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

이 작품은 낭 성 섬유 증 재단 치료제에 의해 지원 되었다, CLANCY14XX0, 번호를 부여 하 고 낭 성 섬유 증 재단을 통해 CLANCY15R0 번호를 부여. 저자는 크리스티나 레이 환자 모집 및 규제 감독 그녀의 지원에 대 한 감사 하 고 싶습니다. 저자 또한 HNE 문화 능력의 세대에 지원 낭 성 섬유 증 재단에서 지 원하는 HNE 작업 그룹을 감사 하 고 싶다: 프레스 톤 Bratcher, 캘빈 면, 마르티나 Gentzsch, 엘리자베스 Joseloff, 마이클 Myerburg, 데이브 니콜스, 스콧 Randell, 스티브 로우, G. 마티 솔로몬, 그리고 캐서린 Tuggle

<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>

저자는 공개 없다.

이 프로토콜 환자 파생 코 셀 회전 타원 체 문화 CFTR 함수의 개별, 구체적인 모델을 생산할 수의 생성을 설명 합니다. 밀접 하 게 어려움을 피하기 위해 참석 해야 하는 과정에서 몇 가지 주요 단계가 있습니다. 먼저 환자의 코에서 좋은 샘플 인수가입니다. 좋은 샘플 있어야 > 50000 세포, 점액/파편, 제한 하 고 (비록 성공 또한 쉽게 달성로 얼음에 배송) 4 h 이내 처리에 대 한 준비. 연습 연구 직원을 ?...

낭 성 섬유 증 (CF) 이상 7 만명 세계적으로1에 영향을 미치는 치명적인, 상 염색체 열성 질병 이다. 이 수명 단축의 유전 질환 돌연변이 낭 성 섬유 증 막 횡단 전도성 레 귤 레이 터 단백질 (CFTR)2에 의해 발생 합니다. CFTR의 멤버인 아데노신 3 인산 염 바인딩 카세트 가족과 기능으로 위장, 땀 샘을 포함 하 여 여러 편광된 epithelia의 꼭대기 막 걸쳐 염화 중 탄산염의 움직임을 허용 하는 이온 채널 그리고 호흡 나무, 다른 사람의 사이에서,34. 따라서, 역 기능적인 CFTR multisystem 상피 부전, 호흡기 질환1에서 형태소 분석 대부분 사망률과 리드. CF 폐, CFTR 구동 기도 표면 액체 (ASL) 규정과 점액 릴리스 리드의 굵은 점액, 기도 방해, 만성 감염, 및 진보적인 기도 개장 손실 및 폐 기능1,5.
질병의 원인으로 CFTR 부전의 식별에 불구 하 고 CF에 치료 증상 (예, 췌 장 효소 대체 요법, 기도 클리어런스 치료)1의 완화에 전통적으로 집중 했다. 이 접근은 최근 "CFTR 변조기," 직접 역 기능적인 CFTR 대상 되 나 새로운 치료제의 출현에 의해 혁명을 일으켰다. 이 방법은 이동 했다 임상 가로 증상 관리에서 질병을 수정 상관 하지만 몇 가지 제한6,7,8,,910. 변조기 활동은 각 CFTR 돌연변이 동반 하는 단백질 결함을 구체적이 고 유전 인구11선택 제한. 이 제한은 단백질 결함의 이질적인 본질 뿐만 아니라 희귀 돌연변이 그룹에서 임상 시험의 비에 의해 구동 됩니다. 또한, 잘 공부 genotypes (예를 들어, F508del/F508del CFTR 가장 일반적인 CFTR 돌연변이), 과목 중 거기에 넓은 가변성 질병 부담 및 변조기 응답6,7,8 ,,911.
모두 이러한 문제를 극복 하기 위해 수 사관 전 임상 테스트12맞춤된 모델의 사용을 제안 했다. 이 개념 맞춤된 패션에서 치료 주제 응답 vivo에서 예측는 개별 비보 전 모델 시스템 복합 테스트 생성 하 환자 관련 조직을 활용 합니다. 일단 확인, 이러한 모델 임상 드라이브 정밀 치료 환자의 기본 CFTR 유전자 형에 의해 사용 될 수 있습니다.
폐 이식 시 explant 조직에서 얻은 인간 기관지 상피 (HBE) 셀 CF13,14에 대 한 이러한 모델의 가능성을 설립. 공기 액체 인터페이스 (알리)에서 성장 하는 HBEs 기능 CFTR 정량화 electrophysiologic 테스트를 통해 직접 또는 간접적으로 ASL 항상성13,15의 조치를 통해 하실 수 있습니다. 이 모델은 CFTR 생물학을 이해 하는 중요 한 되었고 CFTR 변조기16의 개발의 핵심 드라이버 했다. 불행히도, HBE 모델 희귀 돌연변이 또는 가벼운 질병에 대 한 맞춤된 모델 수집 (폐 이식 또는 기관지 솔 질)의 침략 적인 본질과 샘플의 부족으로 유지할 수 없습니다. 반대로, 장 조직, 직장 또는 십이지 장 생 검 표본에서 얻은를 사용할 수 있는 장 전류 측정 (ICM) 또는 붓기 기반 organoid 분석 결과 대 한 개별적인된 CFTR 함수17,18, 연구 19. Organoid 분석 실험, 특히 CFTR 활동20,,2122의 매우 중요 한 모델 이다. 두 모델은 조직 소스 (장 조직, 대부분 질병 병 리는 호흡) 및 수집의 반 침략 적 방법에 의해 제한 됩니다. 또는, 여러 조사 모델 CFTR 복원23,,2425인간의 비 강 상피 (HNE) 세포를 공부 했다. HNEs는 브러쉬 또는 소파 어떤 나이의 주제에 의해 안전 하 게 수확 하실 수 있습니다 그리고, 알리, 경작 하는 때 HBEs25,26,,2728의 많은 특성을 정리. HNE 단층 문화는 전통적으로 편평 변화와 성숙29에 오랜 시간에 의해 제한 되었습니다. 또한, 보고 단락 회로 전류 측정 HNEs에는 HBEs, 치료 효능25를 감지 하는 작은 창을 제안에 그 보다 작은.
CFTR 기능의 개인, 비-침략 적, 호흡기 조직 문화 모델에 대 한 필요성을 감안할 때, 우리 HNEs의 비-침략 적 및 호흡기 자연과 붓기-기반, organoid 분석 결과의 감도 병합 하고자 했다. 여기, 붓기 기반 분석 결과30에 개별된 CFTR 연구에 대 한 HNEs의 3 차원 "회전 타원 체" 문화를 생성 하는 우리의 방법을 설명 합니다. HNE spheroids 구 중심, 또는 루멘으로 상피 정점으로 안정적으로 분극. 이 모델은 낮은 호흡기 상피의 수많은 특성이 고 알리 문화 보다 더 빨리 성숙. 기능 시험으로 HNE spheroids 안정적으로 CFTR 범위 기능 뿐만 아니라, 잘 공부 돌연변이 그룹 (예를 들어, F508del CFTR)에 변조를 계량. 이 붓기 기반 분석 결과 직접 측정 하지 유체 유입은 회전 타원 체로 물 꼭대기 소금 경과 다음과 같이 CFTR의 이온/소금 전송 속성에 대문자로 바꿉니다. 이 패션에 완벽 하 게 기능적인 CFTR와 자극된 spheroids 팽창 견고 하 게, 그 역 기능 CFTR 덜 팽창 또는 수축 하는 동안. 이 사전 이미지 분석을 통해 정량은 고 1 시간 후 자극 spheroids, luminal 영역을 측정 하 고 결정 하는 백분율 변경 합니다. 이 측정 후 실험 그룹 환자 전용 패션에 약 bioactivity에 대 한 화면을 통해 비교할 수 있습니다.

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

generation of human nasal epithelial cell spheroids for individualized cystic fibrosis transmembrane conductance regulator study

낭 성 섬유 증 막 횡단 전도성 레 귤 레이 터 (CFTR) 변조기 약물의 도입 치료 낭 성 섬유 증 (CF)에 혁명 하고있다, 그러나 현재 사용 되 고 있는 유전자 이동 치료 모델은 몇 가지 제한이 있습니다. 첫 번째, 희귀 또는 understudied 돌연변이 그룹 확실 한 임상 시험에서 제외 됩니다. 또한, 추가 변조기 마약 시장에 진입, 그것은 변조기 선택 개별 주제에 대 한 최적화 하기가 될 것입니다. CFTR 기능 및 변조, 개별 환자 파생 전 임상 모델 시스템의 사용으로 이러한 문제를 모두 해결 합니다. 인간의 비 강 상피 세포 (HNEs)는 이러한 모델에 대 한 호흡기 조직의 쉽게 접근할 수 있는 소스입니다. 여기는 CFTR 기능과 기본 HNEs를 사용 하 여 변조의 3 차원 회전 타원 체 모델의 생성에 설명 합니다. HNEs 과목 조건부 재활 조건에서 확장 및 회전 타원 체 문화에 시드, 최소 침 습 방식 으로부터 격리 됩니다. 뿌리기의 2 주 안에 회전 타원 체 문화 HNE spheroids와 3', 5'-주기적인 아데노신 monophosphate (캠프) 자극 될 수 있는 생성-생성 촉진제 CFTR 기능을 활성화. 회전 타원 체 붓기는 CFTR 활동의 프록시 다음 정량 된다. HNE spheroids 질병 사망률 및 사망을 반영 하는 상피에 관련 된 접근, 맞춤 모델을 생성 하 비 강 세포의 침 습, 아직 호흡기 근원에 대문자로 표기 합니다. 공기 액체 인터페이스 HNE 문화에 비해, spheroids는 상대적으로 빨리 성숙, 전체 오염 속도 감소 시키는 있습니다. 그것의 현재 모양에서 모델이 조직 취득 상대적으로 완화에 의해 상쇄 됩니다 비록 낮은 처리량에 의해 제한 됩니다. Spheroids HNE 안정적으로 계량 CFTR 활동 개별 수준에서 특성을 사용할 수 있습니다. Vivo에서 마약 응답을이 정량화를 넥타이 하는 지속적인 연구 HNE spheroids CFTR 변조에 환자의 응답의 진정한 전 임상 예언자 인지 결정 됩니다.

HNEs 문화 접시를 연결 하 고 시드;의 72 h 내의 셀의 작은 섬 형성 한다 1 주일에 좋은 고 가난한 섬 형성의 예는 각각 그림 1A 및 1B에 표시 됩니다. 이 섬 15-30 일의 과정을 통해 요리를 확장 해야 합니다. 작은 또는 차선 샘플 더 오래 걸릴 수 있습니다 및 종종 유용한 spheroids를 생성 하지 것입니다. 전염 성 요원으로 오염 깊은 노란색/흐린 ?...

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

여기 우리 인간의 비 강 상피 세포의 3 차원 회전 타원 체 문화를 생성 하는 방법을 설명 합니다. Spheroids는 다음 드라이브 낭 성 섬유 증 막 횡단 전도성 레 귤 레이 터 (CFTR) 자극-종속 이온 및 액체 분 비, CFTR 함수에 대 한 프록시로 회전 타원 체 luminal 크기의 변화를 측정.

개별된 낭 성 섬유 증 막 횡단 전도성 레 귤 레이 터 연구에 대 한 인간의 비 강 상피 세포 Spheroids의 생성

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

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

Research

JoVE Journal

Medicine

10.6K 조회수.  Cincinnati Children's Hospital Medical Center. 여기 우리 인간의 비 강 상피 세포의 3 차원 회전 타원 체 문화를 생성 하는 방법을 설명 합니다. Spheroids는 다음 드라이브 낭 성 섬유 증 막 횡단 전도성 레 귤 레이 터 (CFTR) 자극-종속 이온 및 액체 분 비, CFTR 함수에 대 한 프록시로 회전 타원 체 luminal 크기의 변화를 측정.

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

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

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

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

어린이에서 차 비강 상피 세포를 배양 및 유도 다 능성 줄기 세포로 재 프로그램

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

연구

발생학

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

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

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

인간 Neuroendocrine 종양 치료의 연구를위한 3 차원 모델로 인간 Neuroendocrine 종양 세포 라인

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 ...

의학

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

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

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

폐 재활 운동 준수를 연구하는 지속적인 데이터 추적 기술을 사용하여

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

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

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

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

인간 다능성 줄기 세포로부터의 기도 상피 세포 공기-액체 계면 배양물의 생성

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

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

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

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

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

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

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

에어 리퀴드 인터페이스에서 인간의 비강 상피 세포의 배양

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

생물학

Culture and Imaging of Human Nasal Epithelial Organoids

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

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

인간 비강 상피 오가노이드의 문화와 이미징

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

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

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

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

섬모 박동 빈도의 정량화를 위한 1차 인간 비강 상피 세포 모델의 수집, 확장 및 분화

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

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

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

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

제노푸스 배아 세포에서 점동 상피 오르가노이드: 생성, 배양 및 고해상도 라이브 이미징

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

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

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

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

일차 인간 비강 상피 세포 : 정밀 의학의 맥락에서 바이오 뱅킹

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

개별된 낭 성 섬유 증 막 횡단 전도성 레 귤 레이 터 연구에 대 한 인간의 비 강 상피 세포 Spheroids의 생성

요약

더 많은 비디오 탐색

이 비디오의 챕터

어린이에서 차 비강 상피 세포를 배양 및 유도 다 능성 줄기 세포로 재 프로그램

인간 Neuroendocrine 종양 치료의 연구를위한 3 차원 모델로 인간 Neuroendocrine 종양 세포 라인

폐 재활 운동 준수를 연구하는 지속적인 데이터 추적 기술을 사용하여

인간 다능성 줄기 세포로부터의 기도 상피 세포 공기-액체 계면 배양물의 생성

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

에어 리퀴드 인터페이스에서 인간의 비강 상피 세포의 배양

인간 비강 상피 오가노이드의 문화와 이미징

섬모 박동 빈도의 정량화를 위한 1차 인간 비강 상피 세포 모델의 수집, 확장 및 분화

제노푸스 배아 세포에서 점동 상피 오르가노이드: 생성, 배양 및 고해상도 라이브 이미징

일차 인간 비강 상피 세포 : 정밀 의학의 맥락에서 바이오 뱅킹