Petaka G3™ Applications: Frequently asked Questions
Why must I hold the Petaka G3™ upright when filling with medium?

The restrictive, respiratory microchannel in the Petaka G3™provides physiological levels of O2
while simultaneously maintaining ideal CO2 and pH
levels for your culture. However, this delicate balance ofinward and outward gas diffusion is not optimal whenthe microchannel is filled with medium. To avoid thisfrom happening, simply keep the Petaka G3™ uprightwhen filling (see Fig. 1). The air path from the culture
chamber to the gas microchannel is on the upper rightcorner of the Petaka, near the injection port. So, if you
are careful to not let liquid into the capillary breakers orthe microchannels your culture will soon enjoy ideal, invivo like gas control. No tri-gas incubators, glove boxes,
The locations of main parts of the Petaka G3™,
or specialty environmental control chambers required!
closed cell culture cassette.

At what level of dissolved oxygen will the Petaka G3™ maintain my cultures?
The proprietary microchannel gas regulator within each Petaka G3™will reduce the dissolved
oxygen from hyperoxic levels (150mmHg pO2) to levels found in native tissues (25-50 mmHgpO2). The exact level of dissolved oxygen will vary depending on cell type, length ofuninterrupted culture and medium conditions. It
is important to understand that oxygen flow into
the Petaka G3™ is controlled by diffusion, so thebigger the difference between the outside air andthe inside culture chamber, the more oxygen will
diffuse in. So, as the internal oxygen level isreduced, more oxygen will automatically andpassively diffuse inward to maintain equilibrium.
As cells divide and grow in Petaka G3™ dissolved
oxygen levels decrease to physioxic levels.

Just as in native tissues, cells in the Petaka G3™
will take what they need and establish theirpreferred local dissolved oxygen levels. The Petaka G3™ does not force an artificial level ofdissolved oxygen on the cells in culture, this is why we state oxygen is “auto-regulated” andcells maintain physiologic levels of oxygen at all times.

Celartia • 974 Checkrein Ave., Columbus, OH 43229, USA
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What types of cells can I culture in the Petaka G3™?
Practically every cell type has been successfully
cultivated in the Petaka G3™, including attached,suspension, immortal, primary and transfectedcells, hybridomas, MSCs, ESCs, iPSCs, etc. ThePetaka G3™ FLAT is untreated for suspension cell
culture while the Petaka G3™ LOT is surface
treated for attached cell culture.
The Petaka G3™ works well with every cell type.

The Petaka G3™ is not just another flask; it corrects
the error of cultivating cells in the harmful, hyperoxic conditions typical with plates, dishes andflasks. Since most cell lines have been cultivated in hyperoxic conditions for decades, Petaka
users may observe differences as the cells re-adapt to their correct, physiological environment.
Practically speaking, this can result in slight morphological changes, changes in doubling time,etc., but this is not a negative effect. It is actually corrective, and in the long-term your cells will
be happier and more reflective of the native tissues when grown inside the Petaka G3™.

How is it possible to grow cells in the Petaka G3™ without supplemental CO2?

Cells naturally generate CO2. However, in plates, dishes and flasks this CO2 rapidly diffuses outof the medium, so a closed CO2 incubator system is required. The Petaka G3™ automaticallyretains CO2 at a level sufficient to maintain pH in a standard bicarbonate-buffered medium.
However, a minimum number of cells in the starting culture must be present to supply
sufficient CO2 to maintain this pH balance. Decades of experience has led Celartia torecommend at least two-million cells (25,000 cells/cm2) be seeded in a Petaka G3™ when
culturing without supplemental CO2. With this number of starting cells, your culture shouldhave no problem maintaining excellent pH of the medium. Much lower cell densities will also
do fine in the Petaka but may require supplemental CO2 for the first 24-hours of until sufficient
cell numbers are reached and sufficient internal CO2 is generated.

How do I perform long-term microscopy with the Petaka G3™?
Because the Petaka G3™ provides a unique, closed, gas-controlled culture environment without
significant evaporation, it acts like a mini-incubator all by itself. Because of this, specialty cellculture and experimental processes may be performed for long periods of time outside of thestandard CO2 incubator environment. This is impossible with plates, dishes, flasks or bags
since medium will evaporate leading to loss of osmolarity and the dissolved CO2 will rapidly belost resulting in cell death.

Celartia • 974 Checkrein Ave., Columbus, OH 43229, USA
www.celartia.com

The Petaka G3™ supports functions like time-lapse
microscopy without any special (and expensive)
microscope incubator equipment. All one needs toprovide is warming for the Petaka G3™, everything else
is automatically maintained by the device (see our longterm benchtop microscopy setup, right). At Celartia, weroutinely use small heating films to maintain cultures at37°C on the microscope to record multi-day microscopy
observations or to record pO2 in the medium on thebenchtop. These films are available from Celartia, justask!

Bench-top, time-lapse microscopy set-up with
Petaka G3™ requires no incubator.

How do I preserve cells at ambient temperature in the Petaka G3™?
One of the amazing features of the Petaka
G3™ is its ability to preserve cells at room
temperature for extended periods of time.
Many labs use the Petaka G3™to eliminate
cryopreservation for processes that benefit
from cell storage for up to 2-weeks. Whencells are maintained at low dissolved
oxygen, ideal pH levels, and ambient
temperature (15-23°C), cells simply godormant, pausing their cell division andreducing their metabolic rate significantly.

Viability of common cell lines during dormancy in Petaka G3™.

Most cells in this state are healthy and remain viable for 2-weeks ormore. To wake these cells, simply return them to a 37°C incubatorand allow them to warm…that’s all. This is only possible becauseeven outside of an incubator the Petaka G3™ maintains low oxygenand balanced CO2 levels. Combined with a sub-euthermic
temperature and the cells are gently and perfectly maintained in adormant state. Labs use this free process time to temporarily store
cells or ship cells anywhere in the world.

Cells in Petaka G3™, in mailer.

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How do I cryopreserve cells in a monolayer in the Petaka G3™, and why is itsuperior to standard freezing in cryovials?
The Petaka G3™ is the only cell culture device that also allows for convenientand direct cryopreservation inside the primary culture device. Cryo vials
feature a low surface-to-volume ratio. This means that the operator mustuse a slow freezing process while the glass transition progresses from theoutside to the center of the vial. During this slow freezing period ice crystals
may form inside of cells and damage them, leading to poor post-thawviability. A preferred method is thin-film cryopreservation, only possible inthe Petaka G3™. To freeze cells grown in a monolayer in the Petaka G3™
simply remove culture medium, rinse cells briefly in freezing medium, thenaspirated out excess freezing medium. This leaves only a thin film of freezing
medium over a layer of cells no more than 30um thick. This thin film maythen be immediately flash frozen (vitrified) for permanent cryopreservation.
This process prevents trypsin exposure, and all risks and damage associatedwith the traditional slow-freezing process.

Thin film productionin Petaka G3™.

To thaw vials, they must be warmed in an unsanitary water bath for 1-2 minutes. This poses amajor risk to sterility and this extremely slow thawing technique leads to even more cell death.
In contrast, thawing cells in a Petaka G3™ is achieved simply by filling it with warm medium.
This means each cell thaws in less than a second as the warm medium covers the frozen cell; nowater baths, running water, or sample transfers required. After adding warm medium, cells in
the Petaka G3™ go directly to culture, with no reattachment time or trypsin damage.
Moreover, up to 25 million attached cells
may be frozen in a single Petaka G3™, using
just 1ml of residual freezing medium. Thismeans that when 24ml of warm medium isadded to a Petaka G3™, the standard
10%DMSO is diluted to around 0.4%. Thislevel of DMSO is no longer damaging to cells
in culture, so cells thawed in a Petaka G3™
never need to be spun-down or washedduring initial recovery and incubation.
Cryopreservation in the Petaka G3™ means
the whole process is faster, easier, carriesless contamination risk, and leads to muchhigher post-thaw cell viability.

Attached cells in Petaka G3™, frozen in a thin film results insuperior cell viability compared to cryofreezing with vials.

Celartia • 974 Checkrein Ave., Columbus, OH 43229, USA
www.celartia.com

How is it possible to wash and centrifuge cells without transfer to conical tubes?
The Petaka G3™ is the only cell culture device that can becentrifuged directly. This allows the user to pellet, washand separate cells without any transfers into conicaltubes. Unlike open dishes, plates, and flasks, the PetakaG3™ is designed and manufactured to withstand up to650g during routine centrifugation. So, instead ofaspirating and transferring cells for centrifugation, theuser can simply place the Petaka G3™ into the centrifugeand spin directly. This results in a cell pellet in thebottom corner of the Petaka G3™. These cells stay put asmedia is aspirated off and fresh media is added.
Likewise, the pelleted cells may be selectively harvested
while the old media is retained. This eliminates sample
transfers into in and out of centrifuge tubes, andpractically eliminates contamination risk. Old Traditional
limitations and cell culture dogma no longer apply;
everything is now possible with the Petaka G3™.

oldCentrifugation in the Petaka G3™rotor.

What do the colored ports on the Petaka G3™ indicate?

The Petaka G3™ LOT for adherent cells features a blue silicone injection port. The Petaka G3™
FLAT for suspension cells features a white silicone injection port. The orange color port on thePetaka G3™ HOT is for when you want slightly higher oxygen tension in your cultures.

Should I culture with the Petaka G3™ in an upright or horizontal configuration?
Suspension cells must be cultured in the Petaka G3™ in ahorizontal configuration, similar to a flask because thesecells will clump on the bottom when the Petaka G3™ iscultured upright. After adherent cells attach, (typically 12 hours), the Petaka G3™ may be left in the horizontal
position or it may be switched to an upright position forthe duration of the culture. However, when the Petaka
G3™ is kept “horizontal” it is strongly advised to elevate
the top end of the Petaka G3™ slightly higher than thebottom (5°-15°) to limit the amount of medium that may
enter the capillary breakers at the top of the Petaka G3™.

The preferred horizontal configuration with thetop end slightly higher than the bottom end.

Celartia • 974 Checkrein Ave., Columbus, OH 43229, USA
www.celartia.com

How can I measure the amount of dissolved oxygen inside my Petaka G3™?
The Petaka G3™ is unique in that it is now possible to measure thedissolved oxygen (pO2) in your culture in real time, using a sterilize,
off-the-shelf setup. The Petaka G3™ DO Sense features an oxygen
sensing dot manufactured by Ocean Insights, that when paired with
the NeoFox™ reader, allows the user to measure and record thedissolved oxygen in the culture medium with precision and hightemporal resolution. With the Petaka G3™ DO Sense it is easy toperform simple bioprocess monitoring or to carry out sophisticatedexperiments determining single cell oxygen consumption rates.
The setup is simple. Simply fill the Petaka G3™ DO Sense with your cell culture and incubate on
any 37°C warming surface. This is only possible in the Petaka G3™ because every other culture
device requires a humidified CO2 incubator. After filling, position the NeoFox™ reader to alignwith the sensing dot and start measuring. That’s
all there is to it. The user will then observe theclassic pO2 reduction curve as the cells consume
excess dissolved oxygen. The user can observeand record the culture progressing in real timefrom an artificial, hyperoxic state to a morenatural and relevant, physioxic condition. Labs
can maintain this culture in open air for days
since there is practically no media loss due toevaporation and the pH of the culture is perfectly
maintained without supplemental CO2.

How is it possible to eliminate the water tray from my incubator?
Standard plates, dishes and flasks must be open to allow open CO2 gas exchange. The Petaka
G3™ is functionally closed and retains CO2 in culture automatically. All liquid transfers areperformed through a closed, sterile silicone port. In addition to benefit of reduced
contamination risk during processing, the closed
design means that media evaporation is practicallyeliminated. This is why there is no need tomaintain a water tray inside the incubator. Thiseliminates a second major contamination source.
The Petaka G3™ has been shown to minimize
evaporation loss in culture even at extremely low
ambient humidity levels.
Celartia • 974 Checkrein Ave., Columbus, OH 43229, USA
www.celartia.com

What are the basic features of the Petaka G3™?
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Class VI medical grade resin
Optically clear polystyrene growth surface
Sterile (SAL 10-6)
Standard SBS footprint
0.2µm PTFE air filter
24ml filling volume
95kPa tested
Free of detectable endotoxin
Sterile silicone port access75cm2 surface (one-sided culture)
150cm2 surface (double-sided culture)
Functionally closed processing
Practically no media loss due to evaporation
Single-use disposable

Celartia • 974 Checkrein Ave., Columbus, OH 43229, USA
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