CN103405759B - Method for preparing tumor-specific DC vaccine by applying CD34+ cells of umbilical cord blood - Google Patents
Method for preparing tumor-specific DC vaccine by applying CD34+ cells of umbilical cord blood Download PDFInfo
- Publication number
- CN103405759B CN103405759B CN201310310485.XA CN201310310485A CN103405759B CN 103405759 B CN103405759 B CN 103405759B CN 201310310485 A CN201310310485 A CN 201310310485A CN 103405759 B CN103405759 B CN 103405759B
- Authority
- CN
- China
- Prior art keywords
- cell
- tumor
- blood
- vaccine
- ucb
- 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.)
- Active
Links
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 40
- 229940029030 dendritic cell vaccine Drugs 0.000 title claims abstract description 24
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 title claims abstract description 19
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 title claims abstract description 19
- 238000000034 method Methods 0.000 title claims abstract description 13
- 210000004700 fetal blood Anatomy 0.000 title abstract description 9
- 210000004027 cell Anatomy 0.000 claims abstract description 69
- 239000002243 precursor Substances 0.000 claims abstract description 21
- 230000003321 amplification Effects 0.000 claims abstract description 14
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 14
- 230000006698 induction Effects 0.000 claims abstract description 11
- 210000004369 blood Anatomy 0.000 claims description 23
- 239000008280 blood Substances 0.000 claims description 23
- 238000002360 preparation method Methods 0.000 claims description 13
- 210000004881 tumor cell Anatomy 0.000 claims description 13
- 210000003754 fetus Anatomy 0.000 claims description 12
- UZOVYGYOLBIAJR-UHFFFAOYSA-N 4-isocyanato-4'-methyldiphenylmethane Chemical compound C1=CC(C)=CC=C1CC1=CC=C(N=C=O)C=C1 UZOVYGYOLBIAJR-UHFFFAOYSA-N 0.000 claims description 11
- 239000001963 growth medium Substances 0.000 claims description 11
- 210000001519 tissue Anatomy 0.000 claims description 11
- 239000013592 cell lysate Substances 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 9
- 102000004169 proteins and genes Human genes 0.000 claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 9
- 238000011002 quantification Methods 0.000 claims description 9
- 238000000926 separation method Methods 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 8
- 239000011324 bead Substances 0.000 claims description 7
- 230000001939 inductive effect Effects 0.000 claims description 7
- 238000000746 purification Methods 0.000 claims description 7
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 claims description 6
- 206010003445 Ascites Diseases 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 229910000831 Steel Inorganic materials 0.000 claims description 6
- 108060008682 Tumor Necrosis Factor Proteins 0.000 claims description 6
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 claims description 6
- 210000004381 amniotic fluid Anatomy 0.000 claims description 6
- 210000000481 breast Anatomy 0.000 claims description 6
- 239000006285 cell suspension Substances 0.000 claims description 6
- 230000000249 desinfective effect Effects 0.000 claims description 6
- 239000000284 extract Substances 0.000 claims description 6
- 230000001605 fetal effect Effects 0.000 claims description 6
- 239000006166 lysate Substances 0.000 claims description 6
- 239000010959 steel Substances 0.000 claims description 6
- 239000000725 suspension Substances 0.000 claims description 6
- 210000003954 umbilical cord Anatomy 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 208000030507 AIDS Diseases 0.000 claims description 3
- 108010029697 CD40 Ligand Proteins 0.000 claims description 3
- 102100032937 CD40 ligand Human genes 0.000 claims description 3
- 208000031404 Chromosome Aberrations Diseases 0.000 claims description 3
- 208000035473 Communicable disease Diseases 0.000 claims description 3
- 208000027205 Congenital disease Diseases 0.000 claims description 3
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 claims description 3
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 claims description 3
- 101000946889 Homo sapiens Monocyte differentiation antigen CD14 Proteins 0.000 claims description 3
- 108090000978 Interleukin-4 Proteins 0.000 claims description 3
- 102100035877 Monocyte differentiation antigen CD14 Human genes 0.000 claims description 3
- 208000002787 Pregnancy Complications Diseases 0.000 claims description 3
- 208000006399 Premature Obstetric Labor Diseases 0.000 claims description 3
- 208000003107 Premature Rupture Fetal Membranes Diseases 0.000 claims description 3
- 206010036600 Premature labour Diseases 0.000 claims description 3
- 230000001464 adherent effect Effects 0.000 claims description 3
- 238000010241 blood sampling Methods 0.000 claims description 3
- 210000000988 bone and bone Anatomy 0.000 claims description 3
- 201000011510 cancer Diseases 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 201000010099 disease Diseases 0.000 claims description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 3
- 230000009977 dual effect Effects 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 208000006454 hepatitis Diseases 0.000 claims description 3
- 231100000283 hepatitis Toxicity 0.000 claims description 3
- 208000015181 infectious disease Diseases 0.000 claims description 3
- 210000004698 lymphocyte Anatomy 0.000 claims description 3
- 230000035800 maturation Effects 0.000 claims description 3
- 210000001006 meconium Anatomy 0.000 claims description 3
- 239000002609 medium Substances 0.000 claims description 3
- 230000002093 peripheral effect Effects 0.000 claims description 3
- 210000002826 placenta Anatomy 0.000 claims description 3
- 230000003169 placental effect Effects 0.000 claims description 3
- 230000035935 pregnancy Effects 0.000 claims description 3
- 208000012113 pregnancy disease Diseases 0.000 claims description 3
- 208000026440 premature labor Diseases 0.000 claims description 3
- 230000029058 respiratory gaseous exchange Effects 0.000 claims description 3
- 229920006395 saturated elastomer Polymers 0.000 claims description 3
- 238000003860 storage Methods 0.000 claims description 3
- 208000006379 syphilis Diseases 0.000 claims description 3
- 210000003606 umbilical vein Anatomy 0.000 claims description 3
- 239000013589 supplement Substances 0.000 claims 1
- 210000005087 mononuclear cell Anatomy 0.000 abstract description 3
- 239000000427 antigen Substances 0.000 description 12
- 102000036639 antigens Human genes 0.000 description 12
- 108091007433 antigens Proteins 0.000 description 12
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 230000001413 cellular effect Effects 0.000 description 8
- 230000006870 function Effects 0.000 description 8
- 238000002560 therapeutic procedure Methods 0.000 description 8
- 229960005486 vaccine Drugs 0.000 description 8
- 238000011282 treatment Methods 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 230000003053 immunization Effects 0.000 description 6
- 238000002649 immunization Methods 0.000 description 6
- 230000003248 secreting effect Effects 0.000 description 6
- 102000013462 Interleukin-12 Human genes 0.000 description 5
- 108010065805 Interleukin-12 Proteins 0.000 description 5
- 210000001744 T-lymphocyte Anatomy 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 238000007799 mixed lymphocyte reaction assay Methods 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 229920002307 Dextran Polymers 0.000 description 3
- 102000003814 Interleukin-10 Human genes 0.000 description 3
- 108090000174 Interleukin-10 Proteins 0.000 description 3
- 238000005138 cryopreservation Methods 0.000 description 3
- 210000001165 lymph node Anatomy 0.000 description 3
- 210000005259 peripheral blood Anatomy 0.000 description 3
- 239000011886 peripheral blood Substances 0.000 description 3
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 3
- 108090001005 Interleukin-6 Proteins 0.000 description 2
- 102000004889 Interleukin-6 Human genes 0.000 description 2
- 230000000735 allogeneic effect Effects 0.000 description 2
- 239000003146 anticoagulant agent Substances 0.000 description 2
- 229940127219 anticoagulant drug Drugs 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000009169 immunotherapy Methods 0.000 description 2
- 230000001665 lethal effect Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000002255 vaccination Methods 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 101000934338 Homo sapiens Myeloid cell surface antigen CD33 Proteins 0.000 description 1
- 101000767631 Human papillomavirus type 16 Protein E7 Proteins 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 102100025243 Myeloid cell surface antigen CD33 Human genes 0.000 description 1
- 208000015634 Rectal Neoplasms Diseases 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 230000006044 T cell activation Effects 0.000 description 1
- 230000006052 T cell proliferation Effects 0.000 description 1
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 1
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 1
- 230000030741 antigen processing and presentation Effects 0.000 description 1
- 210000000612 antigen-presenting cell Anatomy 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 201000008275 breast carcinoma Diseases 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 210000003109 clavicle Anatomy 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000002784 cytotoxicity assay Methods 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011337 individualized treatment Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 206010038038 rectal cancer Diseases 0.000 description 1
- 201000001275 rectum cancer Diseases 0.000 description 1
- 230000005070 ripening Effects 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for preparing a tumor-specific DC vaccine by applying CD34+ cells in umbilical cord blood. The method comprises (1) a step of preparing autologous tumor-related holoantigen; (2) a step of obtaining the umbilical cord blood; (3) a step of obtaining mononuclear cells derived from the umbilical cord blood; (4) a step of purifying CD34+ cells in the mononuclear cells derived from the umbilical cord blood; (5) performing induction culture for a precursor DC; (6) a step of performing amplification and culture of an immature DC; and (7) a step of preparing the DC vaccine.
Description
Technical field
The present invention relates to a kind of preparation method of vaccine, particularly a kind of application umbilical blood CD34+ cell is prepared tumour-specific DC vaccine method.
Background technology
The main T cellular immunization that relies on DC induction of cellular immunization treatment of tumor.DC is the antigen presenting cell of human body sole duty, thereby in immunotherapy of tumors, is bearing picked-up and processing tumor antigen, antigenic information is offered to the cell to T, thereby start the critical function of tumour-specific T cellular immunization.
The DC cell of clinical practice is at present mainly derived from peripheral blood list shape nucleus (Peripheral blood mononuclear cells, PBMC), and is mainly used in the treatment of individuation cellular immunization.Due to PBMC source DC(PBMC-DC) individuation feature, limited clinical practice, particularly limited the production in enormous quantities of DC vaccine and the industrialization of DC bacterin preparation and produced.
Patent innovation of the present invention proposes, application Cord blood list shape nucleus (Umbilical cord blood derived mononuclear cells, UCB-MNCs), sub-elect CD34+ cell (UBC-CD34+), through 2 weeks amplification cultivation, be prepared into immature DC (Immature DC), 5 days load tumor antigens of amplification cultivation, ripe 48 hours (Mature DC), is prepared into tumour-specific DC vaccine.
1., umbilical blood can obtain in a large number as clinical garbage and frozen, can meet clinical practice application UBC-CD34+ cell prepared tumour-specific DC vaccine, possesses following advantage:; 2., from UBC-CD34+ cell induction cultivate, can obtain immature DC and ripe DC(UCB-CD34-DC); 3., UBC-CD34+ cell is through the precursor DC of 14 days inducing culture, the 15-17 that quantitatively can increase doubly, is enough to meet clinical needs; 4., after the precursor DC and ripe DC cryopreservation resuscitation of UBC-CD34+ cell derived, there is not obvious change in its cell phenotype, function, quantity; 5. compare with PBMC-DC, the immature DC of UBC-CD34+ cell induction, antigen uptake, processing, HLA-II antigen are stronger; 6. compare with PBMC-DC, the functional molecular on UBC-CD34-DC surface is expressed higher, and the ability of activated T cell immunity is stronger; 7. compare with PBMC-DC, the CTL that UBC-CD34-DC activates, tumour-specific killing ability is stronger; 8., because the immunogenicity of UBC-CD34-DC is extremely low, for allogeneic ACT treatment (Adoptive cell transfer, ACT), there is not the restriction of HLA phenotype and immunological rejection, thereby can be used for allochthonous cellular immunization treatment (Immunotherapy), the individuation bottleneck of having broken through cellular immunization treatment, makes DC vaccine large-scale industrialized production become possibility.
Summary of the invention
The present invention is according to prior art, and the CD34+ cell that proposes application Cord Blood-Derived is prepared the technology of DC vaccine, has obtained success, and the present invention provides a kind of preparation method of DC vaccine for this reason, described method, and step is as follows:
The preparation of step 1, the relevant holoantigen of autologous tumor:
Obtaining of step 2, umbilical blood:
Obtaining of step 3, Cord blood list shape nucleus:
The purification of step 4, Cord blood list shape nucleus CD34+ cell:
The inducing culture of step 5, precursor DC:
The amplification of step 6, immature DC and cultivation:
The preparation of step 7, DC vaccine:
Wherein each step concrete operation method is as follows:
The preparation of step 1, the relevant holoantigen of autologous tumor:
1., the fresh tumor tissues of excision: get tumor peripheral part tissue (avoiding the slough of tumor center) 0.3-1.0cm
3(can liquid nitrogen or-80 DEG C of Refrigerator stores for subsequent use), wipes out nonneoplastic tissue around, gentamycin-normal saline cyclic washing 3-5 time, shred, 300 order steel meshes grind prepares single cell suspension, and multigelation legal system, for tumor cell lysate, is crossed the rear protein quantification of net for subsequent use.
2. the tumor cell that, breast, ascites obtain: get breast, ascites 1500-2000ml, 1200rpm is centrifugal, and normal saline washs centrifugal 3 times, and 300 order steel meshes obtain single cell suspension after crossing net, multigelation legal system, for tumor cell lysate, is crossed the rear protein quantification of net for subsequent use.
3., with tissue-derived cell strain: multigelation legal system, for tumor cell lysate, is crossed the rear protein quantification of net for subsequent use.
Get above autologous tumor lysate albumen and homologous cell strain lysate albumen each 50%, be mixed with into the relevant holoantigen of autologous tumor.
Obtaining of step 2, umbilical blood:
1., puerpera selects: select mature or premature labor cesarean healthy women, should meet following condition: the age is below 35 years old; Without malignant tumor; Without hereditary; Without infectious diseases such as hepatitis, syphilis, AIDSs; Without serious pregnancy complications; Without family's sex-controlled inheritance disease; Without pregnancy period blood transfusion history.
2., fetus is selected: fetal weight exceedes 3000g; Without congenital diseases or deformity;
3., blood sampling contraindication: placental separation exceedes 12 hours; Premature rupture of fetal membrane exceedes 24 hours; Amniotic fluid detects finds chromosomal abnormality; When production, puerpera has infection, heating; Fetal respiration is poverty-stricken; In amniotic fluid, there is meconium.
4., fetus cuts in latter 5 minutes, cuts off the 75% alcohol disinfecting broken ends of fractured bone apart from fetus 5-7cm place dual ligation fetus side umbilical cord and in line-to-line.Capable 75% alcohol disinfecting of Placenta Hominis side umbilical cord 2-3cm, disposable blood taking device (containing anticoagulant) umbilical vein puncture extracts umbilical blood (approximately 100-120ml).
5., taken a blood sample after, in 18 DEG C of storage boxes, be transported to GMP laboratory as early as possible, and carried out cell separation in 6 hour, can not exceed 12 hours the latest.
Step 3, UCB-MNCs obtain:
Above-mentioned umbilical blood is added to PBS dilution (0.01M by 1:1 volume ratio, PH=7.4), 200 eye mesh screens filter, and are resuspended in the 50ml centrifuge tube containing 15ml lymphocyte separation medium (density 1.077g/ml), 2500r/min gradient density centrifugal 20 minutes, carefully aspirates tunica albuginea layer and obtains UCB-MNCs.
The purification of step 4, UCB-MNCs source CD34+ cell:
The beautiful day small immunomagnetic beads of the anti-CD34+ of Ni of application, by specification extracts the CD34+ cell of purification (after small magnetic bead sorting from UCB-MNCs, cell purity is 97%, cellular morphology, phenotype, amplification ability, biological property etc. do not change, and along with passage, small magnetic bead comes off gradually, thereby does not affect cytologic experiment, and does not affect human body application).
The inducing culture of step 5, precursor DC:
CD34+ cell is row normal saline centrifuge washing 3 times (2000r, 1500r, 1000r/min) respectively, is inoculated in plastic culture bottle, containing in 5% autoserous IMDM culture medium adherent 1.5 hours, removes CD14+ cell.Collect suspension cell, according to 10
6the cell density of/ml is inoculated in culture bottle, is containing FLT3-L 25ng/ml, TPO 10ng/ml, and SCF 20ng/ml, and in 5% autoserous IMDM culture medium, 37 DEG C, 5% CO
2saturated humidity is cultivated 2 weeks, and every 2-3 days changes liquid, and 80% merges time shift bottle, the 14th day results precursor DC(Fig. 1), can be frozen for subsequent use, can continue to induce ripe DC.
The amplification of step 6, immature DC and cultivation:
Precursor DC is inoculated in 6 orifice plates that contain 5% autoserous IMDM culture medium 2ml, and cell density is adjusted into 3 × 10
5/ well, 37 DEG C, 5% CO
2in saturated humidity incubator, adhere-wall culture 1.5 hours, removes suspension cell, and attached cell is immature DC.In immature DC, add GM-CSF 50ng/ml, IL-4 20ng/ml, 37 DEG C, 5% CO
2saturated humidity is cultivated 5 days (within every 2-3 days, half amount is changed liquid), completes the amplification cultivation (Fig. 2) of immature DC.
The preparation of step 7, DC vaccine:
Within the 5th day, add autologous tumor relevant holoantigen 50 μ g/ml, TNF-α 100ng/ml and CD40L 100ng/ml, and supplementary 10% autoserum, cultivate induction DC maturation (Fig. 3) 48 hours, and be prepared into tumour-specific DC vaccine, clinical vaccine therapy or frozen (cryopreservation methods: 10
6/ ml cell, adds in the IMDM culture medium containing 20% autoserum, 10%DMSO, mix, and frozen plate-80 of gradient DEG C refrigerator overnight, unloading is in liquid nitrogen).
Intermediate product and end-product that above-mentioned steps of the present invention obtains detect by the following method, find that it meets the requirements completely in preparation process.
1., UCB-CD34 carrys out source precursor DC cell: UCB-CD34+ cell induction is cultivated the precursor DC obtaining for 14 days and carried out fluidic cell detection discovery, and more than 90% precursor DC has CD33 high expressed, illustrates that precursor DC belongs to myelocytic series.After recovery in frozen 1 week, detect, cell sign thing does not change, and shows that cell function stablizes (table 1).
2., UCB-CD34 source immature DC antigen uptake Function detection: immature DC adds 20 μ g/ml FITC-dextran, 4 DEG C (contrast) and 37 DEG C are cultivated 30 minutes and 60 minutes altogether, PBS centrifuge washing 3 times, fluidic cell detects to be found, 30 minutes antigen uptake rates are within 50%, 60 minute, to be 71%.Show that immature DC has powerful antigen uptake function (Fig. 4).
3., the UCB-CD34 ripe DC(DC vaccine of originating): immature DC amplification adds after antigen ripe cultivation for 5 days, and fluidic cell detects to be found, cell surface important symbol thing all has significantly and raises, and shows that the antigen presentation function of DC significantly improves (table 2).
4., mixed lymphocyte reaction (MLR) detects UCB-CD34 source DC function: get healthy human peripheral blood T cell 10
5/ well, adds round bottom 96 orifice plates, adds ripe DC to cultivate altogether detect discovery in 16 hours by different proportion, and DC can effectively induce Allogeneic T cell proliferation (Fig. 5).
When table 1. inducing culture 14 days, the precursor DC surface marker in UCB-CD34 source
* fresh precursor DC and the frozen precursor DC each mark comparison in surface, respectively P>0.05.
The originate surface marker of ripe DC of table 2. UCB-MNCs
* the each mark comparison of fresh mature DCs and frozen mature DCs, respectively P>0.05.
The UCB-CD34 DC(UCB-CD34-DC that originates) and peripheral blood list shape nucleus source DC(PBMC-DC) comparative study:
1. in immaturity PBMC-DC and UBC-CD34-DC, add, respectively 20 μ g/ml FITC-dextran, 37 DEG C, 5% CO
2saturated humidity is cultivated respectively 30 minutes and 60 minutes.PBS centrifuge washing 3 times, fluidic cell detects to be found, the antigen uptake function of UBC-CD34-DC is higher than PBMC-DC(Fig. 6).
2., UBC-CD34-DC and PBMC-DC are respectively at cultivating the 5th day load tumor cell lysate antigen, add TNF-α to be cultured to the 7th day ELISA and detect discovery, IL-12 and INF-γ secretory volume significantly raise (P<0.01) in PBMC-DC supernatant, in UBC-CD34-DC supernatant, IL-6, IL-10, IL-12 and TNF-α secretory volume significantly raise (P<0.01), the IL-12 secretory volume identical with PBMC-DC (Fig. 7) of UBC-DC.
3., UBC-CD34-DC and PBMC-DC load HPV-16 E7 polypeptide respectively, according to the ratio of T/DC 3:1, with CD8+T co-culture of cells (UBC-CD34-DC, A1-A3; PBMC-DC, B1-B3), ELISApot method detects INF-γ secretory volume.Find that the ability of UBC-DC inducing T cell activation is significantly higher than the inducibility (P<0.01 of PBMC-DC; Fig. 8)
4., UBC-CD34-DC and PBMC-DC induce respectively BGC-823 gastric cancer specificity; and HT-29 colon cancer specific CTL; row in vitro fragmentation test (Cytotoxicity assay) discovery, the CTL of UBC-CD34-DC induction has stronger tumour-specific lethal effect (P<0.05; Fig. 9).
The clinical practice of DC vaccine:
Vaccination ways: according to individualized treatment principle, DC vaccine should take drain regional lymph nodes injection system to carry out vaccine therapy.As: pulmonary carcinoma, take homonymy supraclavicular lymph nodes region vaccine therapy; Breast carcinoma, adopts on homonymy clavicle or axillary gland region vaccine therapy; Rectal cancer, adopts left side inguinal lymph tie region vaccine therapy.
Clinical workflow: before DC vaccination treatment, first applying CTX 800-1000mg disturbs tumor microenvironment, breaks tumour immunity tolerance (effectively reducing Treg, IL-10, TGF-β level), then implement the regional lymph nodes inoculation of DC vaccine, every 2-3 days vaccine therapy once, is for 3-5 time a course for the treatment of; Vein adoptive therapy (ACT) once a day or the next day once.
brief description of the drawings:
Fig. 1. the precursor DC cell (× 200) in UCB-CD34 source.
Fig. 2. the amplification cultivation UCB-CD34 of the 5th day source immature DC cell (× 200).
Fig. 3. the immature DC Antigen after ripening of UCB-CD34 source is cultivated and within 48 hours, is obtained ripe DC(× 200).
Fig. 4. UCB-CD34 source immature DC obviously strengthens (n=3) to the picked-up ability of FITC-dextran.
Fig. 5. mixed lymphocyte reaction (MLR) finds, the DC in UCB-MNCs source and
The DC in UCB-CD34 source, all can effectively activate normal person's periphery blood T cell.
Fig. 6. the antigen uptake function of UCB-CD34 source immature DC is higher than PBMC-DC.
Fig. 7. the IL-12 of PBMC-DC and INF-γ secretory volume obviously raise (P<0.01); IL-6, IL-10, IL-12 and the TNF-α secretory volume of UCB-CD34-DC significantly raise (P<0.01).
Fig. 8. the ability of UCB-CD34-DC activated T cell is significantly higher than PBMC-DC(P<0.01).
Fig. 9. the CTL of UCB-CD34-DC induction, compare with the CTL of PBMC-DC induction, there is stronger tumour-specific lethal effect (P<0.05).
detailed description of the invention:
Further illustrate by the following examples the present invention, but not as limitation of the present invention.
Embodiment 1
The CD34+ cell of Cord Blood-Derived is prepared the preparation method of DC vaccine, and step is as follows:
The preparation of step 1, the relevant holoantigen of autologous tumor:
1., the fresh tumor tissues of excision: get tumor peripheral part tissue (avoiding the slough of tumor center) 0.3-1.0cm
3(can liquid nitrogen or-80 DEG C of Refrigerator stores for subsequent use), wipes out nonneoplastic tissue around, gentamycin-normal saline cyclic washing 3-5 time, shred, 300 order steel meshes grind prepares single cell suspension, and multigelation legal system, for tumor cell lysate, is crossed the rear protein quantification of net for subsequent use.
2. the tumor cell that, breast, ascites obtain: get breast, ascites 1500-2000ml, 1200rpm is centrifugal, and normal saline washs centrifugal 3 times, and 300 order steel meshes obtain single cell suspension after crossing net, multigelation legal system, for tumor cell lysate, is crossed the rear protein quantification of net for subsequent use.
3., with tissue-derived cell strain: multigelation legal system, for tumor cell lysate, is crossed the rear protein quantification of net for subsequent use.
Get above autologous tumor lysate albumen and homologous cell strain lysate albumen each 50%, be mixed with into the relevant holoantigen of autologous tumor.
Obtaining of step 2, umbilical blood:
1., puerpera selects: select mature or premature labor cesarean healthy women, should meet following condition: the age is below 35 years old; Without malignant tumor; Without hereditary; Without infectious diseases such as hepatitis, syphilis, AIDSs; Without serious pregnancy complications; Without family's sex-controlled inheritance disease; Without pregnancy period blood transfusion history.
2., fetus is selected: fetal weight exceedes 3000g; Without congenital diseases or deformity;
3., blood sampling contraindication: placental separation exceedes 12 hours; Premature rupture of fetal membrane exceedes 24 hours; Amniotic fluid detects finds chromosomal abnormality; When production, puerpera has infection, heating; Fetal respiration is poverty-stricken; In amniotic fluid, there is meconium.
4., fetus cuts in latter 5 minutes, cuts off the 75% alcohol disinfecting broken ends of fractured bone apart from fetus 5-7cm place dual ligation fetus side umbilical cord and in line-to-line.Capable 75% alcohol disinfecting of Placenta Hominis side umbilical cord 2-3cm, disposable blood taking device (containing anticoagulant) umbilical vein puncture extracts umbilical blood (approximately 100-120ml).
5., taken a blood sample after, in 18 DEG C of storage boxes, be transported to GMP laboratory as early as possible, and carried out cell separation in 6 hour, can not exceed 12 hours the latest.
Step 3, UCB-MNCs obtain:
Above-mentioned umbilical blood is added to PBS dilution (0.01M by 1:1 volume ratio, PH=7.4), 200 eye mesh screens filter, and are resuspended in the 50ml centrifuge tube containing 15ml lymphocyte separation medium (density 1.077g/ml), 2500r/min gradient density centrifugal 20 minutes, carefully aspirates tunica albuginea layer and obtains UCB-MNCs.
The purification of step 4, UCB-MNCs source CD34+ cell:
The beautiful day small immunomagnetic beads of the anti-CD34+ of Ni of application, by specification extracts the CD34+ cell of purification (after small magnetic bead sorting from UCB-MNCs, cell purity is 97%, cellular morphology, phenotype, amplification ability, biological property etc. do not change, and along with passage, small magnetic bead comes off gradually, thereby does not affect cytologic experiment, and does not affect human body application).
The inducing culture of step 5, precursor DC:
CD34+ cell is row normal saline centrifuge washing 3 times (2000r, 1500r, 1000r/min) respectively, is inoculated in plastic culture bottle, containing in 5% autoserous IMDM culture medium adherent 1.5 hours, removes CD14+ cell.Collect suspension cell, according to 10
6the cell density of/ml is inoculated in culture bottle, is containing FLT3-L 25ng/ml, TPO 10ng/ml, and SCF 20ng/ml, and in 5% autoserous IMDM culture medium, 37 DEG C, 5% CO
2saturated humidity is cultivated 2 weeks, and every 2-3 days changes liquid, and 80% merges time shift bottle, the 14th day results precursor DC(Fig. 1), can be frozen for subsequent use, can continue to induce ripe DC.
The amplification of step 6, immature DC and cultivation:
Precursor DC is inoculated in 6 orifice plates that contain 5% autoserous IMDM culture medium 2ml, and cell density is adjusted into 3 × 10
5/ well, 37 DEG C, 5% CO
2in saturated humidity incubator, adhere-wall culture 1.5 hours, removes suspension cell, and attached cell is immature DC.In immature DC, add GM-CSF 50ng/ml, IL-4 20ng/ml, 37 DEG C, 5% CO
2saturated humidity is cultivated 5 days (within every 2-3 days, half amount is changed liquid), completes the amplification cultivation (Fig. 2) of immature DC.
The preparation of step 7, DC vaccine:
Within the 5th day, add autologous tumor relevant holoantigen 50 μ g/ml, TNF-α 100ng/ml and CD40L 100ng/ml, and supplementary 10% autoserum, cultivate induction DC maturation (Fig. 3) 48 hours, and be prepared into tumour-specific DC vaccine, clinical vaccine therapy or frozen (cryopreservation methods: 10
6/ ml cell, adds in the IMDM culture medium containing 20% autoserum, 10%DMSO, mix, and frozen plate-80 of gradient DEG C refrigerator overnight, unloading is in liquid nitrogen).
Claims (1)
1. application umbilical blood CD34+ cell is prepared a tumour-specific DC vaccine method, described method, and step is as follows:
The preparation of step 1, the relevant holoantigen of autologous tumor:
1., the fresh tumor tissues of excision: get tumor peripheral part and organize 0.3-1.0cm
3, wipe out nonneoplastic tissue around, gentamycin-normal saline cyclic washing 3-5 time, shreds, and 300 order steel meshes grind prepares single cell suspension, and multigelation legal system, for tumor cell lysate, is crossed the rear protein quantification of net for subsequent use;
2. the tumor cell that, breast, ascites obtain: get breast, ascites 1500-2000ml, 1200rpm is centrifugal, and normal saline washs centrifugal 3 times, and 300 order steel meshes obtain single cell suspension after crossing net, multigelation legal system, for tumor cell lysate, is crossed the rear protein quantification of net for subsequent use;
3., with tissue-derived cell strain: multigelation legal system, for tumor cell lysate, is crossed the rear protein quantification of net for subsequent use;
Get above autologous tumor lysate albumen and homologous cell strain lysate albumen each 50%, be mixed with into the relevant holoantigen of autologous tumor;
Obtaining of step 2, umbilical blood:
1., puerpera selects: select mature or premature labor cesarean healthy women, should meet following condition: the age is below 35 years old; Without malignant tumor; Without hereditary; Without infectious diseases such as hepatitis, syphilis, AIDSs; Without serious pregnancy complications; Without family's sex-controlled inheritance disease; Without pregnancy period blood transfusion history;
2., fetus is selected: fetal weight exceedes 3000g; Without congenital diseases or deformity;
3., blood sampling contraindication: placental separation exceedes 12 hours; Premature rupture of fetal membrane exceedes 24 hours; Amniotic fluid detects finds chromosomal abnormality; When production, puerpera has infection, heating; Fetal respiration is poverty-stricken; In amniotic fluid, there is meconium;
4., fetus cuts in latter 5 minutes, cut off apart from fetus 5-7cm place dual ligation fetus side umbilical cord and in line-to-line, the 75% alcohol disinfecting broken ends of fractured bone, capable 75% alcohol disinfecting of Placenta Hominis side umbilical cord 2-3cm, disposable blood taking device umbilical vein puncture extracts umbilical blood 100-120ml;
5., taken a blood sample after, in 18 DEG C of storage boxes, be transported to GMP laboratory as early as possible, and carried out cell separation in 6 hour, can not exceed 12 hours the latest;
Step 3, UCB-MNCs obtain:
Above-mentioned umbilical blood is added to 0.01M by 1:1 volume ratio, the PBS dilution of PH=7.4,200 eye mesh screens filter, and are resuspended in containing in 15ml lymphocyte separation medium 50ml centrifuge tube, and 2500r/min gradient density centrifugal 20 minutes, carefully aspirates tunica albuginea layer and obtains UCB-MNCs;
The purification of step 4, UCB-MNCs source CD34+ cell:
The beautiful day small immunomagnetic beads of the anti-CD34+ of Ni of application, by specification extracts the CD34+ cell of purification from UCB-MNCs;
The inducing culture of step 5, precursor DC:
CD34+ cell row normal saline centrifuge washing 3 times respectively, is inoculated in plastic culture bottle, containing in 5% autoserous IMDM culture medium adherent 1.5 hours, removes CD14+ cell, collects suspension cell, according to 10
6the cell density of/ml is inoculated in culture bottle, is containing FLT3-L 25ng/ml, TPO 10ng/ml, and SCF 20ng/ml, and in 5% autoserous IMDM culture medium, 37 DEG C, 5% CO
2saturated humidity is cultivated 2 weeks, and every 2-3 days changes liquid, and 80% merges time shift bottle, and the 14th day results precursor DC, can be frozen for subsequent use, can continue to induce ripe DC;
The amplification of step 6, immature DC and cultivation:
Precursor DC is inoculated in 6 orifice plates that contain 5% autoserous IMDM culture medium 2ml, and cell density is adjusted into 3 × 10
5/ well, 37 DEG C, 5% CO
2in saturated humidity incubator, adhere-wall culture 1.5 hours, removes suspension cell, and attached cell is immature DC, adds GM-CSF 50ng/ml in immature DC, IL-4 20ng/ml, 37 DEG C, 5% CO
2saturated humidity is cultivated 5 days, completes the amplification cultivation of immature DC;
The preparation of step 7, DC vaccine:
Within the 5th day, add autologous tumor relevant holoantigen 50 μ g/ml, TNF-α 100ng/ml and CD40L 100ng/ml, and supplement 10% autoserum, cultivate 48 hours, induction DC maturation, and be prepared into tumour-specific DC vaccine, frozen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310310485.XA CN103405759B (en) | 2013-07-23 | 2013-07-23 | Method for preparing tumor-specific DC vaccine by applying CD34+ cells of umbilical cord blood |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310310485.XA CN103405759B (en) | 2013-07-23 | 2013-07-23 | Method for preparing tumor-specific DC vaccine by applying CD34+ cells of umbilical cord blood |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103405759A CN103405759A (en) | 2013-11-27 |
| CN103405759B true CN103405759B (en) | 2014-12-10 |
Family
ID=49598832
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310310485.XA Active CN103405759B (en) | 2013-07-23 | 2013-07-23 | Method for preparing tumor-specific DC vaccine by applying CD34+ cells of umbilical cord blood |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103405759B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104845934B (en) * | 2015-06-08 | 2018-05-11 | 河北彤苓榕生物科技有限公司 | Bleeding of the umbilicus CD34+Derived from hematopoietic precursor cells Dendritic Cells is prepared on a large scale method |
| CN106434557B (en) * | 2016-11-25 | 2019-08-13 | 博雅干细胞科技有限公司 | The method for preparing CD34 positive cell by umbilical cord mesenchymal stem cells |
| CN109954133A (en) * | 2017-12-22 | 2019-07-02 | 上海市浦东医院（复旦大学附属浦东医院） | Tumor vaccine and preparation method thereof |
| 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 |
| CN111166876B (en) * | 2018-11-13 | 2022-10-11 | 北京启辰生生物科技有限公司 | Immunopotentiator combination, encoding nucleic acid and application thereof |
| WO2023078279A1 (en) * | 2021-11-04 | 2023-05-11 | 澄交生物科技股份有限公司 | Immunogenic composition and use thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102091327A (en) * | 2010-12-27 | 2011-06-15 | 蔡建辉 | Preparation method of dendritic cell (DC) vaccine loaded with autologous tumor associated holoantigen |
| CN102307989A (en) * | 2008-11-14 | 2012-01-04 | 生物载体株式会社 | Method for producing dendritic cells |
| CN102596234A (en) * | 2009-07-24 | 2012-07-18 | 罗得岛医院 | Dendritic cell vaccines for asparaginyl-beta-hydroxylase expressing tumors |
-
2013
- 2013-07-23 CN CN201310310485.XA patent/CN103405759B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102307989A (en) * | 2008-11-14 | 2012-01-04 | 生物载体株式会社 | Method for producing dendritic cells |
| CN102596234A (en) * | 2009-07-24 | 2012-07-18 | 罗得岛医院 | Dendritic cell vaccines for asparaginyl-beta-hydroxylase expressing tumors |
| CN102091327A (en) * | 2010-12-27 | 2011-06-15 | 蔡建辉 | Preparation method of dendritic cell (DC) vaccine loaded with autologous tumor associated holoantigen |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103405759A (en) | 2013-11-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103405759B (en) | Method for preparing tumor-specific DC vaccine by applying CD34+ cells of umbilical cord blood | |
| CN102091327B (en) | Preparation method of dendritic cell vaccine loaded with autologous tumor-associated whole antigen | |
| CN105176927A (en) | Preparation method of cytotoxicity-enhanced efficient target killing NK/CIK (Natural Killer)/( Cytokine Induced Killer) cells | |
| CN106177931B (en) | The double preparation methods for blocking CTL high efficiency killing cell preparation of immune detection point | |
| KR101732532B1 (en) | Method for obtaining monocytes or nk cells | |
| CN108379569B (en) | DC vaccine for efficiently loading tumor antigen and method for inducing and amplifying tumor antigen specific CTL (cytotoxic T lymphocyte) | |
| CN101514333B (en) | Immunologic tolerance dendritic cell, preparation method thereof and special culture medium | |
| CN104762257A (en) | Method for preparing mesenchymal stem cell from umbilical cord | |
| WO2015081741A1 (en) | Method and kit for preparing immunological cells | |
| CN105112371A (en) | Preparation method for DC-CIK cells originated from umbilical cord blood mononuclear cells and preparation | |
| Sabado et al. | Preparation of tumor antigen-loaded mature dendritic cells for immunotherapy | |
| CN109402057A (en) | A kind of cultural method for the DC-CTL cell loading tumour cell excretion body | |
| CN103405758B (en) | Method for preparing tumor-specific DC vaccine by applying mononuclear cells in umbilical cord blood | |
| CN108841790A (en) | A kind of method of the mononuclearcell induction CIK cell in placenta source | |
| CN114480277B (en) | A kind of method and medium for stimulating dendritic cell maturation | |
| CN113943704B (en) | Preparation method of tumor neoantigen specific T cells | |
| CN105132373A (en) | Antigen sensitized dendritic cell preparing method | |
| KR101440102B1 (en) | Manufacturing method of strong antigen-presenting cells using hydrogel coating and electroporation | |
| CN114807031A (en) | Construction method of human peripheral blood immune cell bank and stem cell bank | |
| WO2022061811A1 (en) | Pharmaceutical composition, and preparation method therefor and application thereof | |
| CN105664155A (en) | DC (dendritic cell) based breast cancer therapeutic vaccine and preparation method thereof | |
| CN105219728A (en) | A kind of for activating the immunoreactive test kit of Breast Cancer-Specific | |
| CN111286490A (en) | Method for constructing in vitro model of tumor microenvironment, model constructed therefrom, and use | |
| CN110872575A (en) | In-vitro amplification method of iNKT cells | |
| CN105219727A (en) | A kind of test kit for activating colorectal cancer specific immune response |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| ASS | Succession or assignment of patent right |
Owner name: CAI YING Free format text: FORMER OWNER: CAI JIANHUI Effective date: 20131105 Free format text: FORMER OWNER: LI CAIZHEN Effective date: 20131105 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| C53 | Correction of patent for invention or patent application | ||
| CB03 | Change of inventor or designer information |
Inventor after: Cai Jianhui Inventor after: Liu Gang Inventor after: Chen Baoping Inventor after: Yuan Can Inventor after: Feng Ruicheng Inventor before: Cai Jianhui Inventor before: Gao Fei Inventor before: Liu Gang |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: CAI JIANHUI GAO FEI LIU GANG TO: CAI JIANHUI LIU GANG CHEN BAOPING YUAN CAN FENG RUICHENG |
|
| TA01 | Transfer of patent application right |
Effective date of registration: 20131105 Address after: 050000 Heping West Road, Hebei, Shijiazhuang, No. 348 Applicant after: Cai Ying Address before: 050000 Heping West Road, Hebei, Shijiazhuang, No. 348 Applicant before: Cai Jianhui Applicant before: Li Caizhen |
|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: HEBEI LITONGKANG BIOLOGICAL TECHNOLOGY CO., LTD. Free format text: FORMER OWNER: CAI YING Effective date: 20150413 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| C53 | Correction of patent for invention or patent application | ||
| CB03 | Change of inventor or designer information |
Inventor after: Cai Jianhui Inventor after: Liu Gang Inventor after: Chen Baoping Inventor after: Li Hanlin Inventor before: Cai Jianhui Inventor before: Liu Gang Inventor before: Chen Baoping Inventor before: Yuan Can Inventor before: Feng Ruicheng |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: CAI JIANHUI LIU GANG CHEN BAOPING YUAN CAN FENG RUICHENG TO: CAI JIANHUI LIU GANG CHEN BAOPING LI HANLIN |
|
| TR01 | Transfer of patent right |
Effective date of registration: 20150413 Address after: 050000 No. 219, Qinshan street, hi tech Zone, Hebei, Shijiazhuang 1-206 Patentee after: Li Tongkang bio tech ltd, Hebei Address before: 050000 Heping West Road, Hebei, Shijiazhuang, No. 348 Patentee before: Cai Ying |
|
| CP03 | Change of name, title or address | ||
| CP03 | Change of name, title or address |
Address after: 050000 Floor 1, Building 238 Changjiang Avenue, Shijiazhuang High-tech Zone, Hebei Province Patentee after: Hebei Fu Fu Yu Biological Technology Co., Ltd. Address before: 050000 1-206, No. 219 Qinshan Street, Shijiazhuang High-tech Zone, Hebei Province Patentee before: Li Tongkang bio tech ltd, Hebei |
|
| TR01 | Transfer of patent right |
Effective date of registration: 20211126 Address after: 050000 a2-145, No. 22, yuanboyuan street, South Zone, Zhengding County high tech Industrial Development Zone, Zhengding District, China (Hebei) pilot Free Trade Zone, Shijiazhuang City, Hebei Province Patentee after: Yuetenong biotechnology Hebei Co.,Ltd. Address before: 050000 floor 1, building 1, 238 Changjiang Avenue, high tech Zone, Shijiazhuang City, Hebei Province Patentee before: HEBEI NONGFOYU BIOLOGY TECHNOLOGY Co.,Ltd. |
|
| TR01 | Transfer of patent right |