Spinner-flask culture
The protocol used for spinner flask culture was the one followed in
Santos et al 2011. Suspension culture experiments were performed in 100
mL flat bottom spinner-flasks (Bellco) with 80 mL working volume.
Spinner-flasks were siliconized using Sigmacote (Sigma, Germany) to
avoid cell attachment. After applying the reagent to all the glass
surface and aspirating it, vessels were let to dry for 24 hours in a
fume hood. After, vessels were rinsed three times with distilled water
(Gibco, UK). Plastic and Plastic Plus microcarriers were used for
spinner-flask culture with a total surface area of 518.4
cm2. Microcarriers were prepared by autoclaving in
distilled water (Gibco, UK) for 121 ºC for 15 min. After, the distilled
water was aspirated as much as possible and complete medium was added.
Microcarriers were let to equilibrate for 1 hour in a standard
incubator. 60-80% confluent PA5 hOMCs were detached as described in
section “hOMC cell culture” and cells were added to the tube
containing the microcarriers suspension in complete medium with a
seeding density of 6000 cells/cm2. After transferring
the microcarrier and cell suspension to spinner-flasks containing
adequate amounts of complete medium, an agitation of 40 rpm was started.
Spinner-flasks were maintained on a Bell-EnniumTMCompact 5 position magnetic stirrer platform (BellCo) in a standard
incubator. In order to allow for air exchange, a side-arm of the
spinner-flask was kept loosened (half a turn of the cap). Cell expansion
was maintained for 7 days. Feeding was performed every two days by
removing 50% of the expanded medium and adding the same amount of fresh
complete medium. Sampling, also performed every two days, was achieved
by retrieving 2 mL of a homogeneous microcarriers suspension for manual
cell counting and immunocytochemistry. Cell counting and viability
measurements were performed through the trypan blue method, using a
haemocytometer.