Name: generation of human nasal epithelial cell spheroids for individualized cystic fibrosis transmembrane conductance regulator study
Uploaded: 2018-04-11T14:45:00.000+00:00
Duration: 8 min
Description: Watch this Scientific Journal Video about Generation of Human Nasal Epithelial Cell Spheroids for Individualized Cystic Fibrosis Transmembrane Conductance Regulator Study at JoVE.com

This work was supported by Cystic Fibrosis Foundation Therapeutics, grant number CLANCY14XX0, and through Cystic Fibrosis Foundation, grant number CLANCY15R0. The authors wish to thank Kristina Ray for her assistance in patient recruitment and regulatory oversight. The authors also wish to thank the HNE working group, supported by the Cystic Fibrosis Foundation, who assisted in generation of HNE culture capabilities: Preston Bratcher, Calvin Cotton, Martina Gentzsch, Elizabeth Joseloff, Michael Myerburg, Dave Nichols, Scott Randell, Steve Rowe, G. Marty Solomon, and Katherine Tuggle.

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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+and+safety+of+ivacaftor+in+patients+with+cystic+fibrosis+who+have+an+Arg117His-CFTR+mutation:+a+double-blind,+randomised+controlled+trial.">Efficacy and safety of ivacaftor in patients with cystic fibrosis who have an Arg117His-CFTR mutation: a double-blind, randomised controlled trial.</a> Lancet Respir Med. 3 (7), 524-533 (2015).</li><li>Wainwright, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lumacaftor-Ivacaftor+in+Patients+with+Cystic+Fibrosis+Homozygous+for+Phe508del+CFTR.">Lumacaftor-Ivacaftor in Patients with Cystic Fibrosis Homozygous for Phe508del CFTR.</a> N Engl J Med. , (2015).</li><li>Brewington, J. J., McPhail, G. L., Clancy, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lumacaftor+alone+and+combined+with+ivacaftor:+preclinical+and+clinical+trial+experience+of+F508del+CFTR+correction.">Lumacaftor alone and combined with ivacaftor: preclinical and clinical trial experience of F508del CFTR correction.</a> Expert Rev Respir Med. 10 (1), 5-17 (2016).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> CFTR2 Database. , (2017).</li><li>Mou, H., Brazauskas, K., Rajagopal, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Personalized+medicine+for+cystic+fibrosis:+establishing+human+model+systems.">Personalized medicine for cystic fibrosis: establishing human model systems.</a> Pediatr Pulmonol. 50 Suppl 40, S14-S23 (2015).</li><li>Randell, S. H., Fulcher, M. L., O'Neal, W., Olsen, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+epithelial+cell+models+for+cystic+fibrosis+research.">Primary epithelial cell models for cystic fibrosis research.</a> Methods Mol Biol. 742, 285-310 (2011).</li><li>Whitcutt, M. J., Adler, K. B., Wu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biphasic+chamber+system+for+maintaining+polarity+of+differentiation+of+cultured+respiratory+tract+epithelial+cells.">A biphasic chamber system for maintaining polarity of differentiation of cultured respiratory tract epithelial cells.</a> In Vitro Cell Dev Biol. 24 (5), 420-428 (1988).</li><li>Worthington, E. 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The authors have nothing to disclose.

This protocol describes the generation of patient-derived nasal cell spheroid cultures able to produce an individualized, specific model of CFTR function. There are several key steps in the process that should be closely attended to avoid difficulty. First is a good sample acquisition from the patient's nose. A good sample should have &gt;50,000 cells, limited mucus/debris, and be ready for processing within 4 h (though success is also easily achievable with overnight shipping on ice). Practice acquiring samples with cur...

Cystic Fibrosis (CF) is a fatal, autosomal recessive disease affecting over 70,000 people worldwide1. This life-shortening genetic disease is caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator protein (CFTR)2. CFTR is a member of the adenosine triphosphate-binding cassette family, and functions as an ion channel allowing movement of chloride and bicarbonate across the apical membranes of multiple polarized epithelia including the gastrointestinal tract, sweat gland, and respiratory tree, among others3,4. As such, dysfunctional CFTR leads to multisystem epithelial dysfunction, with most mortality stemming from the respiratory disease1. In the CF lung, loss of CFTR-driven airway surface liquid (ASL) regulation and mucus release leads to thickened mucus, airway obstruction, chronic infection, and progressive airway remodeling and loss of lung function1,5.
Despite the identification of CFTR dysfunction as the cause of disease, therapies in CF traditionally focused on mitigation of symptoms (e.g., pancreatic enzyme replacement therapy, airway clearance therapies)1. This approach was recently revolutionized by the advent of novel therapeutics, termed &#34;CFTR modulators,&#34; that directly target dysfunctional CFTR. This approach has shifted the clinical landscape from symptom management to disease-modifying care but carries several limitations6,7,8,9,10. Modulator activity is specific to the protein defect accompanying each CFTR mutation and limited to select genetic populations11. This limitation is driven by the heterogeneous nature of protein defects, but also by the impracticality of clinical trials in rare mutation groups. In addition, among subjects with well-studied genotypes (e.g., F508del/F508del CFTR, the most common CFTR mutation), there is wide variability in disease burden and modulator response6,7,8,9,11.
To overcome both of these issues, investigators have proposed the use of personalized models for preclinical testing12. This concept utilizes patient-specific tissue to generate an individualized ex vivo model system for compound testing, predicting in vivo subject response to therapies in a personalized fashion. Once validated, such a model could be used by clinicians to drive precision therapy regardless of the patient&#39;s underlying CFTR genotype.
Human bronchial epithelial (HBE) cells obtained from explant tissue at the time of lung transplant established the possibility of such a model for CF13,14. HBEs grown at an air-liquid interface (ALI) allow for functional CFTR quantification directly through electrophysiologic testing or indirectly through measures of ASL homeostasis13,15. This model has been critical to understanding CFTR biology and was a key driver in the development of CFTR modulators16. Unfortunately, HBE models are not tenable as a personalized model due to the invasive nature of acquisition (lung transplant or bronchial brushing) and lack of samples for those with rare mutations or mild disease. Conversely, intestinal tissue, obtained from rectal or duodenal biopsy specimens, can be used for intestinal current measurement (ICM) or a swelling-based organoid assay to study individualized CFTR function17,18,19. Organoid assays, in particular, are very sensitive models of CFTR activity20,21,22. Both models are limited by the tissue source (intestinal tissue, while most disease pathology is respiratory) and the semi-invasive method of acquisition. Alternatively, several investigators have studied human nasal epithelial (HNE) cells to model CFTR restoration23,24,25. HNEs can be safely harvested by brush or curettage in subjects of any age and, when cultured in ALI, recapitulate many characteristics of HBEs25,26,27,28. HNE monolayer cultures have traditionally been limited by squamous transformation and a long time to maturity29. Moreover, reported short-circuit current measurements in HNEs are smaller than those in HBEs, suggesting a smaller window to detect therapeutic efficacy25.
Given the need for a personalized, non-invasive, respiratory tissue culture model of CFTR function, we sought to merge the sensitivity of a swelling-based, organoid assay with the non-invasive and respiratory nature of HNEs. Here, we describe our method of generating 3-dimensional &#34;spheroid&#34; cultures of HNEs for individualized CFTR study in a swelling-based assay30. HNE spheroids polarize reliably with the epithelial apex towards the sphere center, or lumen. This model recapitulates numerous characteristics of a lower respiratory epithelium and matures more quickly than ALI cultures. As a functional assay, HNE spheroids reliably quantify a range of CFTR function, as well as modulation in well-studied mutation groups (e.g., F508del CFTR). This swelling-based assay capitalizes on the ion/salt transport properties of CFTR, indirectly measuring fluid influx into the spheroid as water follows apical salt efflux. In this fashion, stimulated spheroids with fully functional CFTR swell robustly, while those with dysfunctional CFTR swell less or shrink. This is quantified through image analysis of pre- and 1 h post-stimulation spheroids, measuring luminal area and determining the percent change. This measure can then be compared across experimental groups to screen for drug bioactivity in a patient-specific fashion.

<table><tbody><tr><td>1.5 mL Eppendorf Tube</td><td>USA Scientific&amp;nbsp;</td><td>4036-3204</td><td></td></tr><tr><td>150 mL Filter Flask</td><td>Midsci&amp;nbsp;</td><td>TP99150</td><td>To filter Media</td></tr><tr><td>15 mL Conical Tube</td><td>Midsci&amp;nbsp;</td><td>TP91015</td><td></td></tr><tr><td>1 L Filter Flask</td><td>Midsci&amp;nbsp;</td><td>TP99950</td><td>To filter Media</td></tr><tr><td>35 mm Glass-Bottom Dish</td><td>MatTek Corporation</td><td>P35G-0-20-C</td><td>Optional</td></tr><tr><td>3-Isobutyl-1-Methylxanthine (IBMX)</td><td>Fisher Scientific&amp;nbsp;</td><td>AC228420010&amp;nbsp;</td><td>Prepare a 100 mM stock solution of 22.0 mg in 1 mL of DMSO</td></tr><tr><td>50 mL Conical Tube</td><td>Midsci&amp;nbsp;</td><td>&amp;nbsp;TP91050</td><td></td></tr><tr><td>Accutase</td><td>Innovative Cell Technologies, Inc.</td><td>AT-104</td><td>Cell detachment solution</td></tr><tr><td>Adenine</td><td>Sigma-Aldrich</td><td>A2786-25G</td><td>See Table 1</td></tr><tr><td>Amphotericin B</td><td>Sigma-Aldrich</td><td>A9528-100MG</td><td>See Table 1</td></tr><tr><td>Bovine Brain Extract (9mg/mL)</td><td>Lonza&amp;nbsp;</td><td>CC-4098</td><td>See Table 2</td></tr><tr><td>Ceftazidime hydrate</td><td>Sigma-Aldrich</td><td>C3809-1G</td><td>See Table 1</td></tr><tr><td>Cell Scrapers 20 cm&amp;nbsp;</td><td>Midsci&amp;nbsp;</td><td>TP99010</td><td></td></tr><tr><td>CFTR Inh172</td><td>Tocris Bioscience</td><td>3430</td><td>Prepare a 10 mM stock solution of 4.0 mg in 1 mL of DMSO</td></tr><tr><td>Cholera Toxin B (From Vibrio cholerae)</td><td>Sigma-Aldrich</td><td>C8052-.5MG</td><td>See Table 1</td></tr><tr><td>CYB-1</td><td>Medical Packaging Corporation</td><td>CYB-1</td><td>Cytology brush</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D5879-500ML</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Media (DMEM)/F12 Hepes&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>11330-057</td><td>Base Medium; See Tables 1 and 2</td></tr><tr><td>Epidermal Growth Factor (Recombinant Human Protein, Animal-Origin Free)</td><td>Thermo Fisher Scientific</td><td>PHG6045</td><td>See Table 1</td></tr><tr><td>Epinephrine</td><td>Sigma-Aldrich</td><td>E4250-1G</td><td>See Table 2</td></tr><tr><td>Ethanol</td><td>Fisher Scientific&amp;nbsp;</td><td>2701</td><td></td></tr><tr><td>Ethanolamine</td><td>Sigma-Aldrich</td><td>E0135-500ML</td><td>16.6 mM solution; See Table 2</td></tr><tr><td>Ethylenediaminetetraacetic Acid (EDTA)</td><td>TCI America&amp;nbsp;</td><td>E0084</td><td></td></tr><tr><td>Fetal Bovine Serum (high performance FBS)</td><td>Invitrogen&amp;nbsp;</td><td>10082147</td><td>See Table 1</td></tr><tr><td>Forskolin</td><td>Sigma-Aldrich</td><td>F6886-50</td><td>Prepare a 10 mM stock solution of 4.1 mg in 1 mL of DMSO</td></tr><tr><td>Growth Factor-Reduced Matrigel</td><td>Corning, Inc.</td><td>356231</td><td>Corning Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, Phenol Red-Free, LDEV-Free, 10 mL.</td></tr><tr><td>Hemacytometer</td><td>Hausser Scientific</td><td>1483</td><td></td></tr><tr><td>Human Collagen Solution, Type I (VitroCol; 3 mg/mL)</td><td>Advanced BioMatrix</td><td>5007-A</td><td>Collagen solution</td></tr><tr><td>HyClone (aka FetalClone II)&amp;nbsp;</td><td>GE Healthcare&amp;nbsp;</td><td>SH30066.03HI</td><td>See Table 2</td></tr><tr><td>Hydrocortisone</td><td>StemCell Technologies</td><td>07904</td><td>See Tables 1 and 2</td></tr><tr><td>Insulin, human recombinant, zinc solution</td><td>Life Technologies&amp;nbsp;</td><td>12585014</td><td>4 mg/mL solution; see Table 2</td></tr><tr><td>IVF 4-Well Dish, Non-treated</td><td>NUNC (via Fisher Scientific)</td><td>12566350</td><td>4-well plate for spheroids; similar well size to a 24-well plate</td></tr><tr><td>MEF-CF1-IRR</td><td>Globalstem</td><td>GSC-6001G</td><td>Irradiated murine embryonic fibroblasts</td></tr><tr><td>Metamorph 7.7</td><td>Molecular Devices</td><td></td><td>Analysis Software; https://www.moleculardevices.com/systems/metamorph-research-imaging/metamorph-microscopy-automation-and-image-analysis-software for a quote</td></tr><tr><td>Olympus IX51 Inverted Microscope</td><td>Olympus Corporation</td><td>Discontinued</td><td>Imaging Microscope. Replacment: Olympus IX53, https://www.olympus-lifescience.com/pt/microscopes/inverted/ix53/&amp;nbsp; for a quote</td></tr><tr><td>Pen Strep</td><td>Life Technologies&amp;nbsp;</td><td>15140122</td><td>See Table 2</td></tr><tr><td>Phsophoryletheanolamine</td><td>Sigma-Aldrich</td><td>P0503-5G</td><td>See Table 2</td></tr><tr><td>Retinoic Acid</td><td>Sigma-Aldrich</td><td>R2625-50MG</td><td>See Table 2</td></tr><tr><td>Rhinoprobe</td><td>Arlington Scientific, Inc.</td><td>96-0905</td><td>Nasal curette</td></tr><tr><td>Slidebook 5.5</td><td>3i, Intelligent Imaging Innovations</td><td>Discontinued</td><td>Imaging Software. Replacement: Slidebook 6, https://www.intelligent-imaging.com/slidebook for a quote</td></tr><tr><td>Sterile Phosphate Buffered Saline (PBS)</td><td>Thermo Fisher Scientific</td><td>20012050</td><td></td></tr><tr><td>Sterile Water</td><td>Sigma-Aldrich</td><td>W3500-6X500ML</td><td></td></tr><tr><td>Tissue Culture Dish 100</td><td>Techno Plastic Products</td><td>93100</td><td>Tissue culture dish for expansion</td></tr><tr><td>Tobramycin</td><td>Sigma-Aldrich</td><td>T4014-100MG</td><td>See Table 1</td></tr><tr><td>Transferrin (Human Transferrin 0.5 mL)</td><td>Lonza&amp;nbsp;</td><td>CC-4205</td><td>See Table 2</td></tr><tr><td>Triiodothryonine</td><td>Sigma-Aldrich</td><td>T6397-1G</td><td>3,3&amp;prime;,5-Triiodo-L-thyronine sodium salt&amp;nbsp; [T3]; See Table 2</td></tr><tr><td>Trypsin from Porcine Pancreas</td><td>Sigma-Aldrich</td><td>T4799-10G</td><td></td></tr><tr><td>Ultroser-G</td><td>Crescent Chemical (via Fisher Scientific)</td><td>NC0393024</td><td>20 mL lypophilized powder; See Table 2</td></tr><tr><td>Vancomycin hydrochloride from Streptomyces orientalis</td><td>Sigma-Aldrich</td><td>V2002-5G</td><td>See Table 1</td></tr><tr><td>VX770</td><td>Selleck Chemicals</td><td>S1144</td><td>Prepare a 1 mM stock solution of 0.4 mg in 1 mL of DMSO</td></tr><tr><td>VX809</td><td>Selleck Chemicals</td><td>S1565</td><td>Purchase or prepare a 10 mM stock solution of 4.5 mg in 1 mL of DMSO</td></tr><tr><td>Y-27632 Dihydrochloride&amp;nbsp; ROCK inhibitor&amp;nbsp;</td><td>Enzo LifeSciences&amp;nbsp;</td><td>ALX-270-333-M025&amp;nbsp;</td><td>See Table 1</td></tr></tbody></table>

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.

HNEs should attach to the culture dish and form small islands of cells within 72 h of seeding; examples of good and poor island formation at one week are shown in Figure 1A and 1B, respectively. These islands should expand to cover the dish over the course of 15-30 days. Small or suboptimal samples may take longer, and often will not yield useful spheroids. Contamination with infectious agents is evidenced by deep yellow/cloudy media, failure...

Watch this Scientific Journal Video about Generation of Human Nasal Epithelial Cell Spheroids for Individualized Cystic Fibrosis Transmembrane Conductance Regulator Study at JoVE.com

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

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 ...

generation-human-nasal-epithelial-cell-spheroids-for-individualized

Research

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Medicine

10.6K Views.  Cincinnati Children's Hospital Medical Center. 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.

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

Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They are also involved in many airway diseases through their innate immune response and interaction with immune and airway stromal cells. NECs are of particular interest for studies in children due to their accessibility during clinical visits. Human induced pluripotent stem cells (iPSCs) have been generated from multiple cell types and are a powerful tool for modeling human development and disease, as well as for their potential applications in regenerative medicine. This is the first protocol to lay out methods for successful generation of iPSCs from NECs derived from pediatric participants for research purposes. It describes how to obtain nasal epithelial cells from children, how to generate primary NEC cultures from these samples, and how to reprogram primary NECs into well-characterized iPSCs. Nasal mucosa samples are useful in epidemiological studies related to the effects of air pollution in children, and provide an important tool for studying airway disease. Primary nasal cells and iPSCs derived from them can be a tool for providing unlimited material for patient-specific research in diverse areas of airway epithelial biology, including asthma and COPD research.

This publication demonstrates methods for successful sampling and culture of nasal epithelial mucosa from children, and reprogramming these cells to induced Pluripotent Stem Cells (iPSCs).

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They ...

Developmental Biology

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Using Continuous Data Tracking Technology to Study Exercise Adherence in Pulmonary Rehabilitation

Pulmonary rehabilitation (PR) is an important component in the management of respiratory diseases. The effectiveness of PR is dependent upon adherence to exercise training recommendations. The study of exercise adherence is thus a key step towards the optimization of PR programs. To date, mostly indirect measures, such as rates of participation, completion, and attendance, have been used to determine adherence to PR. The purpose of the present protocol is to describe how continuous data tracking technology can be used to measure adherence to a prescribed aerobic training intensity on a second-by-second basis.
In our investigations, adherence has been defined as the percent time spent within a specified target heart rate range. As such, using a combination of hardware and software, heart rate is measured, tracked, and recorded during cycling second-by-second for each participant, for each exercise session. Using statistical software, the data is subsequently extracted and analyzed. The same protocol can be applied to determine adherence to other measures of exercise intensity, such as time spent at a specified wattage, level, or speed on the cycle ergometer. Furthermore, the hardware and software is also available to measure adherence to other modes of training, such as the treadmill, elliptical, stepper, and arm ergometer. The present protocol, therefore, has a vast applicability to directly measure adherence to aerobic exercise.

Pulmonary rehabilitation is widely recognized in the management of respiratory diseases. A key component to successful pulmonary rehabilitation is adherence to the recommended exercise training. The purpose of the present protocol is to describe how continuous data tracking technology can be used to precisely measure adherence to a prescribed aerobic training intensity.

Pulmonary rehabilitation (PR) is an important component in the management of respiratory diseases. The effectiveness of PR is dependent upon adherence to ...

Generation of Airway Epithelial Cell Air-Liquid Interface Cultures from Human Pluripotent Stem Cells

Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major causes of morbidity and mortality worldwide. In vitro platforms using human bronchial epithelial cells (HBECs) have been instrumental to our understanding of the airway epithelium in health and disease. Access to HBECs from individuals with rare genetic diseases or rare mutations is a bottleneck in lung research.
Induced pluripotent stem cells (iPSCs) are readily generated by "reprogramming" somatic cells and retain the unique genetic background of the individual donor. Recent advances allow for the directed differentiation of iPSCs to lung epithelial progenitor cells, alveolar type 2 cells, as well as the cells of the conducting airway epithelium via basal cells, the major airway stem cells.
Here we outline a protocol for the maintenance and expansion of iPSC-derived airway basal cells (hereafter iBCs) as well as their trilineage differentiation in air-liquid interface (ALI) cultures. iBCs are maintained and expanded as epithelial spheres suspended in droplets of extracellular matrix cultured in a primary basal cell medium supplemented with inhibitors of TGF-&#223; and BMP signaling pathways. iBCs within these epithelial spheres express key basal markers TP63 and NGFR, can be purified by fluorescence activated cell sorting (FACS), and when plated on porous membranes in standard ALI culture conditions, differentiate into a functional airway epithelium. ALI cultures derived from healthy donors are composed of basal, secretory and multiciliated cells and demonstrate epithelial barrier integrity, motile cilia, and mucus secretion. Cultures derived from individuals with CF or PCD recapitulate the dysfunctional CFTR-mediated chloride transport or immotile cilia, the respective disease-causing epithelial defects.
Here, we present a protocol for the generation of human cells that can be applied for modeling and understanding airway diseases.

Recent advances in human induced pluripotent stem cell differentiation protocols allow for the stepwise derivation of organ-specific cell types. Here, we provide detailed steps for the maintenance and expansion of iPSC-derived airway basal cells and their differentiation into a mucociliary epithelium in air-liquid interface cultures.

Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major ...

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 ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Biology

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 ...

Collection, Expansion, and Differentiation of Primary Human Nasal Epithelial Cell Models for Quantification of Cilia Beat Frequency

Measurements of cilia function (beat frequency, pattern) have been established as diagnostic tools for respiratory diseases such as primary ciliary dyskinesia. However, the wider application of these techniques is limited by the extreme susceptibility of ciliary function to changes in environmental factors e.g., temperature, humidity, and pH. In the airway of patients with Cystic Fibrosis (CF), mucus accumulation impedes cilia beating. Cilia function has been investigated in primary airway cell models as an indicator of CF Transmembrane conductance Regulator (CFTR) channel activity. However, considerable patient-to-patient variability in cilia beating frequency has been found in response to CFTR-modulating drugs, even for patients with the same CFTR mutations. Furthermore, the impact of dysfunctional CFTR-regulated chloride secretion on ciliary function is poorly understood. There is currently no comprehensive protocol demonstrating sample preparation of in vitro airway models, image acquisition, and analysis of Cilia Beat Frequency (CBF). Standardized culture conditions and image acquisition performed in an environmentally controlled condition would enable consistent, reproducible quantification of CBF between individuals and in response to CFTR-modulating drugs. This protocol describes the quantification of CBF in three different airway epithelial cell model systems: 1) native epithelial sheets, 2) air-liquid interface models imaged on permeable support inserts, and 3) extracellular matrix-embedded three-dimensional organoids. The latter two replicate in vivo lung physiology, with beating cilia and production of mucus. The ciliary function is captured using a high-speed video camera in an environment-controlled chamber. Custom-built scripts are used for the analysis of CBF. Translation of CBF measurements to the clinic is envisioned to be an important clinical tool for predicting response to CFTR-modulating drugs on a per-patient basis.

This protocol describes nasal epithelial cell collection, expansion, and differentiation to organotypic airway epithelial cell models and quantification of cilia beat frequency via live-cell imaging and custom-built scripts.

Measurements of cilia function (beat frequency, pattern) have been established as diagnostic tools for respiratory diseases such as primary ciliary ...

Mucociliary Epithelial Organoids from Xenopus Embryonic Cells: Generation, Culture and High-Resolution Live Imaging

Mucociliary epithelium provides the first line of defense by removing foreign particles through the action of mucus production and cilia-mediated clearance. Many clinically relevant defects in the mucociliary epithelium are inferred as they occur deep within the body. Here, we introduce a tractable 3D model for mucociliary epithelium generated from multipotent progenitors that were microsurgically isolated from Xenopus laevis embryos. The mucociliary epithelial organoids are covered with newly generated epithelium from deep ectoderm cells and later decorated with distinct patterned multiciliated cells, secretory cells, and mucus-producing goblet cells that are indistinguishable from the native epidermis within 24 h. The full sequences of dynamic cell transitions from mesenchymal to epithelial that emerge on the apical surface of organoids can be tracked by high-resolution live imaging. These in vitro cultured, self-organizing mucociliary epithelial organoids offer distinct advantages in studying the biology of mucociliary epithelium with high-efficiency in generation, defined culture conditions, control over number and size, and direct access for live imaging during the regeneration of the differentiated epithelium.

We describe a simple protocol to develop mucociliary epithelial organoids from deep ectoderm cells isolated from Xenopus laevis embryos. The multipotent progenitors regenerate epithelial goblet cell precursors and allow live tracking of the initiation and progression of the cell transitions on the surface of organoids.

Mucociliary epithelium provides the first line of defense by removing foreign particles through the action of mucus production and cilia-mediated ...

Primary Human Nasal Epithelial Cells: Biobanking in the Context of Precision Medicine

Human nasal epithelial (HNE) cells are easy to collect by simple, non-invasive nasal brushing. Patient-derived primary HNE cells can be amplified and differentiated into a pseudo-stratified epithelium in air-liquid interface conditions to quantify cyclic AMP-mediated Chloride (Cl-) transport as an index of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) function. If critical steps such as quality of nasal brushing and cell density upon cryopreservation are performed efficiently, HNE cells can be successfully biobanked. Moreover, short-circuit current studies demonstrate that freeze-thawing does not significantly modify HNE cells&#39; electrophysiological properties and response to CFTR modulators. In the culture conditions used in this study, when less than 2 x 106 cells are frozen per cryovial, the failure rate is very high. We recommend freezing at least 3 x 106 cells per cryovial. We show that dual therapies combining a CFTR corrector with a CFTR potentiator have a comparable correction efficacy for CFTR activity in F508del-homozygous HNE cells. Triple therapy VX-445 + VX-661 + VX-770 significantly increased correction of CFTR activity compared to dual therapy VX-809 + VX-770. The measure of CFTR activity in HNE cells is a promising pre-clinical biomarker useful to guide CFTR modulator therapy.

Here we describe the isolation, amplification, and differentiation of primary human nasal epithelial (HNE) cells at the air-liquid interface and a biobanking protocol allowing to successfully freeze and then thaw amplified HNE. The protocol analyzes electrophysiological properties of differentiated HNE cells and CFTR-related chloride secretion correction upon different modulator treatments.

Human nasal epithelial (HNE) cells are easy to collect by simple, non-invasive nasal brushing. Patient-derived primary HNE cells can be amplified and ...

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

Summary

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Chapters in this video

Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Using Continuous Data Tracking Technology to Study Exercise Adherence in Pulmonary Rehabilitation

Generation of Airway Epithelial Cell Air-Liquid Interface Cultures from Human Pluripotent Stem Cells

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

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

Culture and Imaging of Human Nasal Epithelial Organoids

Collection, Expansion, and Differentiation of Primary Human Nasal Epithelial Cell Models for Quantification of Cilia Beat Frequency

Mucociliary Epithelial Organoids from Xenopus Embryonic Cells: Generation, Culture and High-Resolution Live Imaging

Primary Human Nasal Epithelial Cells: Biobanking in the Context of Precision Medicine