Induced T-to-natural killer feeder cell as well as preparation method and application thereof
A feeder cell, NK cell technology, applied in the biological field, to achieve the effect of strong tumor killing ability and stable killing function
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Embodiment 1
[0046] Example 1 Construction of reprogrammed NK cells
[0047] (1) Design sgRNA (SEQ ID NO:6:gaccatgaactgctcacttg) according to the Bcl11b gene, and add restriction enzyme sites EcoRI and SalI at the 5' end and 3' end of the sequence after artificial synthesis;
[0048] The synthesized sequence fragment was digested with EcoRI and SalI, then connected to the PX458-gBCL11b vector containing the T7 promoter, and the recombinant plasmid was successfully constructed by sequencing.
[0049] (2) will 1×10 7 T cells were resuspended in 3 mL of T cell medium, seeded in one well of a 6-well plate, and activated by adding a combination of 100 ng / mL anti-human CD3 antibody and 100 ng / mL anti-human CD28 antibody;
[0050] After 3 days, remove the suspended cells for counting, centrifuge at 300×g for 10 min, resuspend the cell pellet in 10 mL of Opti-MEM, centrifuge at 300×g for 10 min, resuspend the cell pellet in 100 μL of Opti-MEM, add CRISPR at a concentration of 40 μM / Cas9 plasmid...
Embodiment 2
[0052] Example 2 Construction of reprogrammed NK feeder cells
[0053] (1) Artificially synthesized T2A-linked nucleic acid molecules containing genes encoding IL-12 and CD8α transmembrane regions, CD19, CD64 and CD86, and added EcoRI and BamHI restriction sites and their protective bases at both ends;
[0054] Use restriction endonucleases EcoRI and BamHI to perform double digestion on nucleic acid molecules, incubate in a water bath at 37°C for 30 minutes, and use 1.5% agarose gel electrophoresis to recover the digested products containing sticky ends;
[0055] The digested product was ligated into the linearized pLVX-EF1-MCS plasmid (containing cohesive ends) digested with EcoRI and BamHI, and the ligation system was shown in Table 1 to obtain a lentiviral vector.
[0056] Table 1
[0057] components Dosage (μL) pLVX-EF1-MCS plasmid 2(50ng) IL-12-CD19-CD64-CD86 nucleic acid molecule 10(150ng) T4 DNA Ligation Buffer 2 T4 DNA Ligase (NEB)...
Embodiment 3
[0061] Example 3 In vitro expansion of reprogrammed NK cells
[0062] In this example, the reprogrammed NK cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, and the cells were counted after 24 hours of culture, and the cell concentration was adjusted to 2×10 6 cells / mL, add an equal proportion of reprogrammed NK feeder cells to the culture medium on day 0 and day 7, and add IL-2 at a concentration of 50 U / mL, at 37°C, 5% CO 2 Co-cultivation in the incubator under 500Gy irradiation conditions.
[0063] Such as figure 1 As shown, after the reprogrammed NK cells were co-cultured with IL-12-CD19-CD64-CD86 reprogrammed NK feeder cells, the number of cells increased significantly. After 2 weeks of culture, the number of reprogrammed NK cells increased by about 120 times, which was significantly higher than that of the control Group.
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