by Brian Coy
Introduction
"Hypoxic" (or gas-controllable) glove boxes have become important tools for studies of cells and tissue cultures. Researchers are discovering that exposing cells to variable oxygen conditions affects their physiology in dramatic ways compared to cells kept in traditional hyperoxic (20 percent oxygen) culture conditions throughout an experiment. Laboratories are utilizing controlled-environment glove boxes that offer maximum flexibility in working in below ambient O
2 atmospheres.
These glove boxes offer investigators the ability to perform sample manipulations without compromising the chamber's gas-controlled environment. Depending on the glove box system employed, researchers also are able to adjust O2, CO2, and temperature and humidity levels as needed during experiments.
"Hypoxic" chambers were introduced in 1992 when COY Laboratory Products manufactured the first oxygen-controlled glove box. Dr. Susan Steinberg and fellow researchers at Columbia University's College of Physicians and Surgeons modified a COY anaerobic chamber to control oxygen levels, prompting other researchers to come to COY for a similar oxygen-control system.
Hypoxic systems from COY are hermetically sealed and operate on a purge procedure, using a microprocessor-based, automatic oxygen controller with sensor. The sensor monitors O2 levels, and the controller purges in either a background gas (usually nitrogen) or oxygen gas, as required, through the appropriate solenoid valve. A leak-free airlock system functions as a transfer chamber for exchanging samples, supplies, pipettes and tools without disrupting the chosen oxygen conditions.
Research using COY glove boxes being conducted at universities is described below.
Hypoxia and inflammation relationship
Understanding the basic relationship between hypoxia and inflammation, particularly in a mucosa, is the research focus of Dr. Sean Colgan, associate professor of cell and molecular pathology, Harvard Medical School, Brigham and Women's Hospital in Boston. His conclusions include the identification of hypoxia as a contributor to inflammatory disease.
Dr. Colgan uses a hypoxic chamber to monitor oxygen concentrations in cell culture media, including endothelial and epithelial cultured cells. He adjusts oxygen content regularly, ranging from normoxia of 21 percent oxygen to approximately 2 percent oxygen (pO2 = 15 torr). He maintains a 5 percent CO2 level and a constant temperature of 37 degrees C. An optional humidified incubation box prevents cell culture media from desiccating.
The glove box also features an automatic purge airlock (standard on all COY units) that matches the chamber's atmosphere before the inner airlock door is opened. The automatic system achieves consistent vacuum and pressure levels, minimizing any potential vacuum damage to cell membranes.
Cellular detection of mild hypoxia
Mechanisms by which cells detect mild levels of hypoxia are being studied by Dr. Paul Schumacker, professor of medicine in the University of Chicago's Pulmonary/Critical Care section. He uses a hypoxic glove box to research O
2 sensing mechanisms that cause cells to activate protective responses, including activation of new genes mediated by HIF-1 and HIF-2 transcription factors. These new genes help the cells to regulate glycolysis, expression of vascular growth factors and expression of glucose transporters in the cell membrane. The transcriptional responses help to sustain oxygen delivery to the tissue and help the cells to survive more severe levels of hypoxia.
The ability to manipulate samples under hypoxic conditions, without compromising the chamber atmosphere, is important. "Some of the proteins that we're studying, especially those in the HIF-1 family, are rapidly degraded once the cells are made normoxic again," Dr. Schumacker explained. "With the glove box, we can be sure the cells are hypoxic when we think they're hypoxic, and we can harvest them and collect the proteins without having to expose the cells to normoxia."
Oxygen concentration within the chamber is lowered from ambient levels to between 1 percent and 10 percent O2 as required for different experiments. Carbon dioxide level is 5 percent and chamber temperature is controlled to 37 degrees C by glove box heaters for culture of living mammalian cells.
The lab was the test site for a new controller that monitors CO2 levels while O2 levels are also controlled. Prior to the controller, the carbon dioxide level was maintained by using tanks of compressed air and compressed nitrogen that contained 5 percent CO2. These tanks were much more expensive than the bulk-compressed, single-mixture single-gas tanks. Multiple experiments involving oxygen tension changes increased costs dramatically.
The lab can now achieve 5 percent CO2 cost-effectively by buying a freestanding CO2 tank, a nitrogen tank and a compressed air tank and programming the two controllers to blend the three elements into a stable control environment. The lab also can achieve 20 percent CO2 level without disconnecting the tanks and installing separate tanks. Hyperoxic (above ambient) conditions can be achieved and controlled by adding a pure O2 tank.
The hypoxic response pathway used by Dr. Schumacker is also the focus of Dr. Richard Bruick, assistant professor of biochemistry at The University of Texas Southwestern Medical Center at Dallas. He employs techniques of biochemistry, structural biology and genetics to study the role of oxygen-dependent HIF prolyl hydroxylase (HPH) enzymes in cellular response to hypoxia.
Dr. Bruick uses a hypoxic chamber equipped with a heated, humidified incubation box to provide the atmosphere needed for incubating tissue culture cell lines. He uses Desiccant Stak-Paks to absorb excess moisture in the glove box.
Looking at the stem cell world "upside down"
"We look at the world upside down" in our stem cell research, says Dr. Marie Csete, associate professor of anesthesiology at Emory University, who uses a glove box to study use of oxygen and reactive oxygen species as signals in stem cell biology.
Dr. Csete departs from traditional stem cell cultivation in an environment of 20 percent O2 (basically room air), which she considers "toxic oxygen conditions." Instead, she grows the cells at low-oxygen conditions that dramatically change the entire gene expression profile.
"We're using the chamber to grow stem cells at oxygen conditions that we think are normal physiologic unstressed conditions," Dr. Csete said. She finds that in low oxygen, stem cells proliferate more, undergo less cell death and can be induced to differentiate down specified lineages. These dramatic changes in the biology of stem cells and patterns of regeneration allow modeling of common disease processes and gene identification that are important drug discovery tools.
The glove box provides flexibility to grow stem cells at oxygen tensions that are characteristic of their particular tissue. Cells are grown at low-oxygen conditions that reflect their normal biology - 6 percent for muscle stem cells, 2 percent for brain stem cells, and 6 percent for marrow stem cells, for example. Small modular incubators are used to seal in individual cell lines at their own oxygen tensions.
The chamber can be used to conduct long culture paradigms, involving changing media and splitting cells, without introducing room air oxygen. CO2 of 5 percent, pH and temperature of 37 degrees C are kept constant. With oxygen tensions the only variant, researchers can confidently attribute phenotype changes in the cells to oxygen.
Liver ischemia/reperfusion research
A University of Oklahoma College of Medicine research team uses a hypoxia glove box to measure liver cell death in cells cultured simultaneously at three different oxygen concentrations. Liver ischemia/reperfusion research, and more specifically the development of in vitro liver culture models using Matrigel and specialized media, is the focus of Department of Surgery investigators Dr. Thomas Broughan, Dr. Kent Teague and Becky Naukam. The hypoxic glove box has been "a very useful and reliable tool" for the team's work, according to Dr. Teague.
The controlled chamber is used as an in vitro model to simulate reduced oxygen scenarios, such as in liver transplants or other liver surgeries. Another purpose of the chamber is to study effects of re-oxygenation (restoration of blood flow to the tissue). The hypoxic chamber utilizes 5 percent CO2/balance nitrogen and 5 percent CO2/balance air to maintain the culture conditions. The temperature is maintained at 37 degrees C. Anoxia (O2 = 0.5 parts per million) is achieved using a palladium catalyst and hydrogen gas option also containing 5 percent CO2.
"In the body, liver cells reside in zones known to have different oxygen concentrations. We are able to simulate liver zonal oxygen concentrations, both long-term and simultaneously. The glove box allows us to precisely control the ambient, and therefore culture media, concentration of oxygen to which the cells are exposed," said Dr. Teague.
An example of Dr. Broughan's team's use of the glove boxes is illustrated in Figure 4. Using primary rat hepatocytes cultured the traditional way on collagen plates, the researchers were able to compare the levels of cell death found in hepatocytes cultured overnight simultaneously at liver zone 1, zone 3 and 0 percent oxygen concentration (see Figure 4). Death was measured by uptake of the fluorescent vital dye propidium iodide (shown in orange bound to nuclei).
When simultaneous oxygen concentrations are needed, the researchers use small chambers to seal individual culture dishes at each oxygen level needed. The hypoxic glove box then "houses" the small chambers and allows them to be opened in a controlled environment to remove dishes for analysis. Another use by some researchers includes studies in which live animals (rats and rabbits) live in the chamber.
Oxygen-controlled chamber systems represent substantial upgrades from the units that merely flush the chamber with nitrogen and which were the only option available a few years ago. Hypoxic chambers are expected to continue to be used by researchers as they discover new relationships between hypoxia and overall biology, disease and regeneration medicine.
About the author
Brian Coy is vice president - marketing and sales for COY Laboratory Products. COY, located near Ann Arbor, Michigan, manufactures oxygen-controlled glove boxes and related products for hypoxic studies. The company can be reached at 734-475-2200, at sales@coylab.com or via the InfoLink information request number below.