Name: Vorming van menselijke thymusorganoïden in driedimensionale fibrinehydrogels (Video) | JoVE Journal
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Description: Leer hoe menselijke thymusorganoïden worden gevormd in driedimensionale fibrinehydrogels voor immunologisch onderzoek. Deze methode ondersteunt de rijping van thymusepitheelcellen en draagt bij aan betere technieken voor T-lymfocytproductie in vitro.

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<meta charset="utf8"></head><body>Het nabootsen van de thymus micro-omgeving met iPSC-afgeleide organoïden

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</ol>

<table><tbody><tr><td>Aprotinin</td><td>Sigma Aldrich</td><td>616370</td><td></td></tr><tr><td>BMP4</td><td>Miltenyi</td><td>130-111-165</td><td></td></tr><tr><td>CCR7 (CD197)</td><td>BD Biosciences</td><td>PE</td><td>Clone: 3D12; Dilutie: 1: 200</td></tr><tr><td>CD14</td><td>BD Biosciences</td><td>FITC</td><td>Clone: M5E2; Dilutie: 1: 200</td></tr><tr><td>CD19</td><td>BD Biosciences</td><td>PE</td><td>Clone: HIB19; Dilutie: 1: 200</td></tr><tr><td>CD205</td><td>BioLegend</td><td>FITC</td><td>Clone: MG38; Dilutie: 1: 200</td></tr><tr><td>CD3</td><td>BD Biosciences</td><td>PE</td><td>Clone: HIT3a; Dilutie: 1: 200</td></tr><tr><td>CD34</td><td>BD Biosciences</td><td>FITC</td><td>Clone: 8G12; Dilutie: 1: 100</td></tr><tr><td>CD4</td><td>BD Biosciences</td><td>PE</td><td>Clone: RPA-T4; Dilutie: 1: 100</td></tr><tr><td>CD4</td><td>BD Biosciences</td><td>BV711</td><td>Clone: L200; Dilutie: 1: 200</td></tr><tr><td>CD45</td><td>BD Biosciences</td><td>PerCP</td><td>Clone: HI30; Dilutie: 1: 200</td></tr><tr><td>CD56</td><td>BD Biosciences</td><td>PE</td><td>Clone: B159; Dilutie: 1: 200</td></tr><tr><td>CD62L</td><td>BD Biosciences</td><td>BV605</td><td>Clone: DREG-56; Dilutie: 1: 200</td></tr><tr><td>CD69</td><td>BD Biosciences</td><td>BV510</td><td>Clone: FN50; Dilutie: 1: 200</td></tr><tr><td>CD7</td><td>BD Biosciences</td><td>APC</td><td>Clone: M-T701; Dilutie: 1: 200</td></tr><tr><td>CD8</td><td>BD Biosciences</td><td>PeCy7</td><td>Clone: RPA-T8; Dilutie: 1: 200</td></tr><tr><td>CD8</td><td>BD Biosciences</td><td>PE</td><td>Clone: HIT8a; Dilutie: 1: 200</td></tr><tr><td>Dynabeads Pan Mouse IgG</td><td>Invitrogen</td><td>11041</td><td></td></tr><tr><td>EGF</td><td>Miltenyi</td><td>130-097-751</td><td></td></tr><tr><td>EPCAM (CD326)</td><td>BD Biosciences</td><td>PE</td><td>Clone: HEA-125; Dilutie: 1: 200</td></tr><tr><td>EPCAM (CD326)</td><td>Miltenyi</td><td>BV711</td><td>Clone: EBA-1; Dilutie: 1: 200</td></tr><tr><td>FGF10</td><td>Miltenyi</td><td>130-127-858</td><td></td></tr><tr><td>FGF8</td><td>Biotechne R&amp;D</td><td>423-F8</td><td></td></tr><tr><td>Fibrinogeen</td><td>Sigma Aldrich</td><td>341578</td><td></td></tr><tr><td>FLT3 L</td><td>Peprotech</td><td>AF-300-19</td><td></td></tr><tr><td>Glutamax</td><td>Gibco</td><td>35050-61</td><td></td></tr><tr><td>IGF1</td><td>Miltenyi</td><td>130-093-886</td><td></td></tr><tr><td>IL7</td><td>Peprotech</td><td>AF-200-07</td><td></td></tr><tr><td>RANK L</td><td>Biotechne R&amp;D</td><td>6449-TEC</td><td></td></tr><tr><td>Rode bloedcel lyseer oplossing</td><td>Miltenyi</td><td>130-094-183</td><td></td></tr><tr><td>RPMI1640</td><td>Gibco</td><td>11875093</td><td></td></tr><tr><td>SCF</td><td>Peprotech</td><td>AF-300-07</td><td></td></tr><tr><td>Trombine</td><td>Sigma Aldrich</td><td>605190</td><td></td></tr><tr><td>TrypLE</td><td>Gibco</td><td>2605010</td><td></td></tr><tr><td>XVIVO10</td><td>Lonza</td><td>LONBE04-380Q</td><td></td></tr></tbody></table>

vorming van menselijke thymusorganoïden in driedimensionale fibrinehydrogels

Formation of Human Thymus Organoids in Three-Dimensional Fibrin Hydrogels

Vorming van menselijke thymusorganoïden in driedimensionale fibrinehydrogels

Generation of a functional and self-tolerant T cell repertoire is a complex process dependent on the thymic microenvironment and, primarily, on the ...

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Research

JoVE Journal

Developmental Biology

Ontwikkelingsbiologie

In deze video de bereiding van 2-dGuo behandelde reaggregate thymus culturen is aangetoond.

Reaggregate Thymus Culturen

Het bestuderen van medullaire thymische epitheelcellen in vitro is grotendeels onsuccesvol geweest, omdat huidige 2D-kweeksystemen het in vivo scenario niet nabootsen. Het 3D-kweeksysteem dat hierin wordt beschreven - een gemodificeerd organotypisch model voor huidcultuur - heeft zich superieur getoond in het nadoen van mTEC-proliferatie, differentiatie en het behoud van promiscue genregulatie.

3D Organotypische Co-cultuur Model Supporting Medullary Thymic epitheelcelproliferatie, Differentiatie en Promiscuous Gene Expression

Deze video toont een protocol voor het verrijken van thymische epitheliale cellen (TEC's) met dichtheidsgradiënt voor FACS-isolatie. Het laat ook het gebruik van EAK16-II/EAKIIH6-peptiden zien om de TEC-aggregatievorming te bevorderen. De micro-omgevingen van EAK16-II/EAKIIH6-hydrogel bieden de 3-D configuratie die nodig is om de overleving en functie van de TEC's te handhaven.

Het bevorderen van 3-D Aggregation van FACS Gezuiverd thymus epitheelcellen met EAK 16-II / EAKIIH6 Self-assembleren Hydrogel

Het doel van dit protocol is om de ontwikkeling van een driedimensionaal (3D) microfluïdisch model van sterk uitgelijnd menselijk hartweefsel, samengesteld uit stamcel-afgeleide cardiomyocyten, samen met hartfibblasten (CFs) in een biomimetische, op collageen gebaseerde hydrogel, uit te leggen en aan te tonen, voor toepassingen in hartweefseltechniek, geneesmiddelenscreening en ziektemodellering.

Het ontwikkelen van 3D georganiseerd menselijk hartweefsel binnen een microfluïdisch platform

Hierin demonstreren we een organoïdemodel in drie stappen (tweedimensionale [2D] expansie, 2D-stimulatie, driedimensionale [3D] rijping) dat een veelbelovend hulpmiddel biedt voor fundamenteel peesonderzoek en een potentiële steigervrije methode voor peesweefselmanipulatie.

Demonstratie van zelf-geassembleerde celbladcultuur en handmatige generatie van een 3D-pees/ligament-achtige organoïde met behulp van menselijke dermale fibroblasten

Dit protocol presenteert een methode voor het genereren van vasculaire organoïden met behulp van menselijke pluripotente stamcellen.

Vasculaire organoïdegeneratie uit door de mens geïnduceerde pluripotente stamcellen

Dit protocol beschrijft een gedefinieerde hydrogel-gebaseerde methode om menselijke borstorganoïden te genereren die belangrijke kenmerken van mammariummorfogenese recapituleren in een gecontroleerd driedimensionaal kweeksysteem.

Een gedefinieerde hydrogel-gebaseerde methode voor het genereren van driedimensionale menselijke borstorganoïden die mammaire morfogenese recapituleren

Met het doel om veranderingen in miRNA's in gedifferentieerde menselijke neurale stamcellen (hNSC's) te karakteriseren, beschrijven we hNSC-differentiatie op een driedimensionaal systeem, de evaluatie van veranderingen in microRNA-expressie door miRNA PCR-array en computationele analyse voor miRNA-doelwitvoorspelling en de validatie ervan door middel van een dual luciferase-assay.

Differentiatie van een menselijke neurale stamcellen Line op Three Dimensional Culturen, Analyse van MicroRNA en vermeende doelwit-genen

Dendritische stekels van piramidale neuronen zijn de sites van de meest prikkelende synapsen in de hersenen van zoogdieren cortex. Deze methode beschrijft een 3D-kwantitatieve analyse van de wervelkolom morfologie in menselijke corticale piramidale glutamaat neuronen afgeleid van geïnduceerde pluripotente stamcellen.

Driedimensionale Kwantificering van Dendritische stekels van Piramidale Neuronen afgeleid van menselijk geïnduceerde pluripotente stamcellen

Dit protocol beschrijft de meting van epitheliale barrière permeabiliteit in real-time volgende farmacologische behandeling in menselijke intestinale organoids met behulp van fluorescentie microscopie en levende cel microscopie.

Real-time meting van epitheliale barrière permeabiliteit in menselijke intestinale Organoids

Leer hoe menselijke thymusorganoïden worden gevormd in driedimensionale fibrinehydrogels voor immunologisch onderzoek. Deze methode ondersteunt de rijping van thymusepitheelcellen en draagt bij aan betere technieken voor T-lymfocytproductie in vitro.

Vorming van menselijke thymusorganoïden in driedimensionale fibrinehydrogels

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