We thank B. Spee for generously providing LD540. This work was supported by a Netherlands Organization for Scientific Research grant (NWO-ZonMW; VIDI 016.146.353) to S.M.

All experimentation using human tissues described herein was approved by the ethical committee at University Medical Center Utrecht (UMCU). Informed consent for tissue collection, generation, storage, and use of the organoids was obtained from patients at the Wilhelmina Children&#39;s Hospital (WKZ)-UMCU.
1. Preparation of Culture Media
NOTE: This protocol should be performed inside a biosafety cabinet. The organoids should be handled according to sta...

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dgat1+and+Dgat2+regulate+enterocyte+triacylglycerol+distribution+and+alter+proteins+associated+with+cytoplasmic+lipid+droplets+in+response+to+dietary+fat.">Dgat1 and Dgat2 regulate enterocyte triacylglycerol distribution and alter proteins associated with cytoplasmic lipid droplets in response to dietary fat.</a> Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids. 1862 (6), 600-614 (2017).</li></ol>

The authors have nothing to disclose.

Here, we provide a protocol to determine LD formation in human intestinal organoids upon incubation with oleic acid. This method is based on the LD-specific fluorescent dye LD54018, which allows for characterization and quantification of the total volume of lipid droplets within an organoid culture. The procedures to establish and maintain human intestinal organoid cultures have been published before13, and a visual guide of this protocol is available as well<sup class="xre...

Lipids are a crucial component of the human diet and play an important role in systemic energy storage and metabolism. When ingested, dietary lipids are degraded into free fatty acids (FFAs) and monoglycerides (MGs) by pancreatic lipases. These substrates are then taken up by the enterocytes of the intestinal epithelium, where they are first re-esterified to diglycerides (DG) by monoglyceride acyltransferases (MGAT) enzymes and subsequently to triglycerides (TG) by diacylglycerol acyltransferase 1 (DGAT1)1. Finally, these TGs are integrated into either chylomicrons for export to the lymph system or cytosolic lipid droplets (LDs) for intracellular storage2,3. Although chylomicrons are needed to distribute dietary lipids to other organs, the importance of intracellular fat storage in LDs is not completely clear. However, LDs have been shown to perform a regulatory function in the intestine, as they slowly release lipids into the circulation up to 16 h after a meal4. Furthermore, LDs have been shown to protect against toxic fatty acid concentrations, such as in mouse adipocytes during lipolytic conditions5.
The DGAT1 protein is located on the endoplasmic reticulum (ER) membrane and plays a crucial role in LD formation in the intestinal epithelium. Homozygous mutations in DGAT1 lead to early-onset severe diarrhea and/or vomiting, hypoalbuminemia, and/or (fatal) protein-losing enteropathy with intestinal failure upon fat intake, illustrating the importance of DGAT1 in lipid homeostasis of the human intestinal epithelium6,7,8,9,10. Since the occurrence of DGAT1-deficiency in humans is rare, access to primary patient-derived cells has been scarce. Furthermore, the long-term culture of intestinal epithelial cells has long been restricted to tumor-derived cell lines which represent the normal physiology only to a limited extend. Therefore, DGAT1-mediated LD formation has mostly been studied in fibroblasts or animal-derived cell lines7,10,11,12. As such, it was recently shown that DGAT1-deficient patient-derived fibroblasts accumulate less LDs compared to healthy control cells after stimulation with oleic acid (OA)8.
Previously, protocols were established to culture epithelial stem cells from any gastrointestinal organ in the form of three-dimensional (3D) organoids13. These intestinal organoids can be kept in culture for a long period of time13, and allow the functional study of patient- and intestinal location-specific epithelial characteristics14. They are genetically and phenotypically stable and can be stored, allowing long-term expansion and biobanking13.
We recently demonstrated that LD formation can be readily measured in human intestinal organoids in a LD formation (LDF) assay6. When exposed to OA for 16 h, organoids generate LDs to protect the cells from lipid-induced toxicity. When OA concentrations are too high, the cells die by caspase-mediated apoptosis6. The LDF assay was previously shown to be largely dependent on DGAT1 as indicated by organoids derived from DGAT1-mutant patients and by the use of DGAT1-specific inhibitors6.
For the LDF assay described in detail here, 3D organoids are cultured from intestinal biopsies and are passaged weekly by disruption into single cells that easily form new organoids. For running the LDF assay, ~7,500 organoid-derived single cells are plated in each well of a 24-well plate. Organoids are formed over several days, incubated overnight with 1 mM OA and stained with LD540, a fluorescent cell-permeable LD-specific dye that facilitates imaging. The LD formation is then quantified by confocal microscopy, fluorescent plate reader, or flow cytometry.
By scaling this LD formation assay to a 96-well format, the assay can also be used for high-throughput analysis of LD formation to screen for novel drugs which affect LD formation in human intestinal organoid cultures, or to study (human genetic) disorders that affect LD metabolism.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Gibco</td>
			<td>12634-028</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement&#160;</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Basement membrane matrix (matrigel)</td>
			<td>BD Biosciences</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DGAT1 inhibitor (AZD 3988)</td>
			<td>Tocris Bioscience</td>
			<td>4837/10</td>
			<td></td>
		</tr>
		<tr>
			<td>Fatty acid free BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Klinipath</td>
			<td>4078-9001</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamin (GlutaMAX, 100X)</td>
			<td>Gibco</td>
			<td>15630-056</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>laser scanning confocal microscope</td>
			<td>Leica</td>
			<td>SP8X</td>
			<td></td>
		</tr>
		<tr>
			<td>LD540</td>
			<td></td>
			<td></td>
			<td>kindly provided by Dr. B. Spee, Utrecht University</td>
		</tr>
		<tr>
			<td>mEGF</td>
			<td>Peprotech</td>
			<td>315-09_500ug</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetyl cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich</td>
			<td>N0636-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Noggin producing cells (HEK293-mNoggin-Fc cells)</td>
			<td></td>
			<td></td>
			<td>MTA with J. den Hertog, Hubrecht Institute</td>
		</tr>
		<tr>
			<td>Oleic acid</td>
			<td>Sigma-Aldrich</td>
			<td>O1008-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>p38 MAPK inhibitor (p38i) (SB202190)</td>
			<td>Sigma-Aldrich</td>
			<td>S7067-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D8662-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS without Ca2+/Mg2+</td>
			<td>Sigma-Aldrich</td>
			<td>D8537-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (5,000 U/ml)</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>R-spondin producing cells (Cultrex HA-R-Spondin1-Fc 293T Cells)</td>
			<td>R&#38;D systems</td>
			<td>3710-001-01</td>
			<td></td>
		</tr>
		<tr>
			<td>TC-treated 24 well plates</td>
			<td>Greiner-One</td>
			<td>662160</td>
			<td></td>
		</tr>
		<tr>
			<td>TC-treated black clear-bottom 96 well plates</td>
			<td>Corning Life Sciences</td>
			<td>353219</td>
			<td></td>
		</tr>
		<tr>
			<td>TGFb type I receptor inhibitor (A83-01)&#160;</td>
			<td>Tocris Bioscience</td>
			<td>2939/10</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin (TrypLE Express)</td>
			<td>Life Technologies</td>
			<td>12604021</td>
			<td></td>
		</tr>
		<tr>
			<td>WNT-3A producing cells (L-Wnt-3A cells)</td>
			<td></td>
			<td></td>
			<td>MTA with J. den Hertog, Hubrecht Institute</td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride (Rho kinase inhibitor)</td>
			<td>Abcam</td>
			<td>ab120129-10</td>
			<td></td>
		</tr>
	</tbody>
</table>

a fluorescence-based assay for characterization and quantification of lipid droplet formation in human intestinal organoids

Dietary lipids are taken up as free fatty acids (FAs) by the intestinal epithelium. These FAs are intracellularly converted into triglyceride (TG) molecules, before they are packaged into chylomicrons for transport to the lymph or into cytosolic lipid droplets (LDs) for intracellular storage. A crucial step for the formation of LDs is the catalytic activity of diacylglycerol acyltransferases (DGAT) in the final step of TG synthesis. LDs are important to buffer toxic lipid species and regulate cellular metabolism in different cell types. Since the human intestinal epithelium is regularly confronted with high concentrations of lipids, LD formation is of great importance to regulate homeostasis. Here we describe a simple assay for the characterization and quantification of LD formation (LDF) upon stimulation with the most common unsaturated fatty acid, oleic acid, in human intestinal organoids. The LDF assay is based on the LD-specific fluorescent dye LD540, which allows for quantification of LDs by confocal microscopy, fluorescent plate reader, or flow cytometry. The LDF assay can be used to characterize LD formation in human intestinal epithelial cells, or to study human (genetic) disorders that affect LD metabolism, such as DGAT1 deficiency. Furthermore, this assay can also be used in a high-throughput pipeline to test novel therapeutic compounds, which restore defects in LD formation in intestinal or other types of organoids.

For proper analysis of LD formation, the organoids should not be seeded too densely prior to stimulation with OA and subsequent staining. This is especially of importance for the confocal and plate reader readout, since overlapping organoids might interfere with the fluorescence. An example of proper organoid seeding density (Figure 1A) and a culture with overlapping organoids is shown (Figure 1B...

Watch this Scientific Journal Video about A Fluorescence-based Assay for Characterization and Quantification of Lipid Droplet Formation in Human Intestinal Organoids at JoVE.com

A Fluorescence-based Assay for Characterization and Quantification of Lipid Droplet Formation in Human Intestinal Organoids

This protocol describes an assay for the characterization of lipid droplet (LD) formation in human intestinal organoids upon stimulation with fatty acids. We discuss how this assay is used for quantification of LD formation, and how it can be used for high throughput screening for drugs that affect LD formation.

Dietary lipids are taken up as free fatty acids (FAs) by the intestinal epithelium. These FAs are intracellularly converted into triglyceride (TG) ...

This protocol describes some methods to quantify lipid droplet formation in human intestinal organoids. It can be used as a high-throughput screening platform to test for drugs that modulate lipid droplet formation. The method we describe is based on a lipid droplet-specific dye and biopsy-derived intestinal organoids.It thereby provides a stable, accurate, and physiologically-relevant model for lipid droplet formation. This protocol can be used to screen for patient-specific novel drug candidates that modulate lipid droplets formation, for example, in DGAT1-deficient patients. This lipid droplet formation assay can also be applied to other types of organoid cultures to study lipid metabolism in other cell types.The assay is to a significant extent dependent on the density of the organoid culture. Try to ensure a consistent organoid density across samples and experiments. Visual demonstration of this assay is critical because it is important to show how the organoid should be cultured and how they should be handled to ensure proper analysis of lipid droplet formation.After preparing the organoids, carefully aspirate the culture medium without disturbing the droplets of basement membrane matrix. Add 500 microliters of cold basal medium to the first well of organoids. Using a P1000 pipette, gently pipette up and down to disrupt the basement membrane matrix droplets with organoids.Repeat this procedures with the same medium in the next well as required, but do not harvest more than two wells per 500 microliters of basal medium. Collect the organoids in a low-binding 1.5 milliliter microcentrifuge tube, and spin them down in a mini-tabletop centrifuge for 15 to 20 seconds. Add 400 microliters of trypsin to the spun-down organoids.Incubate in a water bath at 37 degrees Celsius for five minutes. Next, use a P200 pipette to gently pipette the suspension up and down to disrupt the remaining aggregates of cells. Incubate in a water bath at 37 degrees Celsius for five minutes.When only single cells remain, add one milliliter of basal medium, and spin down the cells in a mini-tabletop centrifuge for 15 to 20 seconds. Aspirate the supernatant completely. Use a P200 pipette to re-suspend the single cells in 200 microliters of fresh hSI-EM, and add an additional 800 microliters of hSI-EM.Then count the number of cells in suspension and calculate the volume of cells needed for the final cell density. Take out the appropriate volume of suspension and adjust the density to 750 cells per microliter. Add basement membrane matrix to the cell suspension in a ratio of two-to-one.Gently pipette the suspension to mix being careful to avoid creating bubbles. In a pre-warmed culture plate, seed out three 10-microliter droplets per well if the plate has 24 wells or one five-microliter droplet per well if the plate has 96 wells. Place the plate in an incubator at 37 degrees Celsius with five percent carbon dioxide for 10 to 15 minutes to solidify the droplets.Meanwhile, pre-warm an appropriate volume of hSI-EM+Y in a water bath at 37 degrees Celsius. After this, carefully add the pre-warmed hSI-EM+Y to each well of the plate. Incubate the cells at 37 degrees Celsius with five percent carbon dioxide.After two to three days, replace the medium with hSI-EM and make sure to refresh it two to three times a week. First, weigh 0.2 grams of liquid oleic acid at room temperature. Add 1.5 milliliters of culture-grade sterile PBS and heat the mixture to 70 degrees Celsius for one hour making sure to vortex intermittently.Next, weigh 5.89 grams of fatty acid-free BSA and dissolve it in 33.9 milliliters of PBS. Warm the mixture in a water bath at 37 degrees Celsius until the BSA is fully dissolved. Vortex the oleic acid mixture again to create an emulsion of fine droplets and immediately add it to the BSA solution using a glass pipette.Keep the mixture at 37 degrees Celsius for 30 minutes until a clear yellowish solution remains. Passage the organoids as previously described and seed out the organoid-derived single cells into a black clear-bottom 96-well plate. On day six of culturing on hSI-EM, replace the culture medium with hSI-EM containing one millimolar of the oleic acid and BSA conjugate.Incubate the cells for 16 to 17 hours at 37 degrees Celsius in the presence or absence of 0.1 micromolar DGAT1 inhibitor. After this, aspirate the medium without disturbing the basement membrane matrix droplets. Fixate the organoids by adding 100 microliters of four percent neutral buffered formaldehyde to the wells for 30 minutes at room temperature.Remove the formaldehyde gently and carefully wash the cells with 150 microliters of PBS per well. Stain the cells for lipid droplets with 025 milligrams per milliliter LD540 and DAPI in PBS for 15 minutes at room temperature in the dark. Then wash the wells carefully with PBS.For overview images of whole organoids use a 40X subjective suited for confocal fluorescent imaging. Set the microscope to image for DAPI and the LD540 dye using the excitation and emission settings shown here. Set the pinhole size to one airy unit for sufficient z-axis resolution.To image one-half of a spherical organoid set a z-stack to approximately 85 micrometers. Using an appropriate image processing system, transform the z-stack of each organoid to a maximum projection by clicking Image, Stacks, Z Project. Set the threshold of the maximum projection to a level in which there is no LD540 signal visible in the BSA vehicle control sample by clicking Image, Adjust, Threshold.Use these settings to threshold each image. Then measure the total area of fluorescence for each maximum projection using the function Analyze, Analyze Particles. For proper analysis of lipid droplet formation, the organoids should not be seeded too densely prior to stimulation with oleic acid and subsequent staining.This is especially of importance for the confocal and plate-reader readout since overlapping organoids might interfere with the fluorescence. After stimulation with one millimolar of oleic acid overnight, lipid droplet formation can be visualized with an inverted bright-field microscope. The accumulation of lipid droplets scatters transmitted light, and therefore the organoids appear darker, whereas non-stimulated organoids have a translucent appearance.Once the oleic acid-stimulated organoids are fixed and stained, the lipid droplet formation can be visualized using a confocal microscope. As the organoids are 3D structures, a regular epifluorescent microscope is not suitable due to the out-of-focus background signal, so a confocal z-stack is used to characterize LDF in organoids. Quantification of the confocal microscopy samples can also be performed using a fluorescent plate reader normalized to the fluorescent Hoechst signal.The plate reader assay indicates a significant decrease in the LD540 signal in organoid cells treated with DGAT1 inhibitor compared to untreated organoids. Quantification of LD formation in individual cells can be achieved using a flow cytometer. The gating strategy shown here is for dissociated human intestinal organoids.Representative histograms for both the SSC-A and the LD540 signal of the final live cell population show that lipid droplet formation will result in an increase in SSC-A due to the formation of intracellular lipid droplets. In addition, LD540 stains for lipids that are stored in the lipid droplets, and the signal will also increase upon the induction of lipid droplet formation. As such, lipid droplet formation is measured as a shift in the mean fluorescent intensity of both SSC-A and LD540.The most crucial steps of this protocol are the culture of human intestinal organoids and the proper corrugation of oleic acid to BSA. To accommodate more conditions, this assay can be analyzed using a fluorescent plate reader. To correct for the organoid number per well, the LD540 signal was normalized to the DAPI signal.With this technique, researchers can study lipid droplet formation in organoid systems, and improve various analysis techniques such as electron microscopy to validate their findings.

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Anatomy and Physiology

Medicine

Unit 1

Biochemistry of the Cell

SCIENTISTS IN ACTION

8.8K visualizações.  University Medical Center Utrecht, Utrecht University. Este protocolo descreve alguns métodos para quantificar a formação de gotículas lipídicas em organoides intestinais humanos. Pode ser usado como uma plataforma de triagem de alto rendimento para testar drogas que modulam a formação de gotículas lipídicas. O método que descrevemos é baseado em um corante específico de gotícula lipídica e organoides intestinais derivados da biópsia.Assim, fornece um modelo estável, preciso e fisiologicamente relevante para a formação de...