The protocol involves cultures initiated at 2.5 × 105 cells/mL in 25 mL in 1 L Vuelife culture bags, with addition of fresh media plus IL2 every 3 days to maintain an optimal density of NK-92 cells for expansion. Daily disruption of cell aggregates enhances NK-92 cells ...
GPC3 CAR-expressing lentivirus was prepared using baboon endogenous retroviral envelope (BaEV) according to our previously described protocol [15, 16]. A human NK cell line (NK92) was transduced with lentiviruses expressing CAR and EGFP, and then CAR-EGFP-expressing NK92 (CAR-GFP NK92) cells ...
NK-92mi does not require exogenous IL-2 administration35. Cell lines were periodically tested to exclude mycoplasma contamination. Human primary sarcoma samples obtained through the human subjects research-approved CW IRB protocol #1347982, as detailed above, were cultured in Dulbecco’s Modified ...
Transfection of Expi293F cells was conducted using the Expi293™ Expression System Kit (A146315; Thermo Fisher Scientific) following the manufacturer’s protocol. The protein was purified from the cell culture supernatant using recombinant Protein A affinity chromatography (HiTram MabSelect SuRe, ...
This study addresses manufacturing-related issues and aimed to develop a GMP-compliant protocol for the generation of NK-92/5.28.z therapeutic doses starting from a well-characterized GMP-compliant master cell bank. Commercially available GMP-grade culture media and supplements (fresh frozen plasma, ...
The cells (K562, A549, A2058, and NK92 cells) were treated with CF (0, 0.1, 1, and 10 μM) for 48 h and measured using Cell Counting Kit-8 assay kit (Beyotime Institute of Biotechnology) following the manufacturer’s general protocol. The absorbance at a wavelength of 450 nm was ...
The human NK cell line NK92 (American Type Culture Collection) was maintained in Minimum Essential Medium-α (MEM-α) (Gibco) containing 20% FBS, 1% penicillin/streptomycin, 1% vitamin solution (Gibco), 0.1 mM β-mercaptoethanol (Gibco) and 200 U/ml rIL-2. REH [Acute Lymphocytic Leukemia...
Cell culture K562 and HepG2 cells were cultured in RPMI-1640 medium (GIBCO/BRL, Grand Island, NY, USA), supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin and 10% fetal bovine serum. The human NK cell line NK-92 was cultured as previously described.15 The HLCZ01 cell line...
To evaluate the stability of these agonists, they were added into the culture medium of the human pancreatic cancer cell line AsPC-1, and the supernatant was collected 5 days later and used to treat NK-92 (Fig. 1h, top). The culture media containing the agonist could all induce phosphoryla...
Furthermore, we confirm that dual-CAR NK-92 cells maintain their functionality after gamma-irradiation, which supports their off-the- shelf clinical applicability. Methods Primary cells, cell lines and culture conditions Human Burkitt's lymphoma cell lines Daudi and Namalwa were purchased from the...