Name: forskolin-induced swelling in intestinal organoids: an in vitro assay for assessing drug response in cystic fibrosis patients
Uploaded: 2017-02-11T14:00:00.000+00:00
Duration: 7 min 4 s
Description: Watch this Scientific Journal Video about Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients at JoVE.com

This work was supported by the HIT-CF program of the Dutch CF foundation (NCFS), ZonMW (40-00812-98-14103), the Wilhelmina Children&#39;s Hospital Research Fund and CZ, and Zilverenkruis/Achmea. We would like to thank S. Heida-Michel, M. Geerdink, K.M. de Winter-de Groot, and G. Berkers (Department of Pediatric Pulmonology, Wilhelmina Children&#39;s Hospital, UMC Utrecht), and R.H.J. Houwen (Department of Pediatric Gastroenterology, Wilhelmina Children&#39;s Hospital, UMC Utrecht) for approaching the patients and getting the biopsies for the generation of a CF Biobank.

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Hans Clevers is an inventor on patents for organoid culture and the Forskolin-induced swelling assay. Jeffrey M Beekman is an inventor on a patent for the Forskolin-induced swelling assay. Sylvia F Boj and Robert Vries are employed by the Foundation Hubrecht Organoid Technology (HUB), which holds the exclusive license to the Organoid Technology. Otherwise, the authors have nothing to disclose.

Here, we provide a complete protocol for the generation, expansion, freezing, and thawing of human colorectal organoids. While we have established human organoid cultures some time ago17, it has sometimes proven difficult to establish the technology in other labs without hands-on training. We anticipate that these protocols will replace such training.
Wnt-3A-conditioned medium is one of the most crucial reagents in order to succeed in establishing and maintaining a long...

CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes an epithelial anion channel. CF affects approximately 85,000 people worldwide1. Over 2,000 CFTR mutations have been identified (<a href="http://www.genet.sickkids.on.ca">www.genet.sickkids.on.ca</a>). This diversity partially explains a wide spectrum of observed disease phenotypes (<a href="http://www.CFTR2.org">www.CFTR2.org</a>)2,3. Six classes of CFTR mutations are defined based on their effect on CFTR protein expression and function: (I) no synthesis, (II) impaired trafficking, (III) defective channel gating, (IV) altered conductance, (V) reduced levels of normally functioning CFTR, and (VI) impaired cell surface stability4. Although the common CFTR mutations are well studied, CFTR function and relation with clinical status remain poorly understood at the level of the individual, particularly for the large group of rare &#34;orphan&#34; mutations (<a href="http://www.CFTR2.org">www.CFTR2.org</a>)1,3.
Recently, drugs have been developed that target the CFTR protein in a mutation-specific fashion. Two classes of CFTR protein-targeting drugs are currently in clinical use and have distinct modes of action. Potentiators, such as VX-770, enhance the open-probability of apically-localized mutant CFTR and act directly upon their addition to cells5. Correctors, such as VX-809, restore the trafficking of endoplasmic reticulum-localized misfolded CFTR and require pre-incubation with cells before the effects are observed6. The CFTR potentiator, VX-770, has been registered for subjects with the G551D mutation7,8, as well as for eight other CFTR gating mutations, including S1251N9; together, these mutations are carried by 5% of all CF subjects. Other clinical trials have indicated that VX-770, combined with the corrector VX-809, has limited yet significant effects on lung function and causes a decrease in exacerbation rates in subjects homozygous for the F508del mutation carried by 45-50% of patients10,11.
Conventional clinical trials to identify drug-responsive subjects within the remaining 50% of CF patients are costly and time-consuming and are not feasible for individuals with extremely rare CFTR genotypes. Novel, cost-effective, personalized methods are crucial to match the increasing number of CFTR modulators to individuals carrying any type of CFTR mutation. Until now, the trial inclusion of patient groups carrying specific CFTR mutations has been guided by studies using mutant CFTR gene transfection in heterologous cell systems, followed by electrophysiological studies in Ussing chambers5,6,12. Due to a lack of adequate CF animal models, drug efficacy studies in air-liquid-interface-differentiated bronchial epithelial cells derived from CF lung explant materials have been used for drug development13,14,15. However, the limited availability of lung explant tissues and the invasive procedures to obtain bronchial cells from subjects without end-stage disease hamper the analysis of less common CFTR mutations and prevent drug testing in a personalized fashion. To overcome these limitations, &#34;easy access&#34; tissues, such as colorectal organoids, nasal airway cells, and airway cells derived from induced pluripotent stem cells, are currently being explored for personalized drug treatments.
Previously, we established protocols to culture epithelial stem cells from any gastrointestinal organ in the form of 3D organoids16,17. For the human colon/rectum, the culture conditions involve defined growth factors (Epithelial Growth Factor (EGF), Gastrin, Wnt-3A, R-spondin 3 (Rspo3), and Noggin) combined with small molecules (Nicotinamide, A83-01, and SB202190) in a basement membrane matrix. Under these conditions, single stem cells or small tissue fragments grow out into closed, cystic, 3D structures formed by highly-polarized epithelium with its basal side oriented towards the outside. All cell types typically appear in their normal ratios and positions. Organoids can be expanded over long time periods by weekly mechanical disruption and re-plating. They are genetically and phenotypically stable and can be stored, allowing long-term expansion and bio-banking17. They are amenable to all standard cell-biological/genetic manipulations and analytical techniques developed for 2D cell lines18.
We recently demonstrated that CFTR function can be readily measured in colorectal organoids in a forskolin-induced swelling (FIS) assay19,20. When exposed to forskolin (Fsk) or, alternatively, to cholera toxin, organoids rapidly increase their cyclic adenosine monophosphate (cAMP) levels, which in turn results in the opening of the CFTR channel19. Organoids from healthy individuals, or from subjects with CFTR mutations associated with residual function, will subsequently swell as a consequence of ion and water transport to the organoid lumen, the in vitro equivalent of secretory diarrhea. The FIS response of colorectal organoids was previously shown to be fully CFTR-dependent, as indicated by organoids derived from CFTR-null individuals and by the use of specific pharmacological CFTR inhibitors19. Large subject-specific data sets can be derived within several weeks after taking a biopsy.
For the FIS assay described in detail here, organoids are cultured from rectal biopsies that can be obtained at any age and with only limited discomfort21. Organoids are passaged weekly by mechanical disruption into single crypts that easily reseal and form new organoids. For running the FIS assay, ~30-80 of these disrupted little organoids are plated in each well of a 96-well plate19. At the day of the assay, the organoids are stained with calcein green, a fluorescent cell-permeable dye that it is retained within living cells, facilitating live imaging. Then, Fsk, which raises intracellular cAMP and thereby activates CFTR, is added in order to stimulate organoid swelling. Potentiators that act upon apical CFTR are added simultaneously with the forskolin, whereas correctors that restore CFTR trafficking are added 24 hr before the addition of Fsk. The organoid swelling is quantified by an automated image analysis that calculates the relative increase in the total area of all fluorescent objects for each time point upon forskolin addition.
3D organoid swelling provides advantages and disadvantages over existing electrophysiological CFTR readouts in 2D cultured airway cells in Ussing chambers. A major advantage is the throughput of the swelling assay. Cells are cultured and assayed using a single type of culture medium, and an experienced technician can culture up to 25 organoid samples on a weekly basis while quantifying approximately 1,200 data points per week in 12 patient samples. We conventionally type a single experimental condition by duplicate or triplicate measurements per plate and repeat such measurements at three independent incubation time points. In total, approximately 300-500 single organoid structures are then measured per experimental condition, which leads to very precise measurements of CFTR function with limited technical variability. This precision allows us to clearly define differences in residual function and response to CFTR modulators and allows us to readily pick up genetic background effects between patients carrying identical CFTR mutations19,22,23,24,25. Data quality can be easily assessed from microscope images. While FIS is fully CFTR-dependent, it is an indirect outcome measure for CFTR function, its read-out caused by the coupling of ion transport to fluid transport. This contrasts with direct CFTR function measurements in Ussing chambers, which measure transepithelial ion currents26. Ussing chambers allow the select stimulation of apical or basolateral compartments (which the organoid assay does not allow); by permeabilizing basolateral membranes, the apical CFTR-dependent anion secretion can be selectively measured27.

<table><tbody><tr><td>Advanced Dulbecco&amp;rsquo;s Modified Eagles Medium with Nutrient Mixture F-12 Hams (Ad-DF) 500 ml&amp;nbsp;</td><td>Thermo Fisher Scientific:&amp;nbsp; Invitrogen</td><td>#12634</td><td>stored at 4 &amp;deg;C</td></tr><tr><td>GlutaMax</td><td>Thermo Fisher Scientific:&amp;nbsp; Invitrogen</td><td>#35050</td><td>stored at 4 &amp;deg;C</td></tr><tr><td>Hepes</td><td>Thermo Fisher Scientific:&amp;nbsp; Invitrogen</td><td># 15630-056</td><td>stored at 4 &amp;deg;C</td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo Fisher Scientific:&amp;nbsp; Invitrogen</td><td>#15140-122</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>96 well culture plate</td><td>Cellstar</td><td>#655180</td><td></td></tr><tr><td>24 well culture plate</td><td>Cellstar</td><td>#662160</td><td></td></tr><tr><td>6 well culture plate</td><td>Cellstar</td><td>#657160</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (-) CaCl&lt;sub&gt;2 &lt;/sub&gt;(-) MgCl&lt;sub&gt;2&lt;/sub&gt;) (DPBS)</td><td>Life Technologies: Gibco</td><td>#14190-094</td><td>stored at 4 &amp;deg;C</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagles Medium&amp;nbsp; (DMEM) 500 ml&amp;nbsp;</td><td>Thermo Fisher Scientific:&amp;nbsp; Invitrogen</td><td>#31966-021</td><td>For Wnt-3A Conditioned Medium Production. Stored at 4 &amp;deg;C</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Bovogen</td><td>#SFBS LOT#11113</td><td>For Wnt-3A Conditioned Medium Production. Stored at -20 &amp;deg;C</td></tr><tr><td>L Wnt3A cell line</td><td>ATCC</td><td>#CRL-2647</td><td>For Wnt-3A Conditioend Medium Production.</td></tr><tr><td>TOP/FOP plasmids</td><td>Millipore&amp;nbsp;</td><td>#17-285</td><td>For measuring Wnt activity</td></tr><tr><td>pTK-Renilla</td><td>Promega&amp;nbsp;</td><td>#E2241</td><td>For measuring Wnt activity</td></tr><tr><td>HEK-293</td><td>ATCC</td><td>#CRL-1573</td><td>For measuring Wnt activity</td></tr><tr><td>Dual-Luciferase Reporter Assay System</td><td>Promega&amp;nbsp;</td><td>#E1910</td><td>For measuring Wnt activity</td></tr><tr><td>Zeocin&amp;nbsp;</td><td>Thermo Fisher Scientific:&amp;nbsp; Invitrogen</td><td>#R250-01</td><td>For Wnt-3A Cell line selection</td></tr><tr><td>B27 supplement&amp;nbsp;</td><td>Thermo Fisher Scientific:&amp;nbsp; Invitrogen</td><td>#17504-044</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>N-Acetylcysteine</td><td>Sigma Aldrich</td><td>#A9165-5G</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>Nicotinamide</td><td>Sigma Aldrich</td><td>#N0636</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>Human Epithelial Growth Factor (hEGF)</td><td>PrepoTech</td><td>#AF-100-15</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>Gastrin</td><td>Sigma Aldrich</td><td>#G9145</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>TGFb type I Receptor inhibitor (A83-01)&amp;nbsp;</td><td>Tocris</td><td>#2939</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>Y-27632 dihydrochloride (RhoKi)</td><td>Selleckchem</td><td>#S1049</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>p38 MAPK inhibitor (p38i) (SB202190)</td><td>Sigma Aldrich</td><td>#S7067</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>Primocin</td><td>InvivoGen</td><td>#ant-pm-1</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>Human Noggin (hNoggin)</td><td>PrepoTech</td><td>#120-10C</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>Human R-spondin 3 (hRspo-3)</td><td>R&amp;amp;D Systems</td><td>#3500-RS/CF</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>Vancomycin</td><td>Sigma Aldrich</td><td>#861987- 250mg</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>Gentamycin</td><td>Life Technologies: Gibco</td><td>#15710-049</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>Ethylenediamine tetraacetic acid (EDTA)</td><td>Sigma Aldrich</td><td>#431788</td><td>Stored at 4 &amp;deg;C</td></tr><tr><td>Matrigel</td><td>Corning</td><td>#354230</td><td>stored at -80 &amp;deg;C</td></tr><tr><td>TryplE Express&amp;nbsp;</td><td>Life Technologies: Gibco</td><td>#12605-010</td><td>for trypsinizing organoids for freezing</td></tr><tr><td>Recovery Cell Culture Freezing Medium</td><td>Life Technologies: Gibco</td><td>#12648010</td><td>for freezing</td></tr><tr><td>Calcein</td><td>Life Technologies: Gibco</td><td>#C3100MP</td><td>stored at -20 &amp;deg;C</td></tr><tr><td>Forskolin</td><td>R&amp;amp;D Systems</td><td>#1099-50 mg</td><td>stored at -80 &amp;deg;C</td></tr><tr><td>Lumacaftor (VX-809)</td><td>Selleckchem</td><td>#s1565</td><td>stored at -80 &amp;deg;C</td></tr><tr><td>Ivacaftor (VX-770)</td><td>Selleckchem</td><td>#s1144</td><td>stored at -80 &amp;deg;C</td></tr><tr><td>&lt;strong&gt;Name of Reagents/Material&lt;/strong&gt;</td><td>&lt;strong&gt;Solvent&lt;/strong&gt;</td><td>&lt;strong&gt;Stock Concentration&lt;/strong&gt;</td><td>&lt;strong&gt;Final Concentration&lt;/strong&gt;</td></tr><tr><td>GlutaMax</td><td></td><td>200 mM</td><td>2 mM</td></tr><tr><td>Hepes</td><td></td><td>1 M</td><td>10 mM</td></tr><tr><td>Penicillin/Streptomycin</td><td></td><td>10K U/ml 10K &amp;micro;g/ml</td><td>100 U/ml 100 &amp;micro;g/ml</td></tr><tr><td>Zeocin&amp;nbsp;</td><td></td><td>100 mg/ml&amp;nbsp;</td><td>125 &amp;micro;g/ml</td></tr><tr><td>B27 supplement&amp;nbsp;</td><td></td><td>100x</td><td>1x</td></tr><tr><td>N-Acetylcysteine</td><td>MiliQ H&lt;sub&gt;2&lt;/sub&gt;O</td><td>500 mM</td><td></td></tr><tr><td>Nicotinamide</td><td>DPBS</td><td>1 M</td><td>10 mM</td></tr><tr><td>Human Epithelial Growth Factor (hEGF)</td><td>DPBS 0.1% BSA</td><td>0.5 mg/ml</td><td>50 ng/ml</td></tr><tr><td>Gastrin</td><td>DPBS</td><td>100 &amp;micro;M</td><td>10 nM</td></tr><tr><td>TGFb type I Receptor inhibitor (A83-01)&amp;nbsp;</td><td>DMSO</td><td>5 mM</td><td>500 nM</td></tr><tr><td>Y-27632 dihydrochloride (RhoKi)</td><td>DMSO</td><td>10 mM</td><td>10 &amp;micro;M</td></tr><tr><td>p38 MAPK inhibitor (p38i) (SB202190)</td><td>DMSO</td><td>30 mM</td><td>10 &amp;micro;M</td></tr><tr><td>Primocin</td><td></td><td>50 mg/ml&amp;nbsp;</td><td>100 &amp;micro;g/ml</td></tr><tr><td>Human Noggin (hNoggin)</td><td>DPBS 0.1%BSA</td><td>100 &amp;micro;g/ml</td><td>100 ng/ml</td></tr><tr><td>Human R-spondin 3 (hRspo-3)</td><td></td><td>varies per lot</td><td>300 ng/ml</td></tr><tr><td>Vancomycin</td><td></td><td>10 mg/ml</td><td>50 &amp;micro;g/ml</td></tr><tr><td>Gentamycin</td><td></td><td>10 mg/ml</td><td>50 &amp;micro;g/ml</td></tr><tr><td>Ethylenediamine tetraacetic acid (EDTA)</td><td>MiliQ H&lt;sub&gt;2&lt;/sub&gt;O</td><td>0.5 M</td><td>2 mM</td></tr><tr><td>Calcein</td><td>DMSO</td><td>10 &amp;micro;g/ml</td><td>3.3 ng/ml</td></tr><tr><td>Forskolin</td><td>DMSO</td><td>10 mM</td><td>variable</td></tr><tr><td>Lumacaftor (VX-809)</td><td>DMSO</td><td>20 mM</td><td>variable</td></tr><tr><td>Ivacaftor (VX-770)</td><td>DMSO</td><td>20 mM</td><td>variable</td></tr></tbody></table>

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.

Figure 1A shows a representative fresh isolation of crypts embedded on BMM. The crypts are from a colorectal biopsy of a CF subject. Usually, an organoid is generated from each crypt (Figure 1A-C). Due to the dysfunction of the CFTR, most of the colon CF organoids are not cystic, but rather are compact and with projections and buddings (Figure 2A-2B). However, some CF organoid cultures, especially with hi...

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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.

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 ...

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19.1K Views.  Foundation Hubrecht Organoid Technology. 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.

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

Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and treatment response to targeted inhibitors in cancer. Pioneering work in gastrointestinal and pancreatic cancers has highlighted the promise of patient-derived organoids (PDOs) as a patient-proximate culture system, with an increasing number of models emerging. Similarly, work in other cancer types has focused on establishing organoid models and optimizing culture protocols. Notably, 3D cancer organoid models maintain the genetic complexity of original tumor specimens and thus translate tumor-derived sequencing data into treatment with genetically informed targeted therapies in an experimental setting. Further, PDOs might foster the evaluation of rational combination treatments to overcome resistance-associated adaptation of tumors in the future. The latter focuses on intense research efforts in non-small-cell lung cancer (NSCLC), as resistance development ultimately limits the treatment success of targeted inhibitors. An early assessment of therapeutically targetable mechanisms using NSCLC PDOs could help inform rational combination treatments. This manuscript describes a standardized protocol for the cell culture plate-based assessment of drug sensitivities to targeted inhibitors in NSCLC-derived 3D PDOs, with potential adaptability to combinational treatments and other treatment modalities.

This protocol describes a standardized evaluation of drug sensitivities to targeted signaling inhibitors in NSCLC patient-derived organoid models.

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and ...

Cancer Research

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

Immunology and Infection

Combining Human Organoids and Organ-on-a-Chip Technology to Model Intestinal Region-Specific Functionality

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, and metabolism of nutrients and xenobiotics while facilitating a symbiotic relationship with microbiota and restricting the invasion of microorganisms. Functional interaction between various cell types and their physical and biochemical environment is vital to establish and maintain intestinal tissue homeostasis. Modeling these complex interactions and integrated intestinal physiology in vitro is a formidable goal with the potential to transform the way new therapeutic targets and drug candidates are discovered and developed.
Organoids and Organ-on-a-Chip technologies have recently been combined to generate human-relevant intestine chips suitable for studying the functional aspects of intestinal physiology and pathophysiology in vitro. Organoids derived from the biopsies of the small (duodenum) and large intestine are seeded into the top compartment of an organ chip and then successfully expand&#160;as monolayers while preserving the distinct cellular, molecular, and functional features of each intestinal region. Human intestine tissue-specific microvascular endothelial cells are incorporated in the bottom compartment of the organ chip to recreate the epithelial-endothelial interface. This novel platform facilitates luminal exposure to nutrients, drugs, and microorganisms, enabling studies of intestinal transport, permeability, and host-microbe interactions.
Here, a detailed protocol is provided for the establishment of intestine chips representing the human duodenum (duodenum chip) and colon (colon chip), and their subsequent culture under continuous flow and peristalsis-like deformations. We demonstrate methods for assessing drug metabolism and CYP3A4 induction in duodenum chip using prototypical inducers and substrates. Lastly, we provide a step-by-step procedure&#160;for the in vitro modeling of interferon gamma (IFN&#947;)-mediated barrier disruption (leaky gut syndrome) in a colon chip, including methods for evaluating the alteration of paracellular permeability, changes in cytokine secretion, and transcriptomic profiling of the cells within the chip.

Biopsy-derived intestinal organoids and organ-on-a-chips technologies are combined into a microphysiological platform to recapitulate region-specific intestinal functionality.

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, ...

Bioengineering

Generation of Human Nasal Epithelial Cell Spheroids for Individualized Cystic Fibrosis Transmembrane Conductance Regulator Study

While the introduction of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulator drugs has revolutionized care in Cystic Fibrosis (CF), the genotype-directed therapy model currently in use has several limitations. First, rare or understudied mutation groups are excluded from definitive clinical trials. Moreover, as additional modulator drugs enter the market, it will become difficult to optimize the modulator choices for an individual subject. Both of these issues are addressed with the use of patient-derived, individualized preclinical model systems of CFTR function and modulation. Human nasal epithelial cells (HNEs) are an easily accessible source of respiratory tissue for such a model. Herein, we describe the generation of a three-dimensional spheroid model of CFTR function and modulation using primary HNEs. HNEs are isolated from subjects in a minimally invasive fashion, expanded in conditional reprogramming conditions, and seeded into the spheroid culture. Within 2 weeks of seeding, spheroid cultures generate HNE spheroids that can be stimulated with 3',5'-cyclic adenosine monophosphate (cAMP)-generating agonists to activate CFTR function. Spheroid swelling is then quantified as a proxy of CFTR activity. HNE spheroids capitalize on the minimally invasive, yet respiratory origin of nasal cells to generate an accessible, personalized model relevant to an epithelium reflecting disease morbidity and mortality. Compared to the air-liquid interface HNE cultures, spheroids are relatively quick to mature, which reduces the overall contamination rate. In its current form, the model is limited by low throughput, though this is offset by the relative ease of tissue acquisition. HNE spheroids can be used to reliably quantify and characterize CFTR activity at the individual level. An ongoing study to tie this quantification to in vivo drug response will determine if HNE spheroids are a true preclinical predictor of patient response to CFTR modulation.

Here we describe a method to generate three-dimensional spheroid cultures of human nasal epithelial cells. Spheroids are then stimulated to drive Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-dependent ion and fluid secretion, quantifying the change in the spheroid luminal size as a proxy for CFTR function.

While the introduction of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulator drugs has revolutionized care in Cystic Fibrosis (CF), the ...

Canine Intestinal Organoids in a Dual-Chamber Permeable Support System

The permeable support system is typically used in conjunction with traditional two-dimensional (2D) cell lines as an in vitro tool for evaluating the oral permeability of new therapeutic drug candidates. However, the use of these conventional cell lines has limitations, such as altered expression of tight junctions, partial cell differentiation, and the absence of key nuclear receptors. Despite these shortcomings, the Caco-2 and MDCK models are widely accepted and validated for the prediction of human in vivo oral permeability.
Dogs are a relevant translational model for biomedical research due to their similarities in gastrointestinal anatomy and intestinal microflora with humans. Accordingly, and in support of parallel drug development, the elaboration of an efficient and accurate in vitro tool for predicting in vivo drug permeability characteristics both in dogs and humans is highly desirable. Such a tool could be the canine intestinal organoid system, characterized by three-dimensional (3D), self-assembled epithelial structures derived from adult stem cells.
The (1) Permeable Support Seeding Protocol describes the experimental methods for dissociating and seeding canine organoids in the inserts. Canine organoid isolation, culture, and harvest have been previously described in a separate set of protocols in this special issue. Methods for general upkeep of the canine intestinal organoid monolayer are discussed thoroughly in the (2) Monolayer Maintenance Protocol. Additionally, this protocol describes methods to assess the structural integrity of the monolayer via transepithelial electrical resistance (TEER) measurements and light microscopy. Finally, the (3) Permeability Experimental Protocol&#160;describes the tasks directly preceding an experiment, including in vitro validation of experimental results.
Overall, the canine organoid model, combined with a dual-chamber cell culture technology, overcomes limitations associated with 2D experimental models, thereby improving the reliability of predictions of the apparent oral permeability of therapeutic drug candidates both in the canine and human patient.

Here, we present a protocol describing the culture of canine intestinal organoids in a dual-chamber, permeable support system. Organoid seeding in the permeable supports, the monolayer maintenance, and subsequent drug permeability experiments are described.

The permeable support system is typically used in conjunction with traditional two-dimensional (2D) cell lines as an in vitro tool for evaluating the oral ...

Evaluating Biofunctional Self-Assembling Peptides for Cell Adhesion and Morphology

Fabrication and Characterization of Colorectal Cancer Organoids from SW1222 Cell Line in Ultrashort Self&#45;Assembling Peptide Matrix

Ultrashort self-assembling peptides (SAPs) can spontaneously form nanofibers that resemble the extracellular matrix. These fibers allow the formation of hydrogels that are biocompatible, biodegradable, and non-immunogenic. We have previously proven that SAPs, when biofunctionalized with protein-derived motifs, can mimic the extracellular matrix characteristics that support colorectal organoid formation. These biofunctional peptide hydrogels retain the original parent peptide's mechanical properties, tunability, and printability while incorporating cues that allow cell-matrix interactions to increase cell adhesion. This paper presents the protocols needed to evaluate and characterize the effects of various biofunctional peptide hydrogels on cell adhesion and lumen formation using an adenocarcinoma cancer cell line able to form colorectal cancer organoids cost-effectively. These protocols will help evaluate biofunctional peptide hydrogel effects on cell adhesion and luminal formation using immunostaining and fluorescence image analysis. The cell line used in this study has been previously utilized for generating organoids in animal-derived matrices.

Author Spotlight: Optimization of Ultrashort Peptide Matrices for Colorectal Cancer Organoids

This protocol aims to evaluate biofunctional self-assembling peptides for cell adhesion, organoid morphology, and gene expression by immunostaining. We will use a colorectal cancer cell line to provide a cost-effective way of obtaining organoids for intensive testing.

Ultrashort self-assembling peptides (SAPs) can spontaneously form nanofibers that resemble the extracellular matrix. These fibers allow the formation of ...

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.

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) ...

Culture and Imaging of Human Nasal Epithelial Organoids

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activity and restoration from small molecule compounds. Our group recently focused on establishing a well-differentiated organoid model directly derived from primary human nasal epithelial cells (HNE). Histology of sectioned organoids, whole-mount immunofluorescent staining, and imaging (using confocal microscopy, immunofluorescent microscopy, and bright field) are essential to characterize organoids and confirm epithelial differentiation in preparation for functional assays. Furthermore, HNE organoids produce lumens of varying sizes that correlate with CFTR activity, distinguishing between CF and non-CF organoids. In this manuscript, the methodology for culturing HNE organoids are described in detail, focusing on the assessment of differentiation using the imaging modalities, including the measurement of baseline lumen area (a method of CFTR activity measurement in organoids that any laboratory with a microscope can employ) as well as the developed automated approach to a functional assay (which requires more specialized equipment).

A detailed protocol is presented here to describe an in vitro organoid model from human nasal epithelial cells. The protocol has options for measurements requiring standard laboratory equipment, with additional possibilities for specialized equipment and software.

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis ...

Rectal Organoid Morphology Analysis (ROMA): A Diagnostic Assay in Cystic Fibrosis

Diagnosis of cystic fibrosis (CF) is not always straightforward, especially when sweat chloride concentration is intermediate and/or less than two disease-causing CFTR mutations can be identified. Physiological CFTR assays (nasal potential difference, intestinal current measurement) have been included in the diagnostic algorithm but are not always readily available or feasible (e.g., in infants). Rectal organoids are 3D structures that grow from stem cells isolated from crypts of a rectal biopsy when cultured under specific conditions. Organoids from non-CF subjects have a round shape and a fluid-filled lumen, as CFTR-mediated chloride transport drives water into the lumen. Organoids with defective CFTR function do not swell, retaining an irregular shape and having no visible lumen. Differences in morphology between CF and non-CF organoids are quantified in the &#39;Rectal Organoid Morphology Analysis&#39; (ROMA) as a novel CFTR physiological assay. For the ROMA assay, organoids are plated in 96-well plates, stained with calcein, and imaged in a confocal microscope. Morphological differences are quantified using two indexes: The circularity index (CI) quantifies the roundness of organoids, and the intensity ratio (IR) is a measure of the presence of a central lumen. Non-CF organoids have a high CI and low IR compared to CF organoids. ROMA indexes perfectly discriminated 167 subjects with CF from 22 subjects without CF, making ROMA an appealing physiological CFTR assay to aid in CF diagnosis. Rectal biopsies can be routinely performed at all ages in most hospitals and tissue can be sent to a central lab for organoid culture and ROMA. In the future, ROMA might also be applied to test the efficacy of CFTR modulators in vitro. The aim of the present report is to fully explain the methods used for ROMA, to allow replication in other labs.

Rectal Organoid Morphology Analysis ROMA: A Diagnostic Assay in Cystic Fibrosis

This protocol describes rectal organoid morphology analysis (ROMA), a novel diagnostic assay for cystic fibrosis (CF). Morphological characteristics, namely the roundness (circularity index, CI) and the presence of a lumen (intensity ratio, IR), are a measure of CFTR function. Analysis of 189 subjects showed perfect discrimination between CF and non-CF.

Diagnosis of cystic fibrosis (CF) is not always straightforward, especially when sweat chloride concentration is intermediate and/or less than two ...

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.

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

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