This method can be used to characterize the physiology of anoxic cells and help answer key questions in host pathogen interactions, stem cell differentiation and cancer cell metastasis and therapeutic susceptibility. This is the first described protocol that enables the long-term anoxic cultivation of mammalian cells. The implications of this technique extend toward targeted, personalized cancer therapy and rejuvenative therapy because, due to oxygen toxicity, multiple cell types prefer low-oxygen environments for growth.

Though this method can provide insight into cell anaerobic respiration and metabolism, it can also be applied to other systems such as stem cell cultivation. Generally, individuals new to this method will struggle because extreme care must be exercised to prevent oxygen contamination. Physical demonstration of this method is critical, as the care needed to exclude oxygen contamination is not intuitive.

To begin, add 0.5 milliliters of nitrite stock solution to low glucose DMEM containing L-glutamine, sodium pyruvate and fetal bovine serum. Then place 20 milliliters of the medium in a sterile 50 milliliter conical centrifuge tube. Next, degas the medium by placing the tube in a vacuum bell jar at a 45 degree angle.

Use a vacuum pump to apply a vacuum at room temperature. Maintain the vacuum for 24 to 48 hours, until bubbles are no longer observed. Remove the tube from the bell jar and close the cap immediately to minimize introduction of air to the medium.

Then place the tube for 24 hours in a prepared anaerobic chamber and loosen the cap. While the tube is in the chamber, transfer 100 microliters of the medium to a sterile, degassed microcentrifuge tube. Then use an oxygen probe to test the tube for dissolved oxygen.

First, grow cells in a T150 flask to 90%confluence. Remove the medium from the flask via aspiration and wash the flask with 10 milliliters of HBSS. Then, replace the removed HBSS with five milliliters of trypsin and agitate the flask until the cells detach from the plastic.

Aspirate most of the trypsin from the flask and tap the flask to dislodge any adhered cells. Then add 10 to 15 milliliters of high glucose DMEM to inactivate the trypsin. Use a 25 milliliter pipette to triturate the cells until all of the cell clumps are disrupted.

Then, using a standard hemocytometer, count the cells and determine the viability. Suspend the cells in high glucose DMEM and add one milliliter of the suspension to the wells of a 24-well tissue culture plate. Then, incubate the cells at 37 degrees Celsius in normoxic conditions for 24 hours.

After the 24 hour incubation period, transfer the plate to the anerobic chamber. Then, immediately change the medium to antibiotic-free, degassed PS-74656 medium. Place the plate in a container with a damp towel and loosely close it.

After the second 24-hour incubation period, continue with further incubation periods, as outlined in the text protocol. Monitor the anaerobic medium for color change over time. When the medium changes from magenta to red, remove the runagate cells via aspiration.

Transfer the aspirated spent media to a prepared microfuge tube. Close the tube and centrifuge it. Replace the aspirated media in the wells of the plate with new degassed PS-74656 medium.

Next, place the microfuge tube with the pelleted cells in the anaerobic chamber before opening it. Aspirate the spent medium and replace it with new degassed PS-74656 medium to a total of one milliliter in the tube. Next, transfer the cells from the microfuge tube to a well in the tissue culture plate.

For microscopy, while in the anaerobic chamber, place the plate in a resealable bag flushed with anaerobic gas mixture. Then close the bag. Seal the bag completely and remove it from the chamber.

Stretch the bag over the plate and use an inverted phase contrast microscope to examine the cells. Return the plate in the sealed bag to the chamber and incubate the plate. Finally, separately analyze the runagate cells in suspension, according to the text protocol.

Using this protocol, established cell lines were successfully cultivated in an anaerobic environment. Even after an extended incubation of several weeks, some cells remain tethered, while the majority displayed rounded runagate morphology. Water droplets can create microscopy artifacts that are frequently mistaken for cells.

Final cell viability for long-term cultivation was dependent on the confluence to which the wells or flask were initially seeded. HeLa 229 cell growth was optimized at 80%initial seeding confluence. Vero cell growth, however, was optimized at 60%initial seeding confluence.

Once mastered, this technique can become a routine procedure for maintaining cells anoxically. While attempting this procedure, it's important to remember to pre-prepare medium so that it's properly degassed and to degas all other materials used, such as microcentrifuge tubes and pipette tips. Following this procedure, other methods, like drug cytotoxicity testing and stem cell cultivation, can be performed in order to answer additional questions, like anaerobic cancer cell drug sensitivity and anoxic cytokine expression.

After it's development, this technique paved the way for researchers in the field of host pathogen interactions, cancer cell chemotherapy and stem cell propagation to explore natural changes in cell physiology that can occur in vivo. After watching this video, you should have a good understanding of how to culture mammalian cells for long periods of time in the absence of oxygen. Don't forget that working with cancer cells can be extremely hazardous and precautions, such as handling of bloodborne pathogens, should always be taken while performing this procedure.