transduction protocol for human CB CD34+ cells

Protocol – preparation of retronectin-coated plates

• thaw retronectin from -20, dilute in PBS to 25-100 g/ml, typical 50 g/ml and pipet 1.5 ml per well of a non-tissue culture 6-well plate
• incubate at RT for 2 hrs
• remove retronectin from 6-well plate, keep at -20 for possible other plate coatings
• block by adding 2 ml PBS/BSA (fridge) onto 6-well plate for 30 min.
• wash plate twice with PBS
• use immediately or seal plate with parafilm and keep at 4°C

Protocol - lentitransduction of CB CD34+ cells

• Isolate CB CD34 cells with MACS column and incubate in HPGM (0.5 million /mL) with KFT (each 100ng/mL) for 18 hours (= ON), ON 37ºC, 5% CO2
• Coat 1 well per group of a 12-wells plate with retronectin.
• Add 500-1000 ul of filtered viral supernatant (fresh or from -80) to a coated well and add: KFT (100 ng/mL), polybrene (2-4 µg/mL)
• Add CB CD34 positive cells (0.2-0.5 x 10⁶ cells per group) and incubate ON 37ºC, 5% CO2
• optional: repeat transduction round 8-12 hrs later (round 2, or even round 3)
• Obtain and wash CB CD34 cells and perform experiments (for sorting/NOD-SCID mice: wash at least three times!)

Protocol – retroviral transduction of CB CD34+ cells

• obtain fresh or cryopreserved CB and positively select CD34+ cells by Miltenyi CD34+ MACS column
• culture cells in sera free media (QBSF, HPGM or X-Vivo 10) with the following recombinant growth factors:
  • Flt-3L (100 ng/ml), SCF (100 ng/ml), Tpo (100 ng/ml), culture CD34+ cells in about 0.5 x 10⁶ cells/ml
• -allow cycling for 48 hrs
• 8-12 hours prior to first transduction round, place 5 ml QBSF, HPGM or X-Vivo 10 on PG13 producer cells grown to 70% confluency in T75 for virus collection (plate I)
• after 8-12 hours of first virus collection:
  • count CD34+ cells, use 0.1-0.5 x 10⁶ cells per group
  • spin cells down (5 min, 1400 rpm)
  • collect 5 ml virus-containing medium from PG13 flask, add polybrene (4 g/ml) and cytokines (KFT), filter and place in fibronectin coated 6-well/12-well plates
  • resuspend CD34+ cells in virus-containing medium and plate in 6-well plate
  • place normal ‘happy’ medium (DMEM 10%) on PG13 producers plate I and QBSF, HPGM or X-Vivo 10 on plate II for second virus harvest 8-12 hours later.
• second transduction round: collect CD34+ cells from 6-well plates, spin down, and resuspend in second virus harvest as for round one.
• repeat for third transduction round

Example

• isolate CD34+ cells from CB on monday, prestimulate on Monday 17.00 hr.
• prepare twp PG13 T75 flasks on monday, plate one should reach 70% confluency on tuesday 17.00 hr, plate should reach 90% confluency on Wednesday 9.00 hr.
• prepare retronectin-coated 6-well plates as needed.
• first PG13 medium change: tuesday 17.00 hr
• first transduction round: wednesday 9.00, change PG13 flask I to happy medium, change PG13 flask II to HPGM
• second transduction round: wednesday 17.00 hrs, change PG13 flask II to happy medium, change PG13 flask I to HPGM
• third transduction round: thursday 9.00, discard PG13 flask I, keep PG13 flask II for further passage
• friday: determine efficiencies by FACS (GFP, DsRED) and start experiments