Name: preparation of human myocardial tissue for long-term cultivation
Uploaded: 2022-06-02T14:10:00
Description: Watch this Scientific Journal Video about Preparation of Human Myocardial Tissue for Long-Term Cultivation at JoVE.com

Forskning blev finansieret af DZHK-tilskud 81Z0600207 (JH, PS og DM) og 81X2600253 (AD og TS).
Forfatterne vil gerne takke Claudia Fahney, Mei-Ping Wu og Matthias Semisch for deres støtte til forberedelsen af opsætningerne samt til regelmæssig vedligeholdelse af vævsdyrkningen.

Vævsindsamling til de eksperimenter, der er beskrevet her, blev godkendt af institutional review boards ved universitetet i München og Ruhr-Universitetet Bochum. Undersøgelserne blev udført i henhold til Helsinki-retningslinjerne. Patienterne gav deres skriftlige informerede samtykke forud for vævsindsamling.
1. Erhvervelse af væv
<ol><li>Få humant væv fra patienter, der gennemgår hjertetransplantation eller hjertekirurgi.</li>
	<li>Før udtagning af vævet skal der...

<ol>
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(160), e60781 (2020).</li><li>Lu, 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=Progressive+stretch+enhances+growth+and+maturation+of+3D+stem-cell-derived+myocardium.">Progressive stretch enhances growth and maturation of 3D stem-cell-derived myocardium.</a> Theranostics. 11 (13), 6138-6153 (2021).</li><li>Pontes Soares, C., 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=2D+and+3D-organized+cardiac+cells+shows+differences+in+cellular+morphology,+adhesion+junctions,+presence+of+myofibrils+and+protein+expression.">2D and 3D-organized cardiac cells shows differences in cellular morphology, adhesion junctions, presence of myofibrils and protein expression.</a> PloS one. 7 (5), 38147 (2012).</li><li>Klumm, M. 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=Long-term+cultivation+of+human+atrial+myocardium.">Long-term cultivation of human atrial myocardium.</a> Frontiers in Physiology. 13, 839139 (2022).</li><li>Krane, 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=Sequential+defects+in+cardiac+lineage+commitment+and+maturation+cause+hypoplastic+left+heart+syndrome.">Sequential defects in cardiac lineage commitment and maturation cause hypoplastic left heart syndrome.</a> Circulation. 144 (17), 1409-1428 (2021).</li><li>Miller, 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=Heart+slice+culture+system+reliably+demonstrates+clinical+drug-related+cardiotoxicity.">Heart slice culture system reliably demonstrates clinical drug-related cardiotoxicity.</a> Toxicology and Applied Pharmacology. , 406 (2020).</li><li>Fischer, C., 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=Long-term+functional+and+structural+preservation+of+precision-cut+human+myocardium+under+continuous+electromechanical+stimulation+in+vitro.">Long-term functional and structural preservation of precision-cut human myocardium under continuous electromechanical stimulation in vitro.</a> Nature Communications. 10 (1), 1-12 (2019).</li><li>Kang, C., 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=Human+organotypic+cultured+cardiac+slices:+new+platform+for+high+throughput+preclinical+human+trials.">Human organotypic cultured cardiac slices: new platform for high throughput preclinical human trials.</a> Scientific Reports. 6 (1), 1-13 (2016).</li><li>Ou, Q., 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=Slicing+and+culturing+pig+hearts+under+physiological+conditions.">Slicing and culturing pig hearts under physiological conditions.</a> Journal of Visualized Experiments: JoVE. (157), e60913 (2020).</li><li>Ou, Q., 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=Physiological+biomimetic+culture+system+for+pig+and+human+heart+slices.">Physiological biomimetic culture system for pig and human heart slices.</a> Circulation research. 125 (6), 628-642 (2019).</li><li>Watson, S. A., 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=Biomimetic+electromechanical+stimulation+to+maintain+adult+myocardial+slices+in+vitro.">Biomimetic electromechanical stimulation to maintain adult myocardial slices in vitro.</a> Nature Communications. 10 (1), 1-15 (2019).</li><li>Abu-Khousa, 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=The+degree+of+t-system+remodeling+predicts+negative+force-frequency+relationship+and+prolonged+relaxation+time+in+failing+human+myocardium.">The degree of t-system remodeling predicts negative force-frequency relationship and prolonged relaxation time in failing human myocardium.</a> Frontiers in Physiology. 11, 182 (2020).</li><li>Bojkova, 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=SARS-CoV-2+infects+and+induces+cytotoxic+effects+in+human+cardiomyocytes.">SARS-CoV-2 infects and induces cytotoxic effects in human cardiomyocytes.</a> Cardiovascular Research. 116 (14), 2207-2215 (2020).</li><li>Esfandyari, D. A. -. O., 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=MicroRNA-365+regulates+human+cardiac+action+potential+duration.">MicroRNA-365 regulates human cardiac action potential duration.</a> Nature Communications. 13 (1), 1-15 (2022).</li><li>Moretti, A., 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=Somatic+gene+editing+ameliorates+skeletal+and+cardiac+muscle+failure+in+pig+and+human+models+of+Duchenne+muscular+dystrophy.">Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy.</a> Nature Medicine. 26 (2), 207-214 (2020).</li><li>Pitoulis, F. G., 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=Remodelling+of+adult+cardiac+tissue+subjected+to+physiological+and+pathological+mechanical+load+in+vitro.">Remodelling of adult cardiac tissue subjected to physiological and pathological mechanical load in vitro.</a> Cardiovascular Research. 118 (3), 814-827 (2021).</li><li>Curtis, T. M., Scholfield, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nifedipine+blocks+Ca2++store+refilling+through+a+pathway+not+involving+L-type+Ca2++channels+in+rabbit+arteriolar+smooth+muscle.">Nifedipine blocks Ca2+ store refilling through a pathway not involving L-type Ca2+ channels in rabbit arteriolar smooth muscle.</a> The Journal of Physiology. 532 (3), 609-623 (2001).</li><li>de Weille, J. R., Schweitz, H., Maes, P., Tartar, A., Lazdunski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calciseptine,+a+peptide+isolated+from+black+mamba+venom,+is+a+specific+blocker+of+the+L-type+calcium+channel.">Calciseptine, a peptide isolated from black mamba venom, is a specific blocker of the L-type calcium channel.</a> Proceedings of the National Academy of Sciences. 88 (6), 2437-2440 (1991).</li><li>Schleifer, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+molecular+modelling+study+of+the+calcium+channel+blockers+nifedipine+and+black+mamba+toxin+FS2.">Comparative molecular modelling study of the calcium channel blockers nifedipine and black mamba toxin FS2.</a> Journal of Computer-Aided Molecular Design. 11 (5), 491-501 (1997).</li><li>Thomas, G., Chung, M., Cohen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dihydropyridine+(Bay-K8644)+that+enhances+calcium+currents+in+guinea+pig+and+calf+myocardial+cells.+A+new+type+of+positive+inotropic+agent.">A dihydropyridine (Bay-K8644) that enhances calcium currents in guinea pig and calf myocardial cells. A new type of positive inotropic agent.</a> Circulation Research. 56 (1), 87-96 (1985).</li><li>Pitoulis, F. G., Watson, S. A., Perbellini, F., Terracciano, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+slices+come+to+age:+an+intermediate+complexity+in+vitro+cardiac+model+for+translational+research.">Myocardial slices come to age: an intermediate complexity in vitro cardiac model for translational research.</a> Cardiovascular Research. 116 (7), 1275-1287 (2020).</li><li>Watson, S. A., 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=Preparation+of+viable+adult+ventricular+myocardial+slices+from+large+and+small+mammals.">Preparation of viable adult ventricular myocardial slices from large and small mammals.</a> Nature Protocols. 12 (12), 2623-2639 (2017).</li></ol>

Tidligere har kardiovaskulær forskning gjort store fremskridt inden for dyrkning af kardiomyocytter. Imidlertid er 3D-dyrkning af kardiomyocytter med intakt geometri endnu ikke veletableret. Sammenlignet med tidligere protokoller, der blev anvendt til ex vivo-dyrkning af myokardievæv, ligner den protokol, som vi beskrev her, vævets in vivo-miljø nærmere. Desuden giver anvendelsen af for- og efterbelastning mulighed for et mere biomimetisk miljø. Vi er i stand til fuldt ud at analysere og forstå d...

I det sidste årti har in vitro-dyrkning af myokardieceller gjort store fremskridt, lige fra 2D og tredimensionelle (3D) teknikker til anvendelse af organoider og inducerede pluripotente stamceller differentieret i hjertemyocytter 1,2,3. Ex vivo og primærcelledyrkning har vist sig at være af stor værdi, især for genetiske undersøgelser og lægemiddeludvikling 4,5,6. Brug af humane væv forbedrer den translationelle værdi af resultaterne. Langsigtet 3D-dyrkning af myokardievæv med intakt geometri er imidlertid ikke veletableret. Den intakte geometri er en nøglefunktion til at efterligne in vivo-forhold, da korrekt hjertefunktion, kommunikation mellem forskellige celler samt cellematrixinteraktioner er forudsætninger. Myokardievævsdyrkning gennemgik forskellige udviklingsfaser. Succesraten og stabiliteten af ex vivo myokardievævsdyrkning var oprindeligt ret lav, men nylige tilgange har givet lovende resultater 7,8,9,10,11.
Blandt disse var Fischer et al. de første til at demonstrere, at levedygtighed og kontraktil ydeevne af humant myokardievæv kan opretholdes i ex vivocelledyrkning i mange uger7. Deres teknik var baseret på tynde vævsskiver skåret fra udplantet humant myokardium, som blev monteret i nyudviklede dyrkningskamre, der gav definerede biomekaniske forhold og kontinuerlig elektrisk stimulering. Denne dyrkningsmetode ligner meget myokardievævets in vivo-funktion og er blevet reproduceret af flere uafhængige forskergrupper 2,12,13,14,15. Det er vigtigt, at de kamre, der blev brugt af Fischer et al., også muliggjorde kontinuerlig registrering af udviklede kræfter i op til 4 måneder og dermed åbnede hidtil usete muligheder for fysiologisk og farmakologisk forskning i intakt humant myokardium7.
Lignende teknikker blev uafhængigt udviklet af andre grupper og anvendt på humant, rotte, svin og kaninmyokardium 7,10,11. Pitoulis et al. udviklede efterfølgende en mere fysiologisk metode, som reproducerer den normale kraftlængderelation under en sammentrækningscyklus, men er mindre egnet til analyse af høj kapacitet16. Som sådan kan den generelle tilgang til biomimetisk dyrkning betragtes som et yderligere skridt i reduktion, raffinement og erstatning (3R) af dyreforsøg.
Udnyttelsen af dette potentiale kræver imidlertid standardiserede procedurer, analyser af højt indhold og et højt gennemløbsniveau. Vi præsenterer en teknik, der kombinerer automatiseret udskæring af levende humant myokardium med in vitro-vedligeholdelse i et biomimetisk dyrkningssystem, der er blevet kommercielt tilgængeligt (se materialetabel). Med den foreslåede tilgang er antallet af individuelle skiver, der kan genereres fra en enkelt transmural myokardieprøve, kun begrænset af behandlingstiden. En prøve af tilstrækkelig størrelse og kvalitet (3 cm x 3 cm) giver ofte 20-40 vævsskiver, der bekvemt skæres med en automatiseret vibratom. Disse skiver kan placeres i dyrkningskamre, der tilhører systemet. Kamrene giver mulighed for elektrisk stimulering, hvis parametre kan moduleres (dvs. pulsvarighed, polaritet, hastighed og strøm) samt justering af for- og efterbelastning ved hjælp af fjedertråde inde i kamrene. Sammentrækningen af hver skive registreres fra bevægelsen af en lille magnet fastgjort til en fjedertråd og vises som en fortolkelig graf. Data kan til enhver tid registreres og analyseres ved hjælp af frit tilgængelig software. Bortset fra den konstante baseline pacing kan planlagte protokoller udføres for funktionelt at vurdere deres ildfaste periode, stimuleringstærskel, post-pause-potensering og kraftfrekvensrelation.
Denne langsigtede biomimetiske dyrkning af flere myokardieskiver fra et individuelt hjerte baner vejen for fremtidig ex vivo-forskning i både humant og animalsk væv og letter screeningen for terapeutiske og kardiotoksiske lægemiddeleffekter i kardiovaskulær medicin. Det er allerede blevet anvendt på forskellige eksperimentelle tilgange 2,12,13,15. Her giver vi en detaljeret trinvis beskrivelse af fremstillingen af humant væv og giver løsninger på ofte opståede dyrkningsproblemer.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose Low melting point</td>
			<td>Roth</td>
			<td>6351.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Bay-K8644</td>
			<td>Cayman Chemical</td>
			<td>19988</td>
			<td></td>
		</tr>
		<tr>
			<td>BDM (2,3-Butanedione monoxime)</td>
			<td>Sigma</td>
			<td>B0753-1kg</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2*H2O</td>
			<td>Merck</td>
			<td>2382.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Calciseptine</td>
			<td>Alomone Labs</td>
			<td>SPC-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose*H2O</td>
			<td>AppliChem</td>
			<td>A3730.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>H2O</td>
			<td>BBraun</td>
			<td>3703452</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>AppliChem</td>
			<td>A1069.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Histoacryl</td>
			<td>BBraun</td>
			<td>1050052</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol 100%</td>
			<td>SAV LP GmbH</td>
			<td>UN1219</td>
			<td></td>
		</tr>
		<tr>
			<td>ITS-X-supplement</td>
			<td>Gibco</td>
			<td>5150056</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Merck</td>
			<td>1.04933.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Gibco</td>
			<td>31150-022</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2*6H2O</td>
			<td>AppliChem</td>
			<td>A1036.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S5886-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4*H2O</td>
			<td>Merck</td>
			<td>1.06346.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Nifedipine</td>
			<td>Sigma</td>
			<td>N7634-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin / streptomycin x100</td>
			<td>Sigma</td>
			<td>P0781-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>AppliChem</td>
			<td>A1108.0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cabinet</td>
			<td>Thermo Scientific</td>
			<td>KS15</td>
			<td></td>
		</tr>
		<tr>
			<td>Frigomix waterpump and cooling + BBraun Thermomix BM</td>
			<td>BBraun</td>
			<td></td>
			<td>In-house made combination of cooling and heating solution.</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Binder</td>
			<td>CB240</td>
			<td></td>
		</tr>
		<tr>
			<td>MyoDish bioreactor system</td>
			<td>InVitroSys GmbH</td>
			<td>MyoDish 1</td>
			<td>Myodish cultute system</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1200s</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath 37 degrees</td>
			<td>Haake</td>
			<td>SWB25</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath 80 degrees</td>
			<td>Daglef Patz KG</td>
			<td>7070</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mL plastic single-use beaker</td>
			<td>Sarstedt</td>
			<td>75.562.105</td>
			<td></td>
		</tr>
		<tr>
			<td>Filtration unit, Steritop Quick Release</td>
			<td>Millipore</td>
			<td>S2GPT05RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles 0.9 x 70 mm 20G</td>
			<td>BBraun</td>
			<td>4665791</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic triangles</td>
			<td></td>
			<td></td>
			<td>In-house made</td>
		</tr>
		<tr>
			<td>Razor Derby premium</td>
			<td>Derby Tokai</td>
			<td>B072HJCFK6</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor Gillette Silver Blue</td>
			<td>Gillette</td>
			<td>7393560010170</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel disposable</td>
			<td>Feather</td>
			<td>02.001.30.020</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 10 mL Luer tip BD Discardit</td>
			<td>BBraun</td>
			<td>309110</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Dish 10 cm</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Dish 3.5 cm</td>
			<td>Falcon</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubes 50 mL</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of human myocardial tissue for long-term cultivation

Kardiomyocytdyrkning har set et stort antal udviklinger, lige fra todimensionel (2D) celledyrkning til iPSC-afledte organoider. I 2019 blev der demonstreret en ex vivo måde at dyrke myokardieskiver opnået fra humane hjerteprøver, mens man nærmer sig in vivo tilstand af myokardiekontraktion. Disse prøver stammer hovedsagelig fra hjertetransplantationer eller placeringer af venstre ventrikulær hjælpeanordning. Ved hjælp af en vibratom og et specielt udviklet dyrkningssystem placeres 300 μm tykke skiver mellem en fast og en fjedertråd, hvilket giver mulighed for stabil og reproducerbar dyrkning i flere uger. Under dyrkning stimuleres skiverne kontinuerligt i henhold til individuelle indstillinger. Sammentrækninger kan vises og registreres i realtid, og farmakologiske midler kan let påføres. Brugerdefinerede stimuleringsprotokoller kan planlægges og udføres for at vurdere vitale sammentrækningsparametre som post-pause-potensering, stimuleringstærskel, kraftfrekvensrelation og ildfast periode. Desuden muliggør systemet en variabel for- og efterbelastningsindstilling for en mere fysiologisk dyrkning.
Her præsenterer vi en trinvis vejledning om, hvordan man genererer en vellykket langsigtet dyrkning af menneskelige venstre ventrikulære myokardieskiver ved hjælp af en kommerciel biomimetisk dyrkningsløsning.

Sammentrækningen af myokardieskiverne blev vist på computerskærmen efter indsættelse af dyrkningskammeret i dets tilsvarende stik (figur 3). Sammentrækning af de menneskelige myokardieskiver startede umiddelbart efter stimulering. Skiverne hyperkontrakterede i 5-10 min. Dette var synligt som en stigning i diastoliske kræfter forårsaget af en tonisk kontraktur af beskadigede vævsfraktioner. Denne proces blev vendt tilbage i varierende grad inden for 1-1,5 time. Efter stabilisering vis...

Watch this Scientific Journal Video about Preparation of Human Myocardial Tissue for Long-Term Cultivation at JoVE.com

Preparation of Human Myocardial Tissue for Long-Term Cultivation

Vi præsenterer en protokol for ex vivo dyrkning af humant ventrikulært myokardievæv. Det giver mulighed for detaljeret analyse af sammentrækningskraft og kinetik samt anvendelse af præ- og efterbelastning for at efterligne det in vivo fysiologiske miljø nærmere.

Forberedelse af humant myokardievæv til langvarig dyrkning

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, ...

Ved hjælp af denne protokol kan humane venstre ventrikulære vævsskiver dyrkes ex vivo i et biomimetisk kammer. Anvendelsen af for- og efterbelastninger forbedrer ligheden mellem det fysiologiske miljø. Denne teknik muliggør kontinuerlig registrering af vævskontraktion.Brugerdefinerede stimuleringsprotokoller giver mulighed for vurdering af vitale sammentrækningsparametre som potensering efter pause, stimuleringstærskel, kraftfrekvensrelation og ildfast periode. Den langsigtede dyrkning af myokardieskiver ved hjælp af denne opsætning vil bane vejen for fremtidig ex vivo-forskning, hvilket letter denne screening for terapeutiske og kardiotoksiske lægemiddeleffekter i kardiovaskulær medicin. Til at begynde med nedsænkes dyrkningskamrene og grafitelektroderne i en liter af en 10% isopropanolopløsning og omrøres natten over.Den følgende dag overføres kamrene til en 100% isopropanolopløsning i 3 minutter, og lad derefter kamrene og grafitelektroderne lufttørre under en laminær flowhætte. Fastgør et printkort til hvert kammer i henhold til de tilgængelige positioner på vippemaskinen. Placer to grafitelektroder på printkortet i henhold til producentens anvisninger, og placer derefter et 35 millimeter petriskållåg på kammeret for at forhindre infektion.Fyld derefter skærebakken op til 90 til 95% med skærebuffer. Overfør vævsprøven til en 100 millimeter petriskål fyldt med kold skærebuffer, og læg fadet på en køleplade ved 4 grader Celsius. Fjern endokardial trabeculae ved at holde endokardiet med pincet, og brug derefter saks til at skære ca. 3 millimeter endokardialt væv væk.Fastgør den afskårne vævsprøve, endokardial side op til en 2 x 2 centimeter gummiplaster ved hjælp af fire 0,9 x 70 millimeter 20-gauge nåle, der er fastgjort i en firkantet position. Sørg for, at den diagonale kant af hver nålespids peger indad, hvilket forbedrer fikseringen og forhindrer beskadigelse af myokardiet. Brug en skalpel til at skære alt overskydende væv væk uden for de fire nålefirkanter.Brug pincet til at placere den trimmede prøve på et sterilt stykke væv i 10 sekunder for at fjerne overskydende skærebuffer. Fjern derefter agarosesprøjten fra vandbadet og nedsænk prøven i agarose. Lad agarosen størkne i fem minutter på en køleplade.Endokardialsiden af prøven skal være synlig i agarosen. Brug pincet til at placere den epikardiale side af prøven indeholdt i agarose oven på det limede område, og tryk derefter forsigtigt den agaroseholdige prøve fra toppen med et stumpt værktøj uden at skære eller beskadige agarosen. Lad limen størkne i 1 minut.Indstil vibrationsamplituden til 1 millimeter, indledende skærehastighed til 0,07 millimeter i sekundet og tykkelsen af skiven til 300 mikron. Når du har tilsluttet dyrkningssystemet til en computer, skal du starte det tilsvarende softwareprogram. Indstil vippehastigheden til 60 o / min, og forudindstil stimuleringsparametrene.For humane hjerteskiver skal du indstille standardstimuleringen til bifasiske impulser med 50 milliamp eller strøm bestående af 3 millisekunder positiv strøm, 1 millisekund pause og en 3-millisekund puls af inverteret strøm med en tempohastighed på 30 slag i minuttet eller bpm, kontroller derefter elektrodeindikatorerne for softwaren og kontroller, at elektroderne i dyrkningskamrene fungerer korrekt. Brug pincet til at adskille agarosen fra vævet. Undgå at røre ved vævet og håndter det omhyggeligt, da enhver skade på vævet vil reducere dyrkningens succesrate.For at fastgøre to plasttrekanter til en prøve skal du placere 1 mikroliter lim på et sterilt petriskållåg. Brug en kroget pincet til at hente en autoklaveret plasttrekant. Dyp hurtigt trekantens forkant i limen og indsæt trekanten på prøven vinkelret på kardiomyocytjusteringen.Gentag for den anden trekant. Brug en skalpel til at trimme væv, der overstiger trekantbredden, og læg skiven med de to monterede trekanter tilbage i skærebakken, der indeholder skærebuffer. Fjern det mellemfyldte dyrkningskammer fra inkubatoren.Vælg en forberedt skive og indsæt den i kammeret ved at forbinde en trekant til hver stift. Juster afstanden mellem monteringsstifterne til prøvestørrelsen. Sørg for, at prøven er nedsænket i mediet.Når du har placeret skålen på vipperen, skal du reducere forspændingen ved at dreje justeringsskruen mod uret, indtil basislinjen for den tilsvarende graf på computerskærmen ikke ændres mere, og derefter forsigtigt øge forspændingen eller spændingen ved at dreje justeringsskruen med uret. For kamre med høj stivhed skal du fortsætte, indtil den tilsvarende basislinje i grafen er steget med 1.000 til 1.200 enheder svarende til en millinewton forspænding. Efter tilberedning af det friske dyrkningsmedium skal du forvarme mediet i et vandbad eller varmluftinkubator ved 37 grader Celsius i 30 til 45 minutter.Fjern medium fra kammeret, efterlad ca. 0,8 ml i kammeret, tilsæt derefter 1,6 ml frisk medium til det samme kammer, hvilket giver et samlet volumen medium til 2,4 ml pr. Kammer. Til sidst skal du placere kammerets dæksel tilbage og placere dyrkningskammeret i sin respektive position. Aflæsningen af sammentrækningen af fem myokardieskiver under en typisk stimulering bestod af fire forskellige sektioner af post-pause potensering, stimuleringstærskel, kraftfrekvensrelation og ildfast periode.Sammenlignet med kontrol blev sammentrækningskraften af skiverne behandlet med calciumantagonister, nifedipin og calciseptin, nedsat inden for 10 minutter. I modsætning hertil øgede den spændingsstyrede calciumkanalagonist, Bay-K8644, sammentrækningskraften. Post-pause potenseringen af kontrol- og behandlede skiver blev vurderet for den intracellulære calciumfrigivelse fra det sarkoplasmatiske retikulum.Kontrolskiven viste ingen ændringer. I nærvær af calciseptin og nifedipin førte inhiberingen af L-type calciumkanaler til potensering af den første sammentrækning efter en pause på 50 sekunder, hvilket afspejler et højere relativt bidrag fra intracellulær calciumfrigivelse til total kontraktilitet. Den modsatte effekt blev observeret med Bay-K8644, som stimulerer indgangen af ekstracellulært calcium via L-type calciumkanaler.I kraftfrekvensrelationsanalyse blev der ikke observeret nogen ændring i kontrolskiven. Calciseptinbehandlingen ændrede ikke skivens evne til at følge stimuli ved en stigning i stimuleringsfrekvensen, når man sammenlignede data før og efter behandling. Sammenlignet med calciseptin forhindrede nifedipin en stigning i kontraktilitet ved højere tempohastigheder og reducerede den maksimale indfangningshastighed ved 80 bpm.Med Bay-K8644 blev der observeret øget sammentrækningskraft ved meget lave stimuleringsfrekvenser. Ved frekvenser højere end 50 bpm var sammentrækningskraften imidlertid lavere end under forbehandlingstilstanden. Udskåret væv skal hurtigt overføres til 4 grader kardioplegi.Efter udskæring skal plasttrekanter fastgøres vinkelret på fiberretningen. Forspændingen må ikke overstige 1500 millinewton. Biomimetisk skivekultur kan hjælpe forskere med at udnytte genetiske manipulationer samt cellebaserede terapier til at studere regenerering og reparation i voksen humant myokardium.

preparation-of-human-myocardial-tissue-for-long-term-cultivation

Research

JoVE Journal

Medicine

Corneal Donor Tissue Preparation for Endothelial Keratoplasty

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full thickness corneal transplantation has been the treatment to restore sight in those limited by corneal disease. Some disadvantages to this approach include a high degree of post-operative astigmatism, lack of predictable refractive outcome, and disturbance to the ocular surface. The development of Descemet's stripping endothelial keratoplasty (DSEK), transplanting only the posterior corneal stroma, Descemet's membrane, and endothelium, has dramatically changed treatment of corneal endothelial disease. DSEK is performed through a smaller incision; this technique avoids 'open sky' surgery with its risk of hemorrhage or expulsion, decreases the incidence of postoperative wound dehiscence, reduces unpredictable refractive outcomes, and may decrease the rate of transplant rejection3-6.
Initially, cornea donor posterior lamellar dissection for DSEK was performed manually1 resulting in variable graft thickness and damage to the delicate corneal endothelial tissue during tissue processing. Automated lamellar dissection (Descemet's stripping automated endothelial keratoplasty, DSAEK) was developed to address these issues. Automated dissection utilizes the same technology as LASIK corneal flap creation with a mechanical microkeratome blade that helps to create uniform and thin tissue grafts for DSAEK surgery with minimal corneal endothelial cell loss in tissue processing.
Eye banks have been providing full thickness corneas for surgical transplantation for many years. In 2006, eye banks began to develop methodologies for supplying precut corneal tissue for endothelial keratoplasty. With the input of corneal surgeons, eye banks have developed thorough protocols to safely and effectively prepare posterior lamellar tissue for DSAEK surgery. This can be performed preoperatively at the eye bank. Research shows no significant difference in terms of the quality of the tissue7 or patient outcomes8,9 using eye bank precut tissue versus surgeon-prepared tissue for DSAEK surgery. For most corneal surgeons, the availability of precut DSAEK corneal tissue saves time and money10, and reduces the stress of performing the donor corneal dissection in the operating room. In part because of the ability of the eye banks to provide high quality posterior lamellar corneal in a timely manner, DSAEK has become the standard of care for surgical management of corneal endothelial disease.
The procedure that we are describing is the preparation of the posterior lamellar cornea at the eye bank for transplantation in DSAEK surgery (Figure 1).

Endothelial corneal transplantation is a surgical technique for treatment of posterior corneal diseases. Mechanical microkeratome dissection to prepare tissue results in thinner, more symmetric grafts with less endothelial cell loss and improved outcomes. Dissections can be performed at the eye bank prior to corneal transplantation surgery.

Corneal donorvæv Forberedelse til Endothelial keratoplasti

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full ...

Working with Human Tissues for Translational Cancer Research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human specimens, precious samples must be handled with the greatest care in order to ensure integrity of organs, tissues, and cells to the highest degree. Unfortunately, tissue processing by definition requires extraction of tissues from the host, a change which can cause great cellular stress and have major repercussions on subsequent analyses. These stresses could result in the specimen being no longer representative of the site from which it was retrieved. Therefore, a strict protocol must be adhered to while processing these specimens to ensure representativeness. The desired assay(s) must also be taken into consideration in order to ensure that an optimal technique is used for sample processing. Outlined here is a protocol for tissue retrieval, processing and various analyses which may be performed on processed tissue in order to maximize downstream production from limited tissue samples.

Translational cancer research is dependent on extraction of human tissues. Much work has gone into optimizing extraction methods for ex vivo analysis. Here, we describe tissue processing methods allowing for maximal data output from limited samples.

Arbejde med Humane væv til Translationel kræftforskning

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human ...

Long-Term Catheterization of the Intestinal Lymph Trunk and Collection of Lymph in Neonatal Pigs

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal (central) efferent lymph. Importantly, the collection of central lymph from the intestine enables researchers to study both the mechanisms and lipid constitutes associated with lipid metabolism, intestinal inflammation and cancer metastasis, as well as cells involved in immune function and immunosurveillance. A ventral mid-line surgical approach permits excellent surgical exposure to the cranial abdomen and relatively easy access to the intestinal lymph trunk vessel that lies near the pancreas and the right ventral segment of the portal vein underneath the visceral aspect of the right liver lobe. The vessel is meticulously dissected and released from the surrounding fascia and then dilated with sutures allowing for insertion and subsequent securing of the catheter into the vessel. The catheter is exteriorized and approximately 1 L/24 hr of lymph is collected over a 7 day period. While this technique enables the collection of large quantities of central lymph over an extended period of time, the success depends on careful surgical dissection, tissue handling and close attention to proper surgical technique. This is particularly important with surgeries in young animals as the lymph vessels can easily tear, potentially leading to surgical and experimental failure. The video demonstrates an excellent surgical technique for the collection of intestinal lymph.

We present a surgical procedure to catheterize the intestinal lymph trunk in neonatal pigs to collect large quantities of lipid metabolism components from efferent lymph.

Langsigtet kateterisation af Intestinal lymfeknuder Trunk og Indsamling af lymfeknuder i neonatale Pigs

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal ...

Surgical Placement of Catheters for Long-term Cardiovascular Exercise Testing in Swine

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early cardiopulmonary dysfunction is difficult to diagnose, particularly in animal models, as cardiopulmonary function is often measured invasively, requiring anesthesia. As many anesthetic agents are cardiodepressive, subtle changes in cardiovascular function may be masked. In contrast, chronic instrumentation allows for measurement of cardiopulmonary function in the awake state, so that measurements can be obtained under quiet resting conditions, without the effects of anesthesia and acute surgical trauma. Furthermore, when animals are properly trained, measurements can also be obtained during graded treadmill exercise.
Flow probes are placed around the aorta or pulmonary artery for measurement of cardiac output and around the left anterior descending coronary artery for measurement of coronary blood flow. Fluid-filled catheters are implanted in the aorta, pulmonary artery, left atrium, left ventricle and right ventricle for pressure measurement and blood sampling. In addition, a 20 G&#160;catheter is positioned in the anterior interventricular vein to allow coronary venous blood sampling.
After a week of recovery, swine are placed on a motor-driven treadmill, the catheters are connected to pressure and flow meters, and swine are subjected to a five-stage progressive exercise protocol, with each stage lasting 3 min. Hemodynamic signals are continuously recorded and blood samples are taken during the last 30 sec of each exercise stage.
The major advantage of studying chronically instrumented animals is that it allows serial assessment of cardiopulmonary function, not only at rest but also during physical stress such as exercise. Moreover, cardiopulmonary function can be assessed repeatedly during disease development and during chronic treatment, thereby increasing statistical power and hence limiting the number of animals required for a study.

Here we present a protocol to assess cardiopulmonary function in awake swine, at rest and during graded treadmill exercise. Chronic instrumentation allows for repeated hemodynamic measurements uninfluenced by cardiodepressive anesthetic agents.

Kirurgisk Placering af Katetre til Langsigtet motionsture Afprøvning i Swine

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early ...

Long-term Blood Pressure Measurement in Freely Moving Mice Using Telemetry

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. Indeed, research focusing on the discovery of new potential therapeutic targets using genetically altered mice requires a reliable, long-term assessment of the systemic arterial pressure variation. Currently, the gold standard for obtaining long-term measurements of blood pressure in ambulatory mice uses implantable radio-transmitters, which require&#160;artery cannulation. This technique eliminates the need for tethering, restraining, or anesthetizing the animals which introduce stress and artifacts during data sampling. However, arterial blood pressure monitoring in mice via catheterization can be rather challenging due to the small size of the arteries. Here we present a step-by-step guide to illustrate the crucial key passages for a successful subcutaneous implantation of radio-transmitters and carotid artery cannulation in mice. We also include examples of long-term blood pressure activity taken from freely moving mice after a period of post-surgery recovery. Following this procedure will allow reliable direct blood pressure recordings from multiple animals simultaneously.

The goal of this protocol is to assess systemic blood pressure in conscious freely moving mice using implantable radio-telemetry devices.

Langsigtet blodtryksmåling i Frit Flytning Mus Brug Telemetri

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. ...

3D Whole-heart Myocardial Tissue Analysis

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other biologicals that enhance angio- or vasculogenesis into the infarct border zone (IBZ), tissue perfusion is improved, and the myocardium can be protected from further damage. For maximum therapeutic effect, it is hypothesized that the regenerative substance is best delivered to the IBZ. This requires accurate injections and has led to the development of new injection techniques. To validate these new techniques, we have designed a validation protocol based on myocardial tissue analysis. This protocol includes whole-heart myocardial tissue processing that enables detailed two-dimensional (2D) and three-dimensional (3D) analysis of the cardiac anatomy and intramyocardial injections. In a pig, myocardial infarction was created by a 90-min occlusion of the left anterior descending coronary artery. Four weeks later, a mixture of a hydrogel with superparamagnetic iron oxide particles (SPIOs) and fluorescent beads was injected in the IBZ using a minimally-invasive endocardial approach. 1 h after the injection procedure, the pig was euthanized, and the heart was excised and embedded in agarose (agar). After the solidification of the agar, magnetic resonance imaging (MRI), slicing of the heart, and fluorescence imaging were performed. After image post-processing, 3D analysis was performed to assess the IBZ targeting accuracy. This protocol provides a structured and reproducible method for the assessment of the targeting accuracy of intramyocardial injections into the IBZ. The protocol can be easily used when the processing of scar tissue and/or validation of the injection accuracy of the whole heart is desired.

This protocol describes a novel method for the 3D comparison of whole-heart myocardial tissue with MRI. This is designed for the accurate assessment of intramyocardial injections in the infarct border zone of a chronic porcine model of myocardial infarction.

3D Whole-hjerte myokardievævet Analyse

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other ...

Improvement of a Closed Chest Porcine Myocardial Infarction Model by Standardization of Tissue and Blood Sampling Procedures

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved drug to prevent or reduce myocardial I/R injury. The study and understanding of the pathophysiological mechanisms of myocardial I/R injury is essential to develop successful treatments. Large animal experiments are an important step in translational methods. The porcine model of acute myocardial infarction has been established and described by ourselves and others. We aimed to further improve the value of the model by focusing in detail on the sampling techniques for use in future experiments. Furthermore, we emphasize small but important steps that can affect the quality of the final results. To mimic the clinical situation of myocardial I/R injury, a percutaneous coronary intervention (PCI) catheter was inserted into the left anterior descending coronary artery (LAD) of an anesthetized pig. &#176;&#176;&#176;This model mimics acute myocardial infarction and PCI treatment in humans with the possibility of accurately determining the area at risk as well as the necrotic- and viable ischemic tissue. Here the model was used to investigate the effect of a bicyclic peptide inhibitor of FXIIa. The model can also be modified to allow longer reperfusion times to study later effects of myocardial infarction.

Here we demonstrate a protocol to standardize sampling procedures of an established porcine model of acute myocardial infarction in order to increase its translational value in the understanding of the pathophysiology of myocardial ischemia/reperfusion injury and to test novel drug candidates.

Forbedring af en lukket brystet svin myokardieinfarkt Model af standardisering af væv og blod prøveudtagningsprocedurer

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved ...

Analyzing Long-Term Electrocardiography Recordings to Detect Arrhythmias in Mice

Arrhythmias are common, affecting millions of patients worldwide. Current treatment strategies are associated with significant side effects and remain ineffective in many patients. To improve patient care, novel and innovative therapeutic concepts causally targeting arrhythmia mechanisms are needed. To study the complex pathophysiology of arrhythmias, suitable animal models are necessary, and mice have been proven to be ideal model species to evaluate the genetic impact on arrhythmias, to investigate fundamental molecular and cellular mechanisms, and to identify potential therapeutic targets.
Implantable telemetry devices are among the most powerful tools available to study electrophysiology in mice, allowing continuous ECG recording over a period of several months in freely moving, awake mice. However, due to the huge number of data points (&gt;1 million QRS complexes per day), analysis of telemetry data remains challenging. This article describes a step-by-step approach to analyze ECGs and to detect arrhythmias in long-term telemetry recordings using the software, Ponemah, with its analysis modules, ECG Pro and Data Insights, developed by Data Sciences International (DSI). To analyze basic ECG parameters, such as heart rate, P wave duration, PR interval, QRS interval, or QT duration, an automated attribute analysis was performed using Ponemah to identify P, Q, and T waves within individually adjusted windows around detected R waves. 
Results were then manually reviewed, allowing adjustment of individual annotations. The output from the attribute-based analysis and the pattern recognition analysis was then used by the Data Insights module to detect arrhythmias. This module allows an automatic screening for individually defined arrhythmias within the recording, followed by a manual review of suspected arrhythmia episodes. The article briefly discusses challenges in recording and detecting ECG signals, suggests strategies to improve data quality, and provides representative recordings of arrhythmias detected in mice using the approach described above.

Here we present a step-by-step protocol for a semiautomated approach to analyze murine long-term electrocardiography (ECG) data for basic ECG parameters and common arrhythmias. Data are obtained by implantable telemetry transmitters in living and awake mice and analyzed using Ponemah and its analysis modules.

Analyse af langsigtede elektrokardiografioptagelser for at detektere arytmier hos mus

Arrhythmias are common, affecting millions of patients worldwide. Current treatment strategies are associated with significant side effects and remain ...

Technique for Obtaining Mesenchymal Stem Cell from Adipose Tissue and Stromal Vascular Fraction Characterization in Long-Term Cryopreservation

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible source for regenerative clinical applications. Different protocols have been used to obtain adipose-derived stem cells. This article describes different steps of an improved time-saving protocol to obtain a more significant amount of ADSC, showing how to cryopreserve and thaw ADSC to obtain viable cells for culture expansion. One hundred milliliters of lipoaspirate were collected, using a 26 cm three-hole and 3 mm caliber syringe liposuction, from the abdominal area of nine patients who subsequently underwent elective abdominoplasty. The stem cells isolation was carried out with a series of washes with Dulbecco&#39;s Phosphate Buffered Saline (DPBS) solution supplemented with calcium and the use of collagenase. Stromal Vascular Fraction (SVF) cells were cryopreserved, and their viability was checked by immunophenotyping. The SVF cellular yield was 15.7 x 105 cells/mL, ranging between 6.1-26.2 cells/mL. Adherent SVF cells reached confluence after an average of 7.5 (&#177;4.5) days, with an average cellular yield of 12.3 (&#177; 5.7) x 105 cells/mL. The viability of thawed SVF after 8 months, 1 year, and 2 years ranged between 23.06%-72.34% with an average of 47.7% (&#177;24.64) with the lowest viability correlating with cases of two-year freezing. The use of DPBS solution supplemented with calcium and bag resting times for fat precipitation with a shorter time of collagenase digestion resulted in an increased stem cell final cellular yield. The detailed procedure for obtaining high yields of viable stem cells was more efficient regarding time and cellular yield than the techniques from previous studies. Even after a long period of cryopreservation, viable ADSC cells were found in the SVF.

The present protocol describes an improved methodology for ADSC isolation resulting in a tremendous cellular yield with time gain compared to the literature. This study also provides a straightforward method for obtaining a relatively large number of viable cells after long-term cryopreservation.

Teknik til opnåelse af mesenkymal stamcelle fra fedtvæv og stromal vaskulær fraktionskarakterisering ved langvarig kryopræservering

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible ...

Long-Term Continuous Measurement of Renal Blood Flow in Conscious Rats

The kidneys play a crucial role in maintaining the homeostasis of body fluids. The regulation of renal blood flow (RBF) is essential to the vital functions of filtration and metabolism in kidney function. Many acute studies have been carried out in anesthetized animals to measure RBF under various conditions to determine mechanisms responsible for the regulation of kidney perfusion. However, for technical reasons, it has not been possible to measure RBF continuously (24 h/day) in unrestrained unanesthetized rats over prolonged periods. These methods allow the continuous determination of RBF over many weeks while also simultaneously recording blood pressure (BP) with implanted catheters (fluid-filled or by telemetry). RBF monitoring is carried out with rats placed in a circular servo-controlled rat cage that enables the unrestrained movement of the rat throughout the study. At the same time, the tangling of cables from the flow probe and arterial catheters is prevented. Rats are first instrumented with an ultrasonic flow probe placement on the left renal artery and an arterial catheter implanted in the right femoral artery. These are routed subcutaneously to the nape of the neck, and connected to the flowmeter and pressure transducer, respectively, to measure RBF and BP. Following surgical implantation, rats are immediately placed in the cage to recover for at least one week and stabilize the ultrasonic probe recordings. Urine collection is also feasible in this system. The surgical and post-surgical procedures for continuous monitoring are demonstrated in this protocol.

The present protocol describes a long-term continuous measurement of renal blood flow in conscious rats and simultaneously recording blood pressure with implanted catheters (fluid-filled or by telemetry).