Lentiviral Vectors for Gene Delivery to the Nervous System - Gene Delivery and Therapy for Neurological Disorders - page 43

Seed 293T
cells

Start Transfection
of 293T cells

Induce
virus

Observe
transfected cells
and harvest
lentiviral vectors

Concentrate
lentiviral vectors
and resuspend
viral pellet

Aliquot virus
and
store at -80C

Day 1 Day 2 Day 3 Day 4 Day 5 Day 6

Seed cells
for titration

Transduce cells
with virus and
incubate for 72 hrs

Carry out
p24 ELISA
and qPCR

Analyze transduction
efficiency by FACS
and calculate
functional titer

Day 1 Day 2 Day 3 Day 4 Day 5

Fig. 2 Timeline of lentiviral vector production and titration

3.2.1 Propagation,
Passaging, and
Preservation of Packaging
Cell Line HEK 293T

Successful lentiviral vector production resulting in high titers is
tightly connected to the state of HEK 293T cells used. It is critical
to use healthy cells at low passage numbers. Thus, a large stock of
HEK 293T cells at a lower passage number should be kept for
future production.

1. Rapidly transfer the cryovial containing the HEK 293T from
the liquid nitrogen storage into a 37 °C water bath.
2. Thaw the cells by gentle agitation until most, but not all, of
the contents is thawed.
3. Remove the vial from the water bath and let the remaining
cells thaw.
4. Rinse the vial with 75 % ethanol before transferring it into the
tissue culture hood.
5. Transfer the thawed cell suspension into a T175 fl ask contain-
ing pre-warmed complete DMEM medium.
6. Transfer the fl ask into the incubator and incubate for 4 h until
the majority of cells attach to the bottom of the fl ask.
7. Four hours later replace the medium with pre-warmed fresh
medium to the cells.
8. Next day examine the cells and check for cell death. Replace
the medium with fresh one.
9. Passage the cells when they are 75-80 % confl uent.
10. Change the medium every 2 days.

Thawing the HEK
293T Cells