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 standard cell culture guidelines.
<ol>
	<li>Prepare basal culture medium. 
	NOTE: Culture medium without growth factors is referred to as basal medium (BM).
	<ol>
		<li>Add 5 mL of HEPES (1 M), 5 mL of L-glutamin (100x) and 5 mL of penicillin-streptomycin (5,000 U/mL) to 500 mL of advanced Dulbecco&#39;s modified Eagle medium with Ham&#39;s nutrient mixture F-12 to prepare BM medium.</li>
		<li>Store the prepared BM medium at 4 &#176;C and use for a maximum of 2 months.</li>
	</ol>
	</li>
	<li>Prepare R-spondin and Noggin conditioned medium (CM) according the manufacturer&#39;s protocol.
	<ol>
		<li>Briefly, grow up the cells to the desired quantity in hyperflasks with 5 x 107&#160;cells in 555 mL medium without selective antibiotic per flask.</li>
		<li>Grow the cells for 4 days until confluent.</li>
		<li>Replace the medium with BM and culture for 8 additional days.</li>
		<li>After 8 days of culture at 37 &#176;C, collect the culture medium and centrifuge for 5 min at 450 x g to pellet any remaining cells.</li>
		<li>Filter sterilize the supernatant, and store aliquots of the R-spondin or Noggin CM at -20 &#176;C for a maximum of 6 months.</li>
	</ol>
	</li>
	<li>Prepare Wnt3A-CM according to Boj et al.15.
	<ol>
		<li>Briefly, grow up the cells to the desired quantity in 145 mm dishes with 2 x 106 cells in 20 mL medium without selective antibiotic per dish. Wrap each dish with plastic foil to avoid evaporation of the culture medium.</li>
		<li>After 8 days of culture at 37 &#176;C, collect the culture medium and centrifuge for 5 min at 450 x g to pellet any remaining cells.</li>
		<li>Filter sterilize the supernatant, and store aliquots of the Wnt3A-CM at 4 &#176;C for a maximum of 2 months.</li>
	</ol>
	</li>
	<li>Prepare organoid expansion medium. 
	NOTE: Human small intestinal organoid expansion medium (see recipe in Supplemental Table 1) is referred to as hSI-EM. The following steps will produce a final volume of 1 L hSI-EM, and can be scaled up or down as required.
	<ol>
		<li>Dissolve 1.46 g of nicotinamide in 12 mL of cell culture grade phosphate-buffered saline (PBS) to create a final dilution of 1 M.</li>
		<li>Dissolve 245 mg of n-acetyl cysteine in 3 mL of cell culture grade PBS to create a final dilution of 500 mM. 
		NOTE: To accelerate the formation of a solution, the nicotinamide and n-acetyl cysteine can be incubated in a water bath at 37 &#176;C. Both solutions can be prepared in batch, aliquoted, and stored at -20 &#176;C for future use.</li>
		<li>Filter sterilize both solutions through a 0.22 &#181;m filter into a sterile 15 mL tube.</li>
		<li>Add 167 mL of BM to a sterile 500 mL culture medium flask. Add 200 mL of R-Spondin-CM, 100 mL of noggin-CM, and 100 &#181;L of recombinant mEGF (500 &#181;g/mL) to a final concentration of 50 ng/mL.</li>
		<li>Add 10 mL of the sterilized nicotinamide solution and 2.5 mL of the sterilized n-acetyl cysteine solution. Add 20 mL of B27 supplement (50x). 
		NOTE: At this stage, the medium is referred to as hSI-EM without WAS (Wnt3A-CM, A83-01, and SB202190), and can be aliquoted and stored at -20 &#176;C. If the medium is aliquoted into smaller volumes, adjust the concentrations of the following steps accordingly.</li>
		<li>To 500 mL of hSI-EM without WAS, add 10 mL of Wnt3A-CM of the last freshly prepared batch and 10 mL of Wnt3A-CM of the second last batch to minimize variability changes in Wnt3A-CM conditions.</li>
		<li>Add A83-01 to a final concentration of 500 nM, and SB202190 to a final concentration of 10 &#181;M.</li>
		<li>Store the prepared hSI-EM (with WAS) at 4 &#176;C and use for a maximum of 2 weeks. 
		NOTE: Add additional Y-27632 to a final concentration of 10 &#181;M (referred to as hSI-EM+Y) when crypts or single cells are cultured or organoids are started from cryopreservation.</li>
	</ol>
	</li>
	<li>Prepare fluorescent activated cell sorting (FACS) buffer.
	<ol>
		<li>Add 10 mL of fetal calf serum (FCS) to 40 mL of PBS without Ca2+/Mg2+ for a final concentration of 10% FCS. 
		NOTE: The FCS prevents cells from adhering to labware.</li>
	</ol>
	</li>
</ol>
2. Culture Procedures for Human Small Intestinal Organoids
NOTE: This protocol should be performed inside a biosafety cabinet. The organoids should be handled according to standard cell culture guidelines. When handling organoids or organoid-derived cells, the cells should be kept on ice whenever possible. The cells will remain viable for a few hours after harvesting when this is ensured. Organoids should be cultured in a standard cell culture incubator at 37 &#176;C with 5% CO2. These conditions apply to all incubation steps with organoids embedded in basement membrane matrix (BMM; i.e., Matrigel) throughout this protocol. The authors have used duodenum-derived organoids for these assays.
<ol>
	<li>Passaging organoids 
	NOTE: Every organoid culture has its own doubling time. Normally, small intestinal organoids can be passaged 1:3&#8722;1:5 every 7&#8722;10 days. When passaged as single cells, passaging efficiency can be up to 1:20, depending on cell density. For establishment and maintenance, organoids are cultured in 24-well plates; for the LDF assay, in either 24- or 96-well plates.
	<ol>
		<li>Preparation
		<ol>
			<li>Pre-warm unpacked 24-well tissue culture plates in an incubator at 37 &#176;C, 5 % CO2 at least overnight and preferably 5 days in advance. 
			NOTE: Pre-warming the tissue culture plates in the cell culture incubator ensures proper formation and attachment of organoid-containing droplets of BMM.</li>
			<li>To reduce the number of freeze-thaw cycles, prepare 1 mL aliquots of BMM and store at -20 &#176;C. Thaw a vial of BMM on ice at least 30 min before starting the organoid passaging procedure.</li>
			<li>Keep a 50 mL tube of BM on ice as washing medium.</li>
			<li>Prepare hSI-EM as described in section 1.4, and prepare an appropriate amount of hSI-EM+Y, for example, 15 mL for a full 24-well plate.</li>
		</ol>
		</li>
		<li>Collect organoids.
		<ol>
			<li>Carefully aspirate the culture medium without disturbing the droplets of BMM.</li>
			<li>Add 500 &#181;L of cold BM to the first well of organoids, and disrupt the BMM droplets with organoids by pipetting gently up and down with a P1000 pipet.</li>
			<li>Repeat this procedure with the same medium in the next well as required, but do not harvest more than 2 wells per 500 &#181;L of BM.</li>
			<li>Collect the organoids in a low-binding 1.5 mL microcentrifuge tube and spin down in a mini tabletop centrifuge for 15&#8722;20 s (max. 2,000 x g). Aspirate the supernatant completely and remove the last bit of medium with a P200 pipet. 
			NOTE: When the organoids cultures look clean under a microscope and do not contain any dead cells or other debris, the BMM drops can be harvested directly in trypsin instead of BM in step 2.1.2.2. After step 2.1.2.3, proceed directly to incubation in step 2.1.3.1.</li>
		</ol>
		</li>
		<li>Dissociate the organoids into single cells.
		<ol>
			<li>Add 400 &#181;L of trypsin to the spun down organoids. Incubate the organoids at 37 &#176;C for 5 min in a water bath.</li>
			<li>Disrupt the remaining aggregates of cells by pipetting up and down gently with a P200 pipet. Again, incubate the organoids at 37 &#176;C for 5 min in a water bath.</li>
			<li>Check the progress of cell dissociation under a microscope using 4x magnification. Repeat manual disruption and incubation when large clumps of cells remain.</li>
			<li>When only single cells remain, add 1 mL of BM and spin down the cells in a mini tabletop centrifuge for 15&#8722;20 s.</li>
			<li>Aspirate the supernatant completely. Resuspend the single cells in 200 &#181;L of fresh hSI-EM using a P200 pipet. Add an additional 800 &#181;L of hSI-EM.</li>
			<li>Count the number of cells in suspension. 
			NOTE: In the authors&#39; lab, manual counting of dissociated organoid cells yields more reliable results than an automated cell counter. When an automated cell counter is used, the cell density for downstream applications should be tested in-house and adjusted if too little or too many organoids grow out.</li>
		</ol>
		</li>
		<li>Seed organoids for maintenance or lipid droplet formation assay.
		<ol>
			<li>Calculate the volume of cells which is needed for the final cell density. 
			NOTE: Approximately 250 cells/&#181;L is an appropriate density. In a 24-well plate, 30 &#181;L is seeded into each well, while 5 &#181;L is seeded into each well of a 96-well plate.</li>
			<li>Take out the appropriate volume of suspension and adjust the density to 750 cells/&#181;L (or spin down and resuspend when the density is too low).</li>
			<li>Add BMM to the cell suspension in a ratio of 2:1. The final cell density in this mixture is 250 cells/&#181;L. Gently mix the suspension by pipetting, carefully avoiding any bubbles.</li>
			<li>In a pre-warmed tissue culture plate, seed out three 10 &#181;L droplets per well in a 24-well plate or a single 5 &#181;L droplet per well in a 96-well plate.</li>
			<li>Place the plate in an incubator at 37 &#176;C, 5% CO2 for 10&#8722;15 min to solidify the BMM droplets. Meanwhile, pre-warm an appropriate amount of hSI-EM+Y in a water bath at 37 &#176;C.</li>
			<li>Carefully add 500 &#181;L of pre-warmed hSI-EM+Y to each well of a 24-well plate or 100 &#181;L to each well of a 96-well plate. Incubate the cells in an incubator at 37 &#176;C, 5% CO2 and after 2&#8722;3 days change the medium to hSI-EM (without Y) and refresh 2&#8722;3 times a week. 
			NOTE: After 7&#8722;10 days, a maintenance culture of organoids should be passaged again.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Lipid Droplet Formation Assay
<ol>
	<li>Preparation of oleic acid conjugate 
	NOTE: Since oleic acid (OA) is hydrophobic and not soluble in water, it is conjugated to bovine serum albumin (BSA). BSA can bind multiple fatty acid molecules and is in this case used in a 1:8 ratio to make oleic acid accessible to the intestinal cells. Free fatty acids and BSA can both bind to plastic labware. To ensure a proper final concentration, use glass vials and glass pipets whenever possible.
	<ol>
		<li>Weigh 0.2 g of liquid oleic acid at room temperature. Add 1.5 mL of culture-grade sterile PBS and heat the mixture to 70 &#176;C for 1 h. Vortex intermittently.</li>
		<li>Weigh 5.89 g of fatty acid-free BSA and dissolve in 33.9 mL of PBS. Warm the mixture in a water bath at 37 &#176;C until the BSA is fully dissolved.</li>
		<li>Vortex the OA mixture again to create an emulsion of fine droplets and immediately add it to the BSA solution using a glass pipet. Keep the final solution close to 37 &#176;C after adding the OA. The final mixture consists of 20 mM OA in 2.5 mM BSA.</li>
		<li>Keep the mixture at 37 &#176;C for 30 min until a clear yellowish solution remains.</li>
		<li>The OA-BSA conjugate can be aliquoted and frozen at -20 &#176;C for at least 6 months. Upon thawing an aliquot, incubate it at 37 &#176;C until the cloudiness dissolves and the mixture is clear again. 
		NOTE: Because of the high protein and lipid content of the final solution, the mixture cannot be filter sterilized or autoclaved. When the components were handled with care, in a laminar flow hood when possible and using sterile PBS, the authors did not experience microbial infections.</li>
	</ol>
	</li>
	<li>LDF confocal assay
	<ol>
		<li>Sample preparation
		<ol>
			<li>Passage organoids as described in section 2.1. Seed out the organoid-derived single cells in a black clear-bottom 96-well plate.</li>
			<li>On day 6 of culture on hSI-EM, replace the culture medium with hSI-EM containing 1 mM OA-BSA conjugate.</li>
			<li>Incubate the cells for 16&#8722;17 h (overnight) at 37 &#176;C in presence or absence of 0.1 &#181;M DGAT1 inhibitor. Include a vehicle control of 2.5 mM BSA.</li>
			<li>After 16&#8722;17 h, aspirate the medium without disturbing the BMM droplets. Fixate the organoids by adding 100 &#181;L of 4% neutral buffered formaldehyde to the wells for 30 min at room temperature. 
			NOTE: The formaldehyde will partially dissolve the BMM, while the organoids sink to the bottom and adhere to the bottom of the plate.</li>
			<li>Remove the formaldehyde gently, and carefully wash the wells with 150 &#181;L of PBS per well.</li>
			<li>Stain the cells for LDs with 0.025 mg/mL LD540 and 4&#8242;,6-diamidino-2-phenylindole (DAPI) in PBS for 15 min at room temperature in the dark.</li>
			<li>Wash the wells carefully with PBS. 
			NOTE: The protocol can be paused here when necessary. Keep the samples covered in PBS in the dark at 4 &#176;C. The LD540 staining will remain stable for up to a week. This assay can also be performed with live cells, to monitor LD formation over a period of time. For this, the fixation has to be omitted from the protocol, and the DAPI should be replaced with Hoechst staining. LD540 can stain LDs in living cells in the same concentration as described.</li>
		</ol>
		</li>
		<li>Confocal imaging 
		NOTE: The imaging can be performed on prepared organoid samples in the black clear-bottom 96-well plate.
		<ol>
			<li>For overview images of whole organoids, use a 40x objective suited for confocal fluorescent imaging.</li>
			<li>Set the microscope to image the DAPI channel at 405 nm excitation and ca. 410&#8722;535 nm emission wavelength. For the LD540 dye choose an excitation laser at 540 nm (543 nm is optimal) and set the emission filters to 545&#8722;700 nm. 
			NOTE: In this protocol, a laser-scanning confocal system with a white light laser, acousto-optical beam splitter (AOBS), 10x/20x objective, and spectral detection system was used. This allows for exact tuning to the specific wavelengths. If a comparable system is not available, choose laser lines and short-/long-pass filters close to the specifications above. For whole-organoid imaging, a resolution of 512 x 512 or 1024 x 1024 is sufficient for downstream image analysis.</li>
			<li>Set the pinhole size to 1 airy unit (AU) for sufficient z-axis resolution.</li>
			<li>To image one half of a spherical organoid, set a z-stack to approximately 85 &#181;m.</li>
		</ol>
		</li>
		<li>Image analysis 
		NOTE: For image analysis, Fiji/ImageJ16,17 was used to generate maximum projections. The analysis can be performed with any image analysis software package which allows for maximum projection, manual thresholding, and particle analysis.
		<ol>
			<li>Using Fiji/ImageJ, transform the z-stack of each organoid to a maximum projection: Image | Stacks | Z-project.</li>
			<li>Set the threshold of the maximum projection to a level in which there is no LD540 signal visible in the BSA vehicle control sample: Image | Adjust | Threshold. Use these settings to threshold each image.</li>
			<li>Measure the total area of fluorescence for each maximum projection using the function Analyze | Analyze particles. 
			NOTE: Although the assay resolution is better using a confocal microscope, this assay can also be analyzed by using a fluorescent plate reader with similar filters. To normalize for the organoid count per well, the LD540 signal should be divided by the DAPI signal. Finally, the signal of the BSA vehicle control should be subtracted to normalize the measurements. This approach allows the assay to be scaled towards a 96-well plate format.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>LDF flow cytometric assay
	<ol>
		<li>Sample preparation
		<ol>
			<li>Passage organoids as described in section 2.1. Seed out the organoid-derived single cells in a 24-well tissue culture plate. Two wells per condition is sufficient for flow cytometry.</li>
			<li>On day 10 of culture on hSI-EM, replace the culture medium with hSI-EM containing 1 mM OA-BSA conjugate. 
			NOTE: In contrast with the confocal analysis, 10 days of growth in EM was chosen for the flow cytometry assay instead of 6 days. The extra 4 days of expansion will result in optically overlapping organoids which would complicate the microscopy-based assay. However, the greater number of cells facilitates flow cytometry analysis.</li>
			<li>Incubate the cells for 16&#8722;17 h (overnight) at 37 &#176;C in presence or absence of 0.1 &#181;M DGAT1 inhibitor. Include a vehicle control of 2.5 mM BSA.</li>
			<li>After 16&#8722;17 h, collect the organoids as described in section 2.1.2.</li>
			<li>Dissociate the organoids into single cells as described in section 2.1.3. 
			NOTE: Although not strictly required, it is recommended to check the cell count in each sample. A total count of 10,000 cells per sample is the absolute minimum required for sufficient resolution. For optimal results use ca. 50,000&#8722;100,000 cells.</li>
			<li>Spin down the cells in a mini tabletop centrifuge for 15&#8722;20 s.</li>
			<li>Stain each sample with 500 &#181;L of 0.025 mg/mL LD540 and 1 &#181;g/mL Hoechst in PBS for 15 min at room temperature in the dark.</li>
			<li>Spin down the cells in a mini tabletop centrifuge for 15&#8722;20 s and wash 3x with PBS.</li>
			<li>Fixate the cells by resuspending them in 500 &#181;L of 4% neutral buffered formaldehyde for 15 min at room temperature.</li>
			<li>Spin down the cells and wash 3x with FACS buffer.</li>
			<li>Pre-rinse FACS tubes with FACS buffer to prevent cells sticking to the tube wall.</li>
			<li>Resuspend the cells in 200 &#181;L of FACS buffer and transfer the cell suspension to the pre-rinsed FACS tubes.</li>
		</ol>
		</li>
		<li>Flow cytometric analysis
		<ol>
			<li>Set the gating parameters to exclude dead cells and clumps of cells (see the representative results section).</li>
			<li>In the final gated population, measure at least 10,000 cells for reliable results. 
			NOTE: From this population, the mean fluorescent intensity (MFI) of LD540 and the mean SSC-A signal together provide a measure of the total volume of LD formation per cell.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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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><tbody><tr><td>Advanced DMEM/F12</td><td>Gibco</td><td>12634-028</td><td></td></tr><tr><td>B27 supplement&amp;nbsp;</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 Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</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&amp;amp;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)&amp;nbsp;</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...

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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) ...

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9.0K Views.  University Medical Center Utrecht, Utrecht University. Dit protocol beschrijft een aantal methoden om lipide druppelvorming in menselijke darmorganoïden te kwantificeren. Het kan worden gebruikt als een high-throughput screening platform om te testen op geneesmiddelen die lipide druppelvorming moduleren. De methode die we beschrijven is gebaseerd op een lipide druppelspecifieke kleurstof en biopsie-afgeleide darmorganoïden.Het biedt daardoor een stabiel, nauwkeurig en fysiologisch relevant model voor lipidedruppelvorming. Dit protocol ka...

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

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.

This protocol describes the measurement of epithelial barrier permeability in real-time following pharmacologic treatment in human intestinal organoids using fluorescent microscopy and live cell microscopy.

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

Developmental Biology

Assessing Hepatic Steatosis with 3D Fluorescence Lipid Droplet Reconstruction

Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment

Lipid droplets (LDs) are specialized organelles that mediate lipid storage and play a very important role in suppressing lipotoxicity and preventing dysfunction caused by free fatty acids (FAs). The liver, given its critical role in the body's fat metabolism, is persistently threatened by the intracellular accumulation of LDs in the form of both microvesicular and macrovesicular hepatic steatosis. The histologic characterization of LDs is typically based on lipid-soluble diazo dyes, such as Oil Red O (ORO) staining, but a number of disadvantages consistently hamper the use of this analysis with liver specimens. More recently, lipophilic fluorophores 493/503 have become popular for visualizing and locating LDs due to their rapid uptake and accumulation into the neutral lipid droplet core. Even though most applications are well-described in cell cultures, there is less evidence demonstrating the reliable use of lipophilic fluorophore probes as an LD imaging tool in tissue samples. Herein, we propose an optimized boron dipyrromethene (BODIPY) 493/503-based protocol for the evaluation of LDs in liver specimens from an animal model of high-fat diet (HFD)-induced hepatic steatosis. This protocol covers liver sample preparation, tissue sectioning, BODIPY 493/503 staining, image acquisition, and data analysis. We demonstrate an increased number, intensity, area ratio, and diameter of hepatic LDs upon HFD feeding. Using orthogonal projections and 3D reconstructions, it was possible to observe the full content of neutral lipids in the LD core, which appeared as nearly spherical droplets. Moreover, with the fluorophore BODIPY 493/503, we were able to distinguish microvesicles (1 &#181;m &lt; d &#8804; 3 &#181;m), intermediate vesicles (3 &#181;m &lt; d &#8804; 9 &#181;m), and macrovesicles (d &gt; 9 &#181;m), allowing the successful discrimination of microvesicular and macrovesicular steatosis. Overall, this BODIPY 493/503 fluorescence-based protocol is a reliable and simple tool for hepatic LD characterization and may represent a complementary approach to the classical histological protocols.

Author Spotlight: Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment  

Herein, we demonstrate an optimized BODIPY 493/503 fluorescence-based protocol for lipid droplet characterization in liver tissue. Through the use of orthogonal projections and 3D reconstructions, the fluorophore allows for successful discrimination between microvesicular and macrovesicular steatosis and may represent a complementary approach to the classical histological protocols for hepatic steatosis assessment.

Lipid droplets (LDs) are specialized organelles that mediate lipid storage and play a very important role in suppressing lipotoxicity and preventing ...

Biochemistry

Exploring the Regulation of Lipid Droplet Catabolism through Lipophagy

Macroautophagy, commonly referred to as autophagy, is a highly conserved cellular process responsible for the degradation of cellular components. This process is particularly prominent under conditions such as fasting, cellular stress, organelle damage, cellular damage, or aging of cellular components. During autophagy, a segment of the cytoplasm is enclosed within double-membrane vesicles known as autophagosomes, which then fuse with lysosomes. Following this fusion, the contents of autophagosomes undergo non-selective bulk degradation facilitated by lysosomes. However, autophagy also exhibits selective functionality, targeting specific organelles, including mitochondria, peroxisomes, lysosomes, nuclei, and lipid droplets (LDs). Lipid droplets are enclosed by a phospholipid monolayer that isolates neutral lipids from the cytoplasm, protecting cells from the harmful effects of excess sterols and free fatty acids (FFAs). Autophagy is implicated in various conditions, including neurodegenerative diseases, metabolic disorders, and cancer. Specifically, lipophagy -- the autophagy-dependent degradation of lipid droplets -- plays a crucial role in regulating intracellular FFA levels across different metabolic states. This regulation supports essential processes such as membrane synthesis, signaling molecule formation, and energy balance. Consequently, impaired lipophagy increases cellular vulnerability to death stimuli and contributes to the development of diseases such as cancer. Despite its significance, the precise mechanisms governing lipid droplet metabolism regulated by lipophagy in cancer cells remain poorly understood. This article aims to describe confocal imaging acquisition and quantitative imaging analysis protocols that enable the investigation of lipophagy associated with metabolic changes in cancer cells. The results obtained through these protocols may shed light on the intricate interplay between autophagy, lipid metabolism, and cancer progression. By elucidating these mechanisms, novel therapeutic targets may emerge for combating cancer and other metabolic-related diseases.

Lipophagy is a selective form of autophagy that involves the degradation of lipid droplets. Dysfunctions in this process are associated with cancer development. However, the precise mechanisms are not yet fully understood. This protocol describes quantitative imaging approaches to better understand the interplay between autophagy, lipid metabolism, and cancer progression.

Macroautophagy, commonly referred to as autophagy, is a highly conserved cellular process responsible for the degradation of cellular components. This ...

Cancer Research

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes all liposomes of the ensemble to be identical. However, new experimental platforms able to observe liposomes at the single-particle level have made it possible to perform highly sophisticated and quantitative studies on protein-membrane interactions or drug carrier properties on individual liposomes, thus avoiding errors from ensemble averaging. Here we present a protocol for preparing, detecting, and analyzing single liposomes using a fluorescence-based microscopy assay, facilitating such single-particle measurements. The setup allows for imaging individual liposomes in a massive parallel manner and is employed to reveal intra-sample size and compositional inhomogeneities. Additionally, the protocol describes the advantages of studying liposomes at the single liposome level, the limitations of the assay, and the important features to be considered when modifying it to study other research questions.

This protocol describes the fabrication of liposomes and how these can be immobilized on a surface and imaged individually in a massive parallel manner using fluorescence microscopy. This allows for the quantification of the size and compositional inhomogeneity between single liposomes of the population.

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes ...

Bioengineering

Investigating Intestinal Barrier Breakdown in Living Organoids

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was not possible in classical cell culture monolayers. Moreover, monolayer cell cultures are good in vitro model systems but do not represent the complex cellular differentiation processes and functions that rely on 3D structure. This has so far only been possible in animal experiments, which are laborious, time consuming, and hard to assess by optical techniques. Here we describe an assay to quantitatively determine the barrier integrity over time in living small intestinal mouse organoids. To validate our model, we applied interferon gamma (IFN-&#947;) as a positive control for barrier destruction and organoids derived from IFN-&#947; receptor 2 knock out mice as a negative control. The assay allowed us to determine the impact of IFN-&#947; on the intestinal barrier integrity and the IFN-&#947; induced degradation of the tight junction proteins claudin-2, -7, and -15. This assay could also be used to investigate the impact of chemical compounds, proteins, toxins, bacteria, or patient-derived probes on the intestinal barrier integrity.

Here we describe a technique to quantify the barrier integrity of small intestinal organoids. The fact that the method is based on living organoids enables the sequential investigation of different barrier integrity modulating substances or combinations thereof in a time-resolved manner.

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was ...

Culture Methods to Study Apical-Specific Interactions using Intestinal Organoid Models

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to external cues. Recent optimization of in vitro culture conditions allows for the re-creation of the intestinal stem cell niche and the development of advanced 3-dimensional (3D) culture systems that recapitulate the cell composition and the organization of the epithelium. Intestinal organoids embedded in an extracellular matrix (ECM) can be maintained for long-term and self-organize to generate a well-defined, polarized epithelium that encompasses an internal lumen and an external exposed basal side. This restrictive nature of the intestinal organoids presents challenges in accessing the apical surface of the epithelium in vitro and limits the investigation of biological mechanisms such as nutrient uptake and host-microbiota/host-pathogen interactions. Here, we describe two methods that facilitate access to the apical side of the organoid epithelium and support the differentiation of specific intestinal cell types. First, we show how ECM removal induces an inversion of the epithelial cell polarity and allows for the generation of apical-out 3D organoids. Second, we describe how to generate 2-dimensional (2D) monolayers from single cell suspensions derived from intestinal organoids, comprised of&#160;mature and differentiated cell types. These techniques provide novel tools to study apical-specific interactions of the epithelium with external cues in vitro and promote the use of organoids as a platform to facilitate precision medicine.

Here we present two protocols that allow the modeling of intestinal apical-specific interactions. Organoid-derived intestinal monolayers and Air-Liquid Interface (ALI) cultures facilitate the generation of well-differentiated epithelia accessible from both luminal and basolateral sides, whereas polarity-inverted intestinal organoids expose their apical side and are amenable to high throughput assays.

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to ...

Biology

Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe

Reactive oxygen species (ROS) play essential roles in intestinal homeostasis. ROS are natural by-products of cell metabolism. They are produced in response to infection or injury at the mucosal level as they are involved in antimicrobial responses and wound healing. They are also critical secondary messengers, regulating several pathways, including cell growth and differentiation. On the other hand, excessive ROS levels lead to oxidative stress, which can be deleterious for cells and favor intestinal diseases like chronic inflammation or cancer. This work provides a straightforward method to detect ROS in the intestinal murine organoids by live imaging and flow cytometry, using a commercially available fluorogenic probe. Here the protocol describes assaying the effect of compounds that modulate the redox balance in intestinal organoids and detect ROS levels in specific intestinal cell types, exemplified here by the analysis of the intestinal stem cells genetically labeled with GFP. This protocol may be used with other fluorescent probes.

The present protocol describes a method to detect reactive oxygen species (ROS) in the intestinal murine organoids using qualitative imaging and quantitative cytometry assays. This work can be potentially extended to other fluorescent probes to test the effect of selected compounds on ROS.

Reactive oxygen species (ROS) play essential roles in intestinal homeostasis. ROS are natural by-products of cell metabolism. They are produced in ...

Rat Intestinal Crypts Isolation and Intestinal Organoids Generation

Generation and Manipulation of Rat Intestinal Organoids

When using organoids to assess physiology and cell fate decisions, it is important to use a model that closely recapitulates in vivo contexts. Accordingly, patient-derived organoids are used for disease modeling, drug discovery, and personalized treatment screening. Mouse intestinal organoids are commonly utilized to understand aspects of both intestinal function/physiology and stem cell dynamics/fate decisions. However, in many disease contexts, rats are often preferred over mice as a model due to their greater physiological similarity to humans in terms of disease pathophysiology. The rat model has been limited by a lack of genetic tools available in vivo, and rat intestinal organoids have proven fragile and difficult to culture long-term. Here, we build upon previously published protocols to robustly generate rat intestinal organoids from the duodenum and jejunum. We provide an overview of several downstream applications utilizing rat intestinal organoids, including functional swelling assays, whole mount staining, the generation of 2D enteroid monolayers, and lentiviral transduction. The rat organoid model provides a practical solution to the need of the field for an in vitro model which retains physiological relevance to humans, can be quickly genetically manipulated, and is easily obtained without the barriers involved in procuring human intestinal organoids.

Author Spotlight: Generation and Manipulation of Rat Intestinal Organoids  

Here, we present a protocol to generate rat intestinal organoids and use them in several downstream applications. Rats are often a preferred preclinical model, and the robust intestinal organoid system fills the need for an in vitro system to accompany in vivo studies.

When using organoids to assess physiology and cell fate decisions, it is important to use a model that closely recapitulates in vivo contexts. ...

Quantifying Cell Death in Human Colonoids in Response to Cytotoxic Perturbagens

Quantification of Cytokine-Induced Cell Death in Human Colonic Organoids Using Live Fluorescence Microscopy

Intestinal epithelial cell (IEC) death is increased in patients with inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn's disease (CD). This can contribute to defects in intestinal barrier function, exacerbation of inflammation, and disease immunopathogenesis. Cytokines and death receptor ligands are partially responsible for this increase in IEC death. IBD-relevant cytokines, such as TNF-&#945; and IFN-&#947;, are cytotoxic to IECs both independently and in combination. This protocol describes a simple and practical assay to quantify cytokine-induced cytotoxicity in CD patient-derived colonic organoids using a fluorescent cell death dye (SYTOX Green Nucleic Acid Stain), live fluorescence microscopy, and open-source image analysis software. We also demonstrate how to use the Bliss independence mathematical model to calculate a coefficient of perturbagen interaction (CPI) based on organoid cytotoxicity. The CPI can be used to determine if interactions between cytokine combinations or other types of perturbagens are antagonistic, additive, or synergistic. This protocol can be implemented to investigate the cytotoxic activity of cytokines and other perturbagens using patient-derived colonic organoids.

Author Spotlight: Understanding Cytokine-Induced Cell Death in Intestinal Epithelial Cells Using Human Organoids

This protocol describes a simple and cost-effective method to investigate and quantify cell death in human colonic organoids in response to cytotoxic perturbagens such as cytokines. The approach employs a fluorescent cell death dye (SYTOX Green Nucleic Acid Stain), live fluorescence microscopy, and open-source image analysis software to quantify single-organoid responses to cytotoxic stimuli.

Intestinal epithelial cell (IEC) death is increased in patients with inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn's disease ...

Fluorescent Probe Imaging Assay: A Technique to Visualize Oxidative Stress in the Reactive Oxygen Species Inducer-Treated Cultured Intestinal Organoid Cells

Source: Stedman, A. et al., <a href="https://app.jove.com/v/62880">Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe.</a> J. Vis. Exp. (2021).
This video describes the technique of visualizing oxidative stress in cultured organoids. This work helps in understanding the level of oxidative stress due to ROS accumulation in the various cell types present in an organoid after treatment with different ROS modulators.

Source: Stedman, A. et al., Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe. J. Vis. ...

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

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