Name: anaerobic growth and maintenance of mammalian cell lines
Uploaded: 2018-07-21T15:00:00.000+00:00
Duration: 7 min 15 s
Description: Watch this Scientific Journal Video about Anaerobic Growth and Maintenance of Mammalian Cell Lines at JoVE.com

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 bovine serum (FBS; heat-inactivated). 
		NOTE: The use of FBS concentrations greater than 10% are toxic. Of note, one cancer cell line tested had a low tolerance for media with nitrite (MDA-MB-231, breast cancer cell line) and, thus, was grown in the absence of nitrite. In addition, although PS-74656 medium supported the growth of all cell lines that were tested (n = 9), certain cell lines (e.g., Vero cells) exhibited higher cell concentrations and a higher viability in PS-74656 with high glucose (4.5 g/L) DMEM25.</li>
		<li>Place 20 mL of medium in a sterile 50 mL conical centrifuge tube. Do not tighten the cap; it should be loose.</li>
		<li>De-gas the medium by placing the tube in a vacuum bell jar at an approximately 45&#176; angle, to maximize the medium surface area.</li>
		<li>Apply a vacuum using a vacuum pump, or its equivalent, at room temperature. Maintain the vacuum until bubbles are no longer observed (24&#8211;48 h).</li>
		<li>Remove the tube from the jar and immediately close the cap, sealing the tube to minimize the introduction of air into the medium.</li>
		<li>Place the tube in the commercial anaerobic chamber.</li>
		<li>Loosen the tube cap and allow the atmosphere in the tube to equilibrate with that of the anaerobic gas mixture in the chamber for at least 24 h. 
		NOTE: The medium should remain in the chamber until it is used up. For speeding up the process, flush the tube at least 3x with the anoxic gas mixture using a sterile transfer pipette filled with anoxic gas, which consists of H2, CO2, and N2. </li>
		<li>Degas all tubes (10&#8211;15 min) placed in the anaerobic chamber as described above before sealing the tube and placing in the chamber. Before use, flush it with the anaerobic gas mixture using 1.5&#8211;2 mL transfer pipettes or pipette tips that have been flushed with anoxic gas at least 3x.</li>
		<li>While in the anaerobic chamber, remove 100 &#181;L of the medium to a sterile degassed micro-centrifuge tube and test it for the dissolved oxygen using an oxygen probe within the chamber. 
		NOTE: The oxygen electrode is calibrated per the manufacturer&#39;s protocol prior to use. Readings of the properly degassed and equilibrated anaerobic gas mixture media are 0. 
		CAUTION: If the readings are above 0.3 ppm of oxygen, continue to incubate the media in the anaerobic chamber since more time to equilibrate the medium is required.</li>
	</ol>
	</li>
</ol>
2. Anoxic Cultivation of Various Mammalian Cell Lines
<ol>
	<li>Designated day -1
	<ol>
		<li>Grow cells (37 &#176;C, 5% CO2) in a T150 flask to 90% confluence for the anoxic and normoxic culture control to provide a sufficient number of cells for three 24-well plates (80% confluence). 
		NOTE: For this study, HeLa 229 and Vero cells were used. This protocol was also tested and proved successful for the growth and maintenance of seven other cell lines25.</li>
		<li>Remove the medium from the flask by aspiration and wash it with 10 mL of HBSS.</li>
		<li>Replace the medium with 5 mL of trypsin. Agitate the flask until the cells visually detach from the plastic. Aspirate most of the trypsin, then tap the flask to dislodge the adherent cells. Add 10&#8211;15 mL of high glucose DMEM (4.5 g/L of glucose, 110 mg/L of L-glutamine, 10% FBS, 50 &#181;g/mL of gentamicin) to inactivate the trypsin.</li>
		<li>Triturate the cells using a 25 mL pipette until all cell clumps are disrupted.</li>
		<li>Count the cells and determine the viability using the standard hemocytometer and the trypan blue dye exclusion method or the automated equivalent.</li>
		<li>Suspend the cells in the high glucose DMEM as mentioned above to a cell density of 2.24 x 105 cells/mL.</li>
		<li>Add 1 mL of the cell suspension to the wells of a 24-well tissue culture plate (2.24 x 105 cells/well; 80% confluence). Seed 1 plate for the anoxic culture and another for a control normoxic culture.</li>
		<li>Incubate the cells at 37 &#176;C in normoxic conditions (5% CO2) for 24 h. 
		NOTE: The cells can be plated in various well-sized plates. However, seeding the cells for anoxic growth in flasks runs the risk of the introduction of oxygen into the system, unless care is exercised to completely flush out atmospheric gas.</li>
	</ol>
	</li>
	<li>Designated day 0
	<ol>
		<li>After 24 h of the normoxic incubation, place the plates for the anoxic growth into the anaerobic chamber (a commercial anaerobic chamber; Figure 1).</li>
		<li>Immediately change the medium to antibiotic-free de-gassed PS-74656 medium which has been acclimated for 24 h to the anaerobic chamber. Place the plate in a container with a damp towel and loosely close it to maintain the moisture. 
		NOTE: The plates for normoxic growth are handled identically with the exception that all the treatments are done under atmospheric conditions.</li>
	</ol>
	</li>
	<li>Designated Day 1
	<ol>
		<li>Day 1 of the anaerobic incubation starts after 24 h of the incubation in the commercial anaerobic chamber which contains the standard anaerobic gas mixture of 10% H2, 10% CO2, and 80% N2.</li>
		<li>Incubate the cells for various time periods.</li>
		<li>Monitor the anaerobic medium for the color change over time. 
		NOTE: A color shift from red-orange to magenta signals the time to change the medium. This can take up to 2 weeks to occur. A color shift in the medium from red-orange to yellow indicates the presence of a bacterial contamination.</li>
		<li>When the medium changes from red to magenta, remove the runagate cells by aspiration.</li>
		<li>Place the aspirated spent media with the runagate cells in degassed and anaerobic gas equilibrated microfuge tubes, close the tubes while making sure that the seal is secure, and then centrifuge them (326 x g; at room temperature for 10 min). Immediately replace the aspirated media in the well with 1 mL of new degassed PS-74656 medium.</li>
		<li>Place the microfuge tube with pelleted cells in the anaerobic chamber prior to opening it. Aspirate the spent medium and replace it with fresh degassed PS-74656 medium to a total volume of 1 ml in the microfuge tube.</li>
		<li>Return the cells from the tube to a well in the 24-well tissue culture plate.</li>
	</ol>
	</li>
</ol>
3. Assessment of Phenotypic Cell Differentiation by Microscopy of Anaerobically Cultured Cells
<ol>
	<li>While in the anaerobic chamber, place the plate in a resealable box flushed with the anaerobic gas mixture and close the box. 
	NOTE: The box must contain a damp paper towel to provide moisture and prevent the plate from drying out. For microscopy, sandwich bags work the best since they are thin.
	<ol>
		<li>To observe the cells under the microscope, completely seal the bag and remove it from the chamber.</li>
		<li>Carefully stretch the plastic bag over the plate, examine cells using an inverted phase contrast microscope with the camera attachment and take photomicrographs at various magnifications.</li>
		<li>Return the plate in the sealed bag to the chamber, and continue the incubation as described in step 2.3.3.</li>
		<li>To separately analyze the runagate cells that are in suspension, follow the steps outlined in step 2.3.5. 
		NOTE: These cells can then be used for aerobic testing or can be returned to the anaerobic chamber for the testing of this specific population.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Cummins, J., Tangney, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteria+and+tumours:+causative+agents+or+opportunistic+inhabitants?.">Bacteria and tumours: causative agents or opportunistic inhabitants?.</a> Infectious Agents and Cancer. 8 (1), 11 (2013).</li><li>Liao, 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=Enhanced+efficiency+of+generating+induced+pluripotent+stem+(iPS)+cells+from+human+somatic+cells+by+a+combination+of+six+transcription+factors.">Enhanced efficiency of generating induced pluripotent stem (iPS) cells from human somatic cells by a combination of six transcription factors.</a> Cell Research. 18 (5), 600-603 (2008).</li><li>Park, I. H., Lerou, P. H., Zhao, R., Huo, H., Daley, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human-induced+pluripotent+stem+cells.">Generation of human-induced pluripotent stem cells.</a> Nature Protocols. 3 (7), 1180-1186 (2008).</li><li>Semenza, G. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+Oxygen+and+Culture+Media+on+Plating+Efficiency+of+Some+Mammalian+Tissue+Cells.">Influence of Oxygen and Culture Media on Plating Efficiency of Some Mammalian Tissue Cells.</a> JNCI: Journal of the National Cancer Institute. 49 (6), 1705-1712 (1972).</li><li>Packer, L., Fuehr, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+oxygen+concentration+extends+the+lifespan+of+cultured+human+diploid+cells.">Low oxygen concentration extends the lifespan of cultured human diploid cells.</a> Nature. 267, 423 (1977).</li><li>Warburg, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+origin+of+cancer.">On the origin of cancer.</a> Science. 123 (3191), 309-314 (1956).</li><li>D&#246;me, B., Hendrix, M. 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. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells+in+breast+cancer+and+metastasis.">Cancer stem cells in breast cancer and metastasis.</a> Breast Cancer Research and Treatment. 118 (2), 241-254 (2009).</li><li>Plotkin, B. 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=A+method+for+the+long-term+cultivation+of+mammalian+cells+in+the+absence+of+oxygen:+Characterization+of+cell+replication,+hypoxia-inducible+factor+expression+and+reactive+oxygen+species+production.">A method for the long-term cultivation of mammalian cells in the absence of oxygen: Characterization of cell replication, hypoxia-inducible factor expression and reactive oxygen species production.</a> Tissue and Cell. 50 (Supplement C), 59-68 (2018).</li><li>Sarmento, B., 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=Cell-based+in+vitro+models+for+predicting+drug+permeability.">Cell-based in vitro models for predicting drug permeability.</a> Expert Opinion on Drug Metabolism &amp; Toxicology. 8 (5), 607-621 (2012).</li><li>Collins, S. J., Gallo, R. C., Gallagher, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+growth+and+differentiation+of+human+myeloid+leukaemic+cells+in+suspension+culture.">Continuous growth and differentiation of human myeloid leukaemic cells in suspension culture.</a> Nature. 270 (5635), 347-349 (1977).</li><li>Plotkin, B. 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=A+method+for+the+long-term+cultivation+of+mammalian+cells+in+the+absence+of+oxygen:+Characterization+of+cell+replication,+hypoxia-inducible+factor+expression+and+reactive+oxygen+species+production.">A method for the long-term cultivation of mammalian cells in the absence of oxygen: Characterization of cell replication, hypoxia-inducible factor expression and reactive oxygen species production.</a> Tissue and Cell. 50, 59-68 (2018).</li></ol>

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 population, and were able to replicate. The secondary cell type remained attached to the substrate; however, the cell morphology reverted to a spindle (dendritic) shape. These findings point out a common error in analyses and observations with regard to anoxic tissue culture cultivation. It is generally accepted that when mammalian cells grown under normoxic conditions (5% CO2) round up and detach, they are dying. The contrary appears to be correct for cells grown in the absence of oxygen, in which case, cells that have rounded up and detached, are simply a morphologically divergent cell subpopulation. 
The optimization of anoxic growth conditions is cell line dependent. Medium PS-74656 overall supported the growth of all cell lines under normoxic and anoxic growth conditions, with the exception of MDA-MB-231, a breast cancer cell line which preferred growth in the absence of nitrite. An encouraging observation is that even with a 98% die-off of cells per well, the remainder replicates, resulting in a highly adapted population. The critical action is to maintain cultures even when only a few cells are directly observed by microscopy. Thus, care must be exercised to ensure that the detached cells are dead, either by trypan blue dye exclusion or by an alternative cell viability detection method. It should be noted that with such a large decrease in cell numbers, sufficient wells must be seeded to have adequate total cell numbers for subsequent experiments. The expected loss of viable cells is both cell line and seeding confluence dependent; thus, optimal cell numbers, over time, must be determined experimentally and cannot be extrapolated from findings with other cell lines. That said, a good starting point is an 80% confluence, which has the greatest chance of optimal growth. It must be noted that any exposure to oxygen during the anaerobic steps in the procedure, no matter how brief, will compromise the findings due to the reported spontaneous cellular HIF-1-alpha response11,12. 
For this procedure, the most critical step is that of the de-gassing of the medium, tubes, and all other apparatuses and materials used. The introduction of even very low levels of oxygen could stop the transition from the fermentation metabolism to the anaerobic respiration. Alternatively, it could prolong the fermentative metabolism so that any experiments result in the measurement of hypoxia, not anoxia. The use of this novel procedure to measure cytotoxicity and chemotherapeutic activity could help to explain drug failure in the treatment of solid tumors.

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><tbody><tr><td>Whitney A35 anaerobic chamber</td><td>Don Whitley Scientific&amp;nbsp;</td><td>Microbiology International</td><td>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><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Fisher Scientific</td><td>3531</td><td></td></tr><tr><td>Tissue Culture Hood</td><td>Labconco</td><td>DO55735</td><td></td></tr><tr><td>pipet aid</td><td>Drummond</td><td>4-000-100</td><td></td></tr><tr><td>sterile transfer pipets</td><td>Santa Cruz</td><td>sc-358867</td><td></td></tr><tr><td>50 cc sterile conical centrifuge tubes</td><td>DOT</td><td>451-PG</td><td></td></tr><tr><td>vaccum jar</td><td>Nalgene 111410862889</td><td>BTA-Mall 5311-0250</td><td></td></tr><tr><td>DMEM high glucose (4.5 g/L)</td><td>CellGro</td><td>10-017-CM</td><td></td></tr><tr><td>DMEM low glucose (1 g/L)</td><td>CellGro</td><td>10-014-CV</td><td></td></tr><tr><td>FBS</td><td>VWR</td><td>1500-500</td><td></td></tr><tr><td>HBSS</td><td>VWR</td><td>20-021-CV</td><td></td></tr><tr><td>trypsin</td><td>VWR</td><td>25-052-CI</td><td></td></tr><tr><td>gentamicin</td><td>Gibco</td><td>15750-060</td><td></td></tr><tr><td>sterile pipets</td><td>CellTreat</td><td>229210B, 229205B</td><td></td></tr><tr><td>Tissue culture flask (T75 or T150)</td><td>Santa Cruz</td><td>sc-200263, sc-200264</td><td></td></tr><tr><td>24 well tissue culture treated dishes</td><td>DOT</td><td>667124</td><td></td></tr><tr><td>glad ziplock sandwich bags</td><td>Ziploc</td><td>Costco</td><td></td></tr><tr><td>inverted phase microscope (10, 20 40x objectives with camera mont)</td><td>Nikon Eclipse</td><td>TS100</td><td></td></tr><tr><td>trypan blue</td><td>Invitrogen</td><td>T10282</td><td></td></tr><tr><td>hemocytometer</td><td>Invitrogen</td><td>C10283</td><td></td></tr><tr><td>Countess Automated Cell Counter</td><td>Life Technologies</td><td>AMQAF1000</td><td></td></tr><tr><td>Rainin microtiter pipets</td><td>Mettler Toledo</td><td>Rainin Classic Pipette PR-100</td><td></td></tr><tr><td>microtiter tips</td><td>Santa Cruz Biotechnology</td><td>sc-213233 &amp;amp; sc-201717&amp;nbsp;</td><td></td></tr><tr><td>test tube rack (50cc tubes)</td><td>The Lab Depot</td><td>HS29050A</td><td></td></tr><tr><td>sodium nitrite</td><td>Sigma</td><td>https://www.sigmaaldrich.com/catalog/product/sigald/237213?lang=en&amp;amp;region=US</td><td></td></tr><tr><td>clear boxes with lids</td><td>Rosti Mepal</td><td>Rubber maid</td><td></td></tr><tr><td>paper towels</td><td>In House</td><td></td><td></td></tr><tr><td>cell scraper</td><td>CellTreat</td><td>229310</td><td></td></tr><tr><td>PBS</td><td>In house prepared</td><td></td><td></td></tr><tr><td>70% Ethanol</td><td>Fisher Scientific</td><td>64-17-5</td><td></td></tr><tr><td>microcentrifuge tubes sterile</td><td>Santa Cruz</td><td>sc-200273</td><td></td></tr><tr><td>biohazard bags with holder (desktop)</td><td>Heathrow Scientific</td><td>HS10320</td><td></td></tr><tr><td>Nitrile Gloves</td><td>VWR</td><td>89428</td><td></td></tr><tr><td>oxygen electrode</td><td>eDAQ</td><td>EPO354</td><td></td></tr><tr><td>pH meter</td><td>Jenway</td><td>3510</td><td></td></tr><tr><td>pH paper/ pHydrion</td><td>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 for either microscopy or other testing are kept in a sealed environment. To date, the range of anoxic survival was 15&#8211;17 days for immortal cancer cell lines and up to 17 days for transformed cell lines (nine cell lines screened for anoxic growth). The levels of viable cells, once these cells were moved to anoxic conditions, declined at least 10%, to a maximum of 90%, before they stabilized. All cell lines reverted to the dual phenotypes (tethered and runagate) with the proportion of cells in the suspension increasing over time. Even after an extended incubation of several weeks, there remained some tethered cells, but the majority of the cells were in the rounded runagate morphology (Figure 2). As reported, these runagate cells are viable and proceeded through their cell cycle. It should be noted that the variables that may need to be adjusted include the glucose levels and the nitrite concentration. That said, from the over 60 different media formulations that were tested for their ability to support both normoxic and anoxic incubation, the PS-74656 medium was able to support the survival and replication of nearly all the cell lines tested.
An essential observation was the differentiation of anoxic cells, regardless of the cell tested, into two distinct phenotypes, tethered and runagate (Figure 2). Analogous to the normoxic monolayer cells, the tethered cells were attached to the tissue culture plastic. However, the attachment was weaker, easier to trypsinize or dislodge by scraping, than the attachment that occurred under the normoxic conditions. In addition, the cell morphology was smaller with more spindle (dendritic) extensions26. The second cellular morphotype observed were cells in suspension (i.e., runagate cells). These cells spontaneously converted into free-floating single cell suspensions in the presence of tissue culture-treated plastic, which were rounded with a smaller cell size and less cytoplasm. They differed from the classical suspension culture cells, which typically require an enzymatic adaptation, grow in aggregates, require non-tissue culture-treated containers, and are larger than their normoxic counterparts27. Over time, the anoxic cell populations could be observed to shift from a predominantly tethered monolayer to that of the runagate cells in suspension. These cells in suspension were viable and replicating; thus, care had to be exercised should the supernatant be removed. Also, of note is that the medium did not require changing over the entire anaerobic incubation period. The medium pH indicator under the anoxic incubation, in contrast to its normoxic control, was also unchanged (i.e., remained red). Should the investigator wish to harvest the runagate cells, or change the medium, the cells can be obtained using anaerobic gas-flushed micro-centrifuge tubes that are closed tightly within the anaerobic chamber. For the cell washing of tethered cells, the procedure must also be performed in the chamber to eliminate any oxygen exposure.
The final cell viability for a long-term cultivation was, to a great extent, dependent on the cell being used and the confluence to which the wells or the flask were initially seeded (Figures 3 and 4). That said, a good starting point is 80%, which had the greatest chance of optimal growth. In addition, a cell line-dependent loss of 10% to 60% of viability can occur in the initial 2&#8211;4 days. However, during the performance of this experiment, the remaining cells cycled through the cell cycle and replicated28. The seeding confluence effect on the longevity of the cell cultivation can be seen in Figures 3 and 4. Both HeLa 229 and Vero cells were seeded in 24-well plates to 40%, 60%, and 80% of initial confluence (100% confluence = 2.8 x 105 cells/well) and then incubated in the absence of oxygen for 17 days. The HeLa 229 cell anoxic growth was optimized at an initial seeding confluence of 80%. In contrast, the Vero cells optimal seeding confluence was 60%. This demonstrates the need to initially optimize the seeding density dependent on the cell line used.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58049/58049fig1.jpg" /> 
Figure 1: Model of anaerobic chamber set-up for culture of mammalian cells. The minimal materials required include tissue culture plates, sterile transfer pipets, biohazard bags, de-gassed media, a tube for the discarding of media or other liquids, and resealable bags for the transfer of the plates to the microscope while maintaining anaerobic conditions.&#160;
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58049/58049fig2.jpg" /> 
Figure 2: Anoxic HeLa cells morphologic differentiation. (A) This panel shows the phase contrast microscopy of the normoxic HeLa cell controls 4 days post-inoculation, with a 20X magnification. (B) This panel shows the anoxic HeLa cells 17 days post-inoculation (with the unchanged from day 1 PS-74656 medium, 80% seeded confluence, and a 20X magnification). (C) This panel shows microscopy artifacts of water droplets on the interior of a sealed plastic bag containing a 24-well anoxic plate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58049/58049fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58049/58049fig3.jpg" /> 
Figure 3: Ratio of anoxic to normoxic HeLa 229 viable cells post-17 days incubation. This panel shows the effect of the seeding density on the HeLa 229 cell viability in the PS-74656 medium as compared to the normoxic control. The results are shown as the mean &#177; SEM.&#160;
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58049/58049fig4.jpg" /> 
Figure 4: Ratio of anoxic to normoxic Vero viable cells 17 days post-incubation. This panel shows the effect of the seeding density on the Vero cell viability in the PS-74656 medium as compared to the normoxic control. The results are shown as the mean &#177; SEM.

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 ...

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10.0K Views.  Midwestern University. Hier beschrijven we een nieuwe methode waarmee de anaërobe op lange termijn teelt van bestaande cellijnen. De maximale overlevingstijd die werd getest is 17 dagen. Deze methode is geschikt voor het testen van cytotoxische agenten en het verkennen van de Fysiologie van de anoxically cellen te repliceren.

Video: Anaerobic Growth and Maintenance of Mammalian Cell Lines

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

Medicine

FIBS-enabled Noninvasive Metabolic Profiling

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be validated for predictive purposes. Ideally such systems would be low volume, which precludes sampling and destructive analyses when time course data are to be obtained. What is needed is an in situ monitoring tool which can report the necessary information in real-time and noninvasively. An interesting option is the use of fluorescent, protein-based in vivo biological sensors as reporters of intracellular concentrations. One particular class of in vivo biosensors that has found applications in metabolite quantification is based on F&#246;rster Resonance Energy Transfer (FRET) between two fluorescent proteins connected by a ligand binding domain. FRET integrated biological sensors (FIBS) are constitutively produced within the cell line, they have fast response times and their spectral characteristics change based on the concentration of metabolite within the cell. In this paper, the method for constructing Chinese hamster ovary (CHO) cell lines that constitutively express a FIBS for glucose and glutamine and calibrating the FIBS in vivo in batch cell culture in order to enable future quantification of intracellular metabolite concentration is described. Data from fed-batch CHO cell cultures demonstrates that the FIBS was able in each case to detect the resulting change in the intracellular concentration. Using the fluorescent signal from the FIBS and the previously constructed calibration curve, the intracellular concentration was accurately determined as confirmed by an independent enzymatic assay.

A description of how to calibrate F&#246;rster Resonance Energy Transfer integrated biological sensors (FIBS) for in situ metabolic profiling is presented.&#160;The FIBS can be used to measure intracellular levels of metabolites noninvasively aiding in the development of metabolic models and high throughput screening of bioprocess conditions.

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

Bioengineering

Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in proteins and generating redox stress. Pathological conditions that lead to metal depletion or accumulation are causal agents of different human diseases. Some examples include anemia, acrodermatitis enteropathica, and Wilson&#8217;s and Menkes&#8217; diseases. It is therefore important to be able to measure the levels and transport of transition metals in biological samples with high sensitivity and accuracy in order to facilitate research exploring how these elements contribute to normal physiological functions and toxicity. Zinc (Zn), for example, is a cofactor in many mammalian proteins, participates in signaling events, and is a secondary messenger in cells. In excess, Zn is toxic and can inhibit absorption of other metals, while in deficit, it can lead to a variety of potentially lethal conditions.

Graphite furnace atomic absorption spectroscopy (GF-AAS) provides a highly sensitive and effective method for determining Zn and other transition metal concentrations in diverse biological samples. Electrothermal atomization via GF-AAS quantifies metals by atomizing small volumes of samples for subsequent selective absorption analysis using wavelength of excitation of the element of interest. Within the limits of linearity of the Beer-Lambert Law, the absorbance of light by the metal is directly proportional to concentration of the analyte. Compared to other methods of determining Zn content, GF-AAS detects both free and complexed Zn in proteins and possibly in small intracellular molecules with high sensitivity in small sample volumes. Moreover, GF-AAS is also more readily accessible than inductively coupled plasma mass spectrometry (ICP-MS) or synchrotron-based X-ray fluorescence. In this method, the systematic sample preparation of different cultured cell lines for analyses in a GF-AAS is described. Variations in this trace element were compared in both whole cell lysates and subcellular fractions of proliferating and differentiated cells as proof of principle.

Cultured primary or established cell lines are commonly used to address fundamental biological and mechanistic questions as an initial approach before using animal models. This protocol describes how to prepare whole cell extracts and subcellular fractions for studies of zinc (Zn) and other trace elements with atomic absorbance spectroscopy.

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in ...

Biochemistry

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized medicine innovations. However, translating these advances into therapeutic applications will require in vitro systems that allow for robust, flexible, and cost effective bioreactor systems. There are several bioreactor systems currently utilized in research and commercial settings; however, many of these systems are not optimal for establishing, expanding, and monitoring the growth of different cell types. The culture parameters most challenging to control in these systems include, minimizing hydrodynamic shear, preventing nutrient gradient formation, establishing uniform culture medium aeration, preventing microbial contamination, and monitoring and adjusting culture conditions in real-time. Using a pneumatic single-use bioreactor system, we demonstrate the assembly and operation of this novel bioreactor for mammalian cells grown on micro-carriers. This bioreactor system eliminates many of the challenges associated with currently available systems by minimizing hydrodynamic shear and nutrient gradient formation, and allowing for uniform culture medium aeration. Moreover, the bioreactor&#8217;s software allows for remote real-time monitoring and adjusting of the bioreactor run parameters. This bioreactor system also has tremendous potential for scale-up of adherent and suspension mammalian cells for production of a variety therapeutic proteins, monoclonal antibodies, stem cells, biosimilars, and vaccines.

Using a pneumatic bioreactor, we demonstrate the assembly, operation, and performance of this single-use bioreactor system for the growth of mammalian cells.

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized ...

Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product to demonstrate how regulating nutrient levels can improve cell yields. In previous studies, bioreactors facilitated increased culture volumes over static cultures, but no increase in cell yields were observed. Limitations in key nutrients such as glucose, which were consumed between batch feedings, can lead to limitations in cell expansion. Large fluctuations in glucose levels were observed, despite the increase in glucose concentrations in the media. The use of continuous feeding systems eliminated fluctuations in glucose levels, and improved cell growth rates when compared with batch fed static and SSB culture methods. Additional increases in growth rates were observed by adjusting the feed rate based on calculated nutrient consumption, which allowed the maintenance of physiological glucose over three weeks in culture. This method can also be adapted for other cell types.

Nutrient regulation using continuous growth adjusted feeding improves growth rates of mammalian cell spheroids compared to intermittent batch feeding for cultures in stirred suspension bioreactors. This study demonstrates the methods required for establishing simple adjusted rate fed cultures.

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product ...

Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture

The glassy-winged sharpshooter (Homalodisca vitripennis) is a highly vagile and polyphagous insect found throughout the southwestern United States. These insects are the predominant vectors of Xylella fastidiosa (X. fastidiosa), a xylem-limited bacterium that is the causal agent of Pierce&#39;s disease (PD) of grapevine. Pierce&#8217;s disease is economically damaging; thus, H. vitripennis have become a target for pathogen management strategies. A dicistrovirus identified as Homalodisca coagulata virus-01 (HoCV-01) has been associated with an increased mortality in H. vitripennis populations. Because a host cell is required for HoCV-01 replication, cell culture provides a uniform environment for targeted replication that is logistically and economically valuable for biopesticide production. In this study, a system for large-scale propagation of H. vitripennis cells via tissue culture was developed, providing a viral replication mechanism. HoCV-01 was extracted from whole body insects and used to inoculate cultured H. vitripennis cells at varying levels. The culture medium was removed every 24 hr for 168 hr, RNA extracted and analyzed with qRT-PCR. Cells were stained with trypan blue and counted to quantify cell survivability using light microscopy. Whole virus particles were extracted up to 96 hr after infection, which was the time point determined to be before total cell culture collapse occurred. Cells were also subjected to fluorescent staining and viewed using confocal microscopy to investigate viral activity on F-actin attachment and nuclei integrity. The conclusion of this study is that H. vitripennis cells are capable of being cultured and used for mass production of HoCV-01 at a suitable level to allow production of a biopesticide.

Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture

Here we present a protocol to propagate Homalodisca vitripennis cells and HoCV-1 in vitro. Medium was removed from HoCV-1 positive cultures and RNA extracted every 24 hr for 168 hr. Cell survivability was quantified by trypan blue staining. Whole virus particles were extracted post-infection. Extracted RNA was quantified by qRT-PCR.

The glassy-winged sharpshooter (Homalodisca vitripennis) is a highly vagile and polyphagous insect found throughout the southwestern United States. These ...

Immunology and Infection

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe a new strategy that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. Two main steps are required. In the first step, adult mature (terminally differentiated) cells are exposed to the epigenetic eraser 5-aza-cytidine to drive them into a pluripotent state. This part of the protocol was developed, based on the increasing understanding of the epigenetic mechanisms controlling cell fate and differentiation, and involves the use of the epigenetic modifier to erase cell differentiated state and then drive into a transient high plasticity window.
In the second step, erased cells are encapsulated in polytetrafluoroethylene (PTFE) micro-bioreactors, also known as Liquid Marbles, to promote 3D cell rearrangement to extend and stably maintain the acquired high plasticity. PTFE is a non-reactive hydrophobic synthetic compound and its use permits the creation of a cellular microenvironment, which cannot be achieved in traditional 2D culture systems. This system encourages and boosts the maintenance of pluripotency though bio-mechanosensing-related cues.
The technical procedures described here are simple strategies to allow for the induction and maintenance of a high plasticity state in adult somatic cells. The protocol allowed the derivation of high plasticity cells in all mammalian species tested. Since it does not involve the use of gene transfection and is free of viral vectors, it may represent a notable technological advance for translational medicine applications. Furthermore, the micro-bioreactor system provides a notable advancement in stem cell organoid technology by in vitro re-creating a specific micro-environment that allows for the long-term culture of high plasticity cells, namely as ESCs, iPSCs, epigenetically erased cells and MSCs.

We here present a method that combines the use of chemical epigenetic erasing with mechanosensing-related cues to efficiently generate mammalian pluripotent cells, without the need of gene transfection or retroviral vectors. This strategy is, therefore, promising for translational medicine and represents a notable advancement in stem cell organoid technology.

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe ...

Harvesting Mouse Skeletal Muscle to Culture Stem Cells and Their Niche

Unfractionated Bulk Culture of Mouse Skeletal Muscle to Recapitulate Niche and Stem Cell Quiescence

Skeletal muscle is the largest tissue of the body and performs multiple functions, from locomotion to body temperature control. Its functionality and recovery from injuries depend on a multitude of cell types and on molecular signals between the core muscle cells (myofibers, muscle stem cells) and their niche. Most experimental settings do not preserve this complex physiological microenvironment, and neither do they allow the ex vivo study of muscle stem cells in quiescence, a cell state that is crucial for them. Here, a protocol is outlined for the ex vivo culture of muscle stem cells with cellular components of their niche. Through the mechanical and enzymatic breakdown of muscles, a mixture of cell types is obtained, which is put in 2D culture. Immunostaining shows that within 1 week, multiple niche cells are present in culture alongside myofibers and, importantly, Pax7-positive cells that display the characteristics of quiescent muscle stem cells. These unique properties make this protocol a powerful tool for cell amplification and the generation of quiescent-like stem cells that can be used to address fundamental and translational questions.

Author Spotlight: Ex Vivo Protocol for Culturing Quiescent Muscle Stem Cells with Niche Components 

Skeletal muscle comprises multiple cell types, including resident stem cells, each with a special contribution to muscle homeostasis and regeneration. Here, the 2D culture of muscle stem cells and the muscle cell niche in an ex vivo setting that preserves many of the physiological, in vivo, and environmental characteristics are described.

Skeletal muscle is the largest tissue of the body and performs multiple functions, from locomotion to body temperature control. Its functionality and ...

Passaging Cells

Cell lines are frequently used in biomedical experiments, as they allow rapid culture and expansion of cell types for experimental analysis. Cell lines are cultured under similar conditions when compared to freshly-isolated, or primary, cells, but with some basic important differences: (i) cell lines require their own specific growth factor cocktails and (ii) their growth must be more closely monitored than primary cells, as the mutations that allow them to be grown indefinitely also can quickly lead to their overgrowth. Therefore, when a cell line reaches the point of growth in culture where it covers most of the bottom of the culture container, or about a 90% confluency, the cells must be resuspended, washed, used experimentally, frozen for later use, or re-seeded for further expansion in new culture containers.
This video will demonstrate how to use media indicators to determine cell culture health, which reagents and equipment are useful for safely removing adherent cell lines from culture, and various methods for transferring these robustly expanding cells into new cultures will be discussed. Also demonstrated are methods for how to culture feeder cells (important for providing essential growth factors to cell lines) and how to expand large numbers of cell line cultures at once.

Passaging Cells: Cell Lines, Subculturing Methods, and Applications

Cell lines are frequently used in biomedical experiments, as they allow rapid culture and expansion of cell types for experimental analysis. Cell lines ...

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Basic Methods in Cellular and Molecular Biology

Enrichment Cultures: Culturing Aerobic and Anaerobic Microbes on Selective and Differential Medias

Source: Christopher P. Corbo1, Jonathan F. Blaize1, Elizabeth Suter1 
1 Department of Biological Sciences, Wagner College, 1 Campus Road, Staten Island NY, 10301
Prokaryotic cells are able to inhabit nearly every environment on this planet. As a kingdom, they possess a great metabolic diversity, allowing them to use a wide variety of molecules for energy generation (1). Therefore, when cultivating these organisms in the lab, all necessary and specific molecules required to make energy must be provided in the growth media. While some organisms are metabolically diverse, others are able to survive in extreme environments such as high or low temperatures, alkaline and acidic pH, reduced or oxygen absent environments, or environments containing high salt (2,3,4). Termed &#34;extremophiles&#34;, these organisms often require these intense environments to proliferate. When scientists look to grow such organisms, the media components as well as any specific environmental conditions all need to be taken into account in order to successfully cultivate the organisms of interest.
Scientists are able to grow culturable organisms in the lab because they understand the specific requirements that those species need to grow. However, culturable organisms account for less than 1% of species estimated to be on the planet (5). Organisms that we have detected by gene sequencing but are not able to grow in the lab are considered unculturable (6). At this time, we do not know enough about the metabolism and growth conditions of these organisms to replicate their environment in the lab.
Fastidious organisms lie somewhere in between the former two. These organisms are culturable, but they require very specific growth conditions, such as specific growth media components and/or specific growth conditions. Two examples of such genera are Neisseria sp. and Haemophilus sp., both of which require partially broken-down red blood cells (also known as chocolate agar), as well as specific growth factors and an environment rich in carbon dioxide (7). Without all of the required specific components, these organisms will not grow at all. Often, even with all of their requirements, these organisms grow poorly.
Unlike eukaryotic cells, which are only able to grow in an aerobic, or oxygen containing, environment, prokaryotic cells are able to grow anaerobically using several fermentation pathways to generate ample energy (8). Other prokaryotes prefer a microaerophilic, or reduced oxygen environment, or even a capnophilic, or high carbon dioxide environment (9). These organisms are more challenging to enrich for, since the atmosphere must be altered. Scientists that frequently work with organisms sensitive to an oxygenated environment would normally work in an anaerobic chamber and incubator, where a heavy, inert gas such as argon is pumped in to displace the oxygen (10). Others make use of conventionally available sealed gas packet systems that use water to generate hydrogen and carbon dioxide, along with a catalyst like palladium to remove all atmospheric oxygen. These commercially available kits can create any of the above-mentioned atmospheric conditions (10).
Whether cultivating a pathogen to determine potential infection or looking to identify a specific species of bacteria present in a natural environment, one problem exists. No one bacterial species inhabits one habitat. Bacteria live as multicellular communities everywhere from the skin of humans to the oceans of our planet (11). When attempting to isolate one species of bacteria, scientists must work to exclude the numerous other organisms that are also inhabiting the isolated area. For this reason, enriched growth media for bacteria often carry out two functions. The first is to make the media selective. A selective agent will prevent some species from growing, while not inhibiting and often even promoting others to grow (12). The second function of media ingredients may be to work as differential agents. Such agents allow for the identification of a particular biochemical feature of an isolated organism. By pairing several different selective and differential medias along with appropriate growth conditions, scientists and diagnosticians are able to identify the presence of specific bacterial species from a particular isolate.
One example of a selective and differential media aiding in to identification is in the case of the clinically significant organism Staphylococcus aureus. This organism is typically cultured on mannitol salt agar. This media not only selects for only organisms which can live in a high salt environment, which include some gram positives like Staphylococcus, but it also inhibits any organisms sensitive to salt. The mannitol sugar is the differential component of this medium. Of all the clinically significant Staphylococcus species, only S. aureus is able to ferment mannitol. This fermentation reaction produces acid as a by-product which causes the red methyl red indicator in the media to turn yellow. Other Staphylococcus species (such as Staphylococcus epidermidis) although able to grow, will leave the media red in color.
This lab exercise demonstrates proper aseptic technique, as well as proper inoculation of growth media from broth. It also introduces the growth of common contaminant organisms on enrichment media, the use of a gas package anaerobic culture system for anaerobic bacteria, and the use of different selective and differential medias for the presumptive identification of gram positive and gram-negative bacteria.

Enrichment Cultures, Selective and Differential Media

Source: Christopher P. Corbo1, Jonathan F. Blaize1, Elizabeth Suter1
1 Department of Biological Sciences, Wagner College, 1 Campus Road, Staten Island NY, ...

Anaerobic Growth and Maintenance of Mammalian Cell Lines

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