Name: establishment of proliferative tetraploid cells from nontransformed human fibroblasts
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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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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.

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	<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 ...

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