CN105713875A - Sorting method for NK cells - Google Patents
Sorting method for NK cells Download PDFInfo
- Publication number
- CN105713875A CN105713875A CN201610143835.1A CN201610143835A CN105713875A CN 105713875 A CN105713875 A CN 105713875A CN 201610143835 A CN201610143835 A CN 201610143835A CN 105713875 A CN105713875 A CN 105713875A
- Authority
- CN
- China
- Prior art keywords
- cell
- sorting
- cells
- platelet extract
- separating
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 210000000822 natural killer cell Anatomy 0.000 title claims abstract description 72
- 238000000034 method Methods 0.000 title claims abstract description 39
- 210000004027 cell Anatomy 0.000 claims abstract description 46
- 239000011324 bead Substances 0.000 claims abstract description 21
- 210000005087 mononuclear cell Anatomy 0.000 claims abstract description 15
- 238000011534 incubation Methods 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims abstract description 4
- 238000000926 separation method Methods 0.000 claims description 19
- -1 ethyl mercaptan phosphate ester Chemical class 0.000 claims description 18
- 238000010790 dilution Methods 0.000 claims description 17
- 239000012895 dilution Substances 0.000 claims description 17
- 210000004698 lymphocyte Anatomy 0.000 claims description 17
- 239000002609 medium Substances 0.000 claims description 15
- 210000005259 peripheral blood Anatomy 0.000 claims description 13
- 239000011886 peripheral blood Substances 0.000 claims description 13
- 239000006228 supernatant Substances 0.000 claims description 13
- 230000002779 inactivation Effects 0.000 claims description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 210000004369 blood Anatomy 0.000 claims description 9
- 239000008280 blood Substances 0.000 claims description 9
- 210000004700 fetal blood Anatomy 0.000 claims description 9
- 230000012447 hatching Effects 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 8
- 239000012530 fluid Substances 0.000 claims description 7
- 238000001802 infusion Methods 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 4
- 239000012679 serum free medium Substances 0.000 claims description 4
- 239000002054 inoculum Substances 0.000 claims description 3
- 239000008363 phosphate buffer Substances 0.000 claims description 3
- 239000002356 single layer Substances 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 2
- 230000003833 cell viability Effects 0.000 abstract description 8
- 238000003756 stirring Methods 0.000 abstract description 7
- 238000002474 experimental method Methods 0.000 abstract description 2
- 230000002147 killing effect Effects 0.000 abstract description 2
- 239000000203 mixture Substances 0.000 abstract description 2
- 210000001772 blood platelet Anatomy 0.000 abstract 3
- FSFWWNFTJHUXGE-UHFFFAOYSA-N ethanethiol;phosphoric acid Chemical compound CCS.OP(O)(O)=O FSFWWNFTJHUXGE-UHFFFAOYSA-N 0.000 abstract 2
- 230000035755 proliferation Effects 0.000 abstract 1
- 230000000052 comparative effect Effects 0.000 description 13
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 206010028980 Neoplasm Diseases 0.000 description 6
- 108010002350 Interleukin-2 Proteins 0.000 description 5
- 239000006173 Good's buffer Substances 0.000 description 3
- JKOQGQFVAUAYPM-UHFFFAOYSA-N amifostine Chemical compound NCCCNCCSP(O)(O)=O JKOQGQFVAUAYPM-UHFFFAOYSA-N 0.000 description 3
- 229960001097 amifostine Drugs 0.000 description 3
- 230000000254 damaging effect Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 101000581981 Homo sapiens Neural cell adhesion molecule 1 Proteins 0.000 description 2
- 206010020751 Hypersensitivity Diseases 0.000 description 2
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 description 2
- 208000026935 allergic disease Diseases 0.000 description 2
- 230000007815 allergy Effects 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 239000002458 cell surface marker Substances 0.000 description 2
- 238000002659 cell therapy Methods 0.000 description 2
- 210000002865 immune cell Anatomy 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 230000001665 lethal effect Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 208000018084 Bone neoplasm Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 208000009329 Graft vs Host Disease Diseases 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 208000002454 Nasopharyngeal Carcinoma Diseases 0.000 description 1
- 206010061306 Nasopharyngeal cancer Diseases 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000002924 anti-infective effect Effects 0.000 description 1
- 201000008275 breast carcinoma Diseases 0.000 description 1
- 230000022534 cell killing Effects 0.000 description 1
- 230000009087 cell motility Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000011393 cytotoxic chemotherapy Methods 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 208000024908 graft versus host disease Diseases 0.000 description 1
- 210000005096 hematological system Anatomy 0.000 description 1
- 210000003677 hemocyte Anatomy 0.000 description 1
- 229940000351 hemocyte Drugs 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 229940088592 immunologic factor Drugs 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 201000011216 nasopharynx carcinoma Diseases 0.000 description 1
- 210000000581 natural killer T-cell Anatomy 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
Classifications
-
- 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
- C12N5/0646—Natural killers cells [NK], NKT cells
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the field of cell biology and discloses a sorting method for NK cells. According to the sorting method, a mononuclear cell is taken, blood platelet extract and ethanethiol phosphate are added, then magnetic bead liquid is added, and the mixture is mixed to be uniform before incubation; the mixed liquor obtained after incubation is taken, the blood platelet extract is added, the mixed liquor is put into magnetic poles corresponding to magnetic beads for sorting, and supernate is collected after standing. According to the sorting method, by adding the blood platelet extract and ethanethiol phosphate, external damage caused by the magnetic beads, stirring and the like can be well buffered and prevented, and meanwhile sorting efficiency of the cells and culture of the NK cells after sorting will not be affected. Experiment results show that the NK cells obtained by the adoption of the sorting method for the NK cells, the cell viability is high, the proliferation speed is high, the high-purity NK cells can be obtained, and the killing effect of the obtained NK cells is obviously higher than that of NK cells obtained after sorting through a conventional sorting method.
Description
Technical field
The invention belongs to cell biology, be specifically related to the method for separating of a kind of NK cell.
Background technology
Natural killer cell (naturalkillercell, NK) is the immunocyte that body is important, not only relevant with antitumor, viral infection resisting and immunomodulating, and participates in the generation of allergy and autoimmune disease in some cases.NK cell is distributed mainly in peripheral blood, accounts for PBMC (mononuclearcell) 5%～10%, lymph node and also have NK activity in bone marrow, but level relatively peripheral blood is low.Owing to the killing activity of NK cell limits without MHC, it is independent of antibody, because of referred to herein as Nk Cell Activity.NK cytosis lethal effect after target cell occurs early, 1 hour in vitro, internal 4 hours i.e. visible lethal effect.The target cell of NK cell mainly has some tumor cell (including part cell line), virus infected cell, some autologous tissue's cell (such as hemocyte), parasite etc., therefore NK cell is body antitumor, the important immune factor of anti-infective, also assists in Type-II allergy and graft versus host disease.
NK cell is the one in immunocyte, clinical immune cell therapy plays a significant role, but the NK cell content in the lymphocyte of separation is very low from blood, only about 10%, even if the NK cell induction of routine is cultivated, its concentration also only maintains about 30%～40%, so can not play the good spectrum of NK cell in immune cell therapy to kill tumor feature.
Cell sorting is a kind of good a kind of method solving the not high problem of NK cell purity.Cell sorting is a kind of a kind of method that cell is separated from many cells group's sample, generally adopts antibody labeled cells, and antibody is fluorescently labeled or connects immunomagnetic beads simultaneously, utilizes the characteristic of fluorescence and immunomagnetic beads to separate cell.General point key player on a team and negative choosing, key player on a team is the cell that labelling needs to obtain, the cell that directly sorting needs, and negative choosing is labelling unwanted cells, sub-elects cell not, thus obtaining the cell needed.
Existing various kinds of cell sorting test kit is applicable to the sorting of NK cell, and existing preferably cell sorting method is as follows:
1. sample is put into.Putting in sample cup by the turbid solution including target cell, labelling magnetic bead and spuious cell, sample cup is away from magnetic field；
2. stirring and evenly mixing: mix head and be downwardly into sample cup, slowly accelerates to rotate, makes magnetic bead fully be combined with target cell；
3. stand sorting.Stop stirring, sample cup is positioned over above the Magnet in screening installation magnetic field, regulate the distance of sample cup and Magnet, adopt magnetometer monitoring sample cup bottom magnetic field intensity, regulate and be suitable for height, stand 5-15 minute, remove supernatant, obtain sorting cells.
But in above-mentioned sorting technology, the addition of magnetic bead, along with the mechanical damage that cell is bigger in particularly stirring, the process such as centrifugal, the target cell motility rate obtained after sorting is on the low side and cytoactive substantially reduces, and directly application or the follow-up cultivation of sorting cells is caused very big impact.
Summary of the invention
In view of this, present invention aims to prior art Problems existing, it is provided that the method for separating of a kind of NK cell.The damaging action that magnetic bead, stirring etc. are extraneous, by adding platelet extract, ethyl mercaptan phosphate ester, can be played good buffer protection function by method for separating of the present invention, simultaneously without influence on the cultivation after the efficiency of separation of cell and NK cell sorting.
In order to realize the purpose of the present invention, the present invention adopts the following technical scheme that
A kind of method for separating of NK cell, including
Step 1, take mononuclearcell and add platelet extract and ethyl mercaptan phosphate ester, hatch after being subsequently adding the mixing of magnetic bead liquid；
Step 2, take the mixed liquor after hatching add platelet extract, put in magnetic bead correspondence magnetic pole and sort, after standing collect supernatant.
Ethyl mercaptan phosphate ester has another name called amifostine, and amifostine belongs to injection, as the auxiliary therapeutical agent of tumor radiotherapy or cytotoxic chemotherapies, can be mainly used in the auxiliary treatment of various cancer.Before the kinds cancer patients such as pulmonary carcinoma, ovarian cancer, breast carcinoma, nasopharyngeal carcinoma, bone tumor, digestive tract tumor, hematological system tumor are carried out chemotherapy, apply ethyl mercaptan phosphate ester, can substantially alleviate kidney produced by chemotherapeutics, bone marrow, heart, ear and neural toxicity.
Method for separating of the present invention is by adding platelet extract and ethyl mercaptan phosphate ester, and the damaging action that magnetic bead, stirring etc. is extraneous can play good buffer protection function.
Wherein, in some embodiments, the addition of platelet extract described in step 1 is by every 1 × 106Mononuclearcell amount adds 0.02mL-0.1mL platelet extract.
In some embodiments, the addition of described ethyl mercaptan phosphate ester is by every 1 × 106Mononuclearcell amount adds the ethyl mercaptan phosphate ester 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, platelet extract described in step 2 with hatch after the volume ratio of mixed liquor be 1:2-1:3.Namely the addition of platelet extract described in step 2 is the 1/3～1/2 of the volume of the mixed liquor after hatching.
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 separated by peripheral blood or Cord blood and obtain.
In some embodiments, the preparation method of described mononuclearcell is peripheral blood or Cord blood addition normal saline dilution, it is slowly added in the centrifuge tube containing lymphocyte separation medium, the blood making dilution is clear with lymphocyte separation medium layering, 400-700g is centrifuged 15-30min, tunica albuginea confluent monolayer cells in the middle of collecting.
Preferably, described normal saline is 1:1 with the volume ratio of peripheral blood or Cord blood.
Preferably, described lymphocyte separation medium is 1:2 with the volume ratio of the blood of normal saline dilution.
Described in method for separating of the present invention, ethyl mercaptan phosphate ester can be commercially available by commercial channel.
Platelet extract described in method for separating of the present invention can be extracted by the platelet bought by commercial channel and obtain.
In some embodiments, after the resuspended platelet of the phosphate buffer that preparation method is pH7.2 of described platelet extract,-80 DEG C/37 DEG C multigelations more than 5 times, then at 4 DEG C the centrifugal 30min of 8000g to remove platelet membrane and other cell debris, collection supernatant.
In some embodiments, described platelet can separate from peripheral blood or Cord blood and obtain.Described hematoblastic preparation method is specially peripheral blood or Cord blood adds normal saline dilution, it is slowly added in the centrifuge tube containing lymphocyte separation medium, the blood making dilution is clear with lymphocyte separation medium layering, 400-700g is centrifuged 15-30min, collect upper plasma, 200-400g is centrifuged 5min, takes supernatant 1200-1500g and is centrifuged, collects the platelet of lower floor's enrichment.
Further, in some embodiments, method for separating of the present invention also includes the incubation step after sorting.
Cultivation after sorting of the present invention is specially the NK cell inoculation X-VIVO15 serum-free medium that sorting obtains, and adds OKT-3, IL-2, IL-15, IL-21 and inactivation filtered plasma cultivation NKT cell.
In some embodiments, the inoculum concentration sorting the NK cell inoculation X-VIVO15 serum-free medium obtained described in the cultivation after described sorting is 1 × 106/mL-2×106/mL。
Cultivation after sorting of the present invention adds some cytokines, including OKT-3, IL-2, IL-15, IL-21.
In some embodiments, the final concentration of 100-1000U/mL of final concentration of 100U/mL-1000U/mL, IL-21 of final concentration of 100U/mL-1000U/mL, IL-15 of final concentration of 100U/mL-1000U/mL, IL-2 of OKT-3 described in the cultivation after sorting of the present invention.
Cultivation after sorting of the present invention also added inactivation filtered plasma.
Described inactivation filtered plasma can be bought by commercial channel or separate from peripheral blood or Cord blood and obtain.
In some embodiments, the preparation method of described inactivation filtered plasma is peripheral blood or Cord blood addition normal saline dilution, it is slowly added in the centrifuge tube containing lymphocyte separation medium, the blood making dilution is clear with lymphocyte separation medium layering, 400-700g is centrifuged 15-30min, collect upper plasma, through 0.22 μm of frit after 56 DEG C of inactivations.
In some embodiments, the addition inactivateing filtered plasma described in the cultivation after sorting of the present invention is 5v/v%.
The incubation time of the cultivation after sorting of the present invention is preferably more than 14 days.
Further, in some embodiments, the cultivation after sorting of the present invention every fluid infusion in 2-3 days once.
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 5v/v% of the final concentration of 100U/mL-1000U/mL of final concentration of 100U/mL-1000U/mL, IL-21 of final concentration of 100U/mL-1000U/mL, IL-15 of final concentration of 100U/mL-1000U/mL, IL-2 of each fluid infusion OKT-3, inactivation filtered plasma.
As shown from the above technical solution, the invention provides the method for separating of a kind of NK cell, take mononuclearcell and add platelet extract and ethyl mercaptan phosphate ester, hatch after being subsequently adding the mixing of magnetic bead liquid；Take the mixed liquor after hatching and add platelet extract, put in magnetic bead correspondence magnetic pole and sort, after standing, collect supernatant.The damaging action that magnetic bead, stirring etc. are extraneous, by adding platelet extract, ethyl mercaptan phosphate ester, can be played good buffer protection function by method for separating of the present invention, simultaneously without influence on the cultivation after the efficiency of separation of cell and NK cell sorting.Experimental result display adopts the NK cell that the method for separating of NK cell of the present invention obtains, Cell viability is high, growth rate is fast, can obtaining the NK cell of higher purity, the NK Cell killing efficacy obtained also obtains NK cell apparently higher than conventional method for separating sorting.
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, the accompanying drawing used required in embodiment or description of the prior art will be briefly described below.
Fig. 1 shows that test example 2 respectively organizes growth curve；WhereinShow comparative example 1 sorting obtain NK cell,Show embodiment 3 sorting obtain NK cell,Show embodiment 4 sorting obtain NK cell,Embodiment 5 sorting obtains NK cell；
Fig. 2 shows that in test example 2, comparative example 1 sorting obtains cell surface marker thing CD3 after NK cell is cultivated 14 days+CD56+Expression figure；
Fig. 3 shows that in test example 2, embodiment 5 sorting obtains cell surface marker thing CD3 after NK cell is cultivated 14 days+CD56+Expression figure；.
Detailed description of the invention
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described, it is clear that described embodiment is only a part of embodiment of the present invention, rather than whole embodiments.Based on the embodiment in the present invention, the every other embodiment that those of ordinary skill in the art obtain under not making creative work premise, broadly fall into the scope of protection of the invention.
In order to be better understood from the present invention, below in conjunction with specific embodiment, the present invention will be described in detail.Wherein said ethyl mercaptan phosphate ester (amifostine) is purchased from Chen Xi bio tech ltd, Shanghai.
Embodiment 1: the separation of mononuclearcell
Gather peripheral blood 20mL, adding 10mL normal saline dilution, be slowly added in the centrifuge tube containing lymphocyte separation medium 15mL, the blood making dilution is clear with lymphocyte separation medium layering, 400g-700g centrifugal force, lifting speed is 0, centrifugal 15-30min, tunica albuginea confluent monolayer cells in the middle of extracting, obtain mononuclearcell, adding normal saline to clean twice, counting, cell concentration is 2 × 107Individual-4 × 107Individual.
Embodiment 2, platelet extract preparation
Gather peripheral blood 20mL, add 10mL normal saline dilution, being slowly added in the centrifuge tube containing lymphocyte separation medium 15mL, the blood making dilution is clear with lymphocyte separation medium layering, 400g-700g centrifugal force, lifting speed is 0, centrifugal 15-30min, collects upper plasma, and 200-400g is centrifuged 5min, take supernatant 1200-1500g to be centrifuged, collect the platelet of lower floor's enrichment.
After the resuspended platelet of phosphate buffer of pH7.2 ,-80 DEG C/37 DEG C multigelations more than 5 times, then the centrifugal 30min of 8000g at 4 DEG C, collects supernatant-80 DEG C frozen standby.
Embodiment 3, NK cell of the present invention method for separating
The mononuclearcell (PBMC) 4 × 10 that Example 1 prepares7Individual, by every 1 × 106Cell concentration adds the ethyl mercaptan phosphate ester of 0.02mL platelet extract and 0.02mL, it is subsequently adding the magnetic bead of Germany U.S. sky girl, the platelet extract of mixed liquor 1/3 volume after hatching is added after hatching 5min, the magnetic pole of the Germany U.S. sky girl putting into correspondence sorts, and collects supernatant and obtain NK cell after standing 15min.
Embodiment 4, NK cell of the present invention method for separating
The mononuclearcell (PBMC) 4 × 10 that Example 1 prepares7Individual, by every 1 × 106Cell concentration adds the ethyl mercaptan phosphate ester of 0.1mL platelet extract and 0.05mL, it is subsequently adding the magnetic bead of Germany U.S. sky girl, the platelet extract of mixed liquor 1/2 volume after hatching is added after hatching 10min, the magnetic pole of the Germany U.S. sky girl putting into correspondence sorts, and collects supernatant and obtain NK cell after standing 5min.
Embodiment 5, NK cell of the present invention method for separating
The mononuclearcell (PBMC) 4 × 10 that Example 1 prepares7Individual, by every 1 × 106Cell concentration adds the ethyl mercaptan phosphate ester of 0.05mL platelet extract and 0.03mL, it is subsequently adding the magnetic bead of Germany U.S. sky girl, adding the platelet extract of 1/3 volume after hatching 8min, the magnetic pole of the Germany U.S. sky girl putting into correspondence sorts, and collects supernatant and obtain NK cell after standing 10min.
Comparative example 1,
Take mononuclearcell (PBMC) 4 × 107Individual, add the magnetic bead of Germany U.S. sky girl, adopt Germany U.S. sky girl to sort test kit and sort, obtain NK cell.
Test example 1, Cell viability and flow cytometer detection
Mononuclearcell, embodiment 3-5 and comparative example 1 sorting that Example 1 prepares obtain NK cell and carry out Cell viability and flow cytometer detection, and result is in Table 1.
Table 1 Cell viability 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% |
Result shows, in the PBMC of initially-separate, NK cell content is 8.6%, and after sorted, NK cell proportion has all reached about 99%；The PBMC Cell viability of initially-separate is up to more than 99%, and after sorted, the motility rate of the NK cell that comparative example 1 obtains reduces substantially, and about 90%, and the Cell viability of the NK cell that embodiment 3-5 obtains still has more than 97%, hence it is evident that be better than comparative example 1.
The inducing culture experiment of NK cell after test example 2, sorting
Embodiment 3-5 and comparative example 1 sorting are obtained NK cell and carry out inducing culture respectively.
Wherein, the method for inducing culture adds 1640 culture medium dilutions particularly as follows: sorting obtains NK cell, and 400g is centrifuged 5min, adds X-VIVO15 serum-free medium resuspended, and according to cell concentration, controlling inoculum density is 1 × 106/mL-2×106/ mL, and add the inactivation filtered plasma of OKT-3100U/mL-1000U/mL, IL-2100U/mL-1000U/mL, IL-15100U/mL-1000U/mL, IL-21100-1000U/ml and 5v/v%；Once, fluid infusion cell density is 0.5 × 10 in fluid infusion in every 3 days6/mL-2×106/ mL, and add the inactivation filtered plasma of OKT-3100U/mL-1000U/mL, IL-2100U/mL-1000U/mL, IL-15100U/mL-1000U/mL, IL-21100U/mL-1000U/mL and 5v/v%, it is cultured to 14 days and collects NK cell.
Within every 3 days, carry out a cell counting, collect cultured cells after 14 days and carry out CD3-CD56+ FCM analysis and the fragmentation test to K562 cell.Result is such as shown in table 2-3 and Fig. 1-3.
Table 2 cell proliferative conditions
The Mortaility results of table 3 cell
Note: effect target is than the ratio for effector lymphocyte's NK cell Yu target cell K562 cell
From table 2 and Fig. 1 result, embodiment 3-5 sorting obtains growth rate after NK cell is cultivated and is substantially better than comparative example 1 sorting and obtains NK cell, and comparative example 1 sorting obtains NK cell and starts being cultured to the 12nd day, and cell starts not rise in value phenomenon.
From table 3 and Fig. 2 and 3 result, embodiment 5 sorting obtains ratio after NK cell is cultivated and obtains NK cell apparently higher than comparative example 1 sorting, and killing-efficiency is also significantly better than comparative example 1 sorting and obtains NK cell.
Embodiment 3 and embodiment 4 sorting obtain NK cell cultivate after killing-efficiency be also significantly better than comparative example 1 sorting obtain NK cell.
Claims (10)
1. the method for separating of a NK cell, it is characterised in that include
Step 1, take mononuclearcell and add platelet extract and ethyl mercaptan phosphate ester, hatch after being subsequently adding the mixing of magnetic bead liquid；
Step 2, take the mixed liquor after hatching add platelet extract, put in magnetic bead correspondence magnetic pole and sort, after standing collect supernatant.
2. method for separating according to claim 1, it is characterised in that the addition of platelet extract described in step 1 is by every 1 × 106Mononuclearcell amount adds 0.02mL-0.1mL platelet extract.
3. method for separating according to claim 1 and 2, it is characterised in that the addition of described ethyl mercaptan phosphate ester is by every 1 × 106Mononuclearcell amount adds the ethyl mercaptan phosphate ester of 0.02mL-0.05mL.
4. the method for separating according to claim 1-3 any one, it is characterised in that incubation time described in step 1 is 5min-10min.
5. the method for separating according to claim 1-4 any one, it is characterised in that platelet extract described in step 2 with hatch after the volume ratio of mixed liquor be 1:2-1:3.
6. the method for separating according to claim 1-5 any one, it is characterised in that time of repose described in step 2 is 5min-15min.
7. the method for separating according to claim 1-6 any one, it is characterized in that, the preparation method of described mononuclearcell is peripheral blood or Cord blood addition normal saline dilution, it is slowly added in the centrifuge tube containing lymphocyte separation medium, the blood making dilution is clear with lymphocyte separation medium layering, 400-700g is centrifuged 15-30min, tunica albuginea confluent monolayer cells in the middle of collecting.
8. the method for separating according to claim 1-7 any one, it is characterized in that, after the resuspended platelet of the phosphate buffer that preparation method is pH7.2 of described platelet extract ,-80 DEG C/37 DEG C multigelations more than 5 times, then the centrifugal 30min of 8000g at 4 DEG C, collects supernatant.
9. method for separating according to claim 8, it is characterized in that, described hematoblastic preparation method is peripheral blood or Cord blood addition normal saline dilution, being slowly added in the centrifuge tube containing lymphocyte separation medium, the blood making dilution is clear with lymphocyte separation medium layering, and 400-700g is centrifuged 15-30min, collect upper plasma, 200-400g is centrifuged 5min, takes supernatant 1200-1500g and is centrifuged, collects the platelet of lower floor's enrichment.
10. the method for separating according to claim 1-9, it is characterised in that also include the incubation step after sorting, being specially the supernatant that obtains of sorting and add 1640 culture medium dilutions, 400g is centrifuged 5min, adds X-VIVO15 serum-free medium resuspended, according to cell concentration, controlling inoculum density is 1 × 106/mL-2×106/ mL, and add the inactivation filtered plasma of OKT-3100U/mL-1000U/mL, IL-2100U/mL-1000U/mL, IL-15100U/mL-1000U/mL, IL-21100-1000U/mL and 5v/v%；Once, fluid infusion cell density is 0.5 × 10 in fluid infusion in every 3 days6/mL-2×106/ mL, and add the inactivation filtered plasma of OKT-3100U/mL-1000U/mL, IL-2100U/mL-1000U/mL, IL-15100U/mL-1000U/mL, IL-21100U/mL-1000U/mL and 5v/v%, it is cultured to 14 days and collects NK cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610143835.1A CN105713875B (en) | 2016-03-14 | 2016-03-14 | The method for separating of NK cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610143835.1A CN105713875B (en) | 2016-03-14 | 2016-03-14 | The method for separating of NK cell |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105713875A true CN105713875A (en) | 2016-06-29 |
| CN105713875B CN105713875B (en) | 2019-05-10 |
Family
ID=56157570
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610143835.1A Active CN105713875B (en) | 2016-03-14 | 2016-03-14 | The method for separating of NK cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105713875B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN111548994A (en) * | 2020-04-24 | 2020-08-18 | 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 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104894065A (en) * | 2015-07-09 | 2015-09-09 | 广州赛莱拉干细胞科技股份有限公司 | NK (natural killer) cell culture medium and culture method of NK cell |
| CN105296426A (en) * | 2015-12-07 | 2016-02-03 | 广州赛莱拉干细胞科技股份有限公司 | Induced culture method for NK cell |
-
2016
- 2016-03-14 CN CN201610143835.1A patent/CN105713875B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104894065A (en) * | 2015-07-09 | 2015-09-09 | 广州赛莱拉干细胞科技股份有限公司 | NK (natural killer) cell culture medium and culture method of NK cell |
| CN105296426A (en) * | 2015-12-07 | 2016-02-03 | 广州赛莱拉干细胞科技股份有限公司 | Induced culture method for NK cell |
Non-Patent Citations (2)
| Title |
|---|
| 宁忠华: "血小板对NK细胞杀伤K652细胞的影响", 《江南大学学报（自然科学版）》 * |
| 谢昶等: "氨磷汀对细胞保护作用的研究现状", 《国外医药抗生素分册》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN111548994A (en) * | 2020-04-24 | 2020-08-18 | 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105713875B (en) | 2019-05-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105713875A (en) | Sorting method for NK cells | |
| CN104928243B (en) | The separation of patients with solid tumor autologous NK cells, activation amplification and activity test method | |
| CN109628397B (en) | Method for in-vitro amplification culture of NK (natural killer) cells | |
| CN102876631B (en) | Method for separating immune cells from blood and application of method to disease treatment | |
| CN102321577B (en) | Preparation method of antitumor adoptive immune cells and prepared immune cells | |
| CN102978161A (en) | Kit for separated culture of DC-CIK cells, and application thereof | |
| CN105087487A (en) | Efficient CIK amplifying method | |
| CN102268405A (en) | Method for auto NK (Natural Killer) cell in-vitro activation and amplification culture and special culture medium thereof | |
| CN103923880A (en) | Efficient multiplication CTL preparation method killing tumors in targeted mode | |
| CN102676454B (en) | Preparation method for CIK (cytokine induced killer) cell of umbilical cord blood source | |
| CN105238752A (en) | Culture system and culture method for efficient amplification in vitro of autologous NK cells | |
| CN108004211B (en) | A kind of method of Activated in Vitro amplifying natural killer cell | |
| CN105754941A (en) | In-vitro induction and proliferation culture method for peripheral blood NK (natural killer) cells | |
| CN105274053B (en) | A kind of preparation method of the CIK cell of high cytotoxic activity | |
| CN105176927A (en) | Preparation method of cytotoxicity-enhanced efficient target killing NK/CIK (Natural Killer)/( Cytokine Induced Killer) cells | |
| CN104450616A (en) | Co-culture method of DC cell and CIK cell | |
| CN109022362A (en) | A kind of method for building up of hyperleucocyte acute leukemia PDX model | |
| CN102174469A (en) | Method for effectively culturing tumor infiltrating lymphocytes (TILs) | |
| CN102676455B (en) | Preparation method for dendritic cell of umbilical cord blood source and dendritic cell vaccine | |
| CN111944754B (en) | Natural killer cell culture method | |
| CN108949685A (en) | Method for in vitro induction and expansion of gamma T cells with high killing activity | |
| CN102533649B (en) | Method for easily and efficiently preparing human gamma/deltaT cells | |
| CN110643574A (en) | Preparation method of feeder cells for rapid culture of tumor infiltrating lymphocytes | |
| CN110272871A (en) | A kind of stimulation induction mononuclearcell amplification is composition and its application of gamma delta T cells | |
| CN105176926A (en) | Method for amplifying NK cells through in-vitro cultivation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |