(b)
Submerge the Alvetex® Scaffold inserts in 70% ethanol for a
minimum of 5 min.
(c)
Move the Alvetex® insert into a fresh 6-well plate and add
sterile PBS to remove all ethanol.
(d)
Aspirate the sterile PBS and add 5 mL of complete DMEM
supplemented with 5 ng/mL TGF-β1 and 100 μg/mL ascor-
bic acid, bringing the liquid to the level of the Alvetex®
membrane.
3.2.3
Seeding HDFn Cells
onto the Alvetex® Scaffold
1. Trypsinize HDFn cells at 80% confluency using 0.25% Trypsin–
EDTA until cells have detached.
2. Neutralize the trypsin–EDTA with complete DMEM.
3. Centrifuge the cells at 200 g for 3 min.
4. Perform cell counts using a trypan blue exclusion assay.
5. Seed 5 105 HDFn cells onto the prepared Alvetex® Scaffold
inserts.
6. Incubate at 37 C in a 5% CO2 humidified incubator in com-
plete DMEM supplemented with 5 ng/mL TGF-β1 and
100 μg/mL ascorbic acid. Culture these for 14 days with a
complete medium change twice weekly.
3.2.4
Seeding Caco2
Cells onto the Stromal
Compartments
1. Trypsinize Caco-2 cells at 70–90% confluency using 0.25%
trypsin–EDTA until cells have detached.
2. Neutralize the trypsin–EDTA with complete DMEM.
3. Centrifuge the cells at 200 g for 3 min.
4. Perform cell counts using a trypan blue exclusion assay.
5. Seed 0.4 106 Caco-2 cells onto the Alvetex® Scaffold inserts
previously cultured with HDFn for 14 days.
6. Incubate at 37 C in a 5% CO2 humidified incubator for
21 days with a complete medium change every 2 days.
3.2.5
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 the lid in the
vented orientation.
(b)
Autoclave the bioreactor for 20 min at 121 C using a stan-
dard clean cycle.
(c)
Immediately upon finish, reverse the lid of the bioreactor to
the sealed orientation and store it until use.
3.2.6
Moving Intestinal
Models into the Perfusion
System
1. Take an
autoclaved
bioreactor and
place
in
a laminar
flood hood.
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