKT-3 100U/mL-1000U/mL, IL-2 100U/mL-1000U/mL, IL-15 100U/mL-1000U/mL, IL-
The inactivation filtered plasma of 21 100-1000U/ml and 5v/v%；Every fluid infusion in 3 days is primary, and fluid infusion cell density is 0.5 × 106/
mL-2×106/ mL, and add OKT-3 100U/mL-1000U/mL, IL-2 100U/mL-1000U/mL, IL-15 100U/
The inactivation filtered plasma of mL-1000U/mL, IL-21 100U/mL-1000U/mL and 5v/v% are cultivated thin to 14 days collection NK
Born of the same parents.
The cell count of progress in every 3 days, collected after 14 days culture cell carry out CD3-CD56+ FCM analysis and
To the fragmentation test of K562 cell.As a result as shown in table 2-3 and Fig. 1-3.
2 cell proliferative conditions of table
The Mortaility results of 3 cell of table
Note: effect target is than the ratio for effector cell NK cell and target cell K562 cell
By table 2 and Fig. 1 result as it can be seen that embodiment 3-5, which sorts growth rate after obtaining NK cell culture, is substantially better than comparison
The sorting of example 1 obtains NK cell, and the sorting of comparative example 1 obtains NK cell and started in culture to the 12nd day, and cell starts not rise in value existing
As.
By table 3 and Fig. 2 and 3 results as it can be seen that ratio is apparently higher than comparative example 1 after the sorting of embodiment 5 obtains NK cell culture
Sorting obtains NK cell, and killing-efficiency is also significantly better than the sorting of comparative example 1 and obtains NK cell.
Embodiment 3 and the sorting of embodiment 4 obtain killing-efficiency after NK cell culture and are also significantly better than the sorting of comparative example 1 obtaining
NK cell.
Claims (9)
1. a kind of method for separating of NK cell, which is characterized in that including
Step 1 takes mononuclearcell that blood platelet extract and Amifostine is added, and is then added after magnetic bead liquid mixes and is incubated for；
Step 2 takes the mixed liquor after being incubated for that blood platelet extract is added, and is put into magnetic bead and corresponds in magnetic pole and is sorted, after standing
Collect supernatant；
Wherein, after blood platelet is resuspended in the phosphate buffer that the preparation method of the blood platelet extract is pH 7.2, -80 DEG C/
37 DEG C multigelation 5 times or more, then 8000g is centrifuged 30min at 4 DEG C, collects supernatant.
2. method for separating according to claim 1, which is characterized in that the additional amount of blood platelet extract described in step 1 is
By every 1 × 1060.02mL-0.1mL blood platelet extract is added in mononuclearcell amount.
3. method for separating according to claim 1 or 2, which is characterized in that the additional amount of the Amifostine is by every 1 × 106
The Amifostine of mononuclearcell amount addition 0.02mL-0.05mL.
4. method for separating according to claim 1 or 2, which is characterized in that incubation time described in step 1 is 5min-
10min。
5. method for separating according to claim 1 or 2, which is characterized in that after blood platelet extract described in step 2 and incubation
Mixed liquor volume ratio be 1:2-1:3.
6. method for separating according to claim 1 or 2, which is characterized in that time of repose described in step 2 is 5min-
15min。
7. method for separating according to claim 1 or 2, which is characterized in that the preparation method of the mononuclearcell is outer
Normal saline dilution is added in all blood or Cord blood, is slowly added into the centrifuge tube containing lymphocyte separation medium, makes diluted blood
Liquid and lymphocyte separation medium layering are clear, and 400-700g is centrifuged 15-30min, collect intermediate tunica albuginea confluent monolayer cells.
8. method for separating according to claim 1, which is characterized in that the preparation method of the blood platelet is peripheral blood or navel
Normal saline dilution is added in band blood, is slowly added into the centrifuge tube containing lymphocyte separation medium, makes diluted blood and lymph
The layering of cell separating liquid is clear, and 400-700g is centrifuged 15-30min, collects upper plasma, and 200-400g is centrifuged 5min, takes supernatant
1200-1500g centrifugation, collects the blood platelet of lower layer's enrichment.
9. method for separating according to claim 1 or 2, which is characterized in that further include the incubation step after sorting, will sort
The dilution of 1640 culture mediums is added in obtained supernatant, and 400g is centrifuged 5min, adds X-VIVO15 serum free medium to be resuspended, according to thin
Born of the same parents' amount, control inoculum density are 1 × 106/mL-2×106/ mL, and add OKT-3 100U/mL-1000U/mL, IL-2 100U/
ML-1000U/mL, IL-15 100U/mL-1000U/mL, IL-21 100-1000U/mL and with volume ratio be 5% inactivation
Hemofiltration slurry；Every fluid infusion in 3 days is primary, and fluid infusion cell density is 0.5 × 106/mL-2×106/ mL, and add OKT-3 100U/mL-
1000U/mL、IL-2 100U/mL-1000U/mL、IL-15 100U/mL-1000U/mL、IL-21 100U/mL-1000U/mL
With the inactivation filtered plasma for being 5% with volume ratio, culture to 14 days collection NK cells.
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| CN107267456A (en) * | 2017-08-23 | 2017-10-20 | 湖南开启时代生物科技有限责任公司 | A kind of preparation method of NK cells |
| CN109321524A (en) * | 2018-11-05 | 2019-02-12 | 中国医学科学院血液病医院（血液学研究所） | A method of isolating cells |
| CN110904040A (en) * | 2019-11-18 | 2020-03-24 | 江苏久腾医学科技有限公司 | Cell stripping method |
| CN111548994B (en) * | 2020-04-24 | 2021-05-25 | 广东华夏健康生命科学有限公司 | Cell culture medium and method for culturing NK cells by using same |
| CN113151169A (en) * | 2021-05-14 | 2021-07-23 | 上海赛笠生物科技有限公司 | Method for separating natural killer cells based on magnetic bead positive selection strategy |
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