Cell culture medium preparation
• Gelatine solution: stock solution: 2% gelatine in H2O
working solution: 0,1% gelatine in H2O
• MEF-Medium (MEFM): 10% foetal bovine serum (FBS), 2mM L-glutamine, 1% penicillin/streptomycin in Dulbecco’s modified Eagle’s medium (DMEM)
• Freezing Medium: 50% MEFM, 40% FBS, 10% DMSO
• FACS Buffer: FBS (3%), EDTA (5mM), Penicillin (40units/ml), Streptomycin (40µg/ml), Glutamine (800µM) in PBS
• Collection medium: 20% FBS, Penicillin (100units/ml), Streptomycin (0,1mg/ml), L-Glutamine (2mM) in DMEM
Extraction of mouse embryonic fibroblasts
• Sacrifice a pregnant mouse by cervical dislocation 12.5 days after positive plug check.
• Disinfect the mouse with 70% ethanol and open the peritoneum.
• Dissect the uterus containing the embryos.
• Isolate the embryos in ice-cold PBS.
• Isolate the limbs of the embryos to obtain murine embryonic fibroblasts (avoid contamination with renal tissue). Collect limb tissue in a well of a 24 well plate (filled with 1ml cold PBS). Besides, collect some tissue for genotyping, e.g. tail of the embryo.
• Mince tissue with a scalpel. Then, transfer the embryonic tissue into a 15ml tube containing 8ml 0.25% Trypsin-EDTA and digest the tissue for 30min at 37°C, vortexing the tube every 10min. Centrifuge the tube with the minced tissue for 5min at 1200rpm
• Coat 10cm dishes with 0.1% gelatine solution for at least 10 min and remove excess gelatine solution directly before plating the cells.
• Remove supernatant and resuspend the cell pellet in 5ml MEFM to stop trypsinisation. Seed onto a 10cm dish and culture in a 37°C 5% CO2 incubator. After two days change medium. When cells are firmly attached, change medium every day carefully without detaching loosely attached cells. When cells are firmly attached, change the medium daily.
• Proceed to next step when cells are 80% confluent, approx. after 2 to 4 days.
Note: For optimal efficiency do not freeze or split MEFs. Infect at P0.
• Aspirate the medium and wash cells with 8ml PBS. Add 2,5ml trypsin und put cells back into the incubator for 2-5 minutes.
• Gently tap the plate after 2-5 minutes so that the cells dissociate from the plate. Add 8ml cold MEFM. Resuspend and gently rinse the plate with the mixture. Then transfer the suspension into a 15ml tube. Centrifuge the cells for 5min at 1200rpm
• Remove supernatant and resuspend the pellet in 1ml freezing medium. Transfer the suspension into a Cryovial and store the vial first on -80°C for 3 days, then transfer the Cryovial into liquid nitrogen for long term storage.
In the morning:
• Coat four 6 well dishes with 0,1% gelatine solution.
• Thaw frozen vial of MEFs at 37°C. As soon as freezing solution is free of ice crystals add 1ml pre-warmed MEFM and transfer MEF solution into 15ml tube already containing 5ml of pre-warmed MEFM. Rinse vial again with MEFM, then centrifuge cells for 5 minutes at 1200rpm
• Remove supernatant and resuspend the pellet in MEFM. Seed one vial (or 1.2x106 cells) onto four 6-well plates (3x105 cells/ 9cm2 in each well) and culture in a 37°C 5% CO2 incubator for 7-8 hours.
Lentiviral Transduction of MEFs
In the evening:
Note: For optimal transduction efficiency do not freeze/thaw virus. Each freeze/thaw cycle will decrease virus titre by 20%.
• For generation of induced renal epithelial cells 4 different lentiviruses should be generated, carrying each of the four transcription factors (i.e. Emx2, Hnf1b, Hnf4a and Pax8). To create lentivirus pWPXLd (backbone), psPax2 (packaging) and pMD2.G (envelope) can be used, all available from Didier Trono via Addgene (#12258; #12260; #12259).
• Produce and concentrate virus (~100X) using polyethylene glycol precipitation as described before1. We produce virus by transfecting 12 15cm cell culture plates (293T cells), harvesting 204ml of virus-containing supernatant and concentrating supernatant to 2.4ml before freezing. Virus concentrate can be frozen at -80°C.
• Thaw virus directly before transduction of cells.
• Mix equal amounts of the 4 viruses.
• Dilute the mixture of viruses 1:100 to 1:1000 in MEFM containing 10µg/ml Polybrene.
• Aspirate cell culture medium covering MEFs, which should be at 20% confluency.
• Add 750µl virus containing MEFM per well of a 6-well plate. Incubate infected MEFs in a 37°C 5% CO2 incubator for 12h.
• After 12h (next morning) remove virus solution. Wash cells once with MEFM and add 2ml fresh MEFM per well.
• Repeat infection cycles on five to seven consecutive days.
Isolation of iRECs
• Cultivate transduced MEFs in MEFM for 14 days without splitting.
• Change medium every second day.
• Wash cells with PBS, add 0,25% Trypsin-EDTA and incubate for 2min at 37°C until the cells detach.
• Resuspend cells in FACS Buffer. Centrifuge for 5min at 1200rpm.
• Remove the supernatant.
• Resuspend in 1ml FACS Buffer or PBS and filter suspension through a 50µm cell strainer in FACS buffer. Use sterile cell strainers or autoclave prior to use if continued culture of sorted cells is planned.
• Isolate GFP+ cells using a sorter detecting green and red fluorescence (e.g. BD FACS AriaFusion) into collection medium.
• Seed sorted cells on gelatin coated 12-well plates.
• Change medium every day and split cells onto a 6-well plate after they reach confluency.