Name: generation of organotypic raft cultures from primary human keratinocytes
Uploaded: 2012-02-22T13:00:00
Description: Watch this Scientific Journal Video about Generation of Organotypic Raft Cultures from Primary Human Keratinocytes at JoVE.com

The authors would like to thank Sally Roberts (University of Birmingham, Birmingham UK) for the kind gift of the E1&circ;E4 antibody. This work was supported by a grant from the National Cancer Institute (4R00CA137160-03).

1. Preparation for Organotypic Raft Cultures
<ol>
	<li>The metal raft grids much first be treated with chromic sulfuric acid to remove any residue that could interfere with the differentiation process. Immerse metal grids in a glass beaker containing sulfuric acid for one hour, then continuously rinsed overnight with tap water. After the overnight rinse, raft grids should be rinsed for 3-5 hours in double distilled water.</li>
	<li>To provide support for the rafts, bend three sides of the raft about 0.5 cm at equal dis...

<ol>
	<li>Andrei, G., Duraffour, S., VandenOord, J., Snoeck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+raft+cultures+for+investigations+of+virus+growth,+pathogenesis+and+efficacy+of+antiviral+agents.">Epithelial raft cultures for investigations of virus growth, pathogenesis and efficacy of antiviral agents.</a> Antiviral Research. 85, 431-449 (2010).</li><li>Asselineau, D., Prunieras, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+'simplified'+skin:+control+of+fabrication.">Reconstruction of 'simplified' skin: control of fabrication.</a> The British Journal of Dermatology. 111, 219-222 (1984).</li><li>Bodily, J., Alam, S., Chen, H., Meyers, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Organotypic epithelial raft cultures and the study of the natural history of human papillomavirus. , (2006).</li><li>Cheng, S., Schmidt-Grimminger, D. C., Murant, T., Broker, T. R., Chow, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation-dependent+up-regulation+of+the+human+papillomavirus+E7+gene+reactivates+cellular+DNA+replication+in+suprabasal+differentiated+keratinocytes.">Differentiation-dependent up-regulation of the human papillomavirus E7 gene reactivates cellular DNA replication in suprabasal differentiated keratinocytes.</a> Genes &#38; Development. 9, 2335-2349 (1995).</li><li>Dollard, S. C., Wilson, J. L., Demeter, L. M., Bonnez, W. R., Reichman, C., Broker, T. R., Chow, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+human+papillomavirus+and+modulation+of+the+infectious+program+in+epithelial+raft+cultures.+OFF.">Production of human papillomavirus and modulation of the infectious program in epithelial raft cultures. OFF.</a> Genes &#38; Development. 6, 1131-1142 (1992).</li><li>Frattini, M. G., Lim, H. B., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+synthesis+of+oncogenic+human+papillomaviruses+requires+episomal+genomes+for+differentiation-dependent+late+expression.">In vitro synthesis of oncogenic human papillomaviruses requires episomal genomes for differentiation-dependent late expression.</a> Proceedings of the National Academy of Sciences of the United States of America. 93, 3062-3067 (1996).</li><li>Kopan, R., Traska, G., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoids+as+important+regulators+of+terminal+differentiation:+examining+keratin+expression+in+individual+epidermal+cells+at+various+stages+of+keratinization.">Retinoids as important regulators of terminal differentiation: examining keratin expression in individual epidermal cells at various stages of keratinization.</a> The Journal of Cell Biology. 105, 427-440 (1997).</li><li>Longworth, M. S., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+human+papillomaviruses+in+differentiating+epithelia.">Pathogenesis of human papillomaviruses in differentiating epithelia.</a> Microbiology and Molecular Biology Review. 68, 362-372 (2004).</li><li>Meyers, C., Frattini, M. G., Hudson, J. B., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosynthesis+of+human+papillomavirus+from+a+continuous+cell+line+upon+epithelial+differentiation.">Biosynthesis of human papillomavirus from a continuous cell line upon epithelial differentiation.</a> Science. 257, 971-973 (1992).</li><li>Wilson, R., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Differentiation of HPV-Containing Cells Using Organotypic Raft Culture or Methylcellulose. , 119-119 (2006).</li><li>zur Hausen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Papillomavirus+infections--a+major+cause+of+human+cancers.">Papillomavirus infections--a major cause of human cancers.</a> Biochimica et Biophysica Acta. 1288, F55-F78 (1996).</li></ol>

We describe here a method that can be used to study epithelial differentiation in general, but can also easily be adapted to study viral pathogenesis, as well as the efficacy of potential therapeutics. Many viruses target epithelial cells either as the primary site of infection, as in the case of HPV, or at some point in the viral life cycle, as with herpesviruses. Although growing organotypic raft cultures is time consuming, the ability to faithfully recapitulate in vivo epithelial differentiation provides an e...

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalog number</td>
		</tr>
		<tr>
			<td>Rat Tail Collagen type 1 (4-5mg/ml)</td>
			<td>BD Biosciences</td>
			<td>354236</td>
		</tr>
		<tr>
			<td>DMEM without NaHC03</td>
			<td>Invitrogen</td>
			<td>12100-061</td>
		</tr>
		<tr>
			<td>NaHC03</td>
			<td>Sigma</td>
			<td>S5761</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Calbiochem</td>
			<td>391338</td>
		</tr>
		<tr>
			<td>Stainless Steel metal grids</td>
			<td>Williams and Mettle Co.</td>
			<td>CR-03063-040-100-S</td>
		</tr>
		<tr>
			<td>E medium11</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor</td>
			<td>BD Biosciences</td>
			<td>354010</td>
		</tr>
	</tbody>
</table>

generation of organotypic raft cultures from primary human keratinocytes

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)1. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium2. Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras6 and modified by Kopan et al.7, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies8. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses9. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.

Watch this Scientific Journal Video about Generation of Organotypic Raft Cultures from Primary Human Keratinocytes at JoVE.com

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

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