Work described in this manuscript was made possible by funding from the Patrick F. Taylor Foundation.

1. Chromosome Harvesting of Adherent Cells
<ol>
	<li>Standard Protocol
	<ol>
		<li>Grow cells according to specific cell culturing conditions. When the cells have reached logarithmic phase (80% confluency), add 10 &#956;l/ml of Colcemid to the cell culture flask. A minimum of 2 x 106 cells is recommended.</li>
		<li>Incubate cells at 37 &#176;C in a 5% CO2 incubator for 45 min. Using a sterile pipette, transfer media from cells into a 15 ml conical tube. Set aside.</li>
		<li>Ge...

<ol>
	<li>Driscoll, D., Gross, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prenatal+screening+for+aneuploidy.">Prenatal screening for aneuploidy.</a> New Engl. J. Med. 360, 2556-2562 (2009).</li><li>Nussbuam, 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=.">.</a> Genetics in Medicine: 7th Edition. , 76 (2007).</li><li>Ljunger, E., 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+abnormalities+in+first-trimester+miscarriages.">Chromosomal abnormalities in first-trimester miscarriages.</a> Acta Obstet. Gynecol. Scand. 84 (11), 1103-1107 (2005).</li><li>Kwinecka-Dmitri, 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=Frequency+of+chromosomal+aberrations+in+material+from+abortions.">Frequency of chromosomal aberrations in material from abortions.</a> Ginecol. Pol. 81 (12), 896-901 (2010).</li><li>Reddy, U., 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=Stillbirth+classification&#8212;Developing+an+international+consensus+for+research:+Executive+summary+of+a+national+institute+of+child+health+and+human+development+workshop.">Stillbirth classification&#8212;Developing an international consensus for research: Executive summary of a national institute of child health and human development workshop.</a> Obstet. Gynecol. 114 (4), 901-914 (2009).</li><li>Pierpoint, 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=Genetic+basis+for+congenital+heart+defects:+Current+knowledge.">Genetic basis for congenital heart defects: Current knowledge.</a> Circulation. 115 (23), 3015 (2007).</li><li>Kaneshiro, N., Zieve, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Down+Syndrome:+Trisomy+21..">Down Syndrome: Trisomy 21..</a> PubMed Health. , (2010).</li><li>Cin, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current Protocols in Human Genetics. , (2003).</li><li>Wang, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodologies+in+cancer+cytogenetics+and+molecular+cytogenetics.">Methodologies in cancer cytogenetics and molecular cytogenetics.</a> Am. J. Med. Genet. 115 (3), 118-124 (2002).</li><li>Miura, 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=Accumulated+Chromosomal+Instability+in+Murine+Bone+Marrow+Mesenchymal+Stem+Cells+Leads+to+Malignant+Transformation.">Accumulated Chromosomal Instability in Murine Bone Marrow Mesenchymal Stem Cells Leads to Malignant Transformation.</a> Stem Cells. 24 (4), 1095-1103 (2006).</li><li>Izadpanah, 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=Long-term+In+vitro+Expansion+Alters+the+Biology+of+Adult+Mesenchymal+Stem+Cells.">Long-term In vitro Expansion Alters the Biology of Adult Mesenchymal Stem Cells.</a> Cancer Res. 68 (11), 4229-4238 (2008).</li><li>Dekel-Naftali, 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=Screening+of+human+pluripotent+stem+cells+using+CGH+and+FISH+reveals+low-grade+mosaic+aneuploidy+and+a+recurrent+amplification+of+chromosome+1q.">Screening of human pluripotent stem cells using CGH and FISH reveals low-grade mosaic aneuploidy and a recurrent amplification of chromosome 1q.</a> Eur. J. Hum. Genet. 20 (12), 1248-1255 (2012).</li><li>Moralli, D., Yusuf, M., Mandegar, M., Khoja, S., Monaco, Z. L., Volpi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Improved+Technique+for+Chromosomal+Analysis+of+Human+ES+and+iPS+Cells.">An Improved Technique for Chromosomal Analysis of Human ES and iPS Cells.</a> Stem Cell Rev. Rep. 7 (2), 471-477 (2011).</li><li>Bayani, J., Squire, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traditional+banding+of+chromosomes+for+cytogenetic+analysis.">Traditional banding of chromosomes for cytogenetic analysis.</a> Curr. Protoc. Cell Biol. 22, (2004).</li><li>Speicher, M. R., Carter, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+new+cytogenetics:+blurring+the+boundaries+with+molecular+biology.">The new cytogenetics: blurring the boundaries with molecular biology.</a> Nat. Rev. Genet. 6 (10), 782-792 (2005).</li><li>Benn, P., Delach, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Lymphocyte+Culture+and+Chromosome+Analysis.">Human Lymphocyte Culture and Chromosome Analysis.</a> Cold Spring Harb Protoc. , (2010).</li><li>Muul, L. M., Heine, G., Silvin, C., James, S. P., Candotti, F., Radbruch, A., Worm, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Measurement of Proliferative Responses of Cultured Lymphocytes, Curr. Protoc. Immunol. , (2011).</li><li>Barch, M. J., Knutsen, T., Spurbeck, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Association of Genetic Technologists Cytogenetics Laboratory Manual. San Francisco: 3rd edition. , (1997).</li><li>Dewald, G. W., Ketterling, R. P., Wyatt, W. A., Stupca, P. J., McClatchey, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytogenetic+studies+in+neoplastic+hematologic+disorders.">Cytogenetic studies in neoplastic hematologic disorders.</a> In Clinical Laboratory Medicine, 2nd edition. , 658-685 (2002).</li><li>Kibe, 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=IL-7R&#945;+deficiency+in+p53(null)+mice+exacerbates+thymocyte+telomere+erosion+and+lymphomagenesis.">IL-7R&#945; deficiency in p53(null) mice exacerbates thymocyte telomere erosion and lymphomagenesis.</a> Cell Death Different. 19 (7), 1139-1151 (2012).</li><li>Tsien, F., Morava, E., Talarski, A., Marble, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+features+of+a+boy+with+trisomy+of+16q22--&gt;qter+due+to+paternal+Y;+16+translocation.">Phenotypic features of a boy with trisomy of 16q22--&gt;qter due to paternal Y; 16 translocation.</a> Clin. Dysmorphol. 4 (4), 177-181 (2005).</li><li>Tsien, F., 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=Prolonged+culture+of+normal+chorionic+villus+cells+yields+ICF+syndrome-like+chromatin+decondensation+and+rearrangements.">Prolonged culture of normal chorionic villus cells yields ICF syndrome-like chromatin decondensation and rearrangements.</a> Cytogenet. Genome Res. 8 (1), 13-21 (2002).</li><li>McGrattan, P., Campbell, S., Cuthbert, R., Jones, F., McMullin, M., Humphreys, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+conventional+cytogenetics+(G-banding),+comparative+genomic+hybridization+(CGH)+and+interphase+fluorescence+in+situ+hybridization+(i-FISH)+for+the+detection+of+genomic+rearrangements+in+acute+leukemia.">Integration of conventional cytogenetics (G-banding), comparative genomic hybridization (CGH) and interphase fluorescence in situ hybridization (i-FISH) for the detection of genomic rearrangements in acute leukemia.</a> J. Clin. Pathol. 61 (8), 903-908 (2008).</li></ol>

The authors have nothing to disclose.

We have utilized the present procedure for chromosome isolation from cells of various organisms, including various cell types obtained from human, Rhesus macaques, rats, and mice11,20-22. The standard protocol is provided, but certain key steps and variables may need to be adjusted for these diverse types of cells. Several specific steps are crucial in both the chromosomal preparation and G-banding to ensure the best possible quality of the results. One of the variables which can affect the assay is the Colcem...

Chromosome analysis is a conventional technique utilized worldwide to diagnose chromosome instability and rearrangements leading to genetic disorders and malignancy1,2,8,9. In addition, a higher resolution for the diagnosis and research of constitutional and cancer-acquired genetic abnormalities can be achieved with the combination of the classical cytogenetic procedures and molecular cytogenetic methodologies such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)14,15. More recently, these techniques have been utilized for the evaluation of chromosome instability associated with stem cell research. Karyotypic abnormalities such as aneuploidy of long-term cultured embryonic cells (ES) and adult stem cells of various organisms have been reported by multiple laboratories. Recent evidence supports that some cell lines are inherently more inclined to chromosome instability regardless of culture conditions. For this reason, when establishing and/or maintaining human, mouse, or Rhesus stem cell lines, chromosome analysis is recommended as part of the quality control process. Many reports describe the increasing interest in the use of routine and molecular cytogenetics to monitor the chromosomal stability of stem cells and malignant cells or various organisms in culture10-13. These protocols are increasingly being used by nongenetic laboratories for rapid chromosomal assessment of their cultured cells13. We present our basic procedures for chromosome preparation from various cell types, which can be applied for both clinical and research purposes and cells derived from various organisms.

<table border="0" cellpadding="0" cellspacing="0" width="662">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:155px;">KaryoMAX Colcemid</td>
			<td style="width:213px;">Gibco</td>
			<td style="width:145px;">15212-012</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS Buffer</td>
			<td>Gibco</td>
			<td>24020-117</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5% Trypsin EDTA 10x</td>
			<td>Gibco</td>
			<td>15400-054</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Permount</td>
			<td>Fisher Scientific</td>
			<td>SP15-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Buffer Tablets &#34;GURR&#34;</td>
			<td>Gibco</td>
			<td>10580-013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Geimsa Stain</td>
			<td>Ricca Chemical Company</td>
			<td>3250-16</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge 5810</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methanol</td>
			<td>Caledon Laboratory Chemicals</td>
			<td>6700130</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glacial Acetic Acid</td>
			<td>Krackeler Scientific Inc.&#160;</td>
			<td>11-9508-05</td>
			<td></td>
		</tr>
	</tbody>
</table>

chromosome preparation from cultured cells

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome&#160;and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births1,2, 60-80% of all miscarriages3,4, 10% of stillbirths2,5, 13% of individuals with congenital heart disease6, 3-6% of infertility cases2, and in many patients with developmental delay&#160;and birth defects7. Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance8,9.&#160; Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents10-13.
Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy&#39;s fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)14,15.

High quality metaphase spreads are essential for chromosome analysis. A successful assay yields chromosomes which are well spread and of suitable chromosome morphology. Properly G-banded chromosomes contain the characteristic light and dark banding patterns.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50203/50203fig1highres.jpg" src="/files/ftp_upload/50203/50203fig1.jpg" /> 
Figure 1.&#160;A successfu...

Watch this Scientific Journal Video about Chromosome Preparation From Cultured Cells at JoVE.com

Chromosome Preparation From Cultured Cells

Chromosomes can be isolated from live cells such as lymphocytes or skin fibroblasts, and from organisms including humans or mice. These chromosome preparations can be further utilized for routine G-banding and molecular cytogenetic procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY).

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

chromosome-preparation-from-cultured-cells

Research

JoVE Journal

Biology

79.8K Views.  Louisiana State University Health Science Center. Chromosomes can be isolated from live cells such as lymphocytes or skin fibroblasts, and from organisms including humans or mice. These chromosome preparations can be further utilized for routine G-banding and molecular cytogenetic procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY).