We thank Mrs. Matsumoto for the technical assistance.

1. Cell Culture
<ol>
	<li>Obtain the cells to induce tetraploidy. To date it has been confirmed that this technique can be applied to the human fibroblast cell lines TIG-1, BJ, IMR-90 and telomerase-immortalized TIG-1 (TIG-hT).</li>
	<li>Grow cells in minimum essential medium with &#945; modification or any other cell culture medium suited for the cell type to be studied supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) by incubating in a 5% (v/v) CO2 atmosphere at 37 &#176;C. Passage cel...

<ol>
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Gastroenterol. 96 (11), 3071-3083 (2001).</li><li>Galipeau, P., 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=NSAIDs+modulate+CDKN2A,+TP53,+and+DNA+content+risk+for+progression+to+esophageal+adenocarcinoma.">NSAIDs modulate CDKN2A, TP53, and DNA content risk for progression to esophageal adenocarcinoma.</a> PLoS Med. 4 (2), e67 (2007).</li><li>Olaharski, A., 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=Tetraploidy+and+chromosomal+instability+are+early+events+during+cervical+carcinogenesis.">Tetraploidy and chromosomal instability are early events during cervical carcinogenesis.</a> Carcinogenesis. 27, 337-343 (2006).</li><li>Liu, 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=p53-independent+abrogation+of+a+postmitotic+checkpoint+contributes+to+human+papillomavirus+E6-induced+polyploidy.">p53-independent abrogation of a postmitotic checkpoint contributes to human papillomavirus E6-induced polyploidy.</a> Cancer Res. 67, 2603-2610 (2007).</li><li>Davoli, T., de Lange, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+causes+and+consequences+of+polyploidy+in+normal+development+and+cancer.">The causes and consequences of polyploidy in normal development and cancer.</a> Annu Rev Cell Dev Biol. 27, 585-610 (2011).</li><li>Fox, D., Duronio, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoreplication+and+polyploidy:+insights+into+development+and+disease.">Endoreplication and polyploidy: insights into development and disease.</a> Development. 140, 3-12 (2013).</li><li>Fujiwara, T., 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=Cytokinesis+failure+generating+tetraploids+promotes+tumorigenesis+in+p53-null+cells.">Cytokinesis failure generating tetraploids promotes tumorigenesis in p53-null cells.</a> Nature. 437, 1043-1047 (2005).</li><li>Ganem, N., 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=Cytokinesis+failure+triggers+hippo+tumor+suppressor+pathway+activation.">Cytokinesis failure triggers hippo tumor suppressor pathway activation.</a> Cell. 158 (4), 833-848 (2014).</li><li>Kuznetsova, A., 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=Chromosomal+instability,+tolerance+of+mitotic+errors+and+multidrug+resistance+are+promoted+by+tetraploidization+in+human+cells.">Chromosomal instability, tolerance of mitotic errors and multidrug resistance are promoted by tetraploidization in human cells.</a> Cell cycle. 14 (17), 2810-2820 (2015).</li><li>Vindel&#248;v, L., Christensen, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detergent+and+proteolytic+enzyme-based+techniques+for+nuclear+isolation+and+DNA+content+analysis.">Detergent and proteolytic enzyme-based techniques for nuclear isolation and DNA content analysis.</a> Methods Cell Biol. 41, 219-229 (1994).</li><li>Darzynkiewicz, Z., Juan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+content+measurement+for+DNA+ploidy+and+cell+cycle+analysis.">DNA content measurement for DNA ploidy and cell cycle analysis.</a> Curr Protoc Cytom. , Chapter 7: Unit 7.5 (2001).</li><li>Knutsen, T., Bixenman, H., Lawce, H., Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+analysis+guidelines+preliminary+report.">Chromosome analysis guidelines preliminary report.</a> Cancer Genet Cytogenet. 52 (1), 11-17 (1991).</li><li>Liehr, T., 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=Multicolor+FISH+probe+sets+and+their+applications.">Multicolor FISH probe sets and their applications.</a> Histol. Histopathol. 19 (1), 229-237 (2004).</li><li>Ohshima, S., Seyama, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+bipolar+spindles+with+two+centrosomes+in+tetraploid+cells+established+from+normal+human+fibroblasts.">Formation of bipolar spindles with two centrosomes in tetraploid cells established from normal human fibroblasts.</a> Hum. Cell. 25 (3), 78-85 (2012).</li><li>Ohshima, S., Seyama, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+proliferative+tetraploid+cells+from+normal+human+fibroblasts.">Establishment of proliferative tetraploid cells from normal human fibroblasts.</a> Front. Oncol. 3, 198 (2013).</li><li>Ohshima, S., Seyama, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+proliferative+tetraploid+cells+from+telomerase-immortalized+normal+human+fibroblasts.">Establishment of proliferative tetraploid cells from telomerase-immortalized normal human fibroblasts.</a> Genes, Chromosome Cancer. 55 (6), 522-530 (2016).</li><li>Di Leonardo, A., 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=DNA+rereplication+in+the+presence+of+mitotic+spindle+inhibitors+in+human+and+mouse+fibroblasts+lacking+either+p53+or+pRb+function.">DNA rereplication in the presence of mitotic spindle inhibitors in human and mouse fibroblasts lacking either p53 or pRb function.</a> Cancer Res. 57, 1013-1019 (1997).</li><li>Andreassen, P., Lohez, O., Lacroix, F., Margolis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetraploid+state+induces+p53-dependent+arrest+of+nontransformed+mammalian+cells+in+G1.">Tetraploid state induces p53-dependent arrest of nontransformed mammalian cells in G1.</a> Mol. Biol. Cell. 12, 1315-1328 (2001).</li><li>Vogel, C., 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=Crosstalk+of+the+mitotic+spindle+assembly+checkpoint+with+p53+to+prevent+polyploidy.">Crosstalk of the mitotic spindle assembly checkpoint with p53 to prevent polyploidy.</a> Oncogene. 23, 6845-6853 (2004).</li><li>Aylon, Y., Oren, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p53:+Guardian+of+ploidy.">p53: Guardian of ploidy.</a> Mol. Oncol. 5 (4), 315-323 (2011).</li><li>Uetake, Y., Sluder, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+progression+after+cleavage+failure+:+mammalian+somatic+cells+do+not+possess+a+&amp;#34;tetraploidy+checkpoint&amp;#34.">Cell cycle progression after cleavage failure : mammalian somatic cells do not possess a &amp;#34;tetraploidy checkpoint&amp;#34.</a> J. Cell Biol. 165, 609-615 (2004).</li><li>Ganem, N., Pellman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limiting+the+proliferation+of+polyploid+cells.">Limiting the proliferation of polyploid cells.</a> Cell. 131, 437-440 (2007).</li><li>Ho, C., Hau, P., Marxer, M., Poon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+requirement+of+p53+for+maintaining+chromosomal+stability+during+tetraploidization.">The requirement of p53 for maintaining chromosomal stability during tetraploidization.</a> Oncotarget. 1 (7), 583-595 (2010).</li></ol>

The authors declare that they have no competing financial interest.

A major problem in induction of tetraploidy from diploid cells by chemical agents, either by cytokinesis inhibitors or by spindle inhibitors, is that cells often become a mixture of diploid and tetraploid populations, and tetraploid cells must be separated from diploid cells. Most common approaches for isolation of a tetraploid population free of diploid cells use FACS or cloning by limiting dilution. However, these procedures are laborious and not easy to perform. In this report, we present a new protocol to establish p...

Polyploidy has been observed not only in specialized tissues of mammalian species but also in a variety of pathological conditions, such as cancer and degenerative diseases. Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues, such as Barrett&#39;s esophagus 1,2 or squamous intraepithelial lesions of the cervix 3,4, and have been considered to be the source of malignant aneuploid cells in those tissues 5,6. Although it is suggested that conversion of tetraploid to aneuploid cells could be a crucial event in the early stages of tumorigenesis, the mechanisms involved in this process are not fully understood. This is partly because no in vitro model has been available where nontransformed polyploid human cells can propagate.
Some researchers have induced tetraploidy in nontransformed human epithelial cells through generation of binucleated cells by inhibiting cytokinesis7-9. In this method, however, unnecessary diploid cells must be eliminated by fluorescence-activated cell sorting (FACS) 7,8 or cloning by limiting dilution 9. Because these procedures are laborious and not easy to perform, simpler methods to establish nontransformed tetraploid cells are desired for research in this field.
In the present report, we describe a protocol to establish proliferative tetraploid cells from normal human fibroblasts or telomerase-immortalized human fibroblasts by relatively simple procedures. The procedures use spindle poison demecolcine (DC) to arrest diploid cells in mitosis, and mitotic cells collected by shaking off are further treated with DC. Diploid mitotic cells treated with DC for prolonged time convert to tetraploid G1 cells, and these cells proliferate as tetraploid cells after growth arrest for several days following drug removal. This protocol provides an efficient method for creating a useful model to study the relationship between chromosome instability and the oncogenic potential of polyploid human cells.

<table border="0" cellpadding="0" cellspacing="0" width="687">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:205px;">MEM-&#945;</td>
			<td style="width:101px;">Sigma-Aldrich</td>
			<td style="width:136px;">M8042-500ML</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:205px;">Trypsin-EDTA</td>
			<td style="width:101px;">Sigma-Aldrich</td>
			<td style="width:136px;">T4174</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:205px;">FBS</td>
			<td style="width:101px;">Sigma-Aldrich</td>
			<td style="width:136px;">172012-500ML</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:205px;">Demecolcine solution (10 &#956;g/mL in HBSS)</td>
			<td style="width:101px;">Sigma-Aldrich</td>
			<td style="width:136px;">D1925-10ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:205px;">BD CycleTES Plus DNA Reagent Kits</td>
			<td style="width:101px;">BD Biosciences</td>
			<td style="width:136px;">#340242</td>
			<td style="width:245px;">For examination of DNA ploidy by flow cytometry</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:205px;">Human chromosome multicolor FISH probe 24XCyte</td>
			<td style="width:101px;">MetaSystems</td>
			<td style="width:136px;">#D-0125-060-DI</td>
			<td style="width:245px;">Specialized filter set and software for mFISH analysis are necessary</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:205px;">Isis imaging system with mFISH&#160; software&#160;</td>
			<td style="width:101px;">MetaSystems</td>
			<td style="width:136px;"></td>
			<td style="width:245px;">Specialized probe kit is necessary</td>
		</tr>
	</tbody>
</table>

establishment of proliferative tetraploid cells from nontransformed human fibroblasts

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to be responsible for carcinogenesis in such tissues. Although proliferative polyploid cells are requisite for analyzing chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is rather challenging. Induction of tetraploidy by chemical agents usually results in a mixture of diploid and tetraploid populations, and most studies employed fluorescence-activated cell sorting or cloning by limiting dilution to separate tetraploid from diploid cells. However, these procedures are time-consuming and laborious. The present report describes a relatively simple protocol to induce proliferative tetraploid cells from normal human fibroblasts with minimum contamination by diploid cells. Briefly, the protocol is comprised of the following steps: arresting cells in mitosis by demecolcine (DC), collecting mitotic cells after shaking off, incubating collected cells with DC for an additional 3 days, and incubating cells in drug-free medium (They resume proliferation as tetraploid cells within several days). Depending on cell type, the collection of mitotic cells by shaking off might be omitted. This protocol provides a simple and feasible method to establish proliferative tetraploid cells from normal human fibroblasts. Tetraploid cells established by this method could be a useful model for studying chromosome instability and the oncogenic potential of polyploid human cells.

In our experience, TIG-1 cells can be made almost completely tetraploid by simple continuous treatment with 0.1 &#181;g/mL DC for 4 days (Figure 2A). In contrast, other fibroblast strains, such as BJ or IMR-90, and TIG-hT cells, became a mixture of diploid and tetraploid populations following the same treatment, and isolation of mitotic cells by the shake-off method is necessary during the course of DC treatment (usually 16 - 18 h after the start of treatment) (Fi...

Watch this Scientific Journal Video about Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts at JoVE.com

Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts

Although proliferative polyploid cells are necessary to analyze chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is not easy. The present report describes relatively simple procedures to establish proliferative tetraploid cells free of a diploid population from normal human fibroblasts.

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to ...

The overall goal of this procedure is to establish proliferative tetraploid cells, free of a diploid population, from normal human fibroblasts. This method can help answer key questions in the field of carcinogenesis research, such as how aneuploid cells develop in the early stages of carcinogenesis. The main advantage of this technique is that proliferative tetraploid cells, free of a diploid population, can be established by relatively simple procedures.While some strains become completely tetraploid by simple treatment with spindle poison, demecolcine, most need to be handled with the shake-off method during that treatment to emulate a diploid population. To begin the induction of tetraploidy without the shake-off method, passage approximately five times 10 to the 1/5 cultured cells into a 100-millimeter dish. Incubate the dish in a 5%carbon dioxide atmosphere at 37 degrees Celsius for 24 hours.After this, treat the exponentially-growing cells with medium containing 0.1 micrograms per milliliter DC.Incubate in a 5%carbon dioxide atmosphere at 37 degrees Celsius for four days. Then replace the DC-containing medium with drug-free medium. Incubate in 5%carbon dioxide at 37 degrees Celsius until proliferation resumes.To induce tetraploidy utilizing the shake-off method, passage the cultured cells into T-75 flasks, making sure that each flask contains at least one times 10 to the 1/6 cells. Incubate the flasks in a 5%carbon dioxide atmosphere at 37 degrees Celsius for 24 hours. The next day, treat the cells with medium containing DC for 16 to 18 hours to arrest them in mitosis as outlined in the text protocol.After incubation, check the cells in a flask under the inverted phase contrast microscope. And then gently shake each flask to begin the collection of loosely-adhered mitotic cells. Careless handling, such as violent shaking or touching the surface of the flask by the pipette tip should be avoided.Do not contaminate the mitotic cells with adhered interface cells. Gently wash the flasks with warm medium, and transfer the cells to centrifuge tubes. Utilize an atomic cell counter to count the cell number.After cell counting, centrifuge at 300 times g for five minutes. Reseed the collected mitotic cells into 16-millimeter culture dishes. Then treat the plated cells with medium containing 0.1 micrograms per milliliter DC.Incubate in 5%carbon dioxide at 37 degrees Celsius for three days.After this, replace the DC-containing medium with drug-free medium. Incubate in a 5%carbon dioxide atmosphere at 37 degrees Celsius until proliferation resumes. For analysis of DNA ploidy, wash the cells with PBS.Then add a solution containing 0.05%trypsin and 0.02%EDTA to the cells. Incubate for 10 minutes at 37 degrees Celsius. After this, transfer the cell suspension to a centrifuge tube.And neutralize the trypsin by adding medium containing 10%fetal bovine serum. Centrifuge the cells in medium at 300 times g for five minutes. Resuspend the cells in five milliliters of PBS.Then centrifuge again at 300 times g for five minutes. Next, resuspend cells in one milliliter of PBS. Add four milliliters of 100%ethanol drop-wise with mixing.Let the suspension rest at room temperature for 30 minutes to fix the cells. After this, centrifuge the cells in fixative solution at 300 times g for five minutes. And resuspend cells in five milliliters of PBS.Centrifuge the suspension at 300 times g for five minutes. Then treat the cells with 475 microliters of 0.5 milligrams per milliliter RNA solution. Add 25 microliters of one milligram per milliliter propidium iodine solution to stain the cells.And then mix. Allow the suspension to rest at room temperature for 30 minutes. Next, use a flow cytometer with a 488 nanometer laser and red channel emission filter to analyze the DNA content of the cells.Analyze a reference sample prepared from genuine diploid cells as well to assess ploidy of target cells. To begin, treat exponentially-growing cells with medium containing 0.1 micrograms per milliliter DC for four hours to arrest the cells in metaphase. After this, harvest at least five times 10 to the 1/5 cells by trypsinization.Suspend the harvested cells in medium containing FBS. Centrifuge the suspension at 300 times g for five minutes. Then add five milliliters of a 0.075 molar potassium chloride solution.Incubate at 37 degrees Celsius for 10 minutes. After incubation is complete, centrifuge the suspension at 300 times g for five minutes. Resuspend the cells in five milliliters of Carnoy's fixative.And then centrifuge the suspension at 300 times g for five minutes. Then aspirate the supernatant and resuspend cells in 0.5 milliliters of Carnoy's fixative. Drop 30 microliters of the cell suspension onto a glass slide.Finally, stain and analyze the cells via either chromosome counts or karyotype analysis. In this study, proliferative tetraploid cells, free of a diploid population, are cultured from normal human fibroblasts. Although some cell strains can become completely tetraploid by continuous treatment with DC, most cells need to be treated with the shake-off method during the course of DC treatment to eliminate a diploid population.The representative results for the induction of tetraploidy in three fibroblast strains are shown here. The strain TIG-1 becomes almost entirely tetraploid through simple continuous treatment with DC.However, this treatment causes the BJ and TIG-hT strains to become a mixture of diploid and tetrapod populations. Therefore, isolation of mitotic cells by the shake-off method is necessary during the treatment process.A chromosome count is performed in the established tetraploid cells. As can be seen, the modal chromosome number is 92 for both TIG-1 and BJ cells, and 91 for TIG-hT cells. Once mastered, this technique can be done in two weeks if it is performed properly.While attempting this procedure, it's important to remember to gently shake the flasks in the shake-off step, not to cause contamination of mitotic cells by adherent interface cells.

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7.2K Görüntüleme.  Saitama Medical University. The overall goal of this procedure is to establish proliferative tetraploid cells, free of a diploid population, from normal human fibroblasts. This method can help answer key questions in the field of carcinogenesis research, such as how aneuploid cells develop in the early stages of carcinogenesis. The main advantage of this technique is that proliferative tetraploid cells, free of a diploid population, can be established by relatively simple procedures.While some strains become comp...