この作品は、嚢胞性線維症財団治療によって支えられた、数 CLANCY14XX0 を付与し、嚢胞性線維症財団を通じて付与数 CLANCY15R0。患者の募集と規制監督で彼女の援助のクリスティーナ レイを感謝致します。また、著者は HNE ワーキング グループは、嚢胞性線維症財団、HNE 文化機能の世代の支援者に支えを感謝したい: プレストン Bratcher カルバン綿、マルティナ ・ Gentzsch、エリザベス Joseloff、マイケル ・ Myerburg、デイブ ・ ニコルズスコット ・ ランデル、スティーブ Rowe、G. マーティ ソロモン、キャサリン Tuggle。

<ol>
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著者が明らかに何もありません。

このプロトコルでは、患者由来鼻細胞スフェロイド培養「CFTR 機能個別の特定モデルを生成することができるの生成について説明します。難しさを避けるために密接に出席する必要がありますプロセスでいくつか重要な手順があります。まずは患者さんの鼻から良いサンプル集録です。良いサンプルがあるはず > 50,000 セルは、粘液/破片の限定および 4 時間以内 (ただし、成功は、一晩氷で送?...

嚢胞性線維症 (CF) は、約 7万人の世界1に影響を与える致命的な常染色体性劣性遺伝性疾患です。この人生短縮遺伝病は、嚢胞性線維症膜コンダクタンス レギュレータ蛋白質 (CFTR)2で突然変異によって引き起こされます。CFTR 胃腸、汗腺を含む複数の偏光上皮細胞の頂端膜を渡る塩化物、重炭酸塩の動きを許容するイオン チャネルとして機能や、アデノシン三リン酸結合カセット家族のメンバーであります。・呼吸器病3,4の木の他の中で。など、「CFTR 機能不全は呼吸器疾患1から生じるほとんどの死亡率と多臓器の上皮障害につながります。CFTR 駆動気道表面液体 (ASL) 規制と粘液のリリースにつながる厚く粘液、気道閉塞、慢性感染症、進行性気道リモデリングと肺機能1、5の損失は、CF の肺。
病気の原因として「CFTR 機能障害の識別、にもかかわらず CF 療法は、伝統的症状 (例えば、膵酵素補充療法、気道クリアランス療法)1軽減焦点を当てた。このアプローチは、最近「CFTR 変調」「CFTR 機能不全を直接対象すると呼ばれる新規の治療法の出現によって革命化されました。このアプローチは、疾患修飾に症状マネジメントから臨床の風景をシフトしている気が、いくつかの制限6,7,8,9,10を運ぶ。変調器の活動は各 CFTR の突然変異に伴うタンパク質の欠陥に特定、限られた遺伝的個体群11を選択します。この制限は、まれな突然変異グループでの臨床試験の実行によっても異種タンパク質の欠陥、性質によって駆動されます。さらに、よく研究遺伝子多型 (例えばF508del/F508del CFTR CFTR の最も一般的な突然変異)、被験者の間ではばらつきが大きく、疾病負担と変調器応答6,7,8 ,9,11。
両方のこれらの問題を克服するため、捜査官は前臨床試験12のパーソナライズされたモデルの使用を提案している.この概念は、パーソナライズされた方法で治療する生体内で件名応答予測複合テスト、個別前のヴィヴォモデル システムを生成する患者特有の組織を利用しています。されましたら、臨床医が患者の基になる CFTR 遺伝子型に関係なくドライブ精度療法はこのようなモデルを使用可能性があります。
人間の肺移植の時に植組織から得られた気管支の上皮 (HBE) 細胞は、CF13,14のためのようなモデルの可能性を設立しました。HBEs 気液界面 (アリ) で成長して電気生理学的検査から直接または間接的 ASL 恒常性13,15の措置を通じて機能の CFTR の定量化を可能にします。このモデルは CFTR の生物学を理解するための重要なている、CFTR 変調器16の開発の重要なドライバーだった。残念ながら、HBE モデルはこれらのまれな変異や軽度の病気 (肺移植または気管支擦過) 買収の侵襲的な性質とサンプルの不足のためのパーソナライズされたモデルとして理不尽ではありません。個別「CFTR 機能17,18,を勉強する腸内電流測定 (ICM) または腫れベースにおけるアッセイの腸組織、直腸または十二指腸生検標本から得られたを使用ことができます逆に、19. Organoid 試金、特に、CFTR 活動20,21,22の非常に機密性の高いモデルであります。両方のモデルは、組織ソース (腸組織、ほとんどの病は呼吸器) と半侵襲的買収法によって制限されます。また、いくつか調査官はモデル CFTR 復元23,24,25に人間の鼻粘膜上皮 (HNE) の細胞を研究しています。HNEs はブラシまたはあらゆる年齢層の被験者で掻爬によって安全に収穫できるし、アリで培養した場合 HBEs25,26,27,28の多くの特徴を要約するでしょう。HNE 単層培養は扁平上皮の変換と成熟度29に長い時間によって伝統的に制限されています。また、報告されて短絡 HNEs で電流の測定が治療効果の25を検出する小さいウィンドウを示唆している HBEs のそれらより小さい。
「CFTR 機能のパーソナライズされた、非侵襲的呼吸器の組織培養モデルの必要性を考える我々 は HNEs の非侵襲的・呼吸器病の自然と腫れベースにおけるアッセイの感度をマージしようとしました。ここでは、腫れを用いた測定30個別 CFTR の研究のための HNEs の 3 次元「回転楕円体」文化を生成する手法について述べる。HNE スフェロイド二極化確実に球の中心、または内腔に向かって上皮の頂点。このモデルは、下気道上皮の多数の特徴を繰り返す、アリの文化よりも早く成熟します。機能アッセイとして HNE 回転楕円体は確実に範囲の「CFTR 機能としてよく研究の突然変異のグループ (例えば、F508del CFTR) で変調を定量化します。この腫れベースの試金を直接測定されない流体流入回転楕円体に水が根尖部の塩排出を通り CFTR のイオン/塩の輸送特性における大文字します。この方法で機能的 CFTR の誘導回転楕円体膨らむ頑健、CFTR 機能不全とのそれら、うねりの少ない縮小中。これは事前のイメージ分析を通して定量化され、1 h 後刺激の回転楕円体、内腔領域を測定し、割合の決定を変更します。この測定は、患者特異的薬物活性のスクリーニング実験群間で比較できます。

<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

一方、嚢胞性線維症膜コンダクタンス レギュレータ (CFTR) 変調器薬の導入ケア嚢胞性線維症 (CF) に革命をもたらしました、現在使用している遺伝子型指向療法モデルはいくつかの制限を持っています。最初、稀なまたは流変異グループは、決定的な臨床試験から除外されます。また、追加の変調器薬市場に参入、個々 の主題の変調器の選択を最適化することは困難になります。両方のこれらの問題は「CFTR 機能変調の患者由来、個別の前臨床モデル システムを使って解決されます。ひと鼻粘膜上皮細胞 (HNEs) は、このようなモデルのための呼吸器組織の簡単にアクセス可能なソースです。ここで、「CFTR 機能と主な HNEs を用いた変調方式の三次元回転楕円体モデルの生成について述べる.HNEs は、低侵襲のファッション、条件付きのリプログラミング条件に拡大し、回転楕円体の文化にシードの被験者から分離されています。種まきの 2 週間以内に回転楕円体の文化生成 3', 5'-環状アデノシン一リン酸 (cAMP) を刺激することができます HNE スフェロイド-アゴニスト「CFTR 機能アクティブするを生成します。回転楕円体の腫れは CFTR の活性のプロキシとして、定量化されます。HNE 回転楕円体は、低侵襲、まだ呼吸疾患の罹患率と死亡率を反映した上皮に関連するアクセス可能なパーソナライズされたモデルを生成する鼻の細胞の起源を活用します。気液界面 HNE 文化に比べると、回転楕円体は、成熟する比較的簡単な汚染率を減らします。現在の形態でモデルは組織の買収の相対的な容易さは相殺されて、低スループットによって制限されます。確実に定量化し、個々 のレベルで「CFTR 活動を特徴付ける HNE 回転楕円体を使用できます。CFTR 変調に対する患者の反応の真の臨床予測かどうか HNE 回転楕円体は、生体内で薬剤応答にこの数量を結ぶ継続的な研究を決定します。

HNEs が培養皿に添付し、細胞播種; の 72 時間以内の小さな島々 を形成それぞれ図 1 a 1 b、1 週間で良いと貧しい島形成の例のとおりです。これらの島々 を 15-30 日のコース料理をカバーするため展開する必要があります。小規模または次善のサンプルは、かかることがあり、しばしば便利な回転楕円体は得られません。感染性病原体?...

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

ここで人間の鼻粘膜上皮細胞の三次元回転楕円体文化を生成する手法について述べる。回転楕円体は、嚢胞性線維症膜コンダクタンス レギュレータ (CFTR) ドライブに刺激される-依存イオンと液分泌、「CFTR 機能のプロキシとして回転楕円体内腔サイズ変化を定量化します。

個別の嚢胞性線維症膜コンダクタンス レギュレータ研究のひと鼻腔上皮細胞スフェロイドの生成

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 ビュー.  Cincinnati Children's Hospital Medical Center. ここで人間の鼻粘膜上皮細胞の三次元回転楕円体文化を生成する手法について述べる。回転楕円体は、嚢胞性線維症膜コンダクタンス レギュレータ (CFTR) ドライブに刺激される-依存イオンと液分泌、「CFTR 機能のプロキシとして回転楕円体内腔サイズ変化を定量化します。

ビデオ: 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).

子供からプライマリ鼻上皮細胞を培養し、人工多能性幹細胞に再プログラム

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 ...

発生生物学

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.

人間の神経内分泌腫瘍療法の研究のための三次元モデルとしての人間の神経内分泌腫瘍細胞株

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 ...

医学

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.

呼吸リハビリテーションに運動アドヒアランスを研究する継続的なデータ追跡技術を使用して

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.

ヒト多能性幹細胞からの気道上皮細胞気液界面培養物の生成

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.

フォルスコリン誘発性腸オルガノイドに腫れ：<em&gt;インビトロ</em&gt;アッセイ嚢胞性線維症患者における薬物反応を評価するための

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.

エア·液界面での人間の鼻上皮細胞の培養

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

生物学

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.

ヒト鼻上皮オルガノイドの培養とイメージング

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.

繊毛拍動頻度の定量化のための初代ヒト鼻上皮細胞モデルの収集、拡大、および分化

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.

ゼノプス胚細胞由来粘膜上皮上オルガノイド:生成、培養、高解像度ライブイメージング

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.

初代ヒト鼻上皮細胞:精密医療の文脈におけるバイオバンキング

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

個別の嚢胞性線維症膜コンダクタンスレギュレータ研究のひと鼻腔上皮細胞スフェロイドの生成

要約

さらに動画を探す

この動画の章

子供からプライマリ鼻上皮細胞を培養し、人工多能性幹細胞に再プログラム

人間の神経内分泌腫瘍療法の研究のための三次元モデルとしての人間の神経内分泌腫瘍細胞株

呼吸リハビリテーションに運動アドヒアランスを研究する継続的なデータ追跡技術を使用して

ヒト多能性幹細胞からの気道上皮細胞気液界面培養物の生成

フォルスコリン誘発性腸オルガノイドに腫れ：

エア·液界面での人間の鼻上皮細胞の培養

ヒト鼻上皮オルガノイドの培養とイメージング

繊毛拍動頻度の定量化のための初代ヒト鼻上皮細胞モデルの収集、拡大、および分化

ゼノプス胚細胞由来粘膜上皮上オルガノイド:生成、培養、高解像度ライブイメージング

初代ヒト鼻上皮細胞:精密医療の文脈におけるバイオバンキング

個別の嚢胞性線維症膜コンダクタンス レギュレータ研究のひと鼻腔上皮細胞スフェロイドの生成

要約

さらに動画を探す

この動画の章

子供からプライマリ鼻上皮細胞を培養し、人工多能性幹細胞に再プログラム

人間の神経内分泌腫瘍療法の研究のための三次元モデルとしての人間の神経内分泌腫瘍細胞株

呼吸リハビリテーションに運動アドヒアランスを研究する継続的なデータ追跡技術を使用して

ヒト多能性幹細胞からの気道上皮細胞気液界面培養物の生成

フォルスコリン誘発性腸オルガノイドに腫れ：

エア·液界面での人間の鼻上皮細胞の培養

ヒト鼻上皮オルガノイドの培養とイメージング

繊毛拍動頻度の定量化のための初代ヒト鼻上皮細胞モデルの収集、拡大、および分化

ゼノプス胚細胞由来粘膜上皮上オルガノイド:生成、培養、高解像度ライブイメージング

初代ヒト鼻上皮細胞:精密医療の文脈におけるバイオバンキング

個別の嚢胞性線維症膜コンダクタンスレギュレータ研究のひと鼻腔上皮細胞スフェロイドの生成