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

Dette arbeidet ble støttet av cystisk fibrose Foundation Therapeutics, gi nummer CLANCY14XX0 og gjennom cystisk fibrose Foundation, gi nummer CLANCY15R0. Forfatterne ønsker å takke Kristina Ray for henne hjelp i pasient rekruttering og regulatoriske forglemmelse. Forfatterne også ønsker å takke HNE working group, støttet av cystisk fibrose Foundation, som hjalp i generasjon av HNE kultur: Preston Bratcher, Calvin Cotton, Martina Gentzsch, Elizabeth Joseloff, Michael Myerburg, Dave Nichols, Scott Randell, Steve Rowe, G. Marty Salomo og Katherine Tuggle.

HNE prøver ble anskaffet fra fag rekruttert gjennom Cincinnati Children's Hospital Medical Center CF Research Center. Alle metodene som er beskrevet her er godkjent av institusjonelle Review Board (IRB) til Cincinnati Children's Hospital Medical Center. Skriftlig samtykke ble innhentet fra alle fag før testing.
1. klargjør utvidelse medier og antibiotika medier
<ol><li>Samle media-komponentene som er oppført i tabell 1. Tine frossen materialer ved romtemperatur og foreta...

<ol>
	<li>Rowe, S. M., Miller, S., Sorscher, E. J. <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> N Engl J Med. 352 (19), 1992-2001 (2005).</li><li>Rommens, J. 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=Identification+of+the+cystic+fibrosis+gene:+chromosome+walking+and+jumping.">Identification of the cystic fibrosis gene: chromosome walking and jumping.</a> Science. 245 (4922), 1059-1065 (1989).</li><li>Anderson, M. P., 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=Nucleoside+triphosphates+are+required+to+open+the+CFTR+chloride+channel.">Nucleoside triphosphates are required to open the CFTR chloride channel.</a> Cell. 67 (4), 775-784 (1991).</li><li>Boucher, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+airway+ion+transport.+Part+one.">Human airway ion transport. Part one.</a> Am J Respir Crit Care Med. 150 (1), 271-281 (1994).</li><li>Ehre, C., Ridley, C., Thornton, D. J. <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:+an+inherited+disease+affecting+mucin-producing+organs.">Cystic fibrosis: an inherited disease affecting mucin-producing organs.</a> Int J Biochem Cell Biol. 52, 136-145 (2014).</li><li>Accurso, F. 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=Effect+of+VX-770+in+persons+with+cystic+fibrosis+and+the+G551D-CFTR+mutation.">Effect of VX-770 in persons with cystic fibrosis and the G551D-CFTR mutation.</a> N Engl J Med. 363 (21), 1991-2003 (2010).</li><li>De Boeck, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+and+safety+of+ivacaftor+in+patients+with+cystic+fibrosis+and+a+non-G551D+gating+mutation.">Efficacy and safety of ivacaftor in patients with cystic fibrosis and a non-G551D gating mutation.</a> J Cyst Fibros. 13 (6), 674-680 (2014).</li><li>Moss, R. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+and+safety+of+ivacaftor+in+patients+with+cystic+fibrosis+who+have+an+Arg117His-CFTR+mutation:+a+double-blind,+randomised+controlled+trial.">Efficacy and safety of ivacaftor in patients with cystic fibrosis who have an Arg117His-CFTR mutation: a double-blind, randomised controlled trial.</a> Lancet Respir Med. 3 (7), 524-533 (2015).</li><li>Wainwright, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lumacaftor-Ivacaftor+in+Patients+with+Cystic+Fibrosis+Homozygous+for+Phe508del+CFTR.">Lumacaftor-Ivacaftor in Patients with Cystic Fibrosis Homozygous for Phe508del CFTR.</a> N Engl J Med. , (2015).</li><li>Brewington, J. J., McPhail, G. L., Clancy, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lumacaftor+alone+and+combined+with+ivacaftor:+preclinical+and+clinical+trial+experience+of+F508del+CFTR+correction.">Lumacaftor alone and combined with ivacaftor: preclinical and clinical trial experience of F508del CFTR correction.</a> Expert Rev Respir Med. 10 (1), 5-17 (2016).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> CFTR2 Database. , (2017).</li><li>Mou, H., Brazauskas, K., Rajagopal, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Personalized+medicine+for+cystic+fibrosis:+establishing+human+model+systems.">Personalized medicine for cystic fibrosis: establishing human model systems.</a> Pediatr Pulmonol. 50 Suppl 40, S14-S23 (2015).</li><li>Randell, S. H., Fulcher, M. L., O'Neal, W., Olsen, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+epithelial+cell+models+for+cystic+fibrosis+research.">Primary epithelial cell models for cystic fibrosis research.</a> Methods Mol Biol. 742, 285-310 (2011).</li><li>Whitcutt, M. J., Adler, K. B., Wu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biphasic+chamber+system+for+maintaining+polarity+of+differentiation+of+cultured+respiratory+tract+epithelial+cells.">A biphasic chamber system for maintaining polarity of differentiation of cultured respiratory tract epithelial cells.</a> In Vitro Cell Dev Biol. 24 (5), 420-428 (1988).</li><li>Worthington, E. N., Tarran, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+ASL+measurements+and+mucus+transport+rates+in+cell+cultures.">Methods for ASL measurements and mucus transport rates in cell cultures.</a> Methods Mol Biol. 742, 77-92 (2011).</li><li>Van Goor, 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=Correction+of+the+F508del-CFTR+protein+processing+defect+in+vitro+by+the+investigational+drug+VX-809.">Correction of the F508del-CFTR protein processing defect in vitro by the investigational drug VX-809.</a> Proc Natl Acad Sci U S A. 108 (46), 18843-18848 (2011).</li><li>Hug, M. J., Tummler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+current+measurements+to+diagnose+cystic+fibrosis.">Intestinal current measurements to diagnose cystic fibrosis.</a> J Cyst Fibros. 3 Suppl 2, 157-158 (2004).</li><li>van Barneveld, A., Stanke, F., Ballmann, M., Naim, H. Y., Tummler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+biochemical+analysis+of+CFTR+in+human+rectal+biopsies.">Ex vivo biochemical analysis of CFTR in human rectal biopsies.</a> Biochim Biophys Acta. 1762 (4), 393-397 (2006).</li><li>Dekkers, J. 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=A+functional+CFTR+assay+using+primary+cystic+fibrosis+intestinal+organoids.">A functional CFTR assay using primary cystic fibrosis intestinal organoids.</a> Nat Med. 19 (7), 939-945 (2013).</li><li>Dekkers, J. 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=Optimal+correction+of+distinct+CFTR+folding+mutants+in+rectal+cystic+fibrosis+organoids.">Optimal correction of distinct CFTR folding mutants in rectal cystic fibrosis organoids.</a> Eur Respir J. 48 (2), 451-458 (2016).</li><li>Dekkers, R., 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+bioassay+using+intestinal+organoids+to+measure+CFTR+modulators+in+human+plasma.">A bioassay using intestinal organoids to measure CFTR modulators in human plasma.</a> J Cyst Fibros. 14 (2), 178-181 (2015).</li><li>Zomer-van Ommen, D. D., 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=Limited+premature+termination+codon+suppression+by+read-through+agents+in+cystic+fibrosis+intestinal+organoids.">Limited premature termination codon suppression by read-through agents in cystic fibrosis intestinal organoids.</a> J Cyst Fibros. , (2015).</li><li>Bridges, M. A., Walker, D. C., Davidson, A. G. <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+and+control+nasal+epithelial+cells+harvested+by+a+brushing+procedure.">Cystic fibrosis and control nasal epithelial cells harvested by a brushing procedure.</a> In Vitro Cell Dev Biol. 27a (9), 684-686 (1991).</li><li>Di Lullo, A. 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=An+&amp;#34;ex+vivo+model&amp;#34;+contributing+to+the+diagnosis+and+evaluation+of+new+drugs+in+cystic+fibrosis.">An &amp;#34;ex vivo model&amp;#34; contributing to the diagnosis and evaluation of new drugs in cystic fibrosis.</a> Acta Otorhinolaryngol Ital. 37 (3), 207-213 (2017).</li><li>Pranke, I. 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=Correction+of+CFTR+function+in+nasal+epithelial+cells+from+cystic+fibrosis+patients+predicts+improvement+of+respiratory+function+by+CFTR+modulators.">Correction of CFTR function in nasal epithelial cells from cystic fibrosis patients predicts improvement of respiratory function by CFTR modulators.</a> Sci Rep. 7 (1), 7375 (2017).</li><li>Devalia, J. L., Davies, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+nasal+and+bronchial+epithelial+cells+in+culture:+an+overview+of+their+characteristics+and+function.">Human nasal and bronchial epithelial cells in culture: an overview of their characteristics and function.</a> Allergy Proc. 12 (2), 71-79 (1991).</li><li>Devalia, J. L., Sapsford, R. J., Wells, C. W., Richman, P., Davies, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+and+comparison+of+human+bronchial+and+nasal+epithelial+cells+in+vitro.">Culture and comparison of human bronchial and nasal epithelial cells in vitro.</a> Respir Med. 84 (4), 303-312 (1990).</li><li>Thavagnanam, S., 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=Nasal+epithelial+cells+can+act+as+a+physiological+surrogate+for+paediatric+asthma+studies.">Nasal epithelial cells can act as a physiological surrogate for paediatric asthma studies.</a> PLoS One. 9 (1), e85802 (2014).</li><li>Jorissen, M., Van der Schueren, B., Van den Berghe, H., Cassiman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+preservation+and+regeneration+of+cilia+on+human+nasal+epithelial+cells+cultured+in+vitro.">The preservation and regeneration of cilia on human nasal epithelial cells cultured in vitro.</a> Arch Otorhinolaryngol. 246 (5), 308-314 (1989).</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=Detection+of+CFTR+function+and+modulation+in+primary+human+nasal+cell+spheroids.">Detection of CFTR function and modulation in primary human nasal cell spheroids.</a> J Cyst Fibros. , (2017).</li><li>Sun, 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=Tgf-beta+downregulation+of+distinct+chloride+channels+in+cystic+fibrosis-affected+epithelia.">Tgf-beta downregulation of distinct chloride channels in cystic fibrosis-affected epithelia.</a> PLoS One. 9 (9), e106842 (2014).</li><li>Boucher, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+airway+surface+dehydration+as+the+initiating+event+in+CF+airway+disease.">Evidence for airway surface dehydration as the initiating event in CF airway disease.</a> J Intern Med. 261 (1), 5-16 (2007).</li></ol>

Denne protokollen beskriver generering av pasient-avledede nese formet cellekulturer kunne produsere en individualisert, bestemt modell av CFTR-funksjonen. Det er flere viktige trinn i prosessen som skal være tett deltok for å unngå problemer. Først er en god prøven oppkjøp fra pasientens nesen. Et godt eksempel bør ha > 50 000 celler, begrenset Slim/rusk, og være klar for behandling innen 4 timer (men suksess er også lett oppnåelig med overnatting shipping på is). Praksis å anskaffe prøver med curette eller...

Cystisk fibrose (CF) er en dødelig, autosomalt recessiv sykdom påvirker over 70.000 mennesker over hele verden1. Dette livet-forkorte genetisk sykdom er forårsaket av mutasjoner i den cystisk fibrose Transmembrane konduktans Regulator protein (CFTR)2. CFTR er medlem av adenosin trifosfat bindende kassett familien, og funksjoner som en ion kanal tillater bevegelse av klorid og bikarbonat over apikale membraner av flere polarisert epithelia inkludert mage-tarmkanalen, svette kjertel, og respiratoriske treet, blant annet3,4. Som sådan, fører dysfunksjonelle CFTR til multisystem epithelial dysfunksjon, med de fleste dødelighet stammer fra luftveissykdommer1. I CF lungene, tap av CFTR-drevet airway overflaten flytende (Oh) regulering og Slim release fører til tykkere slim, luftveisobstruksjon, kronisk infeksjon, og progressiv airway remodeling og tap av lunge funksjonen1,5.
Til tross for identifisering av CFTR dysfunksjon som forårsaker sykdom terapi i CF tradisjonelt fokusert på klimatiltak symptomer (f.eks, bukspyttkjertelen enzym erstatning terapi, airway klaring terapi)1. Denne tilnærmingen ble nylig revolusjonert av advent av romanen therapeutics, kalt "CFTR modulatorer," som direkte mot dysfunksjonelle CFTR. Denne tilnærmingen har flyttet klinisk landskapet fra symptom ledelse til endring av sykdom omsorg men bærer flere begrensninger6,7,8,9,10. Modulator aktivitet er spesifikke for protein feilen følger hvert CFTR mutasjon og begrenset å velge genetiske bestander11. Denne begrensningen er drevet av heterogene natur protein defekter, men også av upraktisk av kliniske studier i sjeldne mutasjon grupper. I tillegg emner med godt studert genotyper (f.eksF508del/F508del CFTR, den vanligste CFTR mutasjonen), er det bred variasjon i sykdom byrde og modulator svar6,7,8 ,9,11.
For å overvinne begge disse problemene, har etterforskere foreslått bruk av personlige modeller for prekliniske tester12. Dette konseptet benytter pasient-spesifikke vev for å generere en individualisert ex vivo modellsystem for sammensatte testing, forutsi i vivo emnet respons på behandling på en personlig måte. Når godkjent, kan slik modell brukes av klinikere å kjøre presisjon terapi uansett pasientens underliggende CFTR genotype.
Menneskelige bronkial (HBE) epitelceller innhentet fra explant vev ved lungetransplantasjon etablert muligheten for slik modell for CF13,14. HBEs dyrket ved et luft-flytende grensesnitt (ALI) tillate funksjonelle CFTR kvantifisering direkte gjennom electrophysiologic testing, eller indirekte gjennom tiltak ASL homeostase13,15. Denne modellen har vært avgjørende for å forstå CFTR biologi og var en sentral pådriver i utviklingen av CFTR modulatorer16. Dessverre er HBE modeller ikke troverdig som en personlig modell invasiv art på (lungetransplantasjon eller bronkial penselen) og mangel på prøver for de med sjeldne mutasjoner eller mild sykdom. Derimot kan tarmen tissue, fra endetarms eller duodenal biopsi prøver, brukes for intestinal gjeldende måling (ICM) eller en hevelse-baserte organoid analysen for å studere individualisert CFTR funksjonen17,18, 19. Organoid analyser, spesielt er svært følsomme modeller av CFTR aktivitet20,21,22. Begge modellene er begrenset av vev kilden (tarmen tissue, mens de fleste sykdom patologi er åndedretts) og semi invasiv metode for oppkjøpet. Eventuelt har flere etterforskere studert menneskelige nese (HNE) epitelceller modell CFTR restaurering23,24,25. HNEs kan høstes trygt med pensel eller utskrapning i fag i alle aldre og når kultivert i ALI, recapitulate mange egenskaper HBEs25,26,27,28. HNE monolayer kulturer har tradisjonelt vært begrenset av plateepitel transformasjon og lang tid forfall29. Dessuten rapporterte kortslutt gjeldende mål i HNEs er mindre enn de i HBEs, foreslå et mindre vindu å oppdage terapeutiske effekten25.
Gitt behovet for en personlig, ikke-invasiv, luftveier vev kultur modell av CFTR funksjonen, forsøkte vi å flette følsomheten til en hevelse-basert, organoid analysen med HNEs ikke-invasiv og respiratoriske natur. Her beskriver vi vår metode generere 3-dimensjonale "formet" kulturer av HNEs for individuell CFTR studier i en hevelse-baserte analysen30. HNE spheroids polarize pålitelig med epithelial apex mot sfære senter, eller lumen. Denne modellen viser mange kjennetegner en nedre luftveier epitel og modnes raskere enn ALI kulturer. Som en funksjonell analysen kvantifisere HNE spheroids pålitelig en rekkevidde av CFTR funksjonen, samt moduleringshjul i godt studert mutasjon grupper (f.eksF508del CFTR). Denne hevelse-baserte analysen kapitaliserer på ion/salt transport egenskapene for CFTR, indirekte måle væske tilstrømningen til formet som vann følger apikale salt middelklasseinnbyggere. På denne måten, stimulert spheroids med funksjonell CFTR svelle robust, mens de med dysfunksjonelle CFTR hovne mindre eller krymper. Dette er kvantifisert gjennom bildeanalyse av før og 1 time etter stimulering spheroids, måler luminal området og bestemme prosent endre. Dette tiltaket kan deretter sammenlignes over eksperimentell grupper til skjermen for narkotika bioactivity på en pasient-spesifikk måte.

<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

Mens innføring av cystisk fibrose Transmembrane konduktans Regulator (CFTR) modulator narkotika har revolusjonert omsorg i cystisk fibrose (CF), har genotype rettet terapi modellen er i bruk flere begrensninger. Første, sjeldne eller lite studert mutasjon grupper utelates fra definitive kliniske studier. Videre, som ekstra modulator narkotika inn markedet, vil det bli vanskelig å optimalisere modulator valgene for en enkelt emne. Begge disse problemene er adressert med bruk av pasient-avledede, individualisert prekliniske modellsystemer av CFTR funksjon og modulering. Menneskelige nese epitelceller (HNEs) er en lett tilgjengelig åndedretts vev for slik modell. Her beskriver vi generering av en tredimensjonal spheroid modell av CFTR funksjon og moduleringshjul bruker primære HNEs. HNEs er isolert fra fag i en minimalt invasiv måte, utvidet i betinget omprogrammering forhold, og sådd i spheroid kulturen. Innen 2 uker til såing, formet kulturer generere HNE spheroids som stimuleret med 3', 5'-sykliske adenosin monofosfat (cAMP)-generere agonister aktiverer CFTR-funksjonen. Spheroid hevelse er deretter kvantifisert som en proxy av CFTR aktivitet. HNE spheroids kapitalisere på minimal invasiv, men åndedretts opprinnelsen til nasal celler til å generere en tilgjengelig, personlige modell relevant for en epitel reflekterer sykdom sykelighet og dødelighet. Sammenlignet med luft-flytende grensesnitt HNE kulturer, er spheroids relativt rask modne, noe som reduserer den totale forurensning hastigheten. I sin nåværende form, er modellen begrenset av lav overføringshastighet, men dette er oppveid av relativt enkel vev oppkjøpet. HNE spheroids kan brukes til å pålitelig kvantifisere og karakterisere CFTR aktivitet på individnivå. En pågående studie å knytte denne kvantifisering i vivo narkotika svar vil avgjøre hvis HNE spheroids er en sann prekliniske prediktor for pasient svar CFTR modulering.

HNEs bør festes til kultur parabol og danner små øyene celler innen 72 timer av frø; Eksempler på god og dårlig øya formasjon på en uke er vist i figur 1A og 1B, henholdsvis. Disse øyene bør utvides til å dekke parabolen i løpet av 15-30 dager. Små eller suboptimal kan ta lengre tid, og ofte vil ikke gi nyttig spheroids. Forurensning med smittestoffer er dokumentert av dyp gul/skyet media, svikt i cellene knytte til kultur parabo...

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

Her beskriver vi en metode for å generere tredimensjonalt formet kulturer av menneskelig nese epitelceller. Spheroids deretter stimulert til stasjonen cystisk fibrose Transmembrane konduktans Regulator (CFTR)-avhengige ion og væske sekresjon, kvantifisere endringen i spheroid luminal størrelsen som en proxy for CFTR funksjon.

Generering av menneskelig nese tarmepitelet Spheroids for individualisert cystisk fibrose Transmembrane konduktans Regulator studie

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

Research

JoVE Journal

Medicine

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Eksperimentell Generation of Karsinom-Associated Fibroblaster (kafeer) fra Human mammary Fibroblaster

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

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

Menneskelige Nevroendokrine tumorcellelinjer som en tredimensjonal modell for studiet av menneskelig nevroendokrin svulst 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

Bruk av kontinuerlig datasporingsteknologi for å studere Exercise Etterlevelse i lungerehabilitering

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

Isolasjon, Kryopreservering og kultur menneskerettighets amnion Epitelceller for kliniske anvendelser

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

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Menneskelig Dupuytrens<em&gt; Ex Vivo</em&gt; Kultur for studier av myofibroblasts og ekstracellulære matriks Interactions

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from ...

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

En årehinnen Plexus epithelial Cell-basert modell av Human Blood-cerebrospinalvæsken Barrier å studere bakteriell infeksjon fra basolaterale 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 ...

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

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

Forskolin-indusert Hevelse i tarmen Organoids: An<em&gt; In Vitro</em&gt; Analyse for å vurdere Drug Response i pasienter med cystisk fibrose

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

Optimalisert LC--MS-/ MS metoden for høy gjennomstrømming analyse av klinisk prøver av Ivacaftor, store metabolitter og Lumacaftor i biologiske væsker Cystic Fibrosis pasienter

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)

Standardized Measurement of Nasal Membrane Transepithelial Potential Difference NPD

Standardisert måling av nese membran Transepithelial potensielle forskjellen (OD)

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

Generering av lukkede femur brudd i mus: en modell for å studere bein Healing

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