Petaka G3 Singularities

Oxygen Concentration

Oxygen Concentration:

Physioxia Within (down to 2% O2 inside, 21% O2 outside)

Petaka (Hypoxia Chamber) incubated in normal atmosphere (21% Oxygen) provides a gradually decreasing Dissolved Oxygen (DO) concentration in the media where the cells are cultured, always within the physiological limits (Physioxia) of living tissues cells (Graph 1).

Cell cultures begin with a DO concentration equivalent to that of arterial blood (Oxygen Partial Pressure 75 mmHg), high enough to promote exponential cell growth. As cells proliferate the concentration automatically reduces to those DO concentrations close to those levels that the cells receive in normal living mammalian tissues and in embryonic development, in which PO2 is close to 15 mmHg (see references), avoiding cell damage by ROS formation and facilitating cell differentiation.

Without doubt cultured cells require oxygen, but not in excess. When cells are cultured in media, in classic cell culture devices such as flasks and Petri dishes, cells are exposed to an excess concentration of DO. The open atmosphere contains about 20% oxygen. Therefore, the oxygen of the air dissolves in the media up to the limit of its solubility in water, depending on cell culture temperature, atmospheric pressure, and the degree of salinity of the media, resulting in DO concentrations that are far too high for normal cells integrating the living tissues. That is an artifact which can be the source of many misleading results and discrepancies between experimental setting and in vivo setting.

The CelartiaTM cell culture system automatically balances the partial pressure of the dissolved Oxygen in the media at 25 mmHg, reproducing the cell respiratory and growth conditions in the natural environment, that is, as if within a living being. Therefore, the PetakaTM bioreactors (patented) have been designed to provide semi-hermetic cell chambers with physiologic respiration, using a unique system of micro-tubular gas diffusion channels. With this system, the threshold of oxygen in the cellular environment is maintained at physiological levels, similar to the natural bodies of living creatures, producing a perfect environment for stem cells and all other mammalian cell types. Enabled to incubate the cells in an incubator with 21% Oxygen, the Celartia’s cell culture system reduces the Oxygen down to 5% or even as far as 2% under certain specific conditions depending on cell type and level of confluence, which avoids the use of hypoxia chambers or three gas incubators. These conditions of in vitro “Normoxia” have never been available in any other bioreactor on the market.

Petaka LOT Average DO = 1 mg/L (0.5 to 1.1 mg/L depending on cell type), equivalent to average 22.7 mmHg (Torr) in tissues or equivalent to average 3.11 %O2 in hypoxia chambers.
Petaka HOT Average DO = 3 mg/L (2.8 to 3.2 mg/L depending on cell type), equivalent to 66.5 mmHg in tissues or equivalent to 9.33 %O2 in hypoxia chambers.

Physioxia Chambers

Celartia introduces Petaka G3 the most physiologic hypoxia chamber in the world, mimicking as close as possible, how tissue cells in an organism are exposed to oxygen availability deficit.
Inside Celartia’s hypoxia chambers, cells are exposed to a progressive reduction of oxygen availability from 45 mmHg down to the lowest oxygen tensions recorded and published on living vertebrates: 13.32 mmHg.

Celartia’s hypoxia chambers Petaka G3 are completely autonomous.
Without hypoxia disturbances Celartia’s hypoxia chambers allow for bench top and open regular atmosphere use:
1. Continuous measurement of the oxygen reserve in the media.
2. Permanent evaluation of cellular oxygen consumption per cell and time.
3. Cell culture exposed to Hadron beams, electron beams and electromagnetic radiations maintaining the hypoxia level.
4. Celartia’s hypoxia chambers are closed and highly protected against contamination
5. Infinite media changes, sampling and special substances (and investigative drugs) additions to the media are performed in a regular laminar flow hood.
6. Laboratory bench Microscopy observations and time lapse video recording with regular microscopes, without glove boxes or any other ambient controlling devices
7. Can be incubated in any sort of temperature controlled device such as plain incubators, CO2 incubators, heat blocks or even water baths.
8. Celartia’s hypoxia chambers can be piled in arrays with efficient space and energy management
9. Celartia’s hypoxia chambers are disposable.

The scientific community agrees that mammal tissue hypoxia is a progressive onset status produced when the red blood cells can’t replace efficiently the oxygen of the interstitial media consumed by the tissue cells. This is exactly how Celartia’s hypoxia chambers regulate the hypoxia levels: in close interaction with the living cells. The proprietary micro-channel respiratory system incorporated in the device maintains under control the oxygen exchange, stabilizing the minimal level according to the type and number of cultured cells.

The discovery of the stress response to the oxygen deficit, specifically the cell response through the Hypoxia Induced Factor (HIF) and collaterals, opened a broad field of research in cell biology. As a consequence, new technologies were applied to produce environments where culturing cells with limited oxygen availability were possible.
The straight procedure used in industry has been the production of hermetic boxes (hypoxia chambers) filled and/or purged with the required gas mixture. Inside these boxes, traditional cell culture devices (flasks, bags and dishes) are installed and the whole assembly need to be introduced in incubators to achieve the required temperature.

This layer over layer arrangement implies many difficulties:
1. Restricted access to the cell culture device impairing:
a. Culture observation
b. Media changes
c. Adding supplements to the media

2. Gas composition Instability, due to:
a. The box needs to be opened for many required cell culture management operations.
b. Traumatic steps from normoxia to hypoxia in minutes
c. Gas decompression effect inside the cells
d. Uncontrolled gas diffusion through box materials and maintenance requirements

3. Only limited stepwise of hypoxia. There is no continuous transition from normoxia to hypoxia.
4. Exaggerated waste of space (volume)
5. Requirement of gas canisters as part of the assembly

Only a few highly sophisticated glove boxes available include a control unit program that permits gradual hypoxia in the chamber, where the cell culture devices are incubated; however, these complex devices are enormous, need great user training, are time consuming, need huge gas canisters, considerable power supply and laboratory space.

All of these restrictions and limitations are beat by Petaka G3 hypoxia chamber technology.

Outstandingly, Celartia’s Petaka G3 hypoxia chambers achieve this in the open atmosphere, inside a regular incubator, without accessory gas canisters, electronic sensors, feedback electronics or power supply.
Celartia’s hypoxia chamber’s manipulation is even easier than regular multi-well plates, and does not need special training to use them at the top level of efficiency.

Deep Hypoxia Experiments

Petaka offers the researcher complete freedom of sample manipulation even in very low oxygen hypoxic conditions, without breaking the hypoxic environment at any time.

Petaka, when packed in vacuum Mylar bags, it does not allow the passage of oxygen from the ambient atmosphere in the culture and the cells consume the media-dissolved oxygen in hours, creating a deep hypoxic condition. Cells can be grown and incubated in these conditions and a sample of the cell culture eventually withdrawn by punching the port directly through the Mylar bag, avoiding any exposure to higher oxygen concentrations.

To research cellular behavior under hypoxic or normoxic conditions, laboratories use controlled-environment incubators and glove boxes which offer the possibility of working in below ambient O2 concentration.

Glove boxes offer researchers the ability to incubate or perform sample manipulations without compromising the chamber’s gas-controlled environment. However Petaka does not need special incubators, glove boxes or any gas canisters.

No CO2 incubator

Media based on Earle’s salts are buffered with a bicarbonate/carbonic acid system. These buffers rely upon the physiologically relevant pKa for carbonic acid and the equilibration of gaseous and media dissolved carbon dioxide to maintain the pH in a 5 to 10% CO2 incubator.

Petaka is designed to function without an additional CO2 environment. CO2 incubator is therefore not required, avoiding the need for CO2 cartridges. CO2 produced by the cell culture is also controlled. This is achieved by a gas transfer quenching system (GTQS) integrated into the device. As a result, the level of CO2 is maintained within the Petaka, allowing the pH to remain at a level compatible with the growth of 15 to 30 million cells.

Therefore, Petaka can be incubated in normal incubators without CO2 regulation, and cells grow in a closed and stable environment up to the limit of their own metabolism. These yields will vary according to each individual cell metabolism. We recommend that a culture test and a period of adaptation are implemented when a cell line is going to be cultured in Petaka.

Dehydration prevention

Petaka is virtually vapor hermetic so does not require a humidity saturated incubator.

You may store pre-filled Petakas, ready to host cells, and have them prepared for immediate use. Refrigerated storage time is not limited by dehydration.
Elimination of saturated humidity in the incubator, water pan, and mist, reduces the risk of cross contamination in the incubator.

In Petaka, an integrated micro-channel system (MFS) protects the internal environment, avoiding unwanted water evaporation. Dehydration at 37ºC has been minimized, allowing culture incubation for 30 days at RH 10% with a final media dehydration below 10%.

Contamination prevention

Microbial contamination is a severe problem in cell culture. Typical routes of microbial infection in cultures are the ambient air, when flasks are transferred from the hood to the incubator, the water bath, and the humid environment of the CO₂ incubator which provides ideal growing conditions for many strains of bacteria.

Other routes of cell culture infection include: contact with non-sterile surfaces when performing cell culture manipulations, spillage on materials and the work surface, splash-back from pipetting or pouring cell suspensions and microscopic aerosols.

Petaka is a virtually hermetically closed system. Air access is only possible through a 0.2 micron pore filter. Neither lids nor caps are used to access the liquids. Petaka are injected through a silicon port that seals after the removal of the injection tip.
Sterilizing the injection tips and the silicon port minimizes the chances of contamination. In addition, Petaka has an 80 mm diffusion barrier between the port and the culture chamber. This exponentially decreases the chances of microbial progression through the port slit during long incubations.

In Vitro Cell Dormancy

In vitro cell Dormancy or Pausing

Using regular flasks, cells should be subcultured as soon as possible when they reach a confluent monolayer state or saturate the carriers.

With Petaka, grown cell cultures can be maintained alive at room temperature, without dehydration risk, for long time periods.This facilitates cell culture management and maintenance routines; and provides additional advantages for cell biology and toxicology research (see “Petaka Quick Protocols” for conditions and methods, such as protocols 4 and 8 – “Keeping Cells in In-Vitro Dormancy”).

Cell conservation in dormant state is favored by transferring cultures from 37° C to 22° C when cells are in a slow progressing cell cycle. This is achieved in an 80% confluent state.
Under low temperature and low pH, cells reduce DNA replication practically to zero, and remain in a suspended animation-like state (a certain accumulation of cells in G2 phase could be expected).The duration of the dormancy period is dependent on the cell type. At a minimum, 7 days survival is common for most cells, while others could live for more than 200 days.

Cell Transport/Shipping

Cell Transport/Shipping. With the Petaka you can transport live cells for long distances at normal ambient temperatures. For most cell lines, PetakaG3 does not need dry ice or refrigeration to transport living cells for periods of time between 1 and 14 days.

Its slim shape permits easy packaging and protection. Especially designed mailers are ideal for shipping one or two units.

Cells inside Petaka, at normal temperatures, remain in a dormant state for days, even weeks. Viability decreases progressively depending on the cell type. CHO-K1 cells, for example, can travel for 3 to 4 weeks maintaining 75% viability. Some tumor cells can travel for a month or more.

Cells/Media segregation

Segregation of Cells and Supernatant by Petaka Centrifugation

Petaka G3 simplifies operations by performing two functions, cell growth and cell concentration in a single device. Centrifugation of Petaka using the Petaka rotor, allows concentrate the cells in the corner of the media channel facilitating cell recovery with minimal media. Or concentration of the cells in the opposite corner allowing media harvesting avoiding the cell pellet. As a result, cell culture becomes simpler, quicker & more versatile.

Safe Handling

Petaka’s auto-sealing port, a pre-slit, 1 mm thick, silicon diaphragm, allows easy penetration with plastic and metal tips up to 1 mm external diameter.
Petaka’s 0.2 mm Filtered Vent: The internal chamber communicates with the external atmosphere through an in/out vent closed with a 0.2 µm pore filter.
By avoiding caps and lids, the duo vent-port simplifies the repetitive operations of cell culture and handling, and minimizes routine operations. When the user initiates the injection procedure, the fluids flow through the tip into an atrium communicating with the culture chamber through a protective mini-tube. Therefore, during injection, the interior of the cell culture chamber is never exposed to the external atmosphere, through any aperture. In addition the mini-tube breaks any back pressure, avoiding leaks through the port slit.The diaphragm surface is accessible for swabbing with ethanol, and can be exposed to a gas flame in order to guarantee surface sterility.The port assembly allows safe and quick in/out maneuvers with reliable seal, greatly minimizing contamination risk.

Time Savings

Minimized maneuvers = time saving

Inside the Petaka itself, one is able to perform several protocol steps without removing them from the device. This means that by not having to transfer cells to several other instruments or devices, one is able to cut out several steps. Examples of such applications are cell washing, cell & supernatant separation, enzyme-free detachment & harvesting, DNA & RNA extraction, cell pelleting, cell storage and pre-preparation of ready to host Petakas.

Space Savings

Minimum container volume for the maximum number of cells

For scale up cultures, Petaka offers important advantages.

Using Petaka enables you to maximize the efficiency of your incubator.
By minimizing the amount of air space in Petaka and simultaneously offering 150 cm2 surface area for cell attachment per unit, it offers up to 10 times more cell yield per incubator than T75-Flask or T160-Flask.

Moreover, whilst consuming proportionally less media per cm2 of surface saves media and growth factors per million cell yield.

3D cultures & Microgravity

Petaka™ G3 cell culture devices / bioreactors can be used to grow aggregates of cells forming pseudo-tissues from any kind of differentiated cells or stem cells. These agregates , can be implanted into an organisms for regenerative research, cancer research and regenerative therapy experimentation. Differentiated cells and tumor cells forming 3D structures provide important experimental information about cell behavior in real tissues.Petaka™ G3 cell culture devices / bioreactors can be used to grow aggregates of cells forming pseudo-tissues from any kind of differentiated cells or stem cells.

These agregates , can be implanted into an organisms for regenerative research, cancer research and regenerative therapy experimentation. Differentiated cells and tumor cells forming 3D structures provide important experimental information about cell behavior in real tissues.Microgravity 3D cultures can be obtained in Petaka™ bioreactor when this is subjected to a controlled and adaptive rotation, keeping the cells in microgravity conditions that induces the formation of 3D cell clusters (pseudo-tissues).

Cystic 3D cell culture

This system is suitable to investigate intercellular interactions through soluble mediators, using the simple and unique bioreactor Petaka G3. Conceptually this bioreactor allows:

1) Growth of more than one cell type, separated by an inter-space full of media.
2)     Growth of several million cells, letting repeated cell sampling operations without interruption of the culture.
3)     Separate sampling of the different cell types included in the co-culture.
4)     Repeated media renewal and sampling operations without the interruption of the culture.

The biological product of this concept is the “Cystic 3D cell culture” that offers, in a single volume, at least three separate sheets of cells, with or without matrixes, growing with well defined borders, surrounding apocket (cyst) with 15 to 25 mL of shared media (see upper figure). The system is a cube like structure with 6 sides or 3 pairs of opposed walls facing each other. In this system the central cavity or cyst receives everything secreted or released by the surrounding cells including metabolites, extracellular organelles, exosomes, shedding microvesicles, apoptotic blebs and others. Cell shits are attached to the walls, uncoated or coated with specific matrix of single molecules such as Laminin, Vitronectin, Collagen, etc. or molecular mixtures such as Matrigel or others, all favoring cell attachment through integrins. One side of the device may be coated with a thick layer of semisolid gel allowing the formation of cell clusters and cell penetration evaluation (invasion assays).

Soluble secretions and particulate elements released by cells forming the walls circulate randomly through the cyst media, like in the blastocysts, ovary follicles and others. Those suspended elements become in contact with the other wall cells dragged by soft and slow convection streams, generated by controlled temperature gradients,. Many physiologic reactions are controlled by soluble molecules and particulate elements, such as exosomes released from living cells

The bottom side layer of cells is preferably influenced by particulate elements, it receives by gravity sedimentation all those elements having density over 1, released by the upper cells; this influence is even more intensive on all cells living on the side of the cyst when exposed to a multiplied g force vector, if the device is subject to a convenient centrifugation.

With an accurate control of hypoxia, close to physiologic DO (about 2 ppm), CO2 control and dehydration control (Petaka G3 conditions), these cultures could evolve for more than 2 weeks, allowing long term inter-cellular interactions. The cyst culture system (Petaka G3 device) allows for free-enzyme cell release of all the cells or samples of each cell type separately, therefore sampling each side independently for microscopy, FACS, PCR or biochemistry analysi