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
