CN112725273A - NK cell and preparation method and application thereof - Google Patents
NK cell and preparation method and application thereof Download PDFInfo
- Publication number
- CN112725273A CN112725273A CN202110112416.2A CN202110112416A CN112725273A CN 112725273 A CN112725273 A CN 112725273A CN 202110112416 A CN202110112416 A CN 202110112416A CN 112725273 A CN112725273 A CN 112725273A
- Authority
- CN
- China
- Prior art keywords
- cells
- culture
- engineered
- culture solution
- calf serum
- 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.)
- Pending
Links
Images
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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2321—Interleukin-21 (IL-21)
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/50—Cell markers; Cell surface determinants
- C12N2501/505—CD4; CD8
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/50—Cell markers; Cell surface determinants
- C12N2501/51—B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/50—Cell markers; Cell surface determinants
- C12N2501/599—Cell markers; Cell surface determinants with CD designations not provided for elsewhere
-
- 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
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/99—Coculture with; Conditioned medium produced by genetically modified cells
Abstract
The invention relates to an NK cell and a preparation method and application thereof. The preparation method comprises the step of inducing and culturing umbilical cord blood mononuclear cells by utilizing the engineered K562 cells to obtain NK cells. The engineered K562 cells express cytokines CD19, CD86, IL21, CD64 and CD137L, can efficiently amplify and activate umbilical cord blood mononuclear cells, and the prepared NK cells have high amplification quantity and cell activity, are low in usage amount and can effectively reduce cost.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and relates to NK cells and a preparation method and application thereof.
Background
Natural killer cells (NK) are important immune cells of an organism, are related to anti-tumor, anti-virus infection and immune regulation, are the first line of defense of a human body against cancer cells and virus infection, can kill tumor cells in a non-specific manner, have an immune regulation function, and can interact with other immune cells of the organism to regulate the immune state and the immune function of the organism. Clinical researches find that the NK cell adoptive immunotherapy has good application prospect in treating malignant tumors and has certain effect on various solid tumors and hematological malignant tumors.
The existing NK cell amplification culture technology mainly comprises a factor method: extracting peripheral blood or obtaining Peripheral Blood Mononuclear Cells (PBMC) by using a single cell harvester and combining with lymphocyte separation liquid, then directly culturing or sorting NK cells in the PBMC, and amplifying NK by using cytokines or combined feeder cells in a common lymphocyte culture medium; antibody coating method: expanding and culturing PBMC or sorted NK cells by using a common lymphocyte culture medium in an antibody-coated culture flask; special NK culture medium: purchasing a special NK culture medium to specifically amplify the NK cells from the PBMC or amplifying the sorted NK cells; induction of stem cells: the stem cells were induced to differentiate into NK cells, and then expanded.
CN105238754A discloses an in vitro culture method of NK cells with high proliferation and lethality, which comprises the step of amplifying and culturing PBMC by using a cell culture flask coated by an anti-human CD3 monoclonal antibody and an anti-human CD16 monoclonal antibody to obtain the NK cells without using trophoblast cells. However, the clinical requirements of the amplified quantity, purity and NK cell activity of NK cells are difficult to meet.
CN104894072A provides a preparation method of autologous natural killer cell proliferation culture, which comprises the steps of using K562 cells transfected by 3-phosphoinositide-dependent protein kinase 1 and CD122 and stably expressed and interleukin 2 to amplify and activate mononuclear cells from cancer patients to obtain natural killer cells, wherein the natural killer cells have the proliferation multiple of more than 2500 times and have strong killing toxicity, but the using amount of trophoblasts is larger and the cost is higher.
The method for preparing the NK cells is low in cost, and the prepared NK cells have high amplification quantity and cell activity and have important significance in the field of NK cell preparation.
Disclosure of Invention
Aiming at the defects and actual requirements of the prior art, the invention provides the NK cell and the preparation method and application thereof, the preparation method utilizes the engineered K562 cell to induce and culture the cord blood mononuclear cell, the NK cell can be efficiently prepared, and the prepared NK cell has higher amplification quantity and cell activity.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a preparation method of NK cells, which comprises the step of inducing and culturing umbilical cord blood mononuclear cells by using engineered K562 cells to obtain the NK cells.
In the preparation method, the engineered K562 cells can efficiently amplify and activate the umbilical cord blood mononuclear cells, the prepared NK cells have higher amplification quantity and cell activity, the use amount of the engineered K562 cells is low, and the cost can be effectively reduced.
Preferably, the engineered K562 cells express the membrane proteins CD19, CD86, IL21, CD64 and CD 137L.
Preferably, said IL-21 is expressed on the surface of said engineered K562 cells by fusion to the CD8 α transmembrane region.
Preferably, the genome of the engineered K562 cell integrates a CD19 encoding gene, a CD86 encoding gene, an IL21-CD8 α encoding gene, a CD64 encoding gene, and a CD137L encoding gene.
Preferably, the gene encoding CD19 comprises the nucleic acid sequence shown in SEQ ID NO. 1.
SEQ ID NO:1：
atgccacctcctcgcctcctcttcttcctcctcttcctcacccccatggaagtcaggcccgaggaacctctagtggtgaaggtggaagagggagataacgctgtgctgcagtgcctcaaggggacctcagatggccccactcagcagctgacctggtctcgggagtccccgcttaaacccttcttaaaactcagcctggggctgccaggcctgggaatccacatgaggcccctggccatctggcttttcatcttcaacgtctctcaacagatggggggcttctacctgtgccagccggggcccccctctgagaaggcctggcagcctggctggacagtcaatgtggagggcagcggggagctgttccggtggaatgtttcggacctaggtggcctgggctgtggcctgaagaacaggtcctcagagggccccagctccccttccgggaagctcatgagccccaagctgtatgtgtgggccaaagaccgccctgagatctgggagggagagcctccgtgtctcccaccgagggacagcctgaaccagagcctcagccaggacctcaccatggcccctggctccacactctggctgtcctgtggggtaccccctgactctgtgtccaggggccccctctcctggacccatgtgcaccccaaggggcctaagtcattgctgagcctagagctgaaggacgatcgcccggccagagatatgtgggtaatggagacgggtctgttgttgccccgggccacagctcaagacgctggaaagtattattgtcaccgtggcaacctgaccatgtcattccacctggagatcactgctcggccagtactatggcactggctgctgaggactggtggctggaaggtctcagctgtgactttggcttatctgatcttctgcctgtgttcccttgtgggcattcttcatctt。
Preferably, the gene encoding CD86 comprises the nucleic acid sequence shown in SEQ ID NO. 2.
SEQ ID NO:2：
atgggactgagtaacattctctttgtgatggccttcctgctctctggtgctgctcctctgaagattcaagcttatttcaatgagactgcagacctgccatgccaatttgcaaactctcaaaaccaaagcctgagtgagctagtagtattttggcaggaccaggaaaacttggttctgaatgaggtatacttaggcaaagagaaatttgacagtgttcattccaagtatatgggccgcacaagttttgattcggacagttggaccctgagacttcacaatcttcagatcaaggacaagggcttgtatcaatgtatcatccatcacaaaaagcccacaggaatgattcgcatccaccagatgaattctgaactgtcagtgcttgctaacttcagtcaacctgaaatagtaccaatttctaatataacagaaaatgtgtacataaatttgacctgctcatctatacacggttacccagaacctaagaagatgagtgttttgctaagaaccaagaattcaactatcgagtatgatggtattatgcagaaatctcaagataatgtcacagaactgtacgacgtttccatcagcttgtctgtttcattccctgatgttacgagcaatatgaccatcttctgtattctggaaactgacaagacgcggcttttatcttcacctttctctatagagcttgaggaccctcagcctcccccagaccacattccttggattacagctgtacttccaacagttattatatgtgtgatggttttctgtctaattctatggaaatggaagaagaagaagcggcctcgcaactcttataaatgtggaaccaacacaatggagagggaagagagtgaacagaccaagaaaagagaaaaaatccatatacctgaaagatctgatgaagcccagcgtgtttttaaaagttcgaagacatcttcatgcgacaaaagtgatacatgtttttaa。
Preferably, the gene encoding IL21-CD8 alpha comprises the nucleic acid sequence shown in SEQ ID NO. 3.
SEQ ID NO:3：
atgagatccagtcctggcaacatggagaggattgtcatctgtctgatggtcatcttcttggggacactggtccacaaatcaagctcccaaggtcaagatcgccacatgattagaatgcgtcaacttatagatattgttgatcagctgaaaaattatgtgaatgacttggtccctgaatttctgccagctccagaagatgtagagacaaactgtgagtggtcagctttttcctgttttcagaaggcccaactaaagtcagcaaatacaggaaacaatgaaaggataatcaatgtatcaattaaaaagctgaagaggaaaccaccttccacaaatgcagggagaagacagaaacacagactaacatgcccttcatgtgattcttatgagaaaaaaccacccaaagaattcctagaaagattcaaatcacttctccaaaagatgattcatcagcatctgtcctctagaacacacggaagtgaagattccggatcctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgctaa。
Preferably, the gene encoding CD64 comprises the nucleic acid sequence shown in SEQ ID NO. 4.
SEQ ID NO:4：
atgtggttcttgacaactctgctcctttgggttccagttgatgggcaagtggacaccacaaaggcagtgatcactttgcagcctccatgggtcagcgtgttccaagaggaaaccgtaaccttgcattgtgaggtgctccatctgcctgggagcagctctacacagtggtttctcaatggcacagccactcagacctcgacccccagctacagaatcacctctgccagtgtcaatgacagtggtgaatacaggtgccagagaggtctctcagggcgaagtgaccccatacagctggaaatccacagaggctggctactactgcaggtctccagcagagtcttcacggaaggagaacctctggccttgaggtgtcatgcgtggaaggataagctggtgtacaatgtgctttactatcgaaatggcaaagcctttaagtttttccactggaattctaacctcaccattctgaaaaccaacataagtcacaatggcacctaccattgctcaggcatgggaaagcatcgctacacatcagcaggaatatctgtcactgtgaaagagctatttccagctccagtgctgaatgcatctgtgacatccccactcctggaggggaatctggtcaccctgagctgtgaaacaaagttgctcttgcagaggcctggtttgcagctttacttctccttctacatgggcagcaagaccctgcgaggcaggaacacatcctctgaataccaaatactaactgctagaagagaagactctgggttatactggtgcgaggctgccacagaggatggaaatgtccttaagcgcagccctgagttggagcttcaagtgcttggcctccagttaccaactcctgtctggtttcatgtccttttctatctggcagtgggaataatgtttttagtgaacactgttctctgggtgacaatacgtaaagaactgaaaagaaagaaaaagtgggatttagaaatctctttggattctggtcatgagaagaaggtaatttccagccttcaagaagacagacatttagaagaagagctgaaatgtcaggaacaaaaagaagaacagctgcaggaaggggtgcaccggaaggagccccagggggccacgtag。
Preferably, the gene encoding CD137L comprises the nucleic acid sequence shown in SEQ ID NO. 5.
SEQ ID NO:5：
atggaatacgcctctgacgcttcactggaccccgaagccccgtggcctcccgcgccccgcgctcgcgcctgccgcgtactgccttgggccctggtcgcggggctgctgctgctgctgctgctcgctgccgcctgcgccgtcttcctcgcctgcccctgggccgtgtccggggctcgcgcctcgcccggctccgcggccagcccgagactccgcgagggtcccgagctttcgcccgacgatcccgccggcctcttggacctgcggcagggcatgtttgcgcagctggtggcccaaaatgttctgctgatcgatgggcccctgagctggtacagtgacccaggcctggcaggcgtgtccctgacggggggcctgagctacaaagaggacacgaaggagctggtggtggccaaggctggagtctactatgtcttctttcaactagagctgcggcgcgtggtggccggcgagggctcaggctccgtttcacttgcgctgcacctgcagccactgcgctctgctgctggggccgccgccctggctttgaccgtggacctgccacccgcctcctccgaggctcggaactcggccttcggtttccagggccgcttgctgcacctgagtgccggccagcgcctgggcgtccatcttcacactgaggccagggcacgccatgcctggcagcttacccagggcgccacagtcttgggactcttccgggtgacccccgaaatcccagccggactcccttcaccgaggtcggaataa。
Preferably, the preparation method of the NK cells comprises the step of discontinuously utilizing the engineered K562 cells for induction culture.
Preferably, the induction culture of the engineered K562 cells by the discontinuous method comprises the following steps:
(1) adding the umbilical cord blood mononuclear cells and the engineered K562 cells into a culture solution for mixed culture, and adding fetal bovine serum and gentamycin sulfate;
(2) centrifuging and collecting cells on 2-6 days of culture, adding culture solution, fetal calf serum and gentamicin sulfate for resuspension of the cells, and continuing culture;
(3) adding the engineered K562 cells, a culture solution, fetal calf serum and gentamicin sulfate into the culture medium for continuous culture on 7-8 days;
(4) adding culture solution, fetal calf serum and gentamicin sulfate into the culture medium on day 8-14, and continuously culturing;
(5) and collecting cells on the 14 th-16 th day of culture to obtain the NK cells.
Preferably, the culture medium comprises GT-T561.
Preferably, the fetal bovine serum is 1% to 10% by mass in the culture medium, including but not limited to 2%, 4%, 5%, 6%, 8% or 9%.
Preferably, the final concentration of the gentamicin sulfate is 80-100U/mL, including but not limited to 82U/mL, 88U/mL, 90U/mL, 92U/mL, 94U/mL, 96U/mL or 98U/mL.
As a preferred technical scheme, the preparation method of the NK cells comprises the following steps:
(1) separating umbilical cord blood mononuclear cells, adding the umbilical cord blood mononuclear cells and the engineered K562 cells into a culture solution for mixed culture, wherein the culture solution contains 5-10% of fetal calf serum and 80-100U/mL gentamicin sulfate;
(2) centrifuging and collecting cells on 2-3 days of culture, adding GT-T561 culture solution containing 5-10% fetal calf serum and 80-100U/mL gentamicin sulfate to resuspend the cells, and continuing to culture;
(3) adding GT-T561 culture solution containing 1-2% fetal calf serum and 80-100U/mL gentamicin sulfate into the culture solution for continuous culture on the 4-6 th day;
(4) adding the engineered K562 cells in 7-8 days of culture, and continuously culturing by adopting a GT-T561 culture solution containing 1-2% fetal calf serum and 80-100U/mL gentamicin sulfate;
(5) adding GT-T561 culture solution containing 1-2% fetal calf serum and 80-100U/mL gentamicin sulfate into the culture medium on 8-14 days of culture, and continuing to culture;
(6) and collecting cells on the 14 th-16 th day of culture to obtain the NK cells.
In a second aspect, the present invention provides an NK cell prepared by the method of the first aspect.
In a third aspect, the present invention provides the use of the NK cell of the second aspect for the preparation of a medicament for cellular immunotherapy.
Compared with the prior art, the invention has the following beneficial effects:
(1) in the preparation method, the cord blood mononuclear cells are efficiently amplified and activated by using the engineered K562 cells expressing CD19, CD86, IL21, CD64 and CD137L, the prepared NK cells have higher amplification quantity and cell activity, the use amount of the engineered K562 cells is low, and the cost can be effectively reduced;
(2) in the NK cells prepared by the preparation method, the purity of the NK cells with the CD56 phenotype can reach 93.65%, and the purity of the NK cells with the CD16 phenotype can reach 81.76%.
Drawings
FIG. 1 is a graph showing the results of flow assays of CD19 phenotype engineered K562 cells;
FIG. 2 is a graph showing the results of flow assays of CD86 phenotype engineered K562 cells;
FIG. 3 is a graph showing the results of flow assay of IL21 phenotype engineered K562 cells;
FIG. 4 is a graph showing the results of flow assays of CD64 phenotype engineered K562 cells;
FIG. 5 is a graph showing the results of flow assays of CD137L phenotypically engineered K562 cells;
FIG. 6 is a graph showing the results of flow assay of NK cells expressing CD56 phenotype;
FIG. 7 is a graph showing the results of flow assay of NK cells expressing CD16 phenotype.
Detailed Description
To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
Example 1
This example prepares an engineered K562 cell, the preparation method comprising:
(1) artificially synthesizing the amino acid sequence shown in SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO:4 and SEQ ID NO:5 gene sequence (Biotechnology engineering (Shanghai) Co., Ltd.), and converting SEQ ID NO:1 and SEQ ID NO:2 is synthesized into a gene sequence, SEQ ID NO:1 and SEQ ID NO:2 gene sequence is named as KCD19CD86, SEQ ID NO:3 gene sequence is named as KIL21CD8 alpha, SEQ ID NO:4 gene sequence is named as KCD64, SEQ ID NO:5 is named as CD137L, the synthesized gene sequences are respectively connected to an HLU vector, the enzyme cutting sites are BamHI/EcoRI or BamHI/Not I, the enzyme cutting reaction system is shown in Table 1, the enzyme cutting conditions are water bath at 30 ℃ for 1h and water bath at 37 ℃ for 1.5h, and HLU-KCD19CD86, HLU-KIL21CD8 alpha, HLU-KCD64 and HLU-CD137L expression vectors are obtained;
TABLE 1
(2) Respectively taking strains containing HLU-KCD19CD86, HLU-KIL21CD8 alpha, HLU-KCD64, HLU-CD137L or LV5-EGFP vectors, carrying out plate streaking, culturing overnight (12h), picking single clones (3) the next day, carrying out shake culture (7h) in a small volume (5mL), taking bacterial liquid from a system of small shake culture, and carrying out large shake culture according to the volume ratio of 1: 100;
carrying out large-scale extraction on plasmids by taking each bacterial liquid by using a Tiangen DP117 endotoxin-free plasmid large-scale extraction kit according to an instruction to obtain a Lent-EGFP, a Lent-KCD19CD86, a Lent-KIL21CD8 alpha, a Lent-KCD64 and a Lent-CD137L vector;
(3) adopting Beyotime-Calcium phosphor Cell Transfection Kit (product number: C0508) to co-transfect 293T cells with a carrier of Lent-EGFP, Lent-KCD19CD86, Lent-KIL21CD8 alpha, Lent-KCD64 and Lent-CD137L, a packaging carrier of PSPAX2 and PMD2G according to instructions, adopting a centrifugal ultrafiltration mode (Millipore centrifugal Filter Unit Amicn (R) Ultra-15, product number: C910096, 100kD) to collect and concentrate viruses to obtain a Lent-EGFP, Lent-KCD19CD86, Lent-KIL21CD8 alpha, Lent-KCD64 and a Lent-CD137L lentivirus;
(4) and (3) transfecting K562 cells by using Lent-EGFP, Lent-KCD19CD86, Lent-KIL21CD8 alpha, Lent-KCD64 and Lent-CD137L lentiviruses according to the MOI of 150:1 to obtain the engineered K562 cells.
The expression conditions of cell membrane proteins CD19, CD86, IL21, CD64 and CD137L are detected by flow, and the results are shown in figures 1-5, so that the purity of the corresponding phenotype engineered K562 cell can reach more than 98.29 percent and is at most 99.99 percent, which indicates that the engineered K562 cell is successfully prepared.
Example 2
This example prepares an NK cell, which preparation method comprises the following steps:
(1) fully wiping gaps of an anticoagulation tube cover with iodophor, opening the anticoagulation tube cover after drying, pumping umbilical cord blood in the anticoagulation tube by using a pipette, adding the umbilical cord blood into a 50mL centrifuge tube, balancing the centrifuge tube when each tube is not more than 35mL, and centrifuging for 10min at 2900 r/min;
(2) transferring the upper layer of plasma obtained by centrifugation to a 50mL centrifuge tube, carrying out water bath at 40 ℃ for 30min, then centrifuging at 1500r/min for 10min to remove precipitates, transferring the upper layer of plasma to a new 50mL centrifuge tube, and storing in a refrigerator at 4 ℃ for later use (if precipitates are separated out after the refrigerator is placed, centrifuging again);
(3) diluting the cord blood with normal saline according to the volume ratio of 2:1, and mixing and uniformly mixing;
(4) carefully adding the diluted blood to the separation solution, adding 15mL of human lymphocyte separation solution into each 50mL centrifuge tube, wherein the volume ratio of the diluted blood to the separation solution is 2:1 and 1500r/min, and centrifuging for 20min at the lowest speed reduction;
(5) after the centrifugation is finished, obvious layering appears in the centrifugal tube, which is from bottom to top: the erythrocyte layer, the granulocyte layer, the fiaill layer, the mononuclear cell (MNC) layer and the plasma physiological saline layer, and the cell suspension of the mononuclear cell layer is sucked out;
(6) uniformly mixing the physiological saline and the sucked mononuclear cell liquid according to the volume ratio of 2:1, centrifuging at 1700r/min for 10min, and removing the supernatant after centrifugation;
(7) adding 5mL of normal saline into a centrifuge tube, blowing and mixing the cells uniformly, adding the normal saline to 40mL (keeping 0.5mL for counting before culture), centrifuging at 1000r/min for 10min, and discarding the supernatant after centrifugation;
(8) suspending the collected cells in GT-T561 culture medium, and adjusting the cell concentration to 1-2 × 10 according to the count6Per mL, filled to 75cm220mL of GT-T561 medium (containing 200U/mL IL-2), 1000. mu.L of fetal bovine serum (5%), 2 tubes of the engineered K562 cells prepared in example 1 (1 mL/tube) and gentamicin sulfate (80U/mL) were added to the flask and placed flat on CO2Culturing in a constant-temperature incubator;
(9) on the 3 rd day of culture, the culture solution in the culture bottle is centrifuged at 300 Xg for 8min, the supernatant is discarded, 30mL of fresh GT-561 culture solution (containing 200U/mL IL-2) is used for suspension precipitation, 5% fetal calf serum and gentamicin sulfate (80U/mL) are added, and the mixture is placed in an incubator for culture;
(10) observing cells on the 4 th day of culture, wherein the cell state is good, and the culture solution has no obvious yellowing and is not treated;
(11) observing cells on the 5 th day of culture, wherein the cells have good state and high density, the culture solution turns yellow, and 20mL of GT-561 culture solution (containing 200U/mL IL-2), 1% fetal calf serum and gentamicin sulfate (80U/mL) are supplemented;
(12) observing cells on the 6 th day of culture, wherein the cells have good state and high density, the culture solution turns yellow, and 20mL of GT-561 culture solution (containing 200U/mL IL-2), 1% fetal calf serum and gentamicin sulfate (80U/mL) are supplemented;
(13) observing cells on the 7 th day of culture, wherein the cell state is good, and the culture solution has no obvious yellowing and is not treated;
(14) transferring the culture solution to 640cm on the 8 th day of culture2Adding 5 tubes of the engineered K562 cells (1 mL/tube) prepared in example 1 into the culture bag, supplementing GT-561 culture solution (containing 200U/mL IL-2), 1% fetal bovine serum and gentamicin sulfate (80U/mL), wherein the volume ratio of the supplemented culture solution to the original culture solution is 1:1, and continuing to culture;
(15) observing cells on the 9 th day of culture, wherein the cell state is good, and the culture solution has no obvious yellowing and is not treated;
(16) observing cells on the 10 th day of culture, wherein the cell state is good, and the culture solution has no obvious yellowing and is not treated;
(17) observing the color change of the cell suspension on the 11 th day of culture, observing the growth condition of the cells under a microscope, obviously yellowing the cell suspension and cloning more cells, supplementing GT-561 culture solution (containing 200U/mL IL-2), 1% fetal calf serum and gentamicin sulfate (80U/mL), wherein the volume ratio of the supplemented culture solution to the original culture solution is 1:1, and continuing the culture;
(18) observing cells on the 12 th day of culture, wherein the cell state is good, and the culture solution has no obvious yellowing and is not treated;
(19) observing cells on the 13 th day of culture, wherein the cell suspension obviously turns yellow and the cells are more cloned, supplementing GT-561 culture solution (containing 200U/mL IL-2), 1% fetal calf serum and gentamicin sulfate (80U/mL), and continuing the culture, wherein the volume ratio of the supplemented culture solution to the original culture solution is 1: 1;
(20) observing cells on the 14 th day of culture, wherein the cell state is good, and the culture solution has no obvious yellowing and is not treated;
(21) and collecting cells on the 15 th day of culture, and sending the cells to a quality inspection department for bacterial endotoxin, mycoplasma, gram stain and cell surface antibody detection to obtain the NK cells.
As shown in FIGS. 6 and 7, the purity of NK with CD56 phenotype can reach 93.65%, and the purity of NK with CD16 phenotype can reach 81.76%.
In conclusion, the invention utilizes the engineered K562 cells expressing CD19, CD86, IL21, CD64 and CD137L to efficiently amplify and activate umbilical cord blood mononuclear cells, can efficiently prepare NK cells, has higher amplification quantity and cell activity, is low in usage amount of the engineered K562 cells, and can effectively reduce cost.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
SEQUENCE LISTING
<110> Hualong Biotechnology Ltd, Henan province
<120> NK cell and preparation method and application thereof
<130> 20210121
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 939
<212> DNA
<213> Artificial sequence
<400> 1
atgccacctc ctcgcctcct cttcttcctc ctcttcctca cccccatgga agtcaggccc 60
gaggaacctc tagtggtgaa ggtggaagag ggagataacg ctgtgctgca gtgcctcaag 120
gggacctcag atggccccac tcagcagctg acctggtctc gggagtcccc gcttaaaccc 180
ttcttaaaac tcagcctggg gctgccaggc ctgggaatcc acatgaggcc cctggccatc 240
tggcttttca tcttcaacgt ctctcaacag atggggggct tctacctgtg ccagccgggg 300
cccccctctg agaaggcctg gcagcctggc tggacagtca atgtggaggg cagcggggag 360
ctgttccggt ggaatgtttc ggacctaggt ggcctgggct gtggcctgaa gaacaggtcc 420
tcagagggcc ccagctcccc ttccgggaag ctcatgagcc ccaagctgta tgtgtgggcc 480
aaagaccgcc ctgagatctg ggagggagag cctccgtgtc tcccaccgag ggacagcctg 540
aaccagagcc tcagccagga cctcaccatg gcccctggct ccacactctg gctgtcctgt 600
ggggtacccc ctgactctgt gtccaggggc cccctctcct ggacccatgt gcaccccaag 660
gggcctaagt cattgctgag cctagagctg aaggacgatc gcccggccag agatatgtgg 720
gtaatggaga cgggtctgtt gttgccccgg gccacagctc aagacgctgg aaagtattat 780
tgtcaccgtg gcaacctgac catgtcattc cacctggaga tcactgctcg gccagtacta 840
tggcactggc tgctgaggac tggtggctgg aaggtctcag ctgtgacttt ggcttatctg 900
atcttctgcc tgtgttccct tgtgggcatt cttcatctt 939
<210> 2
<211> 972
<212> DNA
<213> Artificial sequence
<400> 2
atgggactga gtaacattct ctttgtgatg gccttcctgc tctctggtgc tgctcctctg 60
aagattcaag cttatttcaa tgagactgca gacctgccat gccaatttgc aaactctcaa 120
aaccaaagcc tgagtgagct agtagtattt tggcaggacc aggaaaactt ggttctgaat 180
gaggtatact taggcaaaga gaaatttgac agtgttcatt ccaagtatat gggccgcaca 240
agttttgatt cggacagttg gaccctgaga cttcacaatc ttcagatcaa ggacaagggc 300
ttgtatcaat gtatcatcca tcacaaaaag cccacaggaa tgattcgcat ccaccagatg 360
aattctgaac tgtcagtgct tgctaacttc agtcaacctg aaatagtacc aatttctaat 420
ataacagaaa atgtgtacat aaatttgacc tgctcatcta tacacggtta cccagaacct 480
aagaagatga gtgttttgct aagaaccaag aattcaacta tcgagtatga tggtattatg 540
cagaaatctc aagataatgt cacagaactg tacgacgttt ccatcagctt gtctgtttca 600
ttccctgatg ttacgagcaa tatgaccatc ttctgtattc tggaaactga caagacgcgg 660
cttttatctt cacctttctc tatagagctt gaggaccctc agcctccccc agaccacatt 720
ccttggatta cagctgtact tccaacagtt attatatgtg tgatggtttt ctgtctaatt 780
ctatggaaat ggaagaagaa gaagcggcct cgcaactctt ataaatgtgg aaccaacaca 840
atggagaggg aagagagtga acagaccaag aaaagagaaa aaatccatat acctgaaaga 900
tctgatgaag cccagcgtgt ttttaaaagt tcgaagacat cttcatgcga caaaagtgat 960
acatgttttt aa 972
<210> 3
<211> 564
<212> DNA
<213> Artificial sequence
<400> 3
atgagatcca gtcctggcaa catggagagg attgtcatct gtctgatggt catcttcttg 60
gggacactgg tccacaaatc aagctcccaa ggtcaagatc gccacatgat tagaatgcgt 120
caacttatag atattgttga tcagctgaaa aattatgtga atgacttggt ccctgaattt 180
ctgccagctc cagaagatgt agagacaaac tgtgagtggt cagctttttc ctgttttcag 240
aaggcccaac taaagtcagc aaatacagga aacaatgaaa ggataatcaa tgtatcaatt 300
aaaaagctga agaggaaacc accttccaca aatgcaggga gaagacagaa acacagacta 360
acatgccctt catgtgattc ttatgagaaa aaaccaccca aagaattcct agaaagattc 420
aaatcacttc tccaaaagat gattcatcag catctgtcct ctagaacaca cggaagtgaa 480
gattccggat cctacatctg ggcgcccttg gccgggactt gtggggtcct tctcctgtca 540
ctggttatca ccctttactg ctaa 564
<210> 4
<211> 1125
<212> DNA
<213> Artificial sequence
<400> 4
atgtggttct tgacaactct gctcctttgg gttccagttg atgggcaagt ggacaccaca 60
aaggcagtga tcactttgca gcctccatgg gtcagcgtgt tccaagagga aaccgtaacc 120
ttgcattgtg aggtgctcca tctgcctggg agcagctcta cacagtggtt tctcaatggc 180
acagccactc agacctcgac ccccagctac agaatcacct ctgccagtgt caatgacagt 240
ggtgaataca ggtgccagag aggtctctca gggcgaagtg accccataca gctggaaatc 300
cacagaggct ggctactact gcaggtctcc agcagagtct tcacggaagg agaacctctg 360
gccttgaggt gtcatgcgtg gaaggataag ctggtgtaca atgtgcttta ctatcgaaat 420
ggcaaagcct ttaagttttt ccactggaat tctaacctca ccattctgaa aaccaacata 480
agtcacaatg gcacctacca ttgctcaggc atgggaaagc atcgctacac atcagcagga 540
atatctgtca ctgtgaaaga gctatttcca gctccagtgc tgaatgcatc tgtgacatcc 600
ccactcctgg aggggaatct ggtcaccctg agctgtgaaa caaagttgct cttgcagagg 660
cctggtttgc agctttactt ctccttctac atgggcagca agaccctgcg aggcaggaac 720
acatcctctg aataccaaat actaactgct agaagagaag actctgggtt atactggtgc 780
gaggctgcca cagaggatgg aaatgtcctt aagcgcagcc ctgagttgga gcttcaagtg 840
cttggcctcc agttaccaac tcctgtctgg tttcatgtcc ttttctatct ggcagtggga 900
ataatgtttt tagtgaacac tgttctctgg gtgacaatac gtaaagaact gaaaagaaag 960
aaaaagtggg atttagaaat ctctttggat tctggtcatg agaagaaggt aatttccagc 1020
cttcaagaag acagacattt agaagaagag ctgaaatgtc aggaacaaaa agaagaacag 1080
ctgcaggaag gggtgcaccg gaaggagccc cagggggcca cgtag 1125
<210> 5
<211> 765
<212> DNA
<213> Artificial sequence
<400> 5
atggaatacg cctctgacgc ttcactggac cccgaagccc cgtggcctcc cgcgccccgc 60
gctcgcgcct gccgcgtact gccttgggcc ctggtcgcgg ggctgctgct gctgctgctg 120
ctcgctgccg cctgcgccgt cttcctcgcc tgcccctggg ccgtgtccgg ggctcgcgcc 180
tcgcccggct ccgcggccag cccgagactc cgcgagggtc ccgagctttc gcccgacgat 240
cccgccggcc tcttggacct gcggcagggc atgtttgcgc agctggtggc ccaaaatgtt 300
ctgctgatcg atgggcccct gagctggtac agtgacccag gcctggcagg cgtgtccctg 360
acggggggcc tgagctacaa agaggacacg aaggagctgg tggtggccaa ggctggagtc 420
tactatgtct tctttcaact agagctgcgg cgcgtggtgg ccggcgaggg ctcaggctcc 480
gtttcacttg cgctgcacct gcagccactg cgctctgctg ctggggccgc cgccctggct 540
ttgaccgtgg acctgccacc cgcctcctcc gaggctcgga actcggcctt cggtttccag 600
ggccgcttgc tgcacctgag tgccggccag cgcctgggcg tccatcttca cactgaggcc 660
agggcacgcc atgcctggca gcttacccag ggcgccacag tcttgggact cttccgggtg 720
acccccgaaa tcccagccgg actcccttca ccgaggtcgg aataa 765
Claims (10)
1. A preparation method of NK cells is characterized by comprising the step of inducing and culturing umbilical cord blood mononuclear cells by using engineered K562 cells to obtain the NK cells.
2. The method of claim 1, wherein the engineered K562 cells express the membrane proteins CD19, CD86, IL21, CD64, and CD 137L;
preferably, said IL-21 is expressed on the surface of said engineered K562 cells by fusion to the CD8 α transmembrane region.
3. The method of claim 1 or 2, wherein the genome of the engineered K562 cell has integrated therein a CD 19-encoding gene, a CD 86-encoding gene, an IL21-CD8 α -encoding gene, a CD 64-encoding gene, and a CD 137L-encoding gene.
4. The method according to claim 3, wherein the gene encoding CD19 comprises the nucleic acid sequence shown in SEQ ID NO. 1;
preferably, the gene encoding CD86 comprises the nucleic acid sequence shown in SEQ ID NO. 2;
preferably, the gene encoding IL21-CD8 alpha comprises the nucleic acid sequence shown in SEQ ID NO. 3;
preferably, the gene encoding CD64 comprises the nucleic acid sequence shown in SEQ ID NO. 4;
preferably, the gene encoding CD137L comprises the nucleic acid sequence shown in SEQ ID NO. 5.
5. The method according to any one of claims 1 to 4, wherein the method for producing NK cells comprises the step of inducing culture by intermittently using engineered K562 cells.
6. The preparation method of claim 5, wherein the step of discontinuously inducing the culture by using the engineered K562 cells comprises the following steps:
(1) adding the umbilical cord blood mononuclear cells and the engineered K562 cells into a culture solution for mixed culture, and adding fetal bovine serum and gentamycin sulfate;
(2) centrifuging and collecting cells on 2-6 days of culture, adding culture solution, fetal calf serum and gentamicin sulfate for resuspension of the cells, and continuing culture;
(3) adding the engineered K562 cells, a culture solution, fetal calf serum and gentamicin sulfate into the culture medium for continuous culture on 7-8 days;
(4) adding culture solution, fetal calf serum and gentamicin sulfate into the culture medium on day 8-14, and continuously culturing;
(5) and collecting cells on the 14 th-16 th day of culture to obtain the NK cells.
7. The method of claim 6, wherein the culture solution comprises a GT-T561 culture solution;
preferably, the mass percentage of the fetal calf serum in the culture solution is 1-10%;
preferably, the final concentration of the gentamicin sulfate is 80-100U/mL.
8. The method for producing NK cells according to any one of claims 1 to 7, wherein the method for producing NK cells comprises the steps of:
(1) separating umbilical cord blood mononuclear cells, adding the umbilical cord blood mononuclear cells and the engineered K562 cells into a culture solution for mixed culture, wherein the culture solution contains 5-10% of fetal calf serum and 80-100U/mL gentamicin sulfate;
(2) centrifuging and collecting cells on 2-3 days of culture, adding GT-T561 culture solution containing 5-10% fetal calf serum and 80-100U/mL gentamicin sulfate to resuspend the cells, and continuing to culture;
(3) adding GT-T561 culture solution containing 1-2% fetal calf serum and 80-100U/mL gentamicin sulfate into the culture solution for continuous culture on the 4-6 th day;
(4) adding the engineered K562 cells in 7-8 days of culture, and continuously culturing by adopting a GT-T561 culture solution containing 1-2% fetal calf serum and 80-100U/mL gentamicin sulfate;
(5) adding GT-T561 culture solution containing 1-2% fetal calf serum and 80-100U/mL gentamicin sulfate into the culture medium on 8-14 days of culture, and continuing to culture;
(6) and collecting cells on the 14 th-16 th day of culture to obtain the NK cells.
9. An NK cell produced by the production method according to any one of claims 1 to 8.
10. Use of the NK cell of claim 9 for the preparation of a medicament for cellular immunotherapy.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110112416.2A CN112725273A (en) | 2021-01-27 | 2021-01-27 | NK cell and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110112416.2A CN112725273A (en) | 2021-01-27 | 2021-01-27 | NK cell and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112725273A true CN112725273A (en) | 2021-04-30 |
Family
ID=75594140
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110112416.2A Pending CN112725273A (en) | 2021-01-27 | 2021-01-27 | NK cell and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112725273A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113755436A (en) * | 2021-07-12 | 2021-12-07 | 河南省遗传资源细胞库有限公司 | In-vitro NK cell amplification method and NK cell |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586185A (en) * | 2012-03-12 | 2012-07-18 | 浙江中赢方舟生物工程股份有限公司 | Method for amplifying and activating NK (Natural Killer) cells by K562 cells |
| CN105524880A (en) * | 2016-01-27 | 2016-04-27 | 上海润泉生物技术有限公司 | Construction method of immune cell bank |
| CN106456733A (en) * | 2014-06-18 | 2017-02-22 | 阿尔伯特·爱因斯坦医学院股份有限公司 | Syntac polypeptides and uses thereof |
| CN106754730A (en) * | 2017-02-22 | 2017-05-31 | 上海科医联创生物科技有限公司 | A kind of method of efficient amplification NK cells |
| CN108034677A (en) * | 2017-12-28 | 2018-05-15 | 河南省华隆生物技术有限公司 | A kind of recombinant vector and its preparation method and application |
| CN108610420A (en) * | 2016-12-13 | 2018-10-02 | 科济生物医药（上海）有限公司 | The humanized antibody of anti-CD19 and the immune effector cell for targeting CD19 |
| CN110680920A (en) * | 2011-09-30 | 2020-01-14 | 中外制药株式会社 | Antigen binding molecules that induce an immune response against a target antigen |
| CN111073856A (en) * | 2019-12-28 | 2020-04-28 | 郑州大学第一附属医院 | Trophoblast, preparation method thereof and application thereof in NK cell amplification |
| CN112063588A (en) * | 2020-08-13 | 2020-12-11 | 南京北恒生物科技有限公司 | Engineered immune cells and uses thereof |
-
2021
- 2021-01-27 CN CN202110112416.2A patent/CN112725273A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110680920A (en) * | 2011-09-30 | 2020-01-14 | 中外制药株式会社 | Antigen binding molecules that induce an immune response against a target antigen |
| CN102586185A (en) * | 2012-03-12 | 2012-07-18 | 浙江中赢方舟生物工程股份有限公司 | Method for amplifying and activating NK (Natural Killer) cells by K562 cells |
| CN106456733A (en) * | 2014-06-18 | 2017-02-22 | 阿尔伯特·爱因斯坦医学院股份有限公司 | Syntac polypeptides and uses thereof |
| CN105524880A (en) * | 2016-01-27 | 2016-04-27 | 上海润泉生物技术有限公司 | Construction method of immune cell bank |
| CN108610420A (en) * | 2016-12-13 | 2018-10-02 | 科济生物医药（上海）有限公司 | The humanized antibody of anti-CD19 and the immune effector cell for targeting CD19 |
| CN106754730A (en) * | 2017-02-22 | 2017-05-31 | 上海科医联创生物科技有限公司 | A kind of method of efficient amplification NK cells |
| CN108034677A (en) * | 2017-12-28 | 2018-05-15 | 河南省华隆生物技术有限公司 | A kind of recombinant vector and its preparation method and application |
| CN111073856A (en) * | 2019-12-28 | 2020-04-28 | 郑州大学第一附属医院 | Trophoblast, preparation method thereof and application thereof in NK cell amplification |
| CN112063588A (en) * | 2020-08-13 | 2020-12-11 | 南京北恒生物科技有限公司 | Engineered immune cells and uses thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113755436A (en) * | 2021-07-12 | 2021-12-07 | 河南省遗传资源细胞库有限公司 | In-vitro NK cell amplification method and NK cell |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP2002535981A (en) | Methods and devices for maintaining and expanding hematopoietic stem cells and / or progenitor cells | |
| JP2021522860A (en) | Method for Producing γδ T Cells Modified with Chimeric Antigen Receptor | |
| CN110172479B (en) | Plasmid capable of simultaneously expressing LMP1 and CD30 double-target CAR, CAR-T cell, construction method and application thereof | |
| CN112251406B (en) | Exosome sorting method in NK cell activation stage | |
| CN112725284A (en) | NK trophoblast cell and application thereof | |
| CN112626027A (en) | Treg cell culture method | |
| CN113293132A (en) | NK cell in-vitro amplification system and culture method | |
| CN112725273A (en) | NK cell and preparation method and application thereof | |
| JP2023522642A (en) | Process for producing genetically modified autologous T cells | |
| CN115109801B (en) | Slow virus transfection transfer aid and application thereof | |
| CN115094034B (en) | Human NKT cell line and application thereof | |
| CN113122579B (en) | Method for transfecting immune cells by lentivirus | |
| CN113913386B (en) | Trophoblast cell and application thereof in amplifying human NK cells | |
| CN112048474B (en) | Method for enhancing hematopoietic stem cell transplantation capability | |
| CN112626028B (en) | Engineering cell for activating NK-like cells and preparation method and application thereof | |
| CN111690607B (en) | Efficient killer cell in-vitro culture kit and culture method | |
| CN113832190A (en) | Preparation method and application of trophoblast modified by genetic engineering | |
| CN113957046A (en) | Culture medium for T cell lentivirus infection and application | |
| CN112424342A (en) | Compositions and methods for culturing and expanding cells | |
| CN112391349A (en) | Trophoblast cell strain, preparation method thereof and method for in vitro induced amplification of NK cells | |
| CN112608901B (en) | Artificial antigen presenting cell, preparation method and application thereof | |
| CN109385437B (en) | DNA molecule, vector containing the same and immortalized cell obtained | |
| CN115947869B (en) | Chimeric antigen receptor targeting human cytomegalovirus, CAR-NK cell and application | |
| CN113249331B (en) | DC cell and CTL cell loaded with Tax antigen as well as preparation methods and applications of DC cell and CTL cell | |
| CN116179483B (en) | Method for rapidly amplifying stem cell-like memory cervical cancer tumor infiltrating lymphocytes in vitro |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |