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 ble finansiert av DZHK-tilskudd 81Z0600207 (JH, PS og DM) og 81X2600253 (AD og TS).
Forfatterne vil gjerne takke Claudia Fahney, Mei-Ping Wu og Matthias Semisch for deres støtte til å forberede oppsettene, samt for regelmessig vedlikehold av vevsdyrkingen.

Vevssamling for eksperimentene beskrevet her ble godkjent av Institutional Review Boards ved Universitetet i München og Ruhr-Universitetet Bochum. Studier ble utført i henhold til Helsinkideklarasjonens retningslinjer. Pasientene ga skriftlig informert samtykke før vevsinnsamling.
1. Oppkjøp av vev
<ol><li>Få menneskelig vev fra pasienter som gjennomgår hjertetransplantasjon eller hjertekirurgi.</li>
	<li>Før du anskaffer vevet, klargjør du 2 liter kardioplegisk løs...

<ol>
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(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 fremskritt i dyrking av kardiomyocytter. Imidlertid er 3D-dyrking av kardiomyocytter med intakt geometri ennå ikke godt etablert. Sammenlignet med tidligere protokoller anvendt for ex vivo dyrking av myokardvev, ligner protokollen som vi beskrev her vevets in vivo-miljø nærmere. Videre gir anvendelsen av for- og etterbelastning et mer biomimetisk miljø. Vi er i stand til fullt ut å analysere og forstå kontinuerlig registrering av kontraksjonsdata...

I løpet av det siste tiåret har in vitro dyrking av myokardceller gjort store fremskritt, alt fra 2D og tredimensjonale (3D) teknikker til bruk av organoider og induserte pluripotente stamceller differensiert til hjertemyocytter 1,2,3. Ex vivo og primærcelledyrking har vist seg å være av stor verdi, spesielt for genetiske studier og legemiddelutvikling 4,5,6. Bruk av menneskelig vev forbedrer translasjonsverdien av resultatene. Langsiktig 3D-dyrking av myokardvev med intakt geometri er imidlertid ikke godt etablert. Den intakte geometrien er en nøkkelfunksjon for å etterligne in vivo-forhold, da riktig hjertefunksjon, kommunikasjon mellom forskjellige celler, samt cellematrise-interaksjoner er forutsetninger. Dyrking av myokardvev gikk gjennom ulike faser av utviklingen. Suksessraten og stabiliteten til ex vivo myokardvevsdyrking var i utgangspunktet ganske lav, men nyere tilnærminger har gitt lovende resultater 7,8,9,10,11.
Blant disse var Fischer og medarbeidere de første som demonstrerte at levedyktighet og kontraktil ytelse av humant myokardvev kan opprettholdes i ex vivo celledyrking i mange uker7. Deres teknikk var basert på tynne vevsskiver skåret fra eksplantert humant myokardium, som ble montert i nyutviklede dyrkingskamre som ga definerte biomekaniske forhold og kontinuerlig elektrisk stimulering. Denne kultiveringsmetoden ligner in vivo-funksjonen til myokardvev, og har blitt reprodusert av flere uavhengige forskningsgrupper 2,12,13,14,15. Det er viktig at kamrene som Fischer og medarbeidere brukte også muliggjorde kontinuerlig registrering av utviklede krefter i opptil 4 måneder, og dermed åpnet enestående muligheter for fysiologisk og farmakologisk forskning på intakt humant myokard7.
Lignende teknikker ble uavhengig utviklet av andre grupper og anvendt på mennesker, rotter, svin og kaninmyokard 7,10,11. Pitoulis og medarbeidere utviklet senere en mer fysiologisk metode, som reproduserer den normale kraftlengderelasjonen under en kontraksjonssyklus, men er mindre egnet for høy gjennomstrømningsanalyse16. Som sådan kan den generelle tilnærmingen til biomimetisk dyrking betraktes som et ytterligere skritt inn i reduksjon, raffinering og erstatning (3R) av dyreforsøk.
Utnyttelse av dette potensialet krever imidlertid standardiserte prosedyrer, analyser med høyt innhold og et høyt gjennomstrømningsnivå. Vi presenterer en teknikk som kombinerer automatisert kutting av levende humant myokardium med in vitro vedlikehold i et biomimetisk dyrkingssystem som har blitt kommersielt tilgjengelig (se Materialtabell). Med den foreslåtte tilnærmingen er antall individuelle skiver som kan genereres fra en enkelt transmural myokardprøve bare begrenset av behandlingstiden. Et eksemplar av tilstrekkelig størrelse og kvalitet (3 cm x 3 cm) gir ofte 20-40 vevsskiver som er beleilig kuttet med en automatisert vibratom. Disse skivene kan plasseres i dyrkingskamre som tilhører systemet. Kamrene tillater elektrisk stimulering, hvis parametere kan moduleres (dvs. pulsvarighet, polaritet, hastighet og strøm), samt justering av for- og etterbelastning ved hjelp av fjærledninger inne i kamrene. Sammentrekningen av hver skive registreres fra bevegelsen av en liten magnet festet til en fjærtråd og vises som en tolkbar graf. Data kan til enhver tid registreres og analyseres ved hjelp av fritt tilgjengelig programvare. Bortsett fra den konstante baseline pacing, kan planlagte protokoller utføres for å funksjonelt vurdere deres ildfaste periode, stimuleringsterskel, post-pause-potensiering og kraftfrekvensforhold.
Denne langsiktige biomimetiske dyrkingen av flere myokardskiver fra et individuelt hjerte baner vei for fremtidig ex vivo-forskning i både menneskelig og animalsk vev, og letter screeningen for terapeutiske og kardiotoksiske legemiddeleffekter i kardiovaskulær medisin. Det har allerede blitt brukt på ulike eksperimentelle tilnærminger 2,12,13,15. Her gir vi en detaljert trinnvis beskrivelse av fremstillingen av menneskelig vev og gir løsninger for ofte oppståtte dyrkingsproblemer.

<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

Kardiomyocyttdyrking har sett et stort antall utviklinger, alt fra todimensjonal (2D) celledyrking til iPSC-avledede organoider. I 2019 ble det vist en ex vivo måte å dyrke myokardskiver oppnådd fra menneskelige hjerteprøver, mens de nærmet seg in vivo tilstand av myokardial sammentrekning. Disse prøvene stammer hovedsakelig fra hjertetransplantasjoner eller plasseringer av venstre ventrikkelhjelpemidler. Ved hjelp av en vibratom og et spesialutviklet dyrkingssystem plasseres 300 μm tykke skiver mellom en fast og en fjærtråd, noe som muliggjør stabil og reproduserbar dyrking i flere uker. Under dyrking stimuleres skivene kontinuerlig i henhold til individuelle innstillinger. Sammentrekninger kan vises og registreres i sanntid, og farmakologiske midler kan lett påføres. Brukerdefinerte stimuleringsprotokoller kan planlegges og utføres for å vurdere vitale kontraksjonsparametere som potensering etter pause, stimuleringsterskel, kraftfrekvensforhold og ildfast periode. Videre muliggjør systemet en variabel innstilling før og etter belastning for en mer fysiologisk dyrking.
Her presenterer vi en trinnvis veiledning om hvordan man genererer en vellykket langsiktig dyrking av menneskelige venstre ventrikulære myokardskiver, ved hjelp av en kommersiell biomimetisk dyrkingsløsning.

Sammentrekningen av myokardskivene ble vist på dataskjermen etter innsetting av dyrkingskammeret i den tilsvarende kontakten (figur 3). Sammentrekning av de menneskelige myokardskivene startet umiddelbart etter stimulering. Skivene hyperkontrakteres i 5-10 min. Dette var synlig som en økning av diastoliske krefter, forårsaket av en tonisk kontraktur av skadede vevsfraksjoner. Denne prosessen ble i varierende grad tilbakeført innen 1-1,5 time. Etter stabilisering viste humane LV-vevsskive...

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 presenterer en protokoll for ex vivo dyrking av humant ventrikkel myokardvev. Det muliggjør detaljert analyse av kontraksjonskraft og kinetikk, samt anvendelse av pre- og etterbelastning for å etterligne det in vivo fysiologiske miljøet nærmere.

Forberedelse av humant myokardvev for langsiktig dyrking

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

Ved hjelp av denne protokollen kan humane venstre ventrikulære vevsskiver dyrkes ex vivo i et biomimetisk kammer. Påføringen av pre- og afterloads forbedrer likheten til det fysiologiske miljøet. Denne teknikken tillater kontinuerlig registrering av vevskontraksjon.Brukerdefinerte stimuleringsprotokoller tillater vurdering av vitale kontraksjonsparametere som potensiering etter pause, stimuleringsterskel, kraftfrekvensforhold og ildfast periode. Den langsiktige dyrkingen av myokardskiver, ved hjelp av dette oppsettet, vil bane vei for fremtidig ex vivo-forskning, noe som letter denne screeningen for terapeutiske og kardiotoksiske legemiddeleffekter i kardiovaskulær medisin. For å begynne, senk dyrkingskamrene og grafittelektrodene i en liter av en 10% isopropanolløsning og omrør over natten.Neste dag, overfør kamrene til en 100% isopropanolløsning i 3 minutter, og la deretter kamrene og grafittelektrodene lufttørke under en laminær strømningshette. Fest et kretskort til hvert kammer i henhold til de tilgjengelige posisjonene på vippemaskinen. Plasser to grafittelektroder på kretskortet i henhold til produsentens instruksjoner, og plasser deretter et 35 millimeter petriskållokk på kammeret for å forhindre infeksjon.Deretter fyller du skjærebrettet opptil 90 til 95% med skivebuffer. Overfør vevsprøven til en 100 millimeter petriskål fylt med kald skivebuffer, og legg fatet på en kjøleplate ved 4 grader Celsius. Fjern endokardtrabeculae ved å holde endokardiet med pinsett, og bruk deretter saks til å kutte bort ca. 3 millimeter endokardvev.Fest den kuttede vevsprøven, endokardial side opp til en 2-til-2-centimeter gummiplaster ved hjelp av fire 0,9 x 70 millimeter 20-gauge nåler som er festet i en firkantet stilling. Forsikre deg om at den diagonale kanten av hver nålespiss peker innover, forbedrer fiksering og forhindrer skade på myokardiet. Bruk en skalpell til å skjære bort alt overflødig vev utenfor de fire nålefirkantene.Bruk pinsett til å plassere den trimmede prøven på et sterilt stykke vev i 10 sekunder for å fjerne overflødig skivebuffer. Fjern deretter agarosesprøyten fra vannbadet og senk prøven i agarose. La agarose størkne i fem minutter på en kjøleplate.Den endokardiale siden av prøven må være synlig i agarose. Bruk pinsett, plasser epikardiale siden av prøven inneholdt i agarose på toppen av det limte området, og trykk deretter forsiktig på den agaroseholdige prøven fra toppen med et stump verktøy uten å kutte eller skade agarose. La limet stivne i 1 minutt.Sett vibrasjonsamplituden til 1 millimeter, innledende skjærehastighet til 0,07 millimeter per sekund, og tykkelsen på skiven til 300 mikron. Etter å ha koblet dyrkingssystemet til en datamaskin, start det tilsvarende programmet. Sett vippehastigheten til 60 o / min og forhåndsinnstilt stimuleringsparametrene.For menneskelige hjerteskiver, sett standardstimuleringen til bifasiske impulser med 50 milliamper eller strøm bestående av 3 millisekunder positiv strøm, 1 millisekund pause og en 3 millisekund puls av invertert strøm med en pacing rate på 30 slag per minutt eller bpm, kontroller deretter elektrodeindikatorene til programvaren og kontroller at elektrodene i dyrkingskamrene fungerer som de skal. Bruk pinsett, skille agarose fra vevet. Unngå å berøre vevet og håndter det forsiktig, da eventuelle skader på vevet vil redusere suksessraten for dyrkingen.For å feste to plasttrekanter til en prøve, legg 1 mikroliter lim på et sterilt petriskållokk. Bruk en kroket pinsett for å plukke opp en autoklavert plasttrekant. Dypp raskt forkanten av trekanten i limet og lim trekanten på prøven vinkelrett på kardiomyocyttjusteringen.Gjenta for den andre trekanten. Bruk en skalpell, trim av vev som overskrider trekantbredden og legg skiven med de to monterte trekantene tilbake i skjærebrettet som inneholder skivebuffer. Fjern det middels fylte dyrkingskammeret fra inkubatoren.Velg en forberedt skive og sett den inn i kammeret ved å koble en trekant til hver pinne. Juster avstanden mellom monteringspinnene til prøvestørrelsen. Kontroller at prøven er nedsenket i mediet.Etter å ha plassert parabolen på vippemaskinen, reduser forspenningen ved å vri justeringsskruen mot klokken til grunnlinjen til den tilsvarende grafen på dataskjermen ikke endres lenger, og øk deretter forspenningen eller spenningen forsiktig ved å vri justeringsskruen med klokken. For kamre med høy stivhet, fortsett til den tilsvarende grunnlinjen i grafen har økt med 1.000 til 1.200 enheter tilsvarende en millinewton forspenning. Etter å ha tilberedt det friske dyrkingsmediet, forvarm mediet i et vannbad eller varmluftinkubator ved 37 grader Celsius i 30 til 45 minutter.Fjern medium fra kammeret, forlater ca 0,8 milliliter i kammeret, deretter legge 1,6 milliliter friskt medium til samme kammer, noe som gjør et totalt volum av medium til 2,4 milliliter per kammer. Til slutt plasserer du dekselet på kammeret tilbake og plasserer dyrkingskammeret i sin respektive posisjon. Avlesningen av sammentrekningen av fem myokardskiver under en typisk stimulering besto av fire forskjellige seksjoner av potensering etter pause, stimuleringsterskel, kraftfrekvensrelasjon og ildfast periode.Sammenlignet med kontroll ble kontraksjonskraften til skivene behandlet med kalsiumantagonister, nifedipin og kalsiseptin, redusert innen 10 minutter. I motsetning til dette økte den spenningsstyrte kalsiumkanalagonisten, Bay-K8644, sammentrekningskraften. Potenseringen av kontroll- og behandlede skiver etter pause ble vurdert for intracellulær kalsiumfrigjøring fra sarkoplasmatisk retikulum.Kontrollstykket viste ingen endringer. I nærvær av kalsiseptin og nifedipin førte inhiberingen av L-type kalsiumkanaler til potensering av den første sammentrekningen etter en pause på 50 sekunder, noe som gjenspeiler et høyere relativt bidrag av intracellulær kalsiumfrigivelse til total kontraktilitet. Den motsatte effekten ble observert med Bay-K8644, som stimulerer innføringen av ekstracellulært kalsium via kalsiumkanalene av L-typen.I kraftfrekvensrelasjonsanalyse ble det ikke observert noen endring i kontrollskiven. Kalsiseptinbehandlingen endret ikke skjærets evne til å følge stimuliene ved en økning av stimuleringsfrekvensen ved sammenligning av data før og etter behandling. Sammenlignet med kalsiseptin forhindret nifedipin en økning i kontraktilitet ved høyere pacinghastigheter og reduserte maksimal fangstrate ved 80 bpm.Med Bay-K8644 ble det observert økt kontraksjonskraft ved svært lave stimuleringsfrekvenser. Ved frekvenser høyere enn 50 slag/minutt var imidlertid kontraksjonskraften lavere enn under tilstanden før behandling. Utskåret vev skal raskt overføres til 4 grader kardioplegi.Etter kutting må plast trekanter festes vinkelrett på fiberretningen. Forhåndsbelastningen bør ikke overstige 1500 millinewton. Biomimetisk skivekultur kan hjelpe forskere til å utnytte genetiske manipulasjoner samt cellebaserte terapier for å studere regenerering og reparasjon i voksen menneskelig myokard.

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 Donor Tissue Forberedelse til endotel keratoplasty

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.

Arbeide med menneskelig vev for translasjonell kreftforskning

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.

Long-Term Kateterisering av Intestinal Lymph Trunk og innkreving av lymfe i 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 ...

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 Plassering av katetre for langsiktig kardiovaskulær trening Testing 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.

Langsiktig blodtrykksmåling i fritt bevegelige Mus Bruke 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 Hjerteinfarkt vevet 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 av en lukket kiste svin hjerteinfarkt modell av standardisering av vev og blod utvalgstrekking

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.

Analysere langsiktige elektrokardiografiopptak for å oppdage 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.

Teknikk for å oppnå mesenkymal stamcelle fra fettvev og stromal vaskulær fraksjonskarakterisering ved langvarig kryopreservering

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).