Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Summary

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

Abstract

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Introduction

Colorectal cancer (CRC) is the third leading cause of cancer related deaths in the United States. Sporadic colon cancer – i.e. that arising later in life (>50 years of age) and with no clear predisposing genetic factors – accounts for ~80% of all cases, with incidence strongly influenced by long term dietary patterns^1,2. These tumors exhibit a metabolic shift towards dependence on oxidative glycolysis, known as the Warburg effect, which may in part make higher concentrations of cellular building blocks and energy available (through glutaminolysis) to permit and perhaps drive high rates of tumor cell proliferation^3-5. Stud....

Protocol

This study was performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Albert Einstein College of Medicine.

1. Crypt Isolation and Culture

1. Isolation of Crypts from the Small Intestine:
   1. Isolate intestinal crypts from any mice model of interest. Euthanize the mice with CO₂ followed by cervical dislocation.
   2. Open up the abdomen longitudinally and fill the small intestine (SI) with ice cold phosphate buffered saline, PBS (-Ca^....

Results

Crypt organoids were established from 8 month old C57BL/6 mice fed purified rodent diet American Institute of Nutrition 76A (AIN76A). Intestinal crypt organoids can be grown in culture for extended periods from a single crypt (Figure 1A, single red arrow). Organoids grow out crypt-like structures in 18-20 days in culture (Figure 1B, red arrows). Crypts were passaged every 3 weeks and organoids efficiently recovered following each passage.

Seahorse bioenergetic.......

Discussion

We tested the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) of crypts isolated from 8-month old mice and grown into organoids ex vivo. After measurement of the basal rate, crypt metabolism was evaluated by adding oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and rotenone, sequentially.

Basal OCR and basal ECAR were recorded from 0 - 29 min (Figure 2A and 2B). At the 29^th min, oligomyci.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BD Matrigel Basement Membrane Matrix, GFR, Phenol Red-free, LDEV-free	BD Biosciences	356231
PBS (phosphate buffered saline), no magnesium, no calcium, pH 7.2	Life Technologies	20012-027
Advanced DMEM/F-12 (1x)	Life Technologies	12634-028
Dulbecco′s Modified Eagle′s Medium w/o glucose, L-glutamine, phenol red, sodium pyruvate, and sodium bicarbonate	Sigma-Aldrich	D5030
Phenol red sodium salt	Sigma-Aldrich	P4758	Final Concentration 15 mg/l in DMEM (D5030) - step 2.2.2
Antibiotic-Antimycotic, 100x, 100 ml	Life Technologies	15240-062	Final concentration 1x or 2x
Penicilin-Streptomycin, liquid	Life Technologies	15140-122	Final concentration 1x
Gibco® GlutaMAX™ supplement	Life Technologies	35050061	Final concentration 1x
Gibco® HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid), 1 M	Life Technologies	15630-080	Final concentration 10 mM
N-acetyl-L-cysteine, 25 g	Sigma-Aldrich	A9165-25G	Final concentration 1 mM
100x N-2 supplement, liquid	Invitrogen	17502-048	Final concentration 1x
50x B-27® supplement minus Vitamin A, liquid	Invitrogen	12587-010	Final concentration 1x
Recombinant Mouse R-Spondin 1, CF, 50 μg	R&D Systems	3474-RS-050	Final concentration 500 ng/ml
Recombinant Murine EGF, 100 μg	Peprotech	315-09	Final concentration 50 ng/ml
Recombinant Murine Noggin, 20 μg	Peprotech	250-38	Final concentration 100 ng/ml
Gibco® L-glutamine, 200 mM	Life Technologies	25030-081	Final concentration 2 mM
Gibco® glucose powder	Life Technologies	15023-021	Final concentration 5 mM
Ambion® 0.5 M EDTA (ethylenediaminetetraacetic acid), pH 8.0	Life Technologies	AM9260G	Final concentration 3 mM for step 1.1.5; 2 mM for step 1.1.8
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DTT (dithiothreitol), 1M	Life Technologies	P2325	Final concentration 3 mM
Albumin from bovine serum (BSA)	Sigma-Aldrich	A2058	0.1% in PBS
Fetal Bovine Serum (FBS)	Life Technologies	16000-044	1% in PBS
Recovery™ Cell Culture Freezing Medium	Life Technologies	12648-010
ROCK inhibitor (Y-27632)	Sigma-Aldrich	Y0503	Final concentration 10 μM
Oligomycin	Sigma-Aldrich	O4876	Final concentration 1 μM
Carbonyl cyanide-p-trifluoro-methoxy-phenyl-hydrazone (FCCP)	Sigma-Aldrich	C2920	Final concentration 1 μM
Rotenone	Sigma-Aldrich	R8875	Final concentration 1 μM
Sodium hydroxide	Sigma-Aldrich	221465	Final concentration 0.1 N in PBS
XF24 Extracellular Flux Analyzer (XF Analyzer)	Seahorse Bioscience