Name: in vitro organoid culture of primary mouse colon tumors
Uploaded: 2013-05-17T12:00:00
Description: Watch this Scientific Journal Video about In vitro Organoid Culture of Primary Mouse Colon Tumors at JoVE.com

This study was supported by grants to Y.M.S from the National Institutes of Health (CA148828), The University of Michigan Gastrointestinal Peptide Center, and Jeffrey A. Colby Colon Cancer Research and the Tom Liu Memorial Funds of the University of Michigan Comprehensive Cancer Center.

1. Colon Tumor Isolation and Cell Dissociation
<ol>
	<li>Intestinal tumors can be isolated from any sporadic or treatment-induced colon cancer model. The mice should be euthanized with CO2. Colons are then collected, flushed with cold phosphate-buffered saline (PBS) and opened longitudinally. Identify regions containing tumors using a stereomicroscope, dissect out with a pair of scissors, and wash with cold PBS.</li>
	<li>Incubate intestinal fragments containing tumors in EDTA chelation buffer (2 mM EDTA,...

<ol>
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The experimental procedures described in this protocol will allow for isolation and culture of primary murine colon tumors. The protocol is adapted from seminal work done by Dr. Clevers group 1,24,27. We optimized the digestion time and collagenase concentration to get a better yield of tumor organoids. The critical steps include tumor cell digestion into single cells, Matrigel resuspension, and selective culture. For tumor cell digestion, in order to obtain efficient dissociation of colon tumors and maintain ...

The intestinal epithelial cells proliferate and turn over at an extraordinary rate, outpacing all other tissues in the vertebrate body 2,3. The dividing cells including intestinal stem cells (ISC) and transit-amplifying cells differentiate into either secretory (goblet, Paneth and enteroendocrine) cells or enterocytes 3. The ISC is located at the base of the crypt. Paneth cells move down to the bottom of crypts and are long-lived, whereas other lineages migrate upwards to the villi 3,4. Here the cells are exposed to the gut contents including microbiota and are shed from the villus tips through an anoikis-induced apoptotic mechanism. Although the colon lacks villi and Paneth cells, the mechanism for maintaining homeostasis is similar 4.
The Wnt signaling pathway has been implicated in playing a crucial role in intestinal proliferation and ISC maintenance 4. Deletion of the transcription factor TCF4, a downstream effector of Wnt signaling, leads to loss of intestinal stem cells and subsequent breakdown of the tissue 5. Similarly, transgenic expression of the Wnt inhibitor DKK1 reduces the epithelial proliferation and depletes secretory cell lineages 6. Conversely, overexpression of the Wnt agonist R-spondin-1 induces potent and rapid proliferation of intestinal crypt cells 7.
Given the importance of Wnt signaling for intestinal homeostasis, Wnt pathway mutations are frequently observed in colon cancer 8. Colon cancer is the third leading cause of death from cancer in United States 9. Excess dietary intake with red meat and alcohol, reduced physical activity, and inherited and somatic mutations are considered to be risk factors of colon cancer 10,11. The adenomatous polyposis coli (Apc) gene, a key Wnt signaling factor, is mutated in a majority of patients with familial, sporadic, and colitis-associated colon cancer 12,13. Mutations of other factors involved in Wnt signaling pathway including Axin2 and &beta;-catenin are also observed in colon cancer 14,15. However, the precise mechanism and effective therapy for colon cancer are still lacking. To facilitate the investigation of the molecular mechanisms for colon cancer, human colon cancer cell lines representing different stages of cancer progression from a benign to an aggressive cell type have been established 16-18. Mouse colon carcinoma cell lines with different metastatic properties are also available 19,20. Nevertheless, primary cells or organoid cultures are preferred over transformed cell lines because they closely mimic the in vivo state and generate more physiologically relevant data 21. Most colon-cancer derived cell lines grow as monolayer attached to the plate or as cell suspensions, lacking of apical-basolateral orientation and tight junctions between cells. Also, normal and tumor intestinal epithelial cells in vivo undergo a spontaneous form of apoptosis termed anoikis as the differentiated cells reach the villus tips and are shed 22. These features are difficult to recapitulate in cell lines but are important in the developmental process of colon cancer 23. These features are maintained in primary organoids. In addition, the tumor organoid cultures provide an efficient system to assess tumor autonomous functions of genes compared to in vivo studies. Genetic manipulation in vivo of the intestine is a time-consuming process mainly through creating transgenic and/or knockout mice using intestine-specific drivers. However, the tumor organoids are readily amenable to viral mediated genetic manipulations and thus a great tool for assessing precise molecular mechanisms. Primary intestinal tumor organoid cultures have been demonstrated to be a feasible and powerful technique. Primary intestinal cell culture can establish functional intestinal organoids with crypt-villi structure in vitro from a single adult Lgr5+ stem cell 24. These organoids can be transplanted and engrafted into damaged colon tissue for regeneration 25. Further adaption of the culture conditions had made similar epithelial organoids from mouse colon and human small intestine and colon feasible 1. For primary normal colon epithelium culture, basal culture medium as well as growth factors including EGF, Noggin, R-spondin and Wnt3a are essential, whereas basal culture medium and EGF is sufficient for growing primary mouse colon tumor organoids 1. Here we describe a detailed protocol to isolate, culture, and generate colon tumor organoids.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Matrigel Basement Membrane Matrix</td>
			<td>BD Biosciences</td>
			<td>356234</td>
			<td>5 mg/ml</td>
		</tr>
		<tr>
			<td>Collagenase Type IV</td>
			<td>Worthington</td>
			<td>LS004188</td>
			<td>375 U/mg</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td>1.8 U/mg</td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Invitrogen</td>
			<td>12634010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor (EGF), Murine, Natural</td>
			<td>Invitrogen</td>
			<td>53003-018</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>N2 Supplement</td>
			<td>Invitrogen</td>
			<td>17502-048</td>
			<td>100 x</td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td>50 x</td>
		</tr>
		<tr>
			<td>Glutamax-I</td>
			<td>Gibco</td>
			<td>35050-079</td>
			<td>100x</td>
		</tr>
		<tr>
			<td>N-Acetylcysteine</td>
			<td>Sigma</td>
			<td>A9165-5G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dulbecco's Modified Eagle Medium</td>
			<td>Invitrogen</td>
			<td>11965-092</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PicoPureTM RNA Isolation Kit</td>
			<td>Invitrogen</td>
			<td>KIT0204</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

in vitro organoid culture of primary mouse colon tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.

The time course of a colon tumor organoid formation from a three-month-old Apcmin/+ mouse is shown in Figure 1. At day 0, single cells could be observed several hours following plating (Figure 1A). At day 1, survived colon tumor epithelial cells with refractory nuclei could be observed. At day 3, the size of cells doubled. At day 6, the size of organoid expanded more than ten-fold and showed signs of apoptosis in the middle. At day 14, the orgnoids would grow into irr...

Watch this Scientific Journal Video about In vitro Organoid Culture of Primary Mouse Colon Tumors at JoVE.com

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

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