In vitro Organoid Culture of Primary Mouse Colon Tumors

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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, 5.6 mmol/L Na₂HPO₄, 8.0 mmol/L KH₂PO₄, 96.2 mmol/L NaCl, 1.6 mmol/L KCl, 43.4 mmol/L sucrose, 54.9 mmol/L....

Results

The time course of a colon tumor organoid formation from a three-month-old Apc^min/+ 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.......

Discussion

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Matrigel Basement Membrane Matrix	BD Biosciences	356234	5 mg/ml
Collagenase Type IV	Worthington	LS004188	375U/mg
Dispase	Gibco	17105-041	1.8U/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