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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Medicine

In vitro Organoid Culture of Primary Mouse Colon Tumors

Published: May 17th, 2013

DOI:

10.3791/50210

1Department of Molecular & Integrative Physiology, University of Michigan , 2Department of Internal Medicine, Division of Gastroenterology, University of Michigan

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.

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 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 mecha....

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1. Colon Tumor Isolation and Cell Dissociation

  1. 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.
  2. Incubate intestinal fragments containing tumors in EDTA chelation buffer (2 mM EDTA,.......

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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.......

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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 .......

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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.

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

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