Name: a fluorescence-based assay for characterization and quantification of lipid droplet formation in human intestinal organoids
Uploaded: 2019-10-13T13:00:00
Description: 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

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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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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JoVE Core

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Anatomy and Physiology

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Unit 1

Biochemistry of the Cell

SCIENTISTS IN ACTION

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one step treatments for acute and chronic skin lesions. Moreover, the management of chronic but also acute wounds resulting from trauma, diabetes, infections and other causes, remains challenging. In this study we describe a new method to create autologous micro-grafts from cutaneous tissue of a single patient and their clinical application. Moreover, in vitro biological characterization of cutaneous tissue derived from skin, de-epidermized dermis (Ded) and dermis of multi-organ and/or multi-tissue donors was also performed. All tissues were disaggregated by this new protocol, allowing us to obtain viable micro-grafts. In particular, we reported that this innovative protocol is able to create bio-complexes composed by autologous micro-grafts and collagen sponges ready to be applied on skin lesions. The clinical application of autologous bio-complexes on a leg lesion was also reported, showing an improvement of both re-epitalization process and softness of the lesion. Additionally, our in vitro model showed that cell viability after mechanical disaggregation with this system is maintained over time for up to seven (7) days of culture. We also observed, by flow cytometry analysis, that the pool of cells obtained from disaggregation is composed of several cell types, including mesenchymal stem cells, that exert a key role in the processes of tissue regeneration and repair, for their high regenerative potential. Finally, we demonstrated in vitro that this procedure maintains the sterility of micro-grafts when cultured in Agar dishes. In summary, we conclude that this new regenerative approach can be a promising tool for clinicians to obtain in one step viable, sterile and ready to use micro-grafts that can be applied alone or in combination with most common biological scaffolds.

The protocol describes a new method to disaggregate human tissues and to create autologous micro-grafts that, combined with collagen sponges, give rise to human bio-complexes ready to use in the treatment of skin lesions. Further, this system preserves cell viability of micro-grafts at different times after mechanical disaggregation.

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one ...

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis1. Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.

This manuscript describes a tube formation assay to quantify the effects of a given compound or condition on angiogenesis by using endothelial cell tube formation in a controlled environment.

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include well-validated targets for therapeutic intervention. Discovery of lead compounds, including small synthetic molecules, that specifically inhibit the receptor&#39;s function, is an important initial step in drug development and relies on sensitive, specific, and robust cell-based assays. Here, we describe a kinetic cellular assay with a fluorescent readout primarily designed to identify receptor-specific antagonists that inhibit the intracellular Ca2+ release evoked upon the activation of the CXC chemokine receptor 4 (CXCR4) by its endogenous ligand, the CXC chemokine ligand 12 (CXCL12). A key advantage of this method is that it also enables screening of compounds endowed with intrinsic agonistic properties (i.e., compounds eliciting an increase in intracellular Ca2+ concentration in the absence of CXCL12) or compounds modulating the receptor&#39;s function via interaction with allosteric binding sites (i.e., positive and negative allosteric modulators (PAMs and NAMs, respectively)). On the down side, autofluorescent compounds might interfere with the assay&#39;s readout, thereby hampering reliable data interpretation. Most likely this assay can be implemented, with minimal adaptations, as a generic drug discovery assay for many other GPCRs of which the activation leads to a release of intracellular Ca2+.

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

The described cellular assay is designed for the identification of CXC chemokine receptor 4 (CXCR4)-interacting agents that inhibit or stimulate, either competitively or allosterically, the intracellular Ca2+ release initiated by CXCR4 activation.

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include ...

Simultaneous Electrocardiography Recording and Invasive Blood Pressure Measurement in Rats

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. Research focusing on cardiovascular disease models, potential cardiovascular therapeutic targets or pharmaceutical agents requires assessment of systemic arterial pressure and heart rhythm changes. In situations where radio telemetry systems are not available or affordable, the technique of femoral artery cannulation is an alternative way to obtain intra-arterial pressure waveform recordings and systemic BP measurements. This technique is economical and can be performed with standard equipment in animal facilities. However, invasive arterial pressure recording requires cannulation of small arteries, which can be a challenging surgical skill. Here, we present step-by-step protocols for femoral artery cannulation procedures. Key procedures include the calibration of the data acquisition system, tissue dissection and femoral artery cannulation, and setup of the arterial cannulation system for pressure recording. Surface electrocardiography recording procedures are also included. We also present examples of BP recordings from normotensive and hypertensive rats. This protocol allows reliable direct recordings of systemic BP with simultaneous electrocardiography.

Here, we describe a setup for simultaneous recording of electrocardiography and intra-arterial blood pressure (BP) in experimental rats, which can be done with standard equipment in animal facilities and can be applied to physiological or pharmacological studies to investigate pathogenic or therapeutic mechanisms in cardiovascular medicine.

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. ...

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 ...