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Cell culture devices with biologic pH auto-control (CO2 dissolved in the media). Don't need CO2 incubator.

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Cell culture devices with CO2 biologic autocontrol. Advantages and limitations. Don't need CO2 incubator.
 
The amount of environmental H+ is one of the most restrictive factors on the cell growth and physiology of cell in cultured in artificial media; therefore, cell culture media is usually buffered in order to maintain a neutral pH. Several buffer mixtures may be added to the media in order to maintain pH neutral, such as sodium phosphates, Tris, bi-carbonate or HEPES. The bi-carbonate buffer system has been found to minimally affect the growth of cells, and therefore has become the most common. H2CO3 dissolved in water is in equilibrium (CO2 + H2O H+ + HCO3− ) with the CO2 component diffusing rapidly out of the media and into the atmosphere. The concentration of bicarbonate can be determined when formulating the media; however, the CO2 concentration is dynamic, varying with (1) temperature, (2) gas pressure and (3) pH of the media at any  precise instant.

There are 2 major technical strategies to keep the CO2 in the media solution at the desired pH level:


a) Using External Exogenous CO2 Sources.
When using T-flasks and dishes with head space (gas space above the media), supplying additional CO2 into the atmosphere is necessary above the buffer, increasing the CO2 partial pressure in the atmosphere. Otherwise the CO2 in the media would dissipate into the head space and the H2CO3 would disappear as a result of the buffering effect. The media would become increasingly alkaline as this reaction progressed. Using a CO2 regulated cell culture incubator maintains a high level of atmospheric CO2 (5% to 10%), and insures that the media will contain enough HCO3- to maintain the desired pH.



b) Using Internal Physiologic CO2 Sources.
Petaka G3® has autonomous CO2 balancing mechanism, invented and patented by Celartia® that maintains cells in physiologic conditions without external CO2 sources. This strategy reduces the CO2 diffusion rate from the media to the atmosphere by interposing two specifically calculated diffusion barriers between the cell culture environment and the external ambient atmosphere.



Because living cells produce and release metabolically derived CO2 into the media, by

holding the diffusion coefficient under control and thereby saving CO2 within the culture chamber, neutral pH can be achieved without an external supply of additional CO2.

The most important factor in maintaining a successful cell culture is keeping the pH within normal physiologic margins.  Commonly, cell culture media is supplemented with phenol red, a molecule which changes color according to pH, ranging from light yellow at pH 6.5 and bluish magenta at pH 8.

In Petaka, the approximate pH level can be monitored by observing the color of the phenol red supplemented media, actually digital color measurements, using for example an ELISA reader, may help providing quantitative pH data.



The pH is auto-regulated in Petaka and varies according to several factors:

1 The CO2 of the ambient atmosphere

2 The pH of the media

3 The number of living cells in the culture chamber


If the ambient CO2 partial pressure is higher than the internal CO2 partial pressure, the ambient CO2 diffuses into the cell culture chamber gradually, due to a low diffusion coefficient. Consequently, the pH drops. Conversely, if the ambient CO2 partial pressure is lower than the internal CO2 partial pressure, the internal CO2 diffuses to the ambient air, with a diffusion coefficient proportional to the differential inside-outside CO2 partial pressure. Such a “CO2 leak” increases the pH of the media and reduces the CO2 solubility in the media, thereby consolidating the pH increase. The CO2 diffusion rates in Petaka are set by the design of the internal diffusion system and are calculated to maintain pH within the normal cellular physiologic margins (Figure 1).
auto pH

Figure 1 – Progression of pH adaptation as a function of the density of the cell population in the culture.






Exceptions:

At the initiation of a cell culture (seeding the Petaka), difficulties can occur if the media used isn’t set at pH 7.0 to 7.2. Failing to do so can cause the LAG phase to be too long or even may

impair cell growth. A frequent cause is the use of old media that has been stored in a refrigerator with a large headspace in the bottle. Solution: Before using any media check the pH and adjust it to 7.1-7.2.

The LAG phase can be too long, or again, an impairment of cell growth can occur, if the initial cell inoculums (seed) are too small (less than 300,000-500,000 cells). Solution: Always seed about 300,000 or more viable cells. 
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