2. Revive and culture HepG2 cells in in complete DMEM, as per
the manufacturer’s instructions.
3.1.2
Preparation of
Alvetex® Strata
The Alvetex® Strata must be treated with ethanol before use, to
render the scaffold hydrophilic.
(a)
Remove the Alvetex® Strata inserts from the packaging using
sterile forceps and place into a sterile petri dish or beaker
containing 70% ethanol.
(b)
Submerge the Alvetex® Strata inserts in the 70% ethanol for a
minimum of 15 min.
(c)
Move the Alvetex® insert into a fresh 6-well plate and add
10 mL of sterile PBS to remove all ethanol.
(d)
Aspirate the sterile PBS and add 5 mL of complete DMEM,
bringing the liquid to the level of the Alvetex® Strata
membrane.
3.1.3
Seeding HepG2
Cells onto the Alvetex®
Strata
1. Trypsinize HepG2 cells using trypsin–EDTA until cell have
detached.
2. Neutralize the trypsin–EDTA with complete DMEM.
3. Centrifuge the cells at 1000 g for 3 min.
4. Perform cell counts using a trypan blue exclusion assay.
5. Seed 2 106 HepG2 cells onto the prepared Alvetex® Strata
inserts.
6. Incubate at 37 C in a 5% CO2 humidified incubator for 7 days
with a complete medium change every 2 days.
3.1.4
Preparation of the
Perfusion Bioreactors
Perfusion bioreactors need to be autoclaved prior to use to main-
tain sterility.
(a)
Take a complete bioreactor system and place an autoclavable
lid to cover the top, or alternatively wrap aluminum foil over
the top of the vessel.
Fig. 8 Protocol for setting up HepG2 bioreactor cultures in in static and perfusion. HepG2 cells are initially
seeded into Alvetex inserts in a 6-well plate and cultured for a week to allow the cells to adhere and migrate
across the membrane. The samples are then moved into bioreactors and placed on the stirrer unit with the
perfused samples on a lane set to 100 rpm and the static samples placed on a lane which is turned off. The
samples are then cultured for a further 7 days before fixation
250
Henry W. Hoyle et al.