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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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. Studies of colon cancer as well as other gastrointestinal cancers including small intestine cancers provide important insight into the cause of tumor formation. Investigating the metabolic differences between normal, pro-tumorigenic and tumorigenic states of gastrointestinal organ systems may assist determination of relative risk for tumor development as well as early detection of neoplasia. Moreover, understanding bioenergetic metabolism involving mitochondrial respiration and glycolysis will provide fundamental insight into how cell physiology, aging and disease state perturbs intestinal homeostasis. Utilization of the bioenergetics assay technology for extracellular flux analysis can assess the rates of mitochondrial respiration and glycolysis simultaneously in cells growing in culture in real time^6,7.

Until recently, in vitro studies of small intestine were limited to cell lines derived from either benign or malignant tumors^8,9 and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. In 2009, Sato et al.¹⁰ introduced an ex vivo culture system to grow three-dimensional (3D) mouse intestinal epithelial organoids, or epithelial “mini-guts”, suitable for experimental, diagnostic and therapeutic investigations^10,11. Moreover, crypts isolated from calorically restricted mice maintain their altered growth properties as organoids in such cultures¹². Compared to transformed cell lines, crypt organoid cultures can be used to generate physiologically relevant data presenting a far better model to understand the in vivo state.

We adapted bioenergetics analysis technology to assay energy metabolism of intestinal crypt organoids. Mouse intestinal crypt organoids were cultured ex vivo to develop the crypt organoid energy metabolism studies presented. The oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) of crypt organoids were measured in the absence and presence of two different metabolic inhibitors (oligomycin, rotenone) and an ion carrier (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone). The crypt organoid metabolic response to these chemical compounds were successfully reflected through changing ECAR and OCR values.

Cellular bioenergetic studies will elucidate the reciprocal interactions between metabolic state and disease risk and phenotype in cancer, obesity, diabetes, metabolic disorders and mitochondrial diseases and help advance screening methods with direct implications for translational medicine. Here, we describe a detailed protocol to isolate small intestinal crypts and to culture crypt organoids. Moreover, we introduce a novel method to use crypt organoid cultures for metabolic assays.

Protokół

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 (-Ca²⁺; -Mg²⁺) with 2x antibiotic–antimycotic (anti–anti). Rapidly isolate small intestine.
   3. Thoroughly dissect free of mesenteric fat using a scalpel. Be careful not to perforate the tissue – at all times keep the tissue moist with ice cold PBS. Open up intestine longitudinally and wash thoroughly with ice cold PBS.
   4. Cut small intestine into two sections and flatten out using a wet cotton tip, gently scrape off the villi using a pre-cooled slide. Wash vigorously in ice-cold PBS several times.
   5. Incubate 3 min in 20 ml 1x PBS per 3 mM ethylenediaminetetraacetic acid (EDTA)/0.5 mM dithiothreitol (DTT) for non-enzymatic dissociation of the tissue.
   6. Cut tissue into small pieces (2-4 mm) using a razor blade on a pre-cooled slide. Transfer the pieces into a 50 ml tube containing 20 ml ice cold PBS.
   7. Pipette up and down gently 10x using a 10 ml sterile disposable pipette. Let tissue fragments sediment by gravity. Remove the supernatant with a pipette. Repeat three additional times; make sure supernatant is clear.
   8. Add 20 ml of PBS per 2 mM EDTA. Swirl and incubate at 4 °C for 30 min with gentle rocking.
   9. Let tissue sediment and discard supernatant. Add 15 ml ice cold PBS and pipette up and down 5x with a 10 ml pipette. Let the tissue fragments sediment and collect the supernatant as Fraction 1 (F1). Repeat collecting F2-F5, each fraction maintained separately.
   10. Add 15 ml ice cold PBS and this time shake vigorously by hand for 15 sec. Collect F6 and repeat for F7, F8. If tissue pieces start to float, tap to help them settle.
   11. Inspect an aliquot of each fraction under the microscope. Pool those fractions containing crypts.
   12. Pass the pooled fractions through a 70 µm nylon cell strainer, collecting crypts in a 50 ml tube.
   13. Centrifuge at 100 x g for 5 min at 4 °C, discard the supernatant and resuspend pellet in 10 ml ice cold PBS with 2x anti–anti and transfer crypts to a 15 ml tube. Repeat the wash one more time.
   14. Wash the crypts once with 10 ml ice-cold ADF, Advanced DMEM/F-12 (Dulbecco"s Modified Eagle Medium/Ham"s F-12) – 2x anti–anti.
   15. Wash the crypts once with 10 ml ADF – 1x anti–anti.
   16. Count the crypts using a hemocytometer. Adjust the crypt solution volume and transfer to a 1.5 ml tube so that the final crypt concentration when resuspended will be 100-500 crypts per 50 µl of gelatinous protein mixture (Matrigel). Centrifuge at 100 x g for 5 min at 4 °C and resuspend crypts in the gelatinous protein mixture.
   17. Plate 50 µl of crypt – gelatinous protein mixture suspension per well in a 24-well plate (growth area: 2 cm²), carefully placing the suspension drop in the center of each well. Maintain the plate in a CO₂, 37 °C incubator for ~30 min until the suspension solidifies.
   18. Add 500 µl ice cold complete culture media (Advanced DMEM/F12 with 1x anti – anti, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1x GlutaMAX, 1x B27 Supplement, 1x N2 Supplement, 1 mM N-Acetyl-L-Cysteine (NAC), 50 ng/ml Epidermal growth factor (EGF), 100 ng/ml Noggin, 500 ng/ml R-Spondin).
       1. Reconstitute growth factors in 0.1% Bovine Serum Albumin (BSA) in PBS.
2. Crypt Organoid Culture and Passage:
   1. Passage crypt organoids 14-21 days post seeding (or as needed) as follows:
      1. Change media every Friday and Monday. On Wednesdays, replace half the media with fresh media.
   2. Remove the culture medium. Wash the sample twice with 500 µl PBS at 5 min intervals.
   3. Remove PBS and gently break up the gelatinous protein mixture using a sterile P1000 micro pipette tip.
   4. Add 1 ml complete culture media to a single well and resuspend the organoids in 1 ml media.
   5. Gently disrupt the organoids using a P1000 by pipetting up and down 20-30x (check under the microscope for proper organoid dissociation with a good crypt yield until a consistent technique is developed).
   6. Transfer the crypts to a 1.5 ml microcentrifuge tube; centrifuge at 100 x g at 4 °C, 5 min.
   7. Wash the pellet twice with 1 ml complete culture media.
   8. Split at a ratio of 1:3 or 1:6 as needed, resuspending crypts in 50 µl Matrigel per well. Proceed as described above (section 1.1.16-18).
3. Crypt Organoid Freezing:
   1. Remove the culture medium. Wash the sample in 500 µl PBS.
   2. Disperse the gelatinous protein mixture using a P1000 pipette tip.
   3. Add 1 ml media to a single well and resuspend the organoids in 1 ml media.
   4. Gently break up the organoids using a P1000 by pipetting up and down 20-30x.
   5. Transfer the organoids to a 1.5 ml tube. Centrifuge at 100 x g at 4 °C, 5 min.
   6. Wash the pellet twice with 1 ml complete culture media.
   7. Resuspend crypts in 500 µl freezing media.
   8. Transfer crypts to a cryotube, place the tube in a freezing container and store in a -80 °C freezer overnight.
   9. Transfer crypts to liquid N₂ tank.
      1. Recover frozen crypt organoids by rapidly thawing in a 37 °C water bath. Wash with 500 µl complete culture media once, centrifuge at 100 x g, 5 min and resuspend in gelatinous protein mixture, and culture as described above. For better recovery, add ROCK inhibitor (Y-27632) to the freezing media.

2. Crypt Organoid Metabolism Assay

1. 24-well Plate Preparation:
   1. Isolate and count crypts according to the protocol (see section 1.1 for crypt isolation and section 1.2 for crypt passage).
   2. Resuspend crypts in gelatinous protein mixture (100-200 crypts per 20 µl).
   3. Plate crypt-gelatinous protein mixture suspension in a 24-well assay plate (make sure there are at least triplicates for each sample) and allow the suspension to solidify at 37 °C in a CO₂ incubator; then add 500 µl complete culture media.
   4. Culture crypts (as described in section 1.1) and observe under microscope until crypts grow into fully developed organoids.
2. Extracellular Flux Assay:
   1. Hydrate cartridge overnight in a non-CO₂ (0% CO₂), 37 °C incubator.
   2. Remove the culture medium and wash organoids twice with 500 µl DMEM (without: glucose, L-glutamine, sodium pyruvate and sodium bicarbonate; and with: phenol red). Wait 5 min.
   3. Add 675 µl per well assay medium (DMEM with 2 mM L-glutamine and 5 mM D-glucose) to each well.
   4. Check crypt organoids microscopic morphology to ensure that organoids and the gelatinous protein mixture are intact after the washes. Incubate 1 hr in a non-CO₂, 37 °C incubator.
   5. Prepare 10 µM injectable compounds (oligomycin, carbonyl cyanide-p-trifluoro-methoxy-phenyl-hydrazone (FCCP), rotenone) in assay medium.
   6. Set-up the cartridge by loading 75 µl of 10 µM injectable compounds into the ports of the cartridge sequentially: Port A - oligomycin; Port B - FCCP; and Port C - rotenone (The final concentrations during the assay will be 1 µM).
   7. Incubate cartridge 30 min – 1 hr at 37 °C in a non-CO₂ incubator.
   8. Simultaneously, turn on the XF Analyzer and create the assay protocol template.
   9. Place cartridge and utility plate into XF Analyzer and run “calibrate” cartridge.
   10. Check crypt organoids microscopically to make sure they are attached to the gelatinous protein mixture.
   11. Load cell culture plate into instrument and run assay protocol.

Wyniki

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...

Dyskusje

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...

Materiały

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
[header]
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