CEll bIOlOGy

Cell biology
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environment. This means that following each
culture manoeuvre, for example, scheduled
sampling, or re-seeding of cell cultures in
fresh media, cells are exposed to undesirably
high (initially atmospheric) concentrations of
dissolved O 2
for a significant period, which may
introduce undesirable experimental alterations
and important physiological effects.
In order to overcome these problems, sealed
glove boxes for manipulation without exposure
to external atmospheric conditions are used 16 .
Cultures in “Normoxia”
Figure 1.- The Petaka auto-controlled gas
diffusion system (GDS), when exposed to
regular atmosphere with 21% O2, automatically
produces a “Progressive adaptive depletion of
O2” dropping the functional partial pressure of
dissolved oxygen from 140 mm Hg to 40 mm Hg,
which is equivalent to the tissue “normoxia”
reduction is required of the environmental O 2
concentration in the incubator to about 5%
(media O 2
partial pressure, ppO 2
= 38 mm Hg).
Today there are many cell culture incubators
which provide control of O 2
levels. However,
those instruments are cumbersome, costly
in maintenance, and limited in space and
use. Moreover, they have several important
functional setbacks: it takes approximately
30 minutes for an oxygen controlled incubator
to displace sufficient air to create a 5% O 2
environment every time it is opened. Within a
common 50 l incubator it takes a minimum of
180 minutes to create a 5% O 2
headspace in
T25 flasks and 420 minutes in T150 flasks 15 . The
deficiencies of these incubators are exacerbated
by a further issues: The rate of O 2
diffusion
into the cell culture media. During culture, cells
adhere to the bottom of the flask, 2 to 4 mm
below the liquid/gas interface between the
media and gas space above, which acts
effectively as a semi-permeable membrane.
The time required for excess of O 2
to diffuse
out of cell culture media at 37 OC. is minimally
80 minutes when cultured cells reach confluent
monolayer and maximally 180 minutes at low
cell densities [11]. It can therefore take more
than three hours for adjusting the desired O 2
concentration at the cellular peripheral micro
Petaka is a unique cell culture device with
auto-controlled in/out gas diffusion system
(GDS) 17 , which exposed to regular atmosphere
with 21 % O 2
automatically produces a
“Progressive adaptive normoxia” dropping the
functional partial pressure of dissolved oxygen
from 140 mm Hg to 40 to 53 mm Hg (Figure 1),
adjusted to the physiological environment of the
majority of mammalian cells (Table 1), in parallel
to the cell growth. DO concentration remains
stable, within an insignificant fluctuation
responding to the feedback between cell
doubling time and oxygen availability. When
cells grow over a certain threshold, the
controlled oxygen supply through the Petaka
GDS, holds the level of DO 2
, slowing the
doubling time and the average O 2
consumption
by the cell population, and vice versa. (Figure 1)
Initial oxygen concentration of the average
bottled media is about 6.8 ppm based on
regular sea level atmospheric pressure (760 mm
Hg) and standard culture temperature (310 ºK).
Beginning the cell growth the dissolved O 2
partial pressure (ppDO 2
) is dictated by the
normal media exposed to the atmospheric air.
Depending on the cell type (CHO cells as an
example), after a short period of growth, when
cell number exceeds 6 to 8 million, the rate of
oxygen consumption is balanced with the rate
of oxygen diffusing through Petaka GDS, and
the culture media ppDO 2
is maintained at the
tissue physiological limits, between 40 and
60 mm Hg (Figure 1). That auto-control normoxia
level remains balanced from the sea level up to
10 000 ft altitude (3000 m, 537 mm Hg).
The Petaka GDS special feature allows
culturing cells in physiological oxygen
concentration inside any regular incubator, with
regular open air environment (with or without
CO 2
enrichment), and allow manipulating the
cultures, with absolute freedom, without risk
VWR International I VWRbioMarke Issue 27 I September 2011 I
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