Name: Isolatie van primaire menselijke proximale tubule-epitheelcellen en hun gebruik bij het maken van een microfysiologisch model van de proximale niertubuli (Video) | JoVE Journal
Uploaded: 2025-05-09T14:30:00.000+00:00
Duration: 7 min 6 s
Description: Leer hoe u primaire humane proximale tubule-epitheelcellen isoleert en cultiveert in een microfysiologisch systeem. Deze methode maakt nauwkeurige in-vitro-evaluatie van medicijntransport en nefrotoxiciteit mogelijk.

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5 minutes

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<li>Weber, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+kidney+on+a+chip+assessment+of+polymyxin+antibiotic+nephrotoxicity%5BTitle%5D)+AND+%22JCI+Insight%22%5BJournal%5D)">Human kidney on a chip assessment of polymyxin antibiotic nephrotoxicity.</a> JCI Insight. 3 (24), e123673(2018).</li>
<li>Imaoka, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bridging+the+gap+between+in+silico+and+in+vivo+by+modeling+opioid+disposition+in+a+kidney+proximal+tubule+microphysiological+system%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Bridging the gap between in silico and in vivo by modeling opioid disposition in a kidney proximal tubule microphysiological system.</a> Sci Rep. 11 (1), 21356(2021).</li>
<li>Van Ness, K. P., Chang, S., Weber, E. J., Zumpano, D., Eaton, D. L., Kelly, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microphysiological+systems+to+assess+nonclinical+toxicity%5BTitle%5D)+AND+%22Curr+Protoc+Toxicol%22%5BJournal%5D)">Microphysiological systems to assess nonclinical toxicity.</a> Curr Protoc Toxicol. 73, 14.18.1-14.18.28 (2017).</li>
<li>Lidberg, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Serum+protein+exposure+activates+a+core+regulatory+program+driving+human+proximal+tubule+injury%5BTitle%5D)+AND+%22J+Am+Soc+Nephrol%22%5BJournal%5D)">Serum protein exposure activates a core regulatory program driving human proximal tubule injury.</a> J Am Soc Nephrol. 33 (5), 949-965 (2022).</li>
<li>Sakolish, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Technology+transfer+of+the+microphysiological+systems%3A+a+case+study+of+the+human+proximal+tubule+tissue+chip%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Technology transfer of the microphysiological systems: a case study of the human proximal tubule tissue chip.</a> Sci Rep. 8 (1), 14882(2018).</li>
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<li>Adashi, E. Y., O'Mahony, D. P., Cohen, I. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+FDA+modernization+act+2.0%3A+drug+testing+in+animals+is+rendered+optional%5BTitle%5D)+AND+%22Am+J+Med%22%5BJournal%5D)">The FDA modernization act 2.0: drug testing in animals is rendered optional.</a> Am J Med. 136 (9), 853-854 (2023).</li>
<li>Qi, W., Johnson, D. W., Vesey, D. A., Pollock, C. A., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation%2C+propagation+and+characterization+of+primary+tubule+cell+culture+from+human+kidney+%28methods+in+renal+research%29%5BTitle%5D)+AND+%22Nephrology%22%5BJournal%5D)">Isolation, propagation and characterization of primary tubule cell culture from human kidney (methods in renal research).</a> Nephrology. 12 (2), 155-159 (2007).</li>
<li>Mihevc, M., Petreski, T., Maver, U., Bevc, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Renal+proximal+tubular+epithelial+cells%3A+review+of+isolation%2C+characterization%2C+and+culturing+techniques%5BTitle%5D)+AND+%22Mol+Biol+Rep%22%5BJournal%5D)">Renal proximal tubular epithelial cells: review of isolation, characterization, and culturing techniques.</a> Mol Biol Rep. 47 (12), 9865-9882 (2020).</li>
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</ol>

<table><tbody><tr><td>1 mL Luer-Lok Tip Syringes</td><td>Becton, Dickinson</td><td>309628</td><td></td></tr><tr><td>1.5 mL microcentrifuge tubes</td><td>CELLTREAT Scientific Products</td><td>229442</td><td></td></tr><tr><td>15 mL steriele conische polypropyleenbuis</td><td>Fisher Scientific</td><td>14-959-53A</td><td></td></tr><tr><td>3D microfysiologisch apparaat</td><td>Nortis</td><td>TCC-001</td><td></td></tr><tr><td>5 mL Luer-Lok Tip Syringes</td><td>Becton, Dickinson</td><td>309646</td><td></td></tr><tr><td>50 mL steriele conische polypropyleenbuis</td><td>Fisher Scientific</td><td>12-565-271</td><td></td></tr><tr><td>Antibiotic-Antimycotic (100x)</td><td>ThermoFisher Scientific</td><td>15240062</td><td></td></tr><tr><td>Collageenase, Type IV, poeder</td><td>ThermoFisher Scientific</td><td>17104019</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>MilliporeSigma</td><td>G8270</td><td></td></tr><tr><td>Gedefinieerd trypsine-remmer</td><td>ThermoFisher Scientific</td><td>R007100</td><td></td></tr><tr><td>Dimethyl sulfaat, Bioreagens, Thermo Scientific Chemicals</td><td>ThermoFisher Scientific</td><td>J66650-AK</td><td></td></tr><tr><td>Dispase II, poeder</td><td>ThermoFisher Scientific</td><td>17105041</td><td></td></tr><tr><td>Dulbecco&rsquo;s MEM (DMEM)/F-12 zonder glucose</td><td>US Biological Life Sciences</td><td>D9807-02</td><td></td></tr><tr><td>Dulbecco's Phosphate Buffered Saline (DPBS), calcium, magnesium</td><td>ThermoFisher Scientific</td><td>14040117</td><td></td></tr><tr><td>Foetaal runderserum (FBS), Premium</td><td>ThermoFisher Scientific</td><td>A5670701</td><td></td></tr><tr><td>Finnpipette F2 Good Laboratory Pipetting (GLP) Kits</td><td>ThermoFisher Scientific</td><td>05-719-511</td><td>Micropipetten</td></tr><tr><td>Fisherbrand SureOne Micropoint Pipette Tips, Universal Fit, niet-gefilterd</td><td>Fisher Scientific</td><td>02707407, 02707410, 02707438</td><td></td></tr><tr><td>Verse volledige menselijke nier</td><td>Novabiosis</td><td>https://www.novabiosis.com/research-organ-allocation-services/</td><td>Menselijke nieren werden verkregen via Novabiosis, een UNOS-goedgekeurde OPO</td></tr><tr><td>Bevochtigde incubator (37 &deg;C en 5% CO2)</td><td>Fisher Scientific</td><td>51033556</td><td></td></tr><tr><td>Hydrocortison</td><td>MilliporeSigma</td><td>H0888</td><td></td></tr><tr><td>Incuberende orbitale shaker</td><td>Avantor</td><td>12620-946</td><td></td></tr><tr><td>Insulin-Transferrin-Selenium (100X) (ITS -G)</td><td>ThermoFisher Scientific</td><td>41400045</td><td></td></tr><tr><td>Cryogene flesjes met binnendraad</td><td>Corning</td><td>430487</td><td></td></tr><tr><td>Kimtech Science Kimwipes</td><td>Kimberly-Clark Professional</td><td>34120</td><td></td></tr><tr><td>Luer Stubs (stompe naald), 22 G x 0,5 inch</td><td>Instech Laboratories, Inc.</td><td>LS22</td><td></td></tr><tr><td>Medium 199, Earle's zouten (10x)</td><td>ThermoFisher Scientific</td><td>21180021</td><td></td></tr><tr><td>Megafuge 8 kleine tafelcentrifuge</td><td>ThermoFisher Scientific</td><td>75007210</td><td></td></tr><tr><td>Metaalkoppeling (stompe)</td><td>Instech Laboratories, Inc.</td><td>SC 20/15</td><td></td></tr><tr><td>Mr. Frosty vriescontainer</td><td>ThermoFisher Scientific</td><td>5100-0036</td><td></td></tr><tr><td>Nikon Eclipse Ti-S Microscoop</td><td>Nikon Instruments</td><td>https://www.thelabworldgroup.com/product/nikon-eclipse-ti-fluorescent-microscope/</td><td>Origineel model uit productie</td></tr><tr><td>Nunc niet-behandelde kolven, T-25</td><td>ThermoFisher Scientific</td><td>169900</td><td></td></tr><tr><td>Nunc niet-behandelde kolven, T-75</td><td>ThermoFisher Scientific</td><td>156800</td><td></td></tr><tr><td>Pipet, 10 mL, gradueel, 1/10 mL, sterieel</td><td>Greiner Bio-One</td><td>607180</td><td></td></tr><tr><td>Siliconcouling, C-flex-slang, (ID: 0,020", OD: 0,083")</td><td>Cole-Parmer</td><td>06422-00</td><td></td></tr><tr><td>Mes met één snede (sterieel)</td><td>Bioseal</td><td>KI-205/50</td><td></td></tr><tr><td>Natriumbicarbonaat</td><td>MilliporeSigma</td><td>S6297</td><td></td></tr><tr><td>Natriumhydroxide</td><td>MilliporeSigma</td><td>S8045</td><td></td></tr><tr><td>Steriele wegwerpfiltereenheden met PES-membranen</td><td>ThermoFisher Scientific</td><td>567-0020</td><td></td></tr><tr><td>Weefselkweek behandelde schotels, 150 mm&nbsp; x 20 mm geventileerd</td><td>Genesee Scientific</td><td>25-203</td><td></td></tr><tr><td>Trypsine-EDTA (0,05%), fenolrood</td><td>ThermoFisher Scientific</td><td>25300054</td><td></td></tr><tr><td>Type I collageen, rattenstaart</td><td>Corning</td><td>354236</td><td></td></tr><tr><td>Type IV Collageen, Muis</td><td>Corning</td><td>354233</td><td></td></tr></tbody></table>

isolatie van primaire menselijke proximale tubule-epitheelcellen en hun gebruik bij het maken van een microfysiologisch model van de proximale niertubuli

Isolation of Primary Human Proximal Tubule Epithelial Cells and Their Use in Creating a Microphysiological Model of the Renal Proximal Tubule

Isolatie van primaire menselijke proximale tubule-epitheelcellen en hun gebruik bij het maken van een microfysiologisch model van de proximale niertubuli

Kidney disease affects over 850 million people globally, including 37 million Americans. Risk factors for&#160;chronic kidney disease include ...

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In this video, we demonstrate the steps to isolate and purify renal tubular epithelial cells for a wide range of molecular biology and cell culture studies.

Murine Renal Cell Isolation: A Technique to Isolate and Generate Primary Culture of Renal Tubular Epithelial Cells

Ionenkanalen tot expressie in renale tubulaire epitheel spelen een belangrijke rol in de pathologie van polycystische nierziekte. Hier beschrijven we de experimentele protocollen die worden gebruikt om patch-clamp-analyse en intracellulaire calciumgehalte metingen uit te voeren in cystic epitheel vers geïsoleerd van knaagdieren nieren.

Implementeren Patch Clamp en Live-fluorescentie microscopie om functionele eigenschappen van vers geïsoleerde PKD Epithelium Monitor

Dit protocol beschrijft de decellularisatie van Sprague Dawley rattennieren door antegrade perfusie van detergenten door het vaatstelsel, waardoor acellulaire nier extracellulaire matrices ontstaan die dienen als sjablonen voor herbevolking met menselijke nier epitheliale cellen. Recellularisatie en het gebruik van de resazurin perfusie assay om groei te monitoren wordt uitgevoerd binnen speciaal ontworpen perfusie bioreactoren.

Epitheelcellen Herbevolking en voorbereiding van knaagdieren extracellulaire matrix Stellingen voor nierweefsel Development

Hier presenteren we een nieuwe klinische rang isolatie en cultuur methode voor nieren gerelateerde stromale cellen (kPSCs) op basis van hele orgel perfusie met spijsverteringsenzymen en NG2-cel verrijking. Met deze methode is het mogelijk om het verwerven van voldoende cel nummers voor cellulaire therapie.

Een nieuwe klinische Grade isolatie methode voor menselijke nieren gerelateerde stromale cellen

Weefsel-engineered renale constructies bieden een oplossing voor het tekort aan organen en de schadelijke effecten van dialyse. Hier beschrijven we een protocol bij micro ontleden lymfkliertest nieren voor isolatie van cortico-Wallenberg segmenten. Deze segmenten zijn steiger-vrije cellulaire constructies, vorming van renale organoids ingeplant.

Microdissection van primaire renale weefsel segmenten en integratie met nieuwe steiger-vrije constructie technologie

Dit protocol biedt een rendabele aanpak om te isoleren en karakteriseren van muis primaire renale tubulaire cellen die vervolgens sub kunnen worden gekweekt om te beoordelen renale biologische functies- ex vivo, met inbegrip van de mitochondriale Bioenergetica.

Isolatie, karakterisering en High Throughput extracellulaire Flux-analyse van muis primaire renale tubulaire epitheliale cellen

Menselijke nier tubuloïde culturen vertegenwoordigen een waardevol in vitro model om nierfysiologie en ziekte te bestuderen. Tubuloïden kunnen worden vastgesteld uit nierweefsel (gezond en ziek) en urine, waarbij de laatste een gemakkelijk te verkrijgen en minder invasieve bron van onderzoeksmateriaal vertegenwoordigt.

Generatie van menselijke nier tubuloïden uit weefsel en urine

Hier presenteren we protocollen met gedetailleerde instructies voor het implementeren van 3D-celculturen met behulp van collageen- en collageen-agarosematrices in een microfysiologisch systeem. Deze protocollen ondersteunen de co-cultuur van spheroïden in de nierproximale tubulus en niercelcarcinoom, waardoor in vivo omstandigheden worden gesimuleerd en geavanceerd onderzoek naar interacties tussen nierkankercellen mogelijk wordt.

Microfluïdische co-cultuur van gezond nier- en tumorepitheel om de progressie van nierkanker te modelleren

Hier beschrijven we de protocollen voor het verkrijgen van primaire culturen van menselijke renale tubulaire epitheelcellen (HRTEC) uit de nierschors om monolagen en driedimensionale culturen te ontwikkelen op een extracellulaire matrix (3D-HRTEC).

Ontwikkeling van primaire culturen van menselijke niertubulaire epitheelcellen in monolagen en driedimensionale omstandigheden

Hier beschrijven we een in vitro protocol om hypoxie/reoxygenatieschade te modelleren in de sterk metabole proximale tububulaire epitheelcellen. Dit model is ontworpen om schade veroorzaakt door metabole stress te induceren, die kan worden gemeten door de expressie van proximale tubuli-schademarkers en de beoordeling van de mitochondriale ademhalingsfunctie.

Modellering van hypoxie/reoxygenatieschade in proximale tubulära epitheelcellen

Leer hoe u primaire humane proximale tubule-epitheelcellen isoleert en cultiveert in een microfysiologisch systeem. Deze methode maakt nauwkeurige in-vitro-evaluatie van medicijntransport en nefrotoxiciteit mogelijk.

Isolatie van primaire menselijke proximale tubule-epitheelcellen en hun gebruik bij het maken van een microfysiologisch model van de proximale niertubuli (Video) | JoVE Journal

Isolatie van primaire menselijke proximale tubule-epitheelcellen en hun gebruik bij het maken van een microfysiologisch model van de proximale niertubuli

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