Hypoxic chamber system for cost-effective and efficient screening of the effect of different oxygen levels on cellular growth and function

Regenerative medicine relies on harnessing the capacity of stem cells to grow, divide and differentiate safely and predictably. This may be in the context of expanding stem cells in vitro or encouraging their expansion, mobilization and capacity to regenerate tissues either locally or remotely in vivo. In either case, understanding the stem cell niche is fundamental to recapitulating or manipulating conditions to enable therapy. Recent studies have shown that hypoxia plays a fundamental role in the maintenance of the stem cell niche. Low oxygen (O2) conditions benefit the self-renewal of human embryonic, hematopoietic, mesenchymal, and neural stem cells, as well as improving the efficiency of genetic reprogramming to induce pluripotency. There is emerging evidence that harnessing or manipulating the hypoxic response can result in safer, more efficacious methodologies for regenerative medicine. In order to accurately reproduce different hypoxic conditions in cell reprogramming research an efficient and cost-effective system that will allow testing of multiple hypoxic conditions for a given reprogramming protocol is required. CO2/O2 incubators, currently available on the market, are too expensive and can be setup for only one hypoxic condition at a time. In addition, the consumption of nitrogen (N2) gas, used to suppress oxygen to desired low levels, is very high and in crowded labs requires the N2 gas cylinder to be replaced every two or three days. Thus, the use of CO2/O2 incubators for running experiments at different O2 levels is time consuming and expensive. Commercially available hypoxic chambers do not include gas mixers and thus rely on gas providers. To conduct multiple experiments at different O2 levels within these hypoxic chambers requires gas cylinders with different gas mixtures, the accuracy of which cannot be guaranteed. Thus, the use of these systems are time consuming and inaccurate. To resolve these problems recently we developed a multi-chamber hypoxic apparatus with remotely controlled air pump injectors that can be used to simultaneously run several experiments with different O2 levels in a single CO2/O2 incubator. The principle method of this system allows filling of the device chambers by the desired O2 concentration by coordinated opening/closing of remotely controlled air pumps as the oxygen concentration is gradually decreased (for example 5%, 4%, 3%, 2%, and 1%) in the incubator. At each O2 level, the air pump associated with each chamber will be remotely switched on for 5 min to allow equilibration of gases in the incubator and chamber. By switching off the air pump after 5 min, the air valves (with a cracking pressure of 0.087 psi) connected to inlet and outlet ports will be shut off due to absence of pump generated pressure. Thus, this will be an easy, cost-effective, and efficient approach for conducting multiple experiments simultaneously at different O2 levels in a single incubator. The goal of this Phase I proposal is to assemble a five chamber hypoxic device with individual air pump injectors that will be operated with an Android operating system based remote controller as well as to test their performance for cell culture studies.Recent studies and observations underscore the importance of oxygen tension as a metabolic regulator of stem cell biology and represent an added dimension of stem cell control that allows cells to maintain self- renewal and multilineage differentiation potential. In order to accurately reproduce different hypoxic conditions in stem cell research, an efficient and a cost-effective system, that will allow testing of multiple hypoxic conditions for a given cell reprogramming protocol, is required. The goal of this Phase I proposal is to develop easy, cost-effective, and efficient approach for conducting simultaneously multiple stem cell biology related experiments at different O2 levels in a single CO2/O2 incubator.