Hyperflask cell culture vessels

For production of recombinant human CD19 peptide, the extracellular domain (amino acids 20-291, Uniprot P15391) was fused to human IgG1 Fc (CD19-Fc) and expressed by transduction in HEK293 cells in a CMV-driven mammalian expression vector. Transfected cells were cultured in DMEM, 5% FBS, and cell culture supernatant containing CD19-Fc harvested after 10 and 20 days of incubation in HYPERFlask® cell culture vessels (Corning). After centrifugation to remove cell debris and 0.22 μm sterile filtration, CD19-Fc was purified by protein A chromatography (HiTrap MabSelect, GE Healthcare) and stored in PBS at 4 °C. Purity was > 97% as determined by SDS-PAGE and Coomassie Blue staining. Identity of CD19-Fc was confirmed by intact mass spectrometry and peptide mass fingerprint analyses after trypsin digestion(Miltenyi Biotec, Bergisch Gladbach, Germany).

All cell lines were cultured at 37°C and 5% CO₂. The HEK293T cell line was maintained in DMEM + GlutaMAX-I (Gibco), supplemented with 10% (v/v) fetal bovine serum (FBS) (Sigma) and 1× antibiotic-antimycotic (AntiAnti, Gibco). Lentivirus-producing cell line WinPac-RD-HR¹² (link) was maintained in DMEM + GlutaMAX-I supplemented with 10% (v/v), 1× AntiAnti, and antibiotics (1 μg/mL puromycin, 100 μg/mL hygromycin, 30 μg/mL phleomycin, and 10 μg/mL blasticidin). WinPac-RD-HR cells were expanded in T175 flasks under antibiotic selection and seeded in 10-layer HYPERFlask Cell Culture Vessels (Corning) to produce higher LV quantities necessary to carry out further experiments.

Immortalization of PTECs was performed using lentiviral vectors encoding for the SV40 large T-ag and mCherry sequence as a reporter gene. Additionally, lentiviral vectors carrying the sequences for shRNAs targeting β2-microglobulin (β2m) and the class II transactivator (CIITA), as well as sequences for a control vector encoding for non-sense shRNA (shNS) were produced as previously described [16 (link)]. Briefly, HEK293T cells were cultured in HYPERFlask Cell Culture Vessels (Corning, New York, NY, USA) in Dulbecco’s modified Eagle´s medium supplemented with 10% FCS (Sigma-Aldrich), 2% penicillin–streptomycin (C.C.Pro), and 1% glutamine (PAN-Biotech GmbH, Aidenbach, Germany). These conditions were used for the production of all three vectors. HEK293T cells were cotransfected with the lentiviral packaging plasmid psPAX2, the VSV-G envelope plasmid pMD2.G, and the shRNA-encoding lentiviral plasmid using 1 mg/mL polyethyleneimine (Polysciences, Warrington, PA, USA). After 48 h of incubation, the cell supernatant was collected and centrifuged for 3 h at 20,000× g at 16 °C. Lentiviral vector pellets were resuspended in Williams’ Media E (Thermo Fisher Scientific, Waltham, MA, USA) and stored at −80 °C until needed. For the titration, a p24 enzyme-linked immunosorbent assay (Cell Biolabs, San Diego, CA, USA) was performed.