Name: Video: 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: 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.

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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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. 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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 border="0" cellpadding="0" cellspacing="0" width="633">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:180px;">1.5 mL Eppendorf Tube</td>
			<td style="width:149px;">USA Scientific&#160;</td>
			<td style="width:131px;">4036-3204</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:180px;">150 mL Filter Flask</td>
			<td style="width:149px;">Midsci&#160;</td>
			<td style="width:131px;">TP99150</td>
			<td style="width:173px;">To filter Media</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:180px;">15 mL Conical Tube</td>
			<td style="width:149px;">Midsci&#160;</td>
			<td style="width:131px;">TP91015</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:180px;">1 L Filter Flask</td>
			<td style="width:149px;">Midsci&#160;</td>
			<td style="width:131px;">TP99950</td>
			<td style="width:173px;">To filter Media</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:180px;">35 mm Glass-Bottom Dish</td>
			<td style="width:149px;">MatTek Corporation</td>
			<td style="width:131px;">P35G-0-20-C</td>
			<td style="width:173px;">Optional</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:180px;">3-Isobutyl-1-Methylxanthine (IBMX)</td>
			<td style="width:149px;">Fisher Scientific&#160;</td>
			<td style="width:131px;">AC228420010&#160;</td>
			<td style="width:173px;">Prepare a 100 mM stock solution of 22.0 mg in 1 mL of DMSO</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:180px;">50 mL Conical Tube</td>
			<td style="width:149px;">Midsci&#160;</td>
			<td style="width:131px;">&#160;TP91050</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:180px;">Accutase</td>
			<td style="width:149px;">Innovative Cell Technologies, Inc.</td>
			<td style="width:131px;">AT-104</td>
			<td style="width:173px;">Cell detachment solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:180px;">Adenine</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:131px;">A2786-25G</td>
			<td style="width:173px;">See Table 1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:180px;">Amphotericin B</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:131px;">A9528-100MG</td>
			<td style="width:173px;">See Table 1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:180px;">Bovine Brain Extract (9mg/mL)</td>
			<td style="width:149px;">Lonza&#160;</td>
			<td style="width:131px;">CC-4098</td>
			<td style="width:173px;">See Table 2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:180px;">Ceftazidime hydrate</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:131px;">C3809-1G</td>
			<td style="width:173px;">See Table 1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:180px;">Cell Scrapers 20 cm&#160;</td>
			<td style="width:149px;">Midsci&#160;</td>
			<td style="width:131px;">TP99010</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:180px;">CFTR Inh172</td>
			<td style="width:149px;">Tocris Bioscience</td>
			<td style="width:131px;">3430</td>
			<td style="width:173px;">Prepare a 10 mM stock solution of 4.0 mg in 1 mL of DMSO</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:180px;">Cholera Toxin B (From Vibrio cholerae)</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:131px;">C8052-.5MG</td>
			<td style="width:173px;">See Table 1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:180px;">CYB-1</td>
			<td style="width:149px;">Medical Packaging Corporation</td>
			<td style="width:131px;">CYB-1</td>
			<td style="width:173px;">Cytology brush</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:180px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:131px;">D5879-500ML</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:180px;">Dulbecco&#39;s Modified Eagle Media (DMEM)/F12 Hepes&#160;</td>
			<td style="width:149px;">Life Technologies&#160;</td>
			<td style="width:131px;">11330-057</td>
			<td style="width:173px;">Base Medium; See Tables 1 and 2</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:180px;">Epidermal Growth Factor (Recombinant Human Protein, Animal-Origin Free)</td>
			<td style="width:149px;">Thermo Fisher Scientific</td>
			<td style="width:131px;">PHG6045</td>
			<td style="width:173px;">See Table 1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:180px;">Epinephrine</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:131px;">E4250-1G</td>
			<td style="width:173px;">See Table 2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:180px;">Ethanol</td>
			<td style="width:149px;">Fisher Scientific&#160;</td>
			<td style="width:131px;">2701</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:180px;">Ethanolamine</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:131px;">E0135-500ML</td>
			<td style="width:173px;">16.6 mM solution; See Table 2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:180px;">Ethylenediaminetetraacetic Acid (EDTA)</td>
			<td style="width:149px;">TCI America&#160;</td>
			<td style="width:131px;">E0084</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:180px;">Fetal Bovine Serum (high performance FBS)</td>
			<td style="width:149px;">Invitrogen&#160;</td>
			<td style="width:131px;">10082147</td>
			<td style="width:173px;">See Table 1</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:180px;">Forskolin</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:131px;">F6886-50</td>
			<td style="width:173px;">Prepare a 10 mM stock solution of 4.1 mg in 1 mL of DMSO</td>
		</tr>
		<tr height="101">
			<td height="101" style="height:101px;width:180px;">Growth Factor-Reduced Matrigel</td>
			<td style="width:149px;">Corning, Inc.</td>
			<td style="width:131px;">356231</td>
			<td style="width:173px;">Corning Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, Phenol Red-Free, LDEV-Free, 10 mL.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:180px;">Hemacytometer</td>
			<td style="width:149px;">Hausser Scientific</td>
			<td style="width:131px;">1483</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:180px;">Human Collagen Solution, Type I (VitroCol; 3 mg/mL)</td>
			<td style="width:149px;">Advanced BioMatrix</td>
			<td style="width:131px;">5007-A</td>
			<td style="width:173px;">Collagen solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:180px;">HyClone (aka FetalClone II)&#160;</td>
			<td style="width:149px;">GE Healthcare&#160;</td>
			<td style="width:131px;">SH30066.03HI</td>
			<td style="width:173px;">See Table 2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:180px;">Hydrocortisone</td>
			<td style="width:149px;">StemCell Technologies</td>
			<td style="width:131px;">07904</td>
			<td style="width:173px;">See Tables 1 and 2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:180px;">Insulin, human recombinant, zinc solution</td>
			<td style="width:149px;">Life Technologies&#160;</td>
			<td style="width:131px;">12585014</td>
			<td style="width:173px;">4 mg/mL solution; see Table 2</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:180px;">IVF 4-Well Dish, Non-treated</td>
			<td style="width:149px;">NUNC (via Fisher Scientific)</td>
			<td style="width:131px;">12566350</td>
			<td style="width:173px;">4-well plate for spheroids; similar well size to a 24-well plate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:180px;">MEF-CF1-IRR</td>
			<td style="width:149px;">Globalstem</td>
			<td style="width:131px;">GSC-6001G</td>
			<td style="width:173px;">Irradiated murine embryonic fibroblasts</td>
		</tr>
		<tr height="160">
			<td height="160" style="height:160px;width:180px;">Metamorph 7.7</td>
			<td style="width:149px;">Molecular Devices</td>
			<td style="width:131px;"></td>
			<td style="width:173px;">Analysis Software; https://www.moleculardevices.com/systems/metamorph-research-imaging/metamorph-microscopy-automation-and-image-analysis-software for a quote</td>
		</tr>
		<tr height="140">
			<td height="140" style="height:140px;width:180px;">Olympus IX51 Inverted Microscope</td>
			<td style="width:149px;">Olympus Corporation</td>
			<td style="width:131px;">Discontinued</td>
			<td style="width:173px;">Imaging Microscope. Replacment: Olympus IX53, https://www.olympus-lifescience.com/pt/microscopes/inverted/ix53/&#160; for a quote</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:180px;">Pen Strep</td>
			<td style="width:149px;">Life Technologies&#160;</td>
			<td style="width:131px;">15140122</td>
			<td style="width:173px;">See Table 2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:180px;">Phsophoryletheanolamine</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:131px;">P0503-5G</td>
			<td style="width:173px;">See Table 2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:180px;">Retinoic Acid</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:131px;">R2625-50MG</td>
			<td style="width:173px;">See Table 2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:180px;">Rhinoprobe</td>
			<td style="width:149px;">Arlington Scientific, Inc.</td>
			<td style="width:131px;">96-0905</td>
			<td style="width:173px;">Nasal curette</td>
		</tr>
		<tr height="101">
			<td height="101" style="height:101px;width:180px;">Slidebook 5.5</td>
			<td style="width:149px;">3i, Intelligent Imaging Innovations</td>
			<td style="width:131px;">Discontinued</td>
			<td style="width:173px;">Imaging Software. Replacement: Slidebook 6, https://www.intelligent-imaging.com/slidebook for a quote</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:180px;">Sterile Phosphate Buffered Saline (PBS)</td>
			<td style="width:149px;">Thermo Fisher Scientific</td>
			<td style="width:131px;">20012050</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:180px;">Sterile Water</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:131px;">W3500-6X500ML</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:180px;">Tissue Culture Dish 100</td>
			<td style="width:149px;">Techno Plastic Products</td>
			<td style="width:131px;">93100</td>
			<td style="width:173px;">Tissue culture dish for expansion</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:180px;">Tobramycin</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:131px;">T4014-100MG</td>
			<td style="width:173px;">See Table 1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:180px;">Transferrin (Human Transferrin 0.5 mL)</td>
			<td style="width:149px;">Lonza&#160;</td>
			<td style="width:131px;">CC-4205</td>
			<td style="width:173px;">See Table 2</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:180px;">Triiodothryonine</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:131px;">T6397-1G</td>
			<td style="width:173px;">3,3&#8242;,5-Triiodo-L-thyronine sodium salt&#160; [T3]; See Table 2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:180px;">Trypsin from Porcine Pancreas</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:131px;">T4799-10G</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:180px;">Ultroser-G</td>
			<td style="width:149px;">Crescent Chemical (via Fisher Scientific)</td>
			<td style="width:131px;">NC0393024</td>
			<td style="width:173px;">20 mL lypophilized powder; See Table 2</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:180px;">Vancomycin hydrochloride from Streptomyces orientalis</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:131px;">V2002-5G</td>
			<td style="width:173px;">See Table 1</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:180px;">VX770</td>
			<td style="width:149px;">Selleck Chemicals</td>
			<td style="width:131px;">S1144</td>
			<td style="width:173px;">Prepare a 1 mM stock solution of 0.4 mg in 1 mL of DMSO</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:180px;">VX809</td>
			<td style="width:149px;">Selleck Chemicals</td>
			<td style="width:131px;">S1565</td>
			<td style="width:173px;">Purchase or prepare a 10 mM stock solution of 4.5 mg in 1 mL of DMSO</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:180px;">Y-27632 Dihydrochloride&#160; ROCK inhibitor&#160;</td>
			<td style="width:149px;">Enzo LifeSciences&#160;</td>
			<td style="width:131px;">ALX-270-333-M025&#160;</td>
			<td style="width:173px;">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

JoVE Journal

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

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 ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The BCSFB functions in separating the cerebrospinal fluid (CSF) from the blood and restricting the molecular exchange to a minimum extent. An in vitro model of the BCSFB is based on cells derived from a human choroid plexus papilloma (HIBCPP). HIBCPP cells display typical barrier functions including formation of tight junctions (TJs), development of a transepithelial electrical resistance (TEER), as well as minor permeabilities for macromolecules. There are several pathogens that can enter the central nervous system (CNS) via the BCSFB and subsequently cause severe disease like meningitis. One of these pathogens is Neisseria meningitidis (N. meningitidis), a human-specific bacterium. Employing the HIBCPP cells in an inverted cell culture filter insert system enables to study interactions of pathogens with cells of the BCSFB from the basolateral cell side, which is relevant in vivo. In this article, we describe seeding and culturing of HIBCPP cells on cell culture inserts. Further, infection of the cells with N. meningitidis along with analysis of invaded and adhered bacteria via double immunofluorescence is demonstrated. As the cells of the CP are also involved in other diseases, including neurodegenerative disorders like Alzheimer`s disease and Multiple Sclerosis, as well as during the brain metastasis of tumor cells, the model system can also be applied in other fields of research. It provides the potential to decipher molecular mechanisms and to identify novel therapeutic targets.

The epithelial cells of the choroid plexus (CP) form the blood-cerebrospinal fluid barrier (BCSFB). An in vitro model of the BCSFB employs human choroid plexus papilloma (HIBCPP) cells. This article describes culturing and basolateral infection of HIBCPP cells using a cell culture filter insert system.

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The ...

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 ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Standardized Measurement of Nasal Membrane Transepithelial Potential Difference (NPD)

We describe a standardized measurement of nasal potential difference (NPD). In this technique, cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial sodium channel (ENaC) function are monitored by the change in voltage across the nasal epithelium after the superfusion of solutions that modify ion channel activity. This is enabled by the measurement of the potential difference between the subcutaneous compartment and the airway epithelium in the nostril, utilizing a catheter in contact with the inferior nasal turbinate.
The test allows the measurement of the stable baseline voltage and the successive net voltage changes after perfusion of 100 &#181;M amiloride, an inhibitor of Na+ reabsorption in Ringer&#39;s solution; a chloride-free solution containing amiloride to drive chloride secretion and 10 &#181;M isoproterenol in a chloride-free solution with amiloride to stimulate the cyclic adenosine monophosphate (cAMP)-dependent chloride conductance related to CFTR.
This technique has the advantage of demonstrating the electrophysiological properties of two key components establishing the hydration of the airway surface liquid of the respiratory epithelium, ENaC, and CFTR. Therefore, it is a useful research tool for phase 2 and proof of concept trials of agents that target CFTR and ENaC activity for the treatment of cystic fibrosis (CF) lung disease. It is also a key follow-up procedure to establish CFTR dysfunction when genetic testing and sweat testing are equivocal. Unlike sweat chloride, the test is relatively more time consuming and costly. It also requires operator training and expertise to conduct the test effectively. Inter- and intra-subject variability has been reported in this technique especially in young or uncooperative subjects. To assist with this concern, interpretation has been improved through a recently validated algorithm.

Standardized Measurement of Nasal Membrane Transepithelial Potential Difference NPD

Here, we present a standardized protocol to measure the nasal potential difference (NPD). Cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) function are evaluated by the change in the voltage across the nasal epithelium after superfusion of solutions that modify ion channel activity, providing an outcome measure.

We describe a standardized measurement of nasal potential difference (NPD). In this technique, cystic fibrosis transmembrane conductance regulator (CFTR) ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...

Generation of Human 3D Lung Tissue Cultures (3D-LTCs) for Disease Modeling

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) used as 3D lung tissue cultures (3D-LTCs) represent an elegant and biologically highly relevant 3D cell culture model, which highly resemble in situ tissue due to their complexity, biomechanics and molecular composition. Tissue slicing is widely applied in various animal models. 3D-LTCs derived from human PCLS can be used to analyze responses to novel drugs, which might further help to better understand the mechanisms and functional effects of drugs in human tissue. The preparation of PCLS from surgically resected lung tissue samples of patients, who experienced lung lobectomy, increases the accessibility of diseased and peritumoral tissue. Here, we describe a detailed protocol for the generation of human PCLS from surgically resected soft-elastic patient lung tissue. Agarose was introduced into the bronchoalveolar space of the resectates, thus preserving lung structure and increasing the tissue&#39;s stiffness, which is crucial for subsequent slicing. 500 &#181;m thick slices were prepared from the tissue block with a vibratome. Biopsy punches taken from PCLS ensure comparable tissue sample sizes and further increase the amount of tissue samples. The generated lung tissue cultures can be applied in a variety of studies in human lung biology, including the pathophysiology and mechanisms of different diseases, such as fibrotic processes at its best at (sub-)cellular levels. The highest benefit of the 3D-LTC ex vivo model is its close representation of the in situ human lung in respect of 3D tissue architecture, cell type diversity and lung anatomy as well as the potential for assessment of tissue from individual patients, which is relevant to further develop novel strategies for precision medicine.

Generation of Human 3D Lung Tissue Cultures 3D-LTCs for Disease Modeling

Here, we present a protocol for the preparation of agarose-filled human precision-cut lung slices from resected patient tissue that are suitable for generating 3D lung tissue cultures to model human lung diseases in biological and biomedical studies.

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) ...

Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. Microbes have traditionally been classified based on their ability to use or tolerate oxygen. Supplemental oxygen is a common medical therapy administered to people with cystic fibrosis (pwCF); however, existing studies on oxygen and the airway microbiome have focused on how hypoxia (low oxygen) rather than hyperoxia (high oxygen) affects the predominantly aerobic and facultative anaerobic lung microbial communities. To address this critical knowledge gap, this protocol was developed using an artificial sputum medium that mimics the composition of sputum from pwCF. The use of filter sterilization, which yields a transparent medium, allows optical methods to follow the growth of single-celled microbes in suspension cultures. To create hyperoxic conditions, this model system takes advantage of established anaerobic culturing techniques to study hyperoxic conditions; instead of removing oxygen, oxygen is added to cultures by daily sparging of serum bottles with a mixture of compressed oxygen and air. Sputum from 50 pwCF underwent daily sparging for a 72-h period to verify the ability of this model to maintain differential oxygen conditions. Shotgun metagenomic sequencing was performed on cultured and uncultured sputum samples from 11 pwCF to verify the ability of this medium to support the growth of commensal and pathogenic microbes commonly found in cystic fibrosis sputum. Growth curves were obtained from 112 isolates obtained from pwCF to verify the ability of this artificial sputum medium to support the growth of common cystic fibrosis pathogens. We find that this model can culture a wide variety of pathogens and commensals in CF sputum, recovers a community highly similar to uncultured sputum under normoxic conditions, and creates different culture phenotypes under varying oxygen conditions. This new approach might lead to a better understanding of unanticipated effects induced by the use of oxygen in pwCF on airway microbial communities and common respiratory pathogens.

The goal of this protocol is to develop a model system for the effect of hyperoxia on cystic fibrosis airway microbial communities. Artificial sputum medium emulates the composition of sputum, and hyperoxic culture conditions model the effects of supplemental oxygen on lung microbial communities.

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. ...

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

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The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Generation of Human 3D Lung Tissue Cultures (3D-LTCs) for Disease Modeling

Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome