This work was supported by the Ovarian Cancer Research Fund [grant LT/UIC/01.2011], the UIC Center for Clinical and Translational Science, the UIC Cancer Center, and the National Institutes of Health grant C06RR15482.
Experiments on animals were performed in accordance with the guidelines and regulations set forth by AAALAC under approved animal care protocols by the Animal Care and Use Committee at UIC. Animals for this project are housed in barrier rooms in the Biological Resources Laboratory (BRL) at the University of Illinois at Chicago. The BRL has a fully veterinary staff that monitors animals at least twice daily and provides advice on animal care.

1. Ovarian dissection and tissue isolation
<ol>
	<li>Prepare a 0.5% (w/v) solution of sodium alginate, prepare from a modified version of the protocol described previously5, using sterile PBS and heat to 37&deg;C. Mix by inversion or rocking, but do not vortex. Draw the alginate into a 1 ml syringe with a 25 G needle and maintain at 37&deg;C.</li>
	<li>Prepare 10mM sterile CaCl2 solution for crosslinking the alginate upon tissue encapsulation and warm to 37&deg;C.</li>
	<li>Prepare 1mL of cult...

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+follicles+produce+live,+fertile+offspring.">Tissue-engineered follicles produce live, fertile offspring.</a> Tissue Eng. 12, 2739-2746 (2006).</li><li>Wilson, R. F., Wiencek, R. G., Balog, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+and+preventing+infection+after+abdominal+vascular+injuries.">Predicting and preventing infection after abdominal vascular injuries.</a> J Trauma. 29, 1371-1375 (1989).</li><li>Xu, M. <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+Oocyte+Maturation+and+Preantral+Follicle+Culture+from+the+Luteal+Phase+Baboon+Ovary+Produce+Mature+Oocytes.">In Vitro Oocyte Maturation and Preantral Follicle Culture from the Luteal Phase Baboon Ovary Produce Mature Oocytes.</a> Biol Reprod. , .</li><li>Xu, M. <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+grown+human+ovarian+follicles+from+cancer+patients+support+oocyte+growth.">In vitro grown human ovarian follicles from cancer patients support oocyte growth.</a> Hum Reprod. 24, 2531-2540 (2009).</li><li>Jen, A. C., Wake, M. C., Mikos, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+Hydrogels+for+cell+immobilization.">Review: Hydrogels for cell immobilization.</a> Biotechnol Bioeng. 50, 357-364 (1996).</li><li>Iwamoto, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+bifunctional+alginate+lyase+from+Alteromonas+sp.+strain+no.+272+and+its+action+on+saturated+oligomeric+substrates.">Purification and characterization of bifunctional alginate lyase from Alteromonas sp. strain no. 272 and its action on saturated oligomeric substrates.</a> Biosci Biotechnol Biochem. 65, 133-142 (2001).</li><li>Auersperg, N., Maines-Bandiera, S. L., Dyck, H. G., Kruk, P. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+surface+epithelium:+biology,+endocrinology,+and+pathology.">Ovarian surface epithelium: biology, endocrinology, and pathology.</a> Endocr Rev. 22, 255-288 (2001).</li><li>Choi, J. H., Wong, A. S., Huang, H. F., Leung, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gonadotropins+and+ovarian+cancer.">Gonadotropins and ovarian cancer.</a> Endocr Rev. 28, 440-461 (2007).</li><li>Fathalla, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incessant+ovulation--a+factor+in+ovarian+neoplasia.">Incessant ovulation--a factor in ovarian neoplasia.</a> Lancet. 2, 163-163 (1971).</li><li>Espey, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+of+the+hypothesis+that+mammalian+ovulation+is+comparable+to+an+inflammatory+reaction.">Current status of the hypothesis that mammalian ovulation is comparable to an inflammatory reaction.</a> Biol Reprod. 50, 233-238 (1994).</li></ol>

There is nothing to disclose.

The development of a three-dimensional organ culture system utilizing alginate hydrogels represents a versatile method for analyzing many different tissue types from a wide variety of organisms. The use of 3D cultures can be extended to the fields of tissue engineering and regenerative medicine as it provides a scaffold on which cells can grow11. Currently, our laboratory is undergoing initial studies using human ovarian and fallopian tube tissue; however, culture of human and non-human primate follicles has b...

<table border="1">
	<tbody>
		<tr>
			<th>
				Name of the reagent</th>
			<th>
				Company</th>
			<th>
				Catalogue number</th>
			<th>
				Comments (optional)</th>
		</tr>
		<tr>
			<td>Leibovitz L-15 medium</td>
			<td>Gibco</td>
			<td>11415</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&alpha;MEM medium</td>
			<td>Gibco</td>
			<td>12000-022</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium alginate</td>
			<td>Novamatrix</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium cacodylate</td>
			<td>Sigma Aldrich</td>
			<td>C0250</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polypropylene mesh fiber</td>
			<td>McMaster Carr</td>
			<td>&nbsp;</td>
			<td>45" wide roll; 0.0041" opening</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alginate lyase</td>
			<td>Sigma Aldrich</td>
			<td>A1603</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>A1603</td>
			<td>Sigma Aldrich</td>
			<td>C9697</td>
			<td>From Clostridium histolyticum</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>MP Biomedicals</td>
			<td>103700</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>Gibco</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ITS solution</td>
			<td>Roche</td>
			<td>11074547001</td>
			<td>Insulin/transferrin/sodium selenite</td>
		</tr>
		<tr>
			<td>Cytokeratin 8 antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>TROMA-1</td>
			<td>Use at 1:200</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Vector Labs</td>
			<td>H1200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bromodeoxyuridine</td>
			<td>Sigma</td>
			<td>B50002-1G</td>
			<td>Final concentration 10 &mu;M</td>
		</tr>
	</tbody>
</table>

alginate hydrogels for three-dimensional organ culture of ovaries and oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Watch this Scientific Journal Video about Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts at JoVE.com

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

alginate-hydrogels-for-three-dimensional-organ-culture-ovaries

Research

JoVE Journal

Bioengineering

21.8K ビュー.  University of Illinois at Chicago. それらの三次元コンテキストで正常な細胞の培養は、細胞の形質転換および腫瘍形成に必要な初期の事象を研究するための代替メソッドを表します。このメソッドは、卵巣癌の形成の初期イベントを研究するために、通常の卵巣と卵管の細胞を成長させるために使用されます。