Conditioning Media to Target Dissolved Oxygen Levels
Summary
Hypoxia is defined as oxygen deficiency in cellular environments. Oxygen (O2) deficiency within in vivo environments is commonly linked to a wide range of co-morbidities, e.g., respiratory failure, Mycobacterium tuberculosis infection and other life threatening ailments (Reference Section; 2, 6). However, certain cell types thrive in low-O2 environments. Angiogenesis and tumor progression in cancer are routinely either crippled or aided by local exposure to hypoxic conditions, depending on a host of abnormal interactions at the sub-cellular level (Reference Section; 4). In vivo, hypoxia can either i) inhibit proliferation or the spread of cancer, stop differentiation, cause apoptosis or cell death; or ii) promote the down regulation of tumor suppressor genes and development of an aggressive phenotype resistant to therapeutic intervention (Reference Section; 4, 8). The latter is more commonplace than the former. Recent studies focusing on stem cells, diabetes, intracranial neuronal pathways, immunology, and metabolism in obesity and aging have finetuned their research strategy to emphasize the importance of preserving hypoxic conditions in cell culture (Reference Section; 3,7).
In short, creating and maintaining hypoxic conditions from media to environment are fundamental to the development of reliable research models. Exposing cells that are native to or challenged by low-O2 (2-8%) environments to normal atmosphere (21% O2) causes abnormal cell interactions and reduces cell viability. In addition, achieving and maintaining workstation and media conditions within nontoxic pH parameters (7.0-7.4) is paramount to the success of cellular-based work. Therefore, gas-controlled incubators and controlled atmosphere workstations are often used to simulate normal conditions in the body’s organ systems and provide a better understanding of some of the complex processes involved in low-O2 culture work.
However, despite the positive influx of anaerobic and modified atmosphere workstations in research facilities, cell and tissue cultures remain prey to oxidative stress. The problem is contrasting concentrations of dissolved oxygen (DO) between the cells and cell growth media. New culture media contains approximately 10-12% DO by mass, whereas certain tissue cells typically are plated best at 1-3% O2 (Reference Section; 5).
Conditioning media to lower DO levels is a common laboratory practice for working with cells and biological tissues in experimental settings. Decreasing the DO concentration in media and within the workstation decreases the level of O2 shock felt by cells exposed to a lack of molecular equilibration.
Furthermore, maintaining congruent, or near-congruent, O2 levels between media and the workable cultures increases success rates – i.e. greater cell yield, gene expression, and predicted metabolism, etc. – in target cell populations. A document search found little evidence of optimal DO for cell culture techniques.
HypoxyCOOLTM, the media conditioning device developed by The Baker Company, is intended to be used by the customer to condition media to a set DO value accurately and efficiently. The HypoxyCOOL aims to a) improve cell yield and gene expression in all tissue culture processes; b) eliminate oxidative stress due to abnormal O2 concentration; c) enhance productivity of the lab by minimizing time spent conditioning media; and d) function compatibly with tri-gas incubators or hypoxia workstations. In performing this proof-of-concept, The Baker Company intended to show the efficacy of media conditioning as a crucial step in preparing media for use in hypoxic conditions. A series of product quality and biological experiments were performed that focused on three primary areas:
- Assessing the variables that impact the media conditioning process to establish an optimal protocol and parameters for accurate and reproducible media conditioning. The variables considered were the impact from carbon dioxide (CO2); agitation and gas exchange; and cooling.
- Establishing the shelf life of conditioned media.
- Comparing conventional practices for media conditioning with the HypoxyCOOL method.
The available environment for performing these tests was a manufacturing space that was difficult to contain. Clean (not sterile) media had to be opened within this less-than-ideal environment in order to measure its DO content. Despite these challenges, preliminary tests were developed that may be repeated in a contained environment to mitigate the issues encountered in this proof-of-concept.