Cell culture inside chamberswith limited oxygen transfer
Concepts and data
Petaka G3

GEN Nov 1, 2012 (Vol. 32, No. 19)

Ducted Respiratory Chamber Bioreactors
Cell Culture in Naturally Produced State Mimics
True Physiologic Conditions••

Emilio Barbera-Guillem, M.D., Ph.D.1,Michael J. Gallagher, M.D.

Developments in stem cell culture, regenerative medicine, and drug
discovery are creating demand for cell culture under true physiologic
conditions mimicking the atmosphere within living beings.
While former goals were just to keep cells alive and genetically stable,
avoid contamination, and grow cells quickly in large numbers,
investigators now know the subtle effects of unnatural cellularenvironments.
Atmospheric oxygen concentrations of in vitro cultures are far higher
than that experienced by in vivo cells, and this difference has far reaching consequences. Room air oxygen levels in cultures increaseoxidation, mitochondrial activity, and forced cell proliferation, which
inhibit other cell functions, causing misleading data as to tumor cellsensitivity to drugs and radiation.
Traditional labware, that is, flasks, bottles, and plates, are ambientatmosphere dependent—they take on the gas characteristics in which
they are incubated. Without complex active intervent ions, cells are
exposed to room air.
Cells in flasks or plates grow as a monolayer below a shallow layer of
media directly exposed to air containing 21% oxygen, rapidly andconstantly dissolving in the media, while true cellular oxygen levels aremuch lower. Room air contains almost no carbon dioxide, so1

Emilio Barbera-Guillem, M.D., Ph.D., is CSO, and Michael Gallagher,.D. (mgallagher@celartia.com), is associate science officer at Celartia.
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supplemental tanked CO2 is supplied, while low relative humidity inthe lab requires a saturated water environment (water pans).
Excluding oxygen is particularly problematic, requiring sealed
incubators, nitrogen flushing, and repeated attention to prevent theingress of omnipresent atmospheric oxygen. A plethora of complex
hypoxia chambers and glove boxes have been developed as active
interventions to bring oxygen, carbon dioxide, and humidity levels inline with true physiologic conditions, but even so, wide fluctuations
and failure of these active interventions is a constant danger.

Novel Bioreactor
Instead of trying to replicate natural conditions, a new strategy
employs engineered devices, called Ducted Respiratory Chamber(DRC) bioreactors. Celartia’s Petaka® G3, instead of attempting toimpose “normal” gas conditions on the cells through active incubator
controls, passively allows cells to maintain their own oxygen, carbon
dioxide, and humidity levels.
These levels are maintained automatically and independently from thelaboratory or incubator conditions. Because the DRC devices act
passively, they are inherently far simpler, more reliable, and lessexpensive than traditional methods of active gas control.
While the passively controlled gas environments within the DRC are
far simpler than the active interventions required
today’sby
incubators, they are actually far more dependable, as they are driven
exclusively by natural laws.
The Petaka G3 DRC design is shown in Figure 1. The cell culture
chamber is isolated on the injection side from the atmosphere by aself-sealing silicone injection port that allows the closed introductionof media and cells, including most eukaryotic cells types, small earlystage embryos, tissue fragments, and even needle biopsies.
On the venting side, the culture chamber is partially isolated from theatmosphere by an engineered, semi-closed respiratory duct having two2

major portions. Closest to the reaction chamber, a series of smallchambers act as water vapor condensers blocking evaporation,
preventing alterations in media osmolarity, even at 37ºC for 10 to 20
days at 10% RH.
The chambers also act as a capillary breaker to prevent media flow
into the second portion, the respiratory duct that controls internal gaslevels. The length and cross-section of the respiratory duct isengineered to partiallyrestrict the diffusion of oxygen from the highlevels of ambient air to create lower, physiologic levels of dissolved
oxygen in the reaction chamber.
Observing Fick’s Law, as oxygen is consumed inside the culture
chamber, decreasing the partial pressure of oxygen in the media,
oxygen diffuses from the outside atmospheric (higher) partialpressure, through the respiratory duct, to the lower partial pressure
inside.
Diffusion is proportional to the concentration gradient, as regulated bythe engineered design of the respiratory duct, and occurs entirely
spontaneously and without outside intervention.
At the same time, the respiratory duct partially retains carbon dioxidefrom cellular metabolism to maintain a physiologically normal mild
acidosis to balance pH. All gas exchange with the outside environment
occurs through a 0.2 micron filtered vent, preserving the internal
sterility of the device but allowing in and out gas movements toprevent pressurization issues when filling and emptying thebioreactors. These physical features of the Petaka G3 are labeledin Figure 2.
Cell culture in DRCs does not need supplemental oxygen-nitrogenbalancing or CO2 and humidity sources, eliminating the entire panoplyof gas tanks, regulators, sensors, microprocessors, and water pans.
This creates a doubled benefit: not only are cells cultured in morenormal physiologic conditions, but the mechanics and costs of cell
culture are greatly simplified and reduced.

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Because the gas environment is automatically maintained, cultures canbe incubated in virtually any kind of chamber providing warmth, andnothing else.
Naturally Produced ConditionsMany genes promoting cellular differentiation and expression aredownregulated at atmospheric oxygen tensions. Naturally induced
oxygen levels and cell growth in the DRC are depicted in Figure 3.
A series of DRC bioreactors were seeded with neonatally derived
cardiomyocytes, 106 cells per DRC in RPNI 1640 media, and incubatedin room air at 37.5ºC. At time zero (left, Figure 3) seed cells and media
at room air O2 (~120 mm Hg) are injected into the DRC. Cells
proliferate, consuming O2, causing the O2 level within the DRC to begin
to fall, but restricted diffusion through the respiratory duct partiallyreplaces O2.
At about 5 x 106 cell count, internal O2 levels enter the upper limits ofphysiologically normal tissues (~50 mm Hg). At about 8 x 10 6cellcount, O2 diffusion through the respiratory duct approaches
compensatory levels for O2 consumption. Cells enter a differentiation
phase, shown by a leveling of the doubling rate, expanding protein
expression and normal gene expression, and are harvested while still
within the band of normal tissue O2 levels (5−50 mm Hg).
Because the cells control their own gas environment, there is littleeffect rom
foutside gas conditions, and the DRC is effective inatmospheres from 500 meters below sea level up to elevations of 4,000
meters, and in relative humidity levels between 10% and 100%.
The DRC changes the paradigm of cell culture, replacing nearly acentury of active attempts to humanly intervene to manipulate gas
exchange with a self-regulating design driven by natural laws ofdiffusion to create dependable, and truly physiologic, gas
environments.

4Figure 1. Schematic of ductedrespiratory bioreactor (DRC).

Figure 2. Petaka G3 DRC (1)cell culturechamber, (2) injection port, (3)respiratory duct, (4) 0.2 micron filter,(5) water vapor condensers andcapillary breakers, (6) unique barcode.
The DRC is shown upright (7) in asilicone stand.

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Figure 3. Self-regulating gas management in the DRC. Cells consume O2, withrestricted O2 ingress, causing a first proliferative cell phase to evolve into a second
differentiating cell phase for protein production and gene expression.

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