The authors thank the Midwestern University Office of Research and Sponsored Programs for their support.

1. Prepare Media for Anaerobic Culture of Various Mammalian Cell Lines
<ol>
	<li>Make the complete PS-74656 medium for an anoxic cell culture25.
	<ol>
		<li>Make the sterile nitrite stock solution (5 M; 100x) by dissolving 17.25 g of nitrite in 50 mL of distilled deionized water, then filter to sterilize it.</li>
		<li>Add 0.5 mL of the nitrite stock solution per 50 mL of low glucose DMEM (1 g/L of glucose) with 110 mg/L of L-glutamine, 584 mg/L of sodium pyruvate, and 10% fetal...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIF-1:+mediator+of+physiological+and+pathophysiological+responses+to+hypoxia.">HIF-1: mediator of physiological and pathophysiological responses to hypoxia.</a> Journal of Applied Physiology. 88 (4), 1474-1480 (2000).</li><li>Biedermann, L., Rogler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intestinal+microbiota:+its+role+in+health+and+disease.">The intestinal microbiota: its role in health and disease.</a> European Journal of Pediatrics. 174 (2), 151-167 (2015).</li><li>Cui, X. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+influences+stem+cell-like+properties+in+multidrug+resistant+K562+leukemic+cells.">Hypoxia influences stem cell-like properties in multidrug resistant K562 leukemic cells.</a> Blood Cells, Molecules, and Diseases. 51 (3), 177-184 (2013).</li><li>Richter, A., Sanford, K. K., Evans, V. 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J. C., Paku, S., T&#243;v&#225;ri, J., T&#237;m&#225;r, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+Vascularization+Mechanisms+in+Cancer.">Alternative Vascularization Mechanisms in Cancer.</a> The American Journal of Pathology. 170 (1), 1-15 (2007).</li><li>Jewell, U. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+HIF-1&#945;+in+response+to+hypoxia+is+instantaneous.">Induction of HIF-1&#945; in response to hypoxia is instantaneous.</a> The FASEB Journal. 15 (7), 1312-1314 (2001).</li><li>Lee, J. W., Bae, S. H., Jeong, J. W., Kim, S. H., Kim, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-inducible+factor+(HIF-1)alpha:+its+protein+stability+and+biological+functions.">Hypoxia-inducible factor (HIF-1)alpha: its protein stability and biological functions.</a> Experimental &amp; Molecular Medicine. 36 (1), 1-12 (2004).</li><li>Pagoulatos, D., Pharmakakis, N., Lakoumentas, J., Assimakopoulou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Etaypoxia-inducible+factor-1alpha,+von+Hippel-Lindau+protein,+and+heat+shock+protein+expression+in+ophthalmic+pterygium+and+normal+conjunctiva.">Etaypoxia-inducible factor-1alpha, von Hippel-Lindau protein, and heat shock protein expression in ophthalmic pterygium and normal conjunctiva.</a> Molecular Vision. 20, 441-457 (2014).</li><li>Park, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fructose-driven+glycolysis+supports+anoxia+resistance+in+the+naked+mole-rat.">Fructose-driven glycolysis supports anoxia resistance in the naked mole-rat.</a> Science. 356 (6335), 307 (2017).</li><li>Johnston, I. A., Bernard, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+the+Ethanol+Pathway+in+Carp+Following+Exposure+to+Anoxia.">Utilization of the Ethanol Pathway in Carp Following Exposure to Anoxia.</a> Journal of Experimental Biology. 104 (1), 73 (1983).</li><li>Takahashi, E., Sato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaerobic+respiration+sustains+mitochondrial+membrane+potential+in+a+prolyl+hydroxylase+pathway-activated+cancer+cell+line+in+a+hypoxic+microenvironment.">Anaerobic respiration sustains mitochondrial membrane potential in a prolyl hydroxylase pathway-activated cancer cell line in a hypoxic microenvironment.</a> American Journal of Physiology-Cell Physiology. 306 (4), C334-C342 (2014).</li><li>Mandziuk, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+hypoxia+on+toxicity+induced+by+anticancer+agents+in+cardiomyocyte+culture.">Effect of hypoxia on toxicity induced by anticancer agents in cardiomyocyte culture.</a> Medycyna Weterynaryjna. 70 (5), 287-291 (2014).</li><li>Arai, H., Kodama, T., Igarashi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+nirQOP+genes+in+energy+conservation+during+anaerobic+growth+of+Pseudomonas+aeruginosa.">The role of the nirQOP genes in energy conservation during anaerobic growth of Pseudomonas aeruginosa.</a> Bioscience, Biotechnology, and Biochemistry. 62 (10), 1995-1999 (1998).</li><li>M&#252;ller, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemistry+and+Evolution+of+Anaerobic+Energy+Metabolism+in+Eukaryotes.">Biochemistry and Evolution of Anaerobic Energy Metabolism in Eukaryotes.</a> Microbiology and Molecular Biology Reviews. 76 (2), 444-495 (2012).</li><li>Gupta, K. J., Igamberdiev, A. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anoxic+plant+mitochondrion+as+a+nitrite:+NO+reductase.">The anoxic plant mitochondrion as a nitrite: NO reductase.</a> Mitochondrion. 11 (4), 537-543 (2011).</li><li>Gupta, K. J., Igamberdiev, A. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+Nitrogen+Species+in+Mitochondria+and+Their+Implications+in+Plant+Energy+Status+and+Hypoxic+Stress+Tolerance.">Reactive Nitrogen Species in Mitochondria and Their Implications in Plant Energy Status and Hypoxic Stress Tolerance.</a> Frontiers in Plant Science. 7 (369), (2016).</li><li>Kozlov, A. V., Staniek, K., Nohl, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitrite+reductase+activity+is+a+novel+function+of+mammalian+mitochondria.">Nitrite reductase activity is a novel function of mammalian mitochondria.</a> FEBS letters. 454 (1-2), 127-130 (1999).</li><li>Nohl, H., Staniek, K., Kozlov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+existence+and+significance+of+a+mitochondrial+nitrite+reductase.">The existence and significance of a mitochondrial nitrite reductase.</a> Redox Report. 10 (6), 281-286 (2005).</li><li>Lawson, J. C., Blatch, G. L., Edkins, A. 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The authors have nothing to disclose.

This method represents first-time mammalian cells that were cultured for extended periods of time in the absence of oxygen. Based on current observations, the anoxic growth capability via a non-fermentative pathway appears to be universal amongst mammalian cells lines28, where the anaerobic growth resulted in phenotype divergence. This was observed for all cell lines tested. With the anaerobic cultivation, increasing proportions of the cells became rounded, developed a suspension-like pop...

Cells from solid tumors, stem cell niches, and those lining the mucosal surfaces exist in environments that experience reduced oxygen levels, including anoxia1,2,3,4.&#160;In normal physiologic states, oxygenation varies beyond that of hypoxia to anoxia (the complete absence of oxygen)5,6.The realization that atmospheric oxygen adversely affects mammalian cell replication and that in vitro cell growth can be optimized under depleted oxygen conditions was reported in the early 1970s.&#160;Richter et al.7 showed that 1&#8211;3% of oxygen levels (hypoxia) enhanced plating efficiency as compared to atmospheric oxygen (20%). The human diploid cell lifespan is also extended in the hypoxic culture conditions8.
In vivo, hypoxic conditions occur when oxygen stores are depleted (e.g.,&#160;during intense exercise), wherein the ATP production is switched in the skeletal muscle from aerobic respiration to fermentation (anaerobic respiration) with the end-product of lactic acid9. Pathologically, in cancerous tumors, the interior of the tumor mass is hypoxic to anoxic due to poor vascularization10. The effect of the limited perfusion on tumor interiors is independently validated by tumor interiors colonized by obligate anaerobes1. Mechanistically, tumor cell survival in hypoxia is thought to be solely dependent on the expression of the hypoxia-inducible factor 1-alpha gene (HIF1-alpha), which is the initial spontaneous response to hypoxia4,11,12. HIF1-alpha is induced under hypoxic conditions by heat shock proteins that bind the HIF1-alpha promoter and upregulate the gene transcription12. These heat shock proteins are believed to aid in the induction of the various phenotypes seen in the tumor hypoxic microenvironment. These phenotypes exhibit an increased expression of the cell membrane glucose transporters and the rate of glycolysis (the Warburg effect)13. The result is a switch from mitochondrial oxidative phosphorylation to lactate fermentation.
Anoxic survival can also utilize alternatives to glucose to support the survival phenomenon14,15. The best studied mammalian example is the mole rat, which can survive for nearly 20 min without oxygen through a fructose-driven glycolytic fermentation pathway14. An alternative adaptation occurs in certain fish (e.g., carp [Carassius sp.], which can survive for significantly longer time periods using glycolysis with ethanol as the terminal by-product)15. In both cases, fermentation drives the metabolism enabling the survival in the absence of oxygen. The current hypothesis for anoxic survival is that so long as HIF1-alpha is activated during hypoxia, mitochondrial respiration, without the need for oxygen, occurs under anaerobic conditions16. Furthermore, it is postulated that the use of a fermentative pathway for hypoxic/anoxic survival enhances tumor survival since the cells avoid oxidative stress which could prove to be detrimental to the cell survival17. This postulate is supported by a recent study which shows that in cardiomyocytes, hypoxia reduces the oxidative stress placed on the tumor cell17.
To date, the essential nature of a fermentative pathway for anoxic mammalian cell survival has been ingrained in the literature, due, in large part, to an inability to culture mammalian cells in the complete absence of oxygen for more than 3 days. However, an alternative to glycolysis for the anaerobic survival occurs in bacteria. In certain bacteria, nitrogen or sulfate (among other compounds) can serve as terminal electron acceptors for the cytochrome oxidase system in the absence of oxygen18. Although the bacterial and eukaryotic evolution occurred in parallel since diverging from the last universal common ancestor, it is estimated that mitochondria were providing energy to cells for 1.542 million years before the oxygenation of the oceans19. Since researchers have shown that the isolated mitochondria can produce ATP in the absence of oxygen, with nitrite as the terminal electron acceptor, it is reasonable to assume that cells could function for periods of time longer than 3 days in the absence of oxygen20,21,22,23. The methodology described in this study has a utility for the anaerobic mammalian cell growth of numerous cell lines.

<table border="0" cellpadding="0" cellspacing="0" style="width:709px;" width="709">
	<tbody>
		<tr height="147">
			<td height="147" style="height:147px;width:255px;">Whitney A35 anaerobic chamber</td>
			<td style="width:149px;">Don Whitley Scientific&#160;</td>
			<td style="width:139px;">Microbiology International</td>
			<td style="width:167px;">The ability to remove the front of the chamber makes it easy to put instruments inside without concern about getting them through the portals.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">CO2 incubator</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:139px;">3531</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tissue Culture Hood</td>
			<td style="width:149px;">Labconco</td>
			<td style="width:139px;">DO55735</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">pipet aid</td>
			<td style="width:149px;">Drummond</td>
			<td style="width:139px;">4-000-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">sterile transfer pipets</td>
			<td style="width:149px;">Santa Cruz</td>
			<td style="width:139px;">sc-358867</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">50cc sterile conical centrifuge tubes</td>
			<td style="width:149px;">DOT</td>
			<td style="width:139px;">451-PG</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">vaccum jar</td>
			<td style="width:149px;">Nalgene 111410862889</td>
			<td style="width:139px;">BTA-Mall 5311-0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">DMEM high glucose (4.5 g/L)</td>
			<td style="width:149px;">CellGro</td>
			<td style="width:139px;">10-017-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">DMEM low glucose (1 g/L)</td>
			<td style="width:149px;">CellGro</td>
			<td style="width:139px;">10-014-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">FBS</td>
			<td style="width:149px;">VWR</td>
			<td style="width:139px;">1500-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">HBSS</td>
			<td style="width:149px;">VWR</td>
			<td style="width:139px;">20-021-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">trypsin</td>
			<td style="width:149px;">VWR</td>
			<td style="width:139px;">25-052-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">gentamicin</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:139px;">15750-060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">sterile pipets</td>
			<td style="width:149px;">CellTreat</td>
			<td style="width:139px;">229210B, 229205B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tissue culture flask (T75 or T150)</td>
			<td style="width:149px;">Santa Cruz</td>
			<td>sc-200263, sc-200264</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">24 well tissue culture treated dishes</td>
			<td style="width:149px;">DOT</td>
			<td style="width:139px;">667124</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">glad ziplock sandwich bags</td>
			<td style="width:149px;">Ziploc</td>
			<td style="width:139px;">Costco</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">inverted phase microscope (10, 20 40x objectives with camera mont)</td>
			<td style="width:149px;">Nikon Eclipse</td>
			<td style="width:139px;">TS100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">trypan blue</td>
			<td style="width:149px;">Invitrogen</td>
			<td style="width:139px;">T10282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">hemocytometer</td>
			<td style="width:149px;">Invitrogen</td>
			<td style="width:139px;">C10283</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Countess Automated Cell Counter</td>
			<td style="width:149px;">Life Technologies</td>
			<td style="width:139px;">AMQAF1000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Rainin microtiter pipets</td>
			<td style="width:149px;">Mettler Toledo</td>
			<td style="width:139px;">Rainin Classic Pipette PR-100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">microtiter tips</td>
			<td style="width:149px;">Santa Cruz Biotechnology</td>
			<td style="width:139px;">sc-213233 &#38; sc-201717&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">test tube rack (50cc tubes)</td>
			<td style="width:149px;">The Lab Depot</td>
			<td>HS29050A</td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:255px;">sodium nitrite</td>
			<td style="width:149px;">Sigma</td>
			<td style="width:139px;">https://www.sigmaaldrich.com/catalog/product/sigald/237213?lang=en&#38;region=US</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">clear boxes with lids</td>
			<td style="width:149px;">Rosti Mepal</td>
			<td style="width:139px;">Rubber maid</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">paper towels</td>
			<td style="width:149px;">In House</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">cell scraper</td>
			<td style="width:149px;">CellTreat</td>
			<td style="width:139px;">229310</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">PBS</td>
			<td style="width:149px;">In house prepared</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">70% Ethanol</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:139px;">64-17-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">microcentrifuge tubes sterile</td>
			<td style="width:149px;">Santa Cruz</td>
			<td style="width:139px;">sc-200273</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">biohazard bags with holder (desktop)</td>
			<td style="width:149px;">Heathrow Scientific</td>
			<td style="width:139px;">HS10320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Nitrile Gloves</td>
			<td style="width:149px;">VWR</td>
			<td style="width:139px;">89428</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">oxygen electrode</td>
			<td style="width:149px;">eDAQ</td>
			<td style="width:139px;">EPO354</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">pH meter</td>
			<td style="width:149px;">Jenway</td>
			<td style="width:139px;">3510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">pH paper/ pHydrion</td>
			<td style="width:149px;">Sigma Aldich</td>
			<td>Z111813</td>
			<td></td>
		</tr>
	</tbody>
</table>

anaerobic growth and maintenance of mammalian cell lines

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that the incubation in normoxic (5% CO2 in air) or hypoxic (low oxygen levels) conditions followed by an anoxic incubation (an absence of free oxygen) results in limited viability (2&#8211;3 days). A novel methodology was developed that enables an anoxic cell cultivation (for at least 17 days; the maximum time tested). The most critical aspect of this methodology is to ensure that no oxygen is introduced into the system. This is obtained by the degassing of media, and by flushing tubes, dishes, flasks, and pipettes with an anaerobic gas mixture (H2, CO2, N2) followed by permitting the materials to equilibrate to the anoxic (non-oxygen) environment prior to usage.&#160;Additional care must be exercised when acquiring photomicrographs to ensure that the micrographs obtained do not contain artifacts. In the absence of oxygen, cell morphology is significantly altered. Two distinct morphotypes are noted for all anaerobically-grown cells. The ability to grow and maintain mammalian cells in the absence of oxygen can be applied to the analysis of cell physiology, polymicrobial interactions, and the characterization of biosynthetic pathways for novel cancer drug development.

The strength of this protocol lies in its support of the longevity and the growth of multiple cell lines and in the recognition that there are a profound alteration and divergence in the cell morphology25. The most critical element of this study is the transfer and maintenance of the cells under strict anaerobiosis. This requires an anaerobic chamber organized to maximize the protocol (Figure 1) and the assurance that the cells removed...

Watch this Scientific Journal Video about Anaerobic Growth and Maintenance of Mammalian Cell Lines at JoVE.com

Anaerobic Growth and Maintenance of Mammalian Cell Lines

Here, we describe a new method that enables the anaerobic long-term cultivation of established cell lines. The maximum survival time that was tested is 17 days. This method is suitable for the testing of cytotoxic agents and exploring the physiology of anoxically replicating cells.

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that ...

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.

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9.9K vistas.  Midwestern University. 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-o...