Since the first attempts at cell culture in the 1930s, cells have been routinely exposed to conditions that are stressful (Carrel and Lindbergh). This isn’t always apparent to the researcher, who is very comfortable in room air. When a person breathes in, room air is mixed in the airways with air that is breathed out, so that normal lung cells experience oxygen levels half that of room air. However, when we expose cultured lung cells to room air oxygen, it changes their behavior in assays, adding artifact to cell-based assays (Kumar et al.).
Normal physiologic oxygen, or physioxia, for healthy cells inside the body is far lower than room air oxygen. Different cell types have different ranges of oxygen that are normal for them, yet cells from all organs are routinely cultured and handled at supraphysioxic conditions (Keeley and Mann). Tri-gas incubators can culture cells under physioxia, but disrupting those conditions to handle cells in room air biosafety cabinets (BSCs), even for 5 minutes, modulates HIF-1a, changing critical cell functions like proliferation and differentiation (Jewell et al.). Also, it takes hours for oxygen to re-equilibrate in cell culture media, so cells are out of optimum far longer than it takes for the incubator gases to recover from a door opening event.
Room air oxygen isn’t the only invisible factor creating problems for cells in traditional cell culture. Changes in CO2 can drive pH fluctuations when cells are taken to the BSC for cell handling. Temperature changes affect cell function as well (Shimoni and Pathange), and drive edge-effect in plate-based assays (Lundholt, Scudder and Pagliaro). Relative humidity is important to keep high to avoid evaporation from culture vessels that require gas exchange with the incubator. However, high humidity levels also enable microbial growth, so low humidity for cell handling can help protect cell cultures (He et al.).
Variability in cell culture conditions like oxygen, temperature, and relative humidity has had a tremendous impact on scientific reproducibility (S. G. Klein et al.). As a result, researchers are calling for better control and reporting of all cell conditions in basic research (Shannon G. Klein et al.).
Looking for constant physioxic conditions for cells, researchers tried to adapt “bug boxes”, crude hypoxic chambers designed for anaerobic bacterial work. However, the lack of separation between cell incubation and cell handling spaces in these all-in-one chambers allows for high humidity in the entire space to feed microbes. If the operator’s hands breach the closed space with open-cuffed sleeves, the risks for contamination increase further. The resulting environment has all the right conditions for microbial contamination of the space and the cell cultures.
Physically separating the cell handling space from cell incubation spaces in a closed system enables control of these conditions separately. Physiologically relevant oxygen levels can be maintained at all times. Oxygen can even be used as a signaling molecule to drive cell behavior in different ways. In the same system, low oxygen can be used to drive induced pluripotent stem cell (iPSC) clone production, while high oxygen can be used to drive differentiation to retinal progenitors (Bohrer et al.). Temperature control of the handling space reduces edge effect in cell-based assays (Darou et al.). Relative humidity can be low where microbial growth is most risky to cells and high where the cultures need it. Constant HEPA filtration of the gases in the cell handling environment reduces risks further.
Most importantly, modular and controlled cell environments allow for verifiably identical conditions to be produced for cells, anywhere, at any time. Continuous cell culture environmental data can be added to every publication, improving transparency and reporting issues. This will not only help address problems with reproducing basic cell science between labs, but also help answer the call for higher standards for the preclinical lab (Begley and Ellis). Constant ISO5/Grade A conditions inside the controlled spaces only require an ISO8/Grade D background outside, allowing for production of clinical-grade cells anywhere. Modular, controllable, and clonable cell conditions are key innovations to speed new cell-based therapeutics to the patients that need them.
BioSpherix, LLC designs and manufactures Cytocentric® cell incubation and handling environments with temperature, oxygen, CO2, particles, and other parameters under constant control. The Xvivo System® has a modular design with cell incubation and handling in separate modules. This provides both low humidity for microbial risk reduction where cells are opened to the environment, and high humidity for where cultures are closed in vented vessels. Constant control of oxygen means that cells can be maintained at unbroken physiologically relevant oxygen levels, preventing modulation of HIF-1a during cell handling. Constant temperature and CO2 control ensures that cells never experience suboptimal conditions. Chambers can also be chilled if needed. While used for Cytocentric research, the Xvivo System can also provide ISO5/Grade A conditions for clinical cGMP cell and tissue processing.
Key TakeawaysNon-physiological conditions: Cultured cells are routinely exposed to stressful, unnatural conditions—especially higher oxygen levels than found in the body—which can affect their behavior and research results.Oxygen levels matter: Most cells in the body experience much lower oxygen than room air, but standard lab practices often expose all cells to the same, higher oxygen, potentially leading to altered cell functions.Environmental fluctuations: Not just oxygen, but changes in CO₂, temperature, and humidity during cell handling (like moving cells to a biosafety cabinet) can significantly impact cell health and experimental consistency.Lasting effects of brief exposure: Even short exposures to room air or environmental changes can disrupt cell function for hours, meaning cells remain out of their optimal conditions longer than researchers may realize.Reproducibility challenges: Variability in cell culture conditions—especially oxygen, temperature, and humidity—has had a tremendous impact on scientific reproducibility, highlighting the need for better environmental control.
Non-physiological conditions: Cultured cells are routinely exposed to stressful, unnatural conditions—especially higher oxygen levels than found in the body—which can affect their behavior and research results.
Oxygen levels matter: Most cells in the body experience much lower oxygen than room air, but standard lab practices often expose all cells to the same, higher oxygen, potentially leading to altered cell functions.
Environmental fluctuations: Not just oxygen, but changes in CO₂, temperature, and humidity during cell handling (like moving cells to a biosafety cabinet) can significantly impact cell health and experimental consistency.
Lasting effects of brief exposure: Even short exposures to room air or environmental changes can disrupt cell function for hours, meaning cells remain out of their optimal conditions longer than researchers may realize.
Reproducibility challenges: Variability in cell culture conditions—especially oxygen, temperature, and humidity—has had a tremendous impact on scientific reproducibility, highlighting the need for better environmental control.
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