Name: collection, expansion, and differentiation of primary human nasal epithelial cell models for quantification of cilia beat frequency
Uploaded: 2021-11-10T13:00:00
Description: Watch this Scientific Journal Video about Collection, Expansion, and Differentiation of Primary Human Nasal Epithelial Cell Models for Quantification of Cilia Beat Frequency at JoVE.com

We thank the study participants and their families for their contributions. We appreciate the assistance from Sydney Children&#39;s Hospitals (SCH) Randwick respiratory department in the organization and collection of patient biospecimens - special thanks to Dr. John Widger, Dr. Yvonne Belessis, Leanne Plush, Amanda Thompson, and Rhonda Bell. We acknowledge the assistance of Iveta Slapetova and Renee Whan from the Katharina Gaus Light Microscopy Facility within the Mark Wainwright Analytical Centre at UNSW Sydney. This work is supported by National Health and Medical Research Council (NHMRC) Australia (GNT1188987), CF Foundation Australia, and Sydney Children&#39;s Hospital Foundation. The authors would like to acknowledge Luminesce Alliance - Innovation for Children&#39;s Health for its contribution and support. Luminesce Alliance - Innovation for Children&#39;s Health is a not-for-profit cooperative joint venture between the Sydney Children&#39;s Hospitals Network, the Children&#39;s Medical Research Institute, and the Children&#39;s Cancer Institute. It has been established with the support of the NSW Government to coordinate and integrate pediatric research. Luminesce Alliance is also affiliated with the University of Sydney and the University of New South Wales Sydney. KMA is supported by an Australian Government Research Training Program Scholarship. LKF is supported by the Rotary Club of Sydney Cove/Sydney Children&#39;s Hospital Foundation and UNSW University postgraduate award scholarships.

Study approval was received from the Sydney Children&#39;s Hospital Network Ethics Review Board (HREC/16/SCHN/120). Written consent was obtained from all participants (or participants&#39; guardian) prior to the collection of biospecimens.
1. Preparations for establishing airway epithelial cell models
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	<li>Prepare nasal cell collection media by combining 80% Dulbecco&#39;s Modified Eagle Medium and 20% Fetal Bovine Serum. Supplement with 1 &#181;L/mL of Penicillin/Strep...

<ol>
	<li>Barbato, A., 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=Primary+ciliary+dyskinesia:+a+consensus+statement+on+diagnostic+and+treatment+approaches+in+children.">Primary ciliary dyskinesia: a consensus statement on diagnostic and treatment approaches in children.</a> European Respiratory Journal. 34 (6), 1264-1276 (2009).</li><li>Cutting, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+fibrosis+genetics:+from+molecular+understanding+to+clinical+application.">Cystic fibrosis genetics: from molecular understanding to clinical application.</a> Nature Reviews Genetics. 16 (1), 45-56 (2015).</li><li>Chioccioli, M., Feriani, L., Kotar, J., Bratcher, P. E., Cicuta, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotyping+ciliary+dynamics+and+coordination+in+response+to+CFTR-modulators+in+Cystic+Fibrosis+respiratory+epithelial+cells.">Phenotyping ciliary dynamics and coordination in response to CFTR-modulators in Cystic Fibrosis respiratory epithelial cells.</a> Nature Communications. 10 (1), 1763 (2019).</li><li>Hirst, R. A., Rutman, A., Williams, G., O'Callaghan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ciliated+air-liquid+cultures+as+an+aid+to+diagnostic+testing+of+primary+ciliary+dyskinesia.">Ciliated air-liquid cultures as an aid to diagnostic testing of primary ciliary dyskinesia.</a> Chest. 138 (6), 1441-1447 (2010).</li><li>Thomas, B., Rutman, A., O'Callaghan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disrupted+ciliated+epithelium+shows+slower+ciliary+beat+frequency+and+increased+dyskinesia.">Disrupted ciliated epithelium shows slower ciliary beat frequency and increased dyskinesia.</a> European Respiratory Journal. 34 (2), 401-404 (2009).</li><li>Coles, J. L., 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=A+revised+protocol+for+culture+of+airway+epithelial+cells+as+a+diagnostic+tool+for+primary+ciliary+dyskinesia.">A revised protocol for culture of airway epithelial cells as a diagnostic tool for primary ciliary dyskinesia.</a> Journal of Clinical Medicine. 9 (11), (2020).</li><li>Pifferi, M., 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=Simplified+cell+culture+method+for+the+diagnosis+of+atypical+primary+ciliary+dyskinesia.">Simplified cell culture method for the diagnosis of atypical primary ciliary dyskinesia.</a> Thorax. 64 (12), 1077-1081 (2009).</li><li>Pifferi, M., 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=Rapid+diagnosis+of+primary+ciliary+dyskinesia:+cell+culture+and+soft+computing+analysis.">Rapid diagnosis of primary ciliary dyskinesia: cell culture and soft computing analysis.</a> European Respiratory Journal. 41 (4), 960-965 (2013).</li><li>Lee, D. D. H., 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=Higher+throughput+drug+screening+for+rare+respiratory+diseases:+Readthrough+therapy+in+primary+ciliary+dyskinesia.">Higher throughput drug screening for rare respiratory diseases: Readthrough therapy in primary ciliary dyskinesia.</a> European Respiratory Journal. 58 (4), 2000455 (2021).</li><li>Saint-Criq, V., 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=Choice+of+differentiation+media+significantly+impacts+cell+lineage+and+response+to+CFTR+modulators+in+fully+differentiated+primary+cultures+of+cystic+fibrosis+human+airway+epithelial+cells.">Choice of differentiation media significantly impacts cell lineage and response to CFTR modulators in fully differentiated primary cultures of cystic fibrosis human airway epithelial cells.</a> Cells. 9 (9), (2020).</li><li>Awatade, N. T., 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=Significant+functional+differences+in+differentiated+Conditionally+Reprogrammed+(CRC)-+and+Feeder-free+Dual+SMAD+inhibited-expanded+human+nasal+epithelial+cells.">Significant functional differences in differentiated Conditionally Reprogrammed (CRC)- and Feeder-free Dual SMAD inhibited-expanded human nasal epithelial cells.</a> Journal of Cystic Fibrosis. 20 (2), 364-371 (2021).</li><li>Dabrowski, M., Bukowy-Bieryllo, Z., Jackson, C. L., Zietkiewicz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+non-aminoglycoside+compounds+used+to+stimulate+translational+readthrough+of+PTC+mutations+in+primary+ciliary+dyskinesia.">Properties of non-aminoglycoside compounds used to stimulate translational readthrough of PTC mutations in primary ciliary dyskinesia.</a> International Journal of Molecular Sciences. 22 (9), (2021).</li><li>Hirst, R. A., 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=Culture+of+primary+ciliary+dyskinesia+epithelial+cells+at+air-liquid+interface+can+alter+ciliary+phenotype+but+remains+a+robust+and+informative+diagnostic+aid.">Culture of primary ciliary dyskinesia epithelial cells at air-liquid interface can alter ciliary phenotype but remains a robust and informative diagnostic aid.</a> PloS One. 9 (2), 89675 (2014).</li><li>Marthin, J. K., Stevens, E. M., Larsen, L. A., Christensen, S. T., Nielsen, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-specific+three-dimensional+explant+spheroids+derived+from+human+nasal+airway+epithelium:+a+simple+methodological+approach+for+ex+vivo+studies+of+primary+ciliary+dyskinesia.">Patient-specific three-dimensional explant spheroids derived from human nasal airway epithelium: a simple methodological approach for ex vivo studies of primary ciliary dyskinesia.</a> Cilia. 6, 3 (2017).</li><li>Chilvers, M. A., O'Callaghan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+ciliary+beat+pattern+and+beat+frequency+using+digital+high+speed+imaging:+comparison+with+the+photomultiplier+and+photodiode+methods.">Analysis of ciliary beat pattern and beat frequency using digital high speed imaging: comparison with the photomultiplier and photodiode methods.</a> Thorax. 55 (4), 314-317 (2000).</li><li>Chilvers, M. A., Rutman, A., O'Callaghan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+cilia+and+ciliated+epithelial+ultrastructure+in+healthy+children+and+young+adults.">Functional analysis of cilia and ciliated epithelial ultrastructure in healthy children and young adults.</a> Thorax. 58 (4), 333-338 (2003).</li><li>Castillon, N., 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=Polarized+expression+of+cystic+fibrosis+transmembrane+conductance+regulator+and+associated+epithelial+proteins+during+the+regeneration+of+human+airway+surface+epithelium+in+three-dimensional+culture.">Polarized expression of cystic fibrosis transmembrane conductance regulator and associated epithelial proteins during the regeneration of human airway surface epithelium in three-dimensional culture.</a> Laboratory Investigation. 82 (8), 989-998 (2002).</li><li>Jorissen, M., Bessems, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+ciliary+beat+frequency+after+ciliogenesis+in+nasal+epithelial+cells+cultured+sequentially+as+monolayer+and+in+suspension.">Normal ciliary beat frequency after ciliogenesis in nasal epithelial cells cultured sequentially as monolayer and in suspension.</a> Acta Oto-Laryngologica. 115 (1), 66-70 (1995).</li><li>Conger, B. T., 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=Comparison+of+cystic+fibrosis+transmembrane+conductance+regulator+(CFTR)+and+ciliary+beat+frequency+activation+by+the+CFTR+Modulators+Genistein,+VRT-532,+and+UCCF-152+in+primary+sinonasal+epithelial+cultures.">Comparison of cystic fibrosis transmembrane conductance regulator (CFTR) and ciliary beat frequency activation by the CFTR Modulators Genistein, VRT-532, and UCCF-152 in primary sinonasal epithelial cultures.</a> JAMA Otolaryngology-Head &amp; Neck Surgery. 139 (8), 822-827 (2013).</li><li>Pique, N., De Servi, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhinosectan((R))+spray+(containing+xyloglucan)+on+the+ciliary+function+of+the+nasal+respiratory+epithelium;+results+of+an+in+vitro+study.">Rhinosectan((R)) spray (containing xyloglucan) on the ciliary function of the nasal respiratory epithelium; results of an in vitro study.</a> Allergy, Asthma &amp; Clinical Immunology. 14, 41 (2018).</li><li>Chen, Q., 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=Host+antiviral+response+suppresses+ciliogenesis+and+motile+ciliary+functions+in+the+nasal+epithelium.">Host antiviral response suppresses ciliogenesis and motile ciliary functions in the nasal epithelium.</a> Frontiers in Cell and Developmental Biology. 8, 581340 (2020).</li><li>Clary-Meinesz, C. F., Cosson, J., Huitorel, P., Blaive, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+effect+on+the+ciliary+beat+frequency+of+human+nasal+and+tracheal+ciliated+cells.">Temperature effect on the ciliary beat frequency of human nasal and tracheal ciliated cells.</a> Biology of the Cell. 76 (3), 335-338 (1992).</li><li>Ballenger, J. J., Orr, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurement+of+human+ciliary+activity.">Quantitative measurement of human ciliary activity.</a> Annals of Otology, Rhinology and Laryngology. 72, 31-39 (1963).</li><li>Mercke, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+varying+air+humidity+on+mucociliary+activity.">The influence of varying air humidity on mucociliary activity.</a> Acta Oto-Laryngologica. 79 (1-2), 133-139 (1975).</li><li>Sutto, Z., Conner, G. E., Salathe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+human+airway+ciliary+beat+frequency+by+intracellular+pH.">Regulation of human airway ciliary beat frequency by intracellular pH.</a> Journal of Physiology. 560, 519-532 (2004).</li><li>Salathe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+mammalian+ciliary+beating.">Regulation of mammalian ciliary beating.</a> Annual Review of Physiology. 69, 401-422 (2007).</li><li>Kempeneers, C., Seaton, C., Garcia Espinosa, B., Chilvers, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ciliary+functional+analysis:+Beating+a+path+towards+standardization.">Ciliary functional analysis: Beating a path towards standardization.</a> Pediatric Pulmonology. 54 (10), 1627-1638 (2019).</li><li>Kempeneers, C., Seaton, C., Chilvers, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+of+ciliary+beat+pattern+in+three+different+beating+planes+in+healthy+subjects.">Variation of ciliary beat pattern in three different beating planes in healthy subjects.</a> Chest. 151 (5), 993-1001 (2017).</li><li>Jackson, C. L., 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=Accuracy+of+diagnostic+testing+in+primary+ciliary+dyskinesia.">Accuracy of diagnostic testing in primary ciliary dyskinesia.</a> European Respiratory Journal. 47 (3), 837-848 (2016).</li><li>Feriani, L., 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=Assessing+the+collective+dynamics+of+motile+cilia+in+cultures+of+human+airway+cells+by+multiscale+DDM.">Assessing the collective dynamics of motile cilia in cultures of human airway cells by multiscale DDM.</a> Biophysical Journal. 113 (1), 109-119 (2017).</li><li>Brewington, J. J., 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=Brushed+nasal+epithelial+cells+are+a+surrogate+for+bronchial+epithelial+CFTR+studies.">Brushed nasal epithelial cells are a surrogate for bronchial epithelial CFTR studies.</a> JCI Insight. 3 (13), (2018).</li><li>Liu, X., 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=ROCK+inhibitor+and+feeder+cells+induce+the+conditional+reprogramming+of+epithelial+cells.">ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells.</a> The American Journal of Pathology. 180 (2), 599-607 (2012).</li><li>Suprynowicz, F. A., 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=Conditionally+reprogrammed+cells+represent+a+stem-like+state+of+adult+epithelial+cells.">Conditionally reprogrammed cells represent a stem-like state of adult epithelial cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (49), 20035-20040 (2012).</li><li>Martinovich, K. M., 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=Conditionally+reprogrammed+primary+airway+epithelial+cells+maintain+morphology,+lineage+and+disease+specific+functional+characteristics.">Conditionally reprogrammed primary airway epithelial cells maintain morphology, lineage and disease specific functional characteristics.</a> Scientific Reports. 7 (1), 17971 (2017).</li><li>Wong, J. Y., Rutman, A., O'Callaghan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+the+ciliated+epithelium+following+acute+bronchiolitis+in+infancy.">Recovery of the ciliated epithelium following acute bronchiolitis in infancy.</a> Thorax. 60 (7), 582-587 (2005).</li><li>Gentzsch, M., 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=Pharmacological+rescue+of+conditionally+reprogrammed+cystic+fibrosis+bronchial+epithelial+cells.">Pharmacological rescue of conditionally reprogrammed cystic fibrosis bronchial epithelial cells.</a> American Journal of Respiratory Cell and Molecular Biology. 56 (5), 568-574 (2017).</li><li>Sommer, J. U., Gross, S., Hormann, K., Stuck, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-dependent+changes+in+nasal+ciliary+beat+frequency.">Time-dependent changes in nasal ciliary beat frequency.</a> European Archives of Oto-Rhino-Laryngology. 267 (9), 1383-1387 (2010).</li><li>Ratjen, F., 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=Cystic+fibrosis.">Cystic fibrosis.</a> Nature Reviews Disease Primers. 1, 15010 (2015).</li><li>Delmotte, P., Sanderson, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ciliary+beat+frequency+is+maintained+at+a+maximal+rate+in+the+small+airways+of+mouse+lung+slices.">Ciliary beat frequency is maintained at a maximal rate in the small airways of mouse lung slices.</a> American Journal of Respiratory Cell and Molecular Biology. 35 (1), 110-117 (2006).</li><li>Smith, C. M., 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=Cooling+of+cilia+allows+functional+analysis+of+the+beat+pattern+for+diagnostic+testing.">Cooling of cilia allows functional analysis of the beat pattern for diagnostic testing.</a> Chest. 140 (1), 186-190 (2011).</li><li>Raidt, J., 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=Ciliary+beat+pattern+and+frequency+in+genetic+variants+of+primary+ciliary+dyskinesia.">Ciliary beat pattern and frequency in genetic variants of primary ciliary dyskinesia.</a> European Respiratory Journal. 44 (6), 1579-1588 (2014).</li></ol>

There are multiple factors that could obscure the quantification of CBF in nasal epithelial sheets. Epithelial sheets should be imaged within 3-9 hrs of sample collection since the ciliary function is most stable during this time37. Less red blood cells and debris are most optimal for imaging since these interfere with data acquisition. When selecting an ROI for imaging, it is important to select an epithelial sheet that edge has not been damaged or disrupted during the collection of the sample, a...

Measurements of Cilia Beat Frequency (CBF) and pattern have been established as diagnostic tools for respiratory diseases such as Primary Ciliary Dyskinesia (PCD)1. In Cystic Fibrosis (CF), dysfunction of the CF Transmembrane conductance Regulator (CFTR) chloride channel causes dehydration of the airway surface liquid and impaired mucociliary clearance2. Ciliary function has been investigated in vitro in primary airway cell models as an indicator of CFTR channel activity3. However, considerable patient-to-patient variability exists in CBF in response to CFTR-modulating drugs, even for patients with the same CFTR mutations3. 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 CBF.
Nasal epithelial sheets isolated from nasal mucosal brushings are directly used for measurements of ciliary function for PCD diagnosis4. Yet, while there is no control over the size or quality of the nasal epithelial sheets obtained, CBF varies depending on whether it is measured on single cells or cell sheets and on epithelial sheet ciliated edges that are disrupted or undisrupted5. As such, secondary dyskinesias caused by damage to cells during the collection of nasal mucosal brushings may influence CBF. Primary cell culture of nasal epithelial cells and their differentiation at Air-Liquid Interface (ALI) or in three-dimensional basement membrane matrix into ciliated airway epithelial organoids give rise to cilia that are free from secondary dyskinesias4,6,7,8. Airway epithelial cells differentiated at ALI (henceforth termed ALI models) have been deemed an important secondary diagnostic aid that replicate the ciliary beat patterns and frequency of ex vivo nasal mucosal brushings6 and enable analysis of ciliary ultrastructure, beat pattern, and beat frequency while retaining patient-specific defects9. Yet, discrepancies exist in the methodologies used to create these pseudostratified, mucociliary differentiated cell models. Different culture expansion or differentiation protocols could induce distinct epithelial phenotypes (ciliated or secretory)10 and result in significant differences in CBF11. CBF has been quantified in nasal epithelial brushings4,6,12,13,14,15,16, airway epithelial organoids14,17,18 and ALI models3,4,6,13,19,20,21. Yet, amongst these protocols, there are large variabilities, and often many parameters are not controlled for. For example, in some studies, CBF is imaged in situ while the cells of the ALI model remain on the permeable support insert3,19,20,21, yet others scrape the cells from the permeable support insert and image them suspended in media4,6,13.
Furthermore, the wider application of techniques that measure ciliary function is limited by the extreme susceptibility of ciliary function to changes in environmental factors. Environmental factors such as temperature22, humidity23,24, and pH25,26 influence ciliary function and must be regulated to quantify CBF accurately. The various physiological parameters used across different laboratories and how they influence CBF has been reviewed previously27.
Various imaging technologies and approaches to CBF measurements are reported in the literature. For PCD diagnostics, video microscopy is used to measure ciliary function28,29. Recently, a video analysis algorithm based on differential dynamic microscopy was used to quantify both CBF and cilia coordination in airway epithelial cell ALI models3,30. This method enables the characterization of ciliary beating in airway epithelial cells in a fast and fully automated manner, without the need to segment or select regions. Various methods for imaging and quantification of CBF may add to the differences reported in CBF in the literature (Supplementary File 1).
A protocol from culture to quantification to streamline existing methods, standardization of culture conditions, and image acquisition, performed in strict environmentally controlled conditions, would enable consistent, reproducible quantification of CBF within and between individuals.
This protocol provides a complete description of the collection of epithelial cells, expansion and differentiation culture conditions, and quantification of CBF in three different airway epithelial cell model systems of nasal origin: 1) native epithelial sheets, 2) ALI models imaged on permeable support inserts and 3) Extracellular Matrix (ECM)-embedded three-dimensional organoids (Figure 1). Nasal epithelial cells obtained from nasal inferior turbinate brushings are used as representatives of the airway epithelium since they are an effective surrogate for bronchial epithelial cells31 while overcoming the invasive procedure associated with collecting bronchial brushings. The Conditional Reprogramming Cell (CRC) method is used to expand primary airway epithelial cells for the creation of ALI models&#160;and three-dimensional organoids. Conditional reprogramming of airway epithelial cells to a stem cell-like state is induced by co-culture with growth-arrested fibroblast feeder cell system and Rho-associated kinase (ROCK) inhibitor32. Importantly, the CRC method increases population doubling in airway epithelial cells while retaining their tissue-specific differentiation potential33,34. In all airway epithelial cell models, the ciliary function is captured in a temperature-controlled chamber using a high-speed video camera with standardized image acquisition settings. Custom-built scripts are employed for the quantification of CBF.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63090/63090fig01.jpg" /> 
Figure 1: Schematic of workflow. Following brushing the participants&#39; nasal inferior turbinate, airway epithelial cells are utilized in one of two ways. Either airway epithelial sheets are isolated, and cilia beat frequency is imaged immediately, or airway epithelial cells are expanded via the conditional reprogramming cell method. CRC-expanded airway epithelial cells are differentiated to establish airway epithelial cells at an air-liquid interface or airway epithelial organoid cultures. Imaging of ciliary beat frequency is acquired using a live-cell imaging microscope with a heating and humidity environmental chamber and a fast frame rate (&#62;100Hz) scientific camera. Data analysis is performed using custom-built scripts. <a href="https://www.jove.com/files/ftp_upload/63090/63090fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adenine</td>
			<td>Sigma-Aldrich</td>
			<td>A2786</td>
			<td>10 mg/mL</td>
		</tr>
		<tr>
			<td>Advanced DMEM/F-12</td>
			<td>Thermo Fisher Scientific</td>
			<td>12634-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Alanyl-glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>G8541</td>
			<td>200 mM</td>
		</tr>
		<tr>
			<td>Andor Zyla 4.2 sCMOS</td>
			<td>Oxford Instruments</td>
			<td></td>
			<td>Fast frame rate (&#62;100 Hz) scientific camera</td>
		</tr>
		<tr>
			<td>Bottle-top vacuum filter system</td>
			<td>Sigma-Aldrich</td>
			<td>CLS431098</td>
			<td></td>
		</tr>
		<tr>
			<td>Ceftazidime hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>A6987</td>
			<td>50 mg/mL</td>
		</tr>
		<tr>
			<td>Cell Culture Microscope</td>
			<td>Olympus</td>
			<td>CKX53</td>
			<td></td>
		</tr>
		<tr>
			<td>CFI S Plan Fluor ELWD 20XC</td>
			<td>Nikon Instruments Inc.</td>
			<td>MRH08230</td>
			<td>Long working distance objective lens. NA0.45 WD 8.2-6.9</td>
		</tr>
		<tr>
			<td>Cholera toxin</td>
			<td>Sigma-Aldrich</td>
			<td>C8052-1MG</td>
			<td>200 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Corning Gel Strainer 40 UM</td>
			<td>Sigma-Aldrich</td>
			<td>CLS431750</td>
			<td>Pore size 40 &#956;m</td>
		</tr>
		<tr>
			<td>Corning Matrigel Matrix (Phenol red-free)</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Extracellular matrix (ECM)</td>
		</tr>
		<tr>
			<td>Corning bottle-top vacuum filter system</td>
			<td>Sigma-Aldrich</td>
			<td>CLS431098</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning CoolCell LX Cell Freezing Container</td>
			<td>Sigma-Aldrich</td>
			<td>CLS432002</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Transwell polyester membrane cell culture inserts</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3470</td>
			<td>Permeable support inserts. 6.5 mm Transwell with 0.4 &#956;m pore polyester membrane insert.</td>
		</tr>
		<tr>
			<td>Countess Cell Counting Chamber Slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>C10228</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II Automated Cell Counter</td>
			<td>ThermoFisher Scientific</td>
			<td>AMQAX1000</td>
			<td>Automated cell counter</td>
		</tr>
		<tr>
			<td>Cytology brushes</td>
			<td>McFarlane Medical</td>
			<td>33009</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12-Ham</td>
			<td>Thermo Fisher Scientific</td>
			<td>11330032</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12-Ham</td>
			<td>Thermo Fisher Scientific</td>
			<td>11330032</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM-High Glucose</td>
			<td>Thermo Fisher Scientific</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8242;s Phosphate Buffered Saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Eclipse Ti2-E</td>
			<td>Nikon</td>
			<td></td>
			<td>Live-cell imaging microscope.</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, certified, heat inactivated, United States</td>
			<td>Thermo Fisher Scientific</td>
			<td>10082147</td>
			<td></td>
		</tr>
		<tr>
			<td>Fungizone (Amphotericin B)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15290018</td>
			<td>250 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Gentamicin solution</td>
			<td>Sigma-Aldrich</td>
			<td>G1397</td>
			<td>50 mg/mL</td>
		</tr>
		<tr>
			<td>Graphpad Prism</td>
			<td>Graphpad</td>
			<td></td>
			<td>Scientific analysis software</td>
		</tr>
		<tr>
			<td>Greiner Cryo.s vials</td>
			<td>Sigma-Aldrich</td>
			<td>V3135</td>
			<td>Cryogenic vials</td>
		</tr>
		<tr>
			<td>HEPES solution</td>
			<td>Sigma-Aldrich</td>
			<td>H0887</td>
			<td>1 M</td>
		</tr>
		<tr>
			<td>HI-FBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>10082-147</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma-Aldrich</td>
			<td>H0888</td>
			<td>3.6 mg/mL</td>
		</tr>
		<tr>
			<td>Incubator NL Ti2 BLACK 2000</td>
			<td>PeCon</td>
			<td></td>
			<td>Microscope environmental chamber. Allows warm air incubation and local CO2 and O2 gassing</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I2643</td>
			<td>2 mg/mL</td>
		</tr>
		<tr>
			<td>Lab Armor 74220 706 Waterless Bead Bath 6L</td>
			<td>John Morris Group</td>
			<td>74220 706</td>
			<td>Bead bath</td>
		</tr>
		<tr>
			<td>Lab Armor Beads</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1254302</td>
			<td>Thermal beads</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td></td>
			<td>Computing software</td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microscoft</td>
			<td></td>
			<td>Spreadsheet software</td>
		</tr>
		<tr>
			<td>NIH/3T3</td>
			<td>American Type Culture Collection</td>
			<td>CRL-1658</td>
			<td>Irradiated NIH-3T3 mouse embryonic feeder cells</td>
		</tr>
		<tr>
			<td>NIS-Elements AR</td>
			<td>Nikon Instruments Inc.</td>
			<td></td>
			<td>Image acquisition software</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td>10,000 units penicillin and 10&#160;mg streptomycin/mL</td>
		</tr>
		<tr>
			<td>Dulbecco&#8242;s Phosphate Buffered Saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>PneumaCult Airway Organoid Kit</td>
			<td>StemCell Technologies</td>
			<td>5060</td>
			<td>Airway Organoid Kit</td>
		</tr>
		<tr>
			<td>PneumaCult-ALI Medium</td>
			<td>StemCell Technologies</td>
			<td>5001</td>
			<td></td>
		</tr>
		<tr>
			<td>PneumaCult-Ex Plus Medium</td>
			<td>StemCell Technologies</td>
			<td>5040</td>
			<td></td>
		</tr>
		<tr>
			<td>PureCol-S</td>
			<td>Advanced BioMatrix</td>
			<td>5015</td>
			<td>Type I Collagen solution</td>
		</tr>
		<tr>
			<td>ReagentPack Subculture Reagents</td>
			<td>Lonza</td>
			<td>CC-5034</td>
			<td></td>
		</tr>
		<tr>
			<td>rhEGF (Epidermal Growth Factor, human)</td>
			<td>Sigma-Aldrich</td>
			<td>E9644</td>
			<td>25 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Y-27632 2HCl (ROCK inhibitor)</td>
			<td>Selleckchem</td>
			<td>S1049</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Tobramycin</td>
			<td>Sigma-Aldrich</td>
			<td>T4014</td>
			<td>100 mg/mL</td>
		</tr>
		<tr>
			<td>Trypan blue solution</td>
			<td>Sigma-Aldrich</td>
			<td>T8154</td>
			<td>0.4%</td>
		</tr>
		<tr>
			<td>UNO Stage Top Incubator</td>
			<td>Okolab</td>
			<td></td>
			<td>Microscope incubator. Allows temperature, humidity and CO2 conditioning</td>
		</tr>
	</tbody>
</table>

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.

To demonstrate the efficiency of this protocol in quantifying CBF, the results of CBF measured in airway epithelial cell ALI models derived from three participants with CF and three healthy control participants are presented. On Day 14 of culture differentiation, beating cilia were present (Figure 6). From Day 14 to 21 of culture differentiation, a statistically significant (P &#60; 0.0345) increase in CBF was observed within both cohorts. On Day 21 of culture differentiation, the mean CBF f...

Watch this Scientific Journal Video about Collection, Expansion, and Differentiation of Primary Human Nasal Epithelial Cell Models for Quantification of Cilia Beat Frequency at JoVE.com

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

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 ...

We describe the collection of airway stem cells from nasal mucosa. These cells are expanded and differentiated into a pseudostratified epithelium in the lab. This is in vitro expansion and differentiation, which is similar to our airways in vivo, which means that there are specialized cells, such as ciliated cells, present that can be used for quantifying cilia beat frequency.To enable consistent and reproducible quantification of cilia beat frequency between different individuals and in response to different drugs, we standardize culture conditions and image acquisition. Cilia beat frequency measurements are used as clinical tools in disease such as primary ciliary dyskinesia. We hope to establish this as a clinical marker for cystic fibrosis.Demonstrating the procedure is Katelin Allan and Laura Fawcett, PhD students from my lab, and Dr.Sharon Wong, a postdoc from my laboratory. Begin by asking the participant to breathe through their mouth. Then taking a cytology brush in the dominant hand and resting the fifth digit on the participant's chin to anchor the hand, insert the cytology brush into the participant's nasal passage at a 45-degree angle to the participant's face, and pass it through the nasal meatus.After pivoting the brush upright, so that it is perpendicular to the participant's face, advance the brush gently, but firmly, against the lateral wall of the nose beneath the inferior turbinate until it is at the mid to posterior part of the inferior turbinate. Rotate the brush 360 degrees up to three times. Then remove it gently, reversing the insertion maneuver, so that the cells are not dislodged from the brush.Place the brush into the prepared collection tube with nasal cell collection media, and place the collection tube on ice. After gentle vortexing to dislodge cells from the brush, transfer the collection tube on ice back to the biosafety cabinet. Using a serological pipette, transfer the media from the collection tube to a new tube, leaving behind the cytology brushes.Then centrifuge the sample. After centrifugation, resuspend the cell pellet in one milliliter of conditional reprogramming cell media. Then using a five-milliliter serological pipette, pass the cells through a cell sieve placed on top of a 50-milliliter tube in a circular motion.To obtain a single cell suspension, collect the suspension from the bottom of the tube, and pass it through the sieve multiple times. Then discard the cell sieve. To seed the airway epithelial cells onto the prepared permeable support inserts, mix the cells well without creating bubbles, ensuring that they are homogenous and in suspension.Then add 150 microliters of the cell suspension to the apical side of the prepared permeable support inserts. To differentiate the airway epithelial cells at the air-liquid interface, culture them in differentiation, or ALI media for two days. Then aspirate the media, and add 750 microliters of ALI media only to the basal compartment to create an air-liquid interface.To seed the airway epithelial cells in ECM domes, resuspend dissociated airway epithelial cells with the appropriate volume of 90%ECM. Then holding the pipette vertically at a 90-degree angle as close to the bottom of the well as possible, dispense 50 microliters of the ECM cell suspension to the center of the well. Incubate the plate at 37 degrees Celsius for 20 minutes until the ECM solidifies.While the ECM is solidifying, warm the Airway Organoid Seeding Media, or AOSM, to room temperature to prevent it from causing re-liquification, and disintegration of the ECM dome upon addition. After the incubation, add 500 microliters of warmed AOSM to each well by dispensing down the wall of the well. Do not pipette media directly onto the ECM dome.Change the media every two days for four to seven days. To aspirate the media, tilt the plate at a 45-degree angle and aspirate from the bottom edge of the well away from the ECM dome. After four to seven days, initiate organoid differentiation by adding 500 microliters of warmed Airway Organoid Differentiation Media, or AODM, to each well, and change media every two days for seven days.Place the culture plate containing the airway epithelial cell models into the microscope plate insert, and close the microscope environmental chamber. Allow the sample to equilibrate in the pre-warmed, 37 degrees Celsius, 5%carbon dioxide-filled microscope chamber for 30 minutes. At the microscope eyepiece, focus on the cell model.Then using the acquisition software, click on L100 to switch the light path to the port where the camera is mounted. Click the green Play button to visualize the microscope field of view via the software. Check that the cilia are in focus, and adjust if required.To acquire time-lapse images from the menu, click on Acquire, and then on Fast TimeLapse. In the pop-up window, select a save location, and file name, acquire 1, 000 frames. To preview the cilia in the microscope field of view, click on Apply, followed by the green Play button.Adjust the Z focus if required. Then click Run now to capture the Fast TimeLapse. After installing the necessary software and toolboxes, Copy the appropriate custom analysis scripts and the support scripts folder to the local drive of the computer.Next, upon the computing software, click on the Home tab, followed by Set Path. In the pop-up window, click on Add with Subfolders. Under the MATLAB search path, select the folder shown.Then click on Save and Close. Confirm that the analysis scripts are linked to the computing software by checking that they appear in the left-hand panel. Next, transfer the example raw image files acquired to the computer's local drive.Then click on the BeatingCiliaBatchOMEfiles_jove. m analysis script file in the computing software. To run the script, click on the Editor tab, followed by the green Play button.In the prompt window that appears, select the raw image files to be analyzed. Enter the exposure time for acquisition time per frame. Then click on OK.Run the GetFirstAmplitude.m script on the folder that contains the AveSpectrum files. Wait for the script to output the FirstAmplitudeStacked. xlsx file, which contains the highest amplitude frequency, and is within the physiological range of airway epithelial cilia beating.Copy the frequency values from the FirstAmplitudeStacked. xlsx file, and plot them using scientific analysis software. The results of cilia beat frequency measured in airway epithelial cell ALI models derived from three participants with cystic fibrosis and three healthy control participants are presented.On day 14 of culture differentiation, beating cilia were present. From day 14, day 21 of culture differentiation, a statistically significant increase in cilia beat frequency was not observed between cohorts. On day 21 of culture differentiation, the mean cilia beat frequency for healthy control participants was significantly higher than that of participants with cystic fibrosis.Further, when cilia beat frequency was imaged in the same cell models following the removal of mucus there was a statistically significant increase in cilia beat frequency when mucus was removed in ALI models of both healthy individuals and those with cystic fibrosis. The imaging environment must be strictly regulated, since cilia function is extremely susceptible to changes in environmental factors. This platform has the potential to be established for patient-specific drug screening to be able to identify drugs that modulate CFTR function.

collection-expansion-differentiation-primary-human-nasal-epithelial

Research

JoVE Journal

Medicine

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Primary Culture of Human Vestibular Schwannomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.

Vestibular Schwannomas (VSs) are non-malignant tumors of Schwann cell (SC) origin, associated with mutations in the NF2 tumor suppressor gene. We report a reproducible, efficient protocol for primary human VS cell culture that allows for molecular and cellular experimental manipulation and analysis and recapitulates the heterogeneous nature of human disease.

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

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 ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the human respiratory tract. NA is a non-invasive technique which absorbs fluid from the inferior turbinate, and causes minimal discomfort. NA has yielded reproducible results with the ability to frequently repeat sampling of the upper airway. 
By comparison, alternative methods of sampling the respiratory mucosa, such as nasopharyngeal aspiration (NPA) and conventional swabbing, are more invasive and may result in greater data variability. Other methods have limitations, for instance, biopsies and bronchial procedures are invasive, sputum contains many dead and dying cells and requires liquefaction, exhaled breath condensate (EBC) contains water and saliva, and lavage samples are dilute and variable.
BA can be performed through the working channel of a bronchoscope in clinic. Sampling is well tolerated and can be conducted at multiple sites in the airway. BA results in MLF samples being less dilute than bronchoalveolar lavage (BAL) samples.
This article demonstrates the techniques of NA and BA, as well as the laboratory processing of the resulting samples, which can be tailored to the desired downstream biomarker being measured. These absorption techniques are useful alternatives to the conventional sampling techniques used in clinical respiratory research.

This manuscript describes the use of nasal and bronchial absorption techniques, specifically using synthetic absorptive matrices (SAM) to sample the mucosal lining fluid (MLF) of the upper and lower airway. These methods provide better standardization and tolerability than existing respiratory sampling techniques.

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the ...

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

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

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

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

Nanoscopic Imaging of Human Tissue Sections via Physical and Isotropic Expansion

In modern pathology, optical microscopy plays an important role in disease diagnosis by revealing microscopic structures of clinical specimens. However, the fundamental physical diffraction limit prevents interrogation of nanoscale anatomy and subtle pathological changes when using conventional optical imaging approaches. Here, we describe a simple and inexpensive protocol, called expansion pathology (ExPath), for nanoscale optical imaging of common types of clinical primary tissue specimens, including both fixed-frozen or formalin-fixed paraffin embedded (FFPE) tissue sections. This method circumvents the optical diffraction limit by chemically transforming the tissue samples into tissue-hydrogel hybrid and physically expanding them isotropically across multiple scales in pure water. Due to expansion, previously unresolvable molecules are separated and thus can be observed using a conventional optical microscope.

Nanoscale imaging of clinical tissue samples can improve understanding of disease pathogenesis. Expansion pathology (ExPath) is a version of expansion microscopy (ExM), modified for compatibility with standard clinical tissue samples, to explore the nanoscale configuration of biomolecules using conventional diffraction limited microscopes.

In modern pathology, optical microscopy plays an important role in disease diagnosis by revealing microscopic structures of clinical specimens. However, ...

Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately understood, thus impeding the establishment of defined treatments. Cell-based additive gene therapies for the increased expression of a protective factor are considered as a promising option to medicate neurodegenerative diseases, such as age-related macular degeneration (AMD). We have developed a method for the stable expression of the gene encoding pigment epithelium-derived factor (PEDF), which is characterized as a neuroprotective and anti-angiogenic protein in the nervous system, into the genome of primary human pigment epithelial (PE) cells using the Sleeping Beauty (SB) transposon system. Primary PE cells were isolated from human donor eyes and maintained in culture. After reaching confluence, 1 x 104 cells were suspended in 11 &#181;L of resuspension buffer and combined with 2 &#181;L of a purified solution containing 30 ng of hyperactive SB (SB100X) transposase plasmid and 470 ng of PEDF transposon plasmid. Genetic modification was carried out with a capillary electroporation system using the following parameters: two pulses with a voltage of 1,100 V and a width of 20 ms. Transfected cells were transferred into culture plates containing medium supplemented with fetal bovine serum; antibiotics and antimycotics were added with the first medium exchange. Successful transfection was demonstrated in independently performed experiments. Quantitative polymerase chain reaction (qPCR) showed the increased expression of the PEDF transgene. PEDF secretion was significantly elevated and remained stable, as evaluated by immunoblotting, and quantified by enzyme-linked immunosorbent assay (ELISA). SB100X-mediated transfer allowed for a stable PEDF gene integration into the genome of PE cells and ensured the continuous secretion of PEDF, which is critical for the development of a cell-based gene addition therapy to treat AMD or other retinal degenerative diseases. Moreover, analysis of the integration profile of the PEDF transposon into human PE cells indicated an almost random genomic distribution.

We have developed a protocol to transfect primary human pigment epithelial cells by electroporation with the gene encoding pigment epithelium-derived factor (PEDF) using the Sleeping Beauty (SB) transposon system. Successful transfection was demonstrated by quantitative polymerase chain reaction (qPCR), immunoblotting, and enzyme-linked immunosorbent assay (ELISA).