Name: microdissection of primary renal tissue segments and incorporation with novel scaffold-free construct technology
Uploaded: 2018-03-27T13:30:00
Description: Watch this Scientific Journal Video about Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology at JoVE.com

NIH institutionele postdoctorale opleiding Grant, NIH-HL-007260

Alle dierlijke chirurgische procedures die hieronder worden beschreven zijn door de institutionele Animal Care en gebruiken Comité (IACUC) op de medische universiteit van South Carolina voorafgaand aan elk dier chirurgie of het gebruik van alle dierlijke weefsels goedgekeurd.
1. lymfkliertest Nefrectomie
<ol><li>Don een chirurgisch masker en bouffant kap voor het minimaliseren van het risico van besmetting. Steriliteit handhaven tijdens de set-up van het chirurgische gebied.<ol>
<li>Plaats ...

<ol>
	<li>Jha, V., 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=Chronic+kidney+disease:+global+dimension+and+perspectives.">Chronic kidney disease: global dimension and perspectives.</a> Lancet. 382 (9888), 260-272 (2013).</li><li>2016 USRDS Annual Data Report: Epidemiology of Kidney Disease in the United States. U.S.R.D Available from: <a target="_blank" href="https://www.usrds.org/2016/download/v2_ESRD_16.pdf">https://www.usrds.org/2016/download/v2_ESRD_16.pdf</a> (2016)</li><li>Hart, 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=OPTN/SRTR+2015+Annual+Data+Report:+Kidney.">OPTN/SRTR 2015 Annual Data Report: Kidney.</a> American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 17, 21-116 (2017).</li><li>de Vries, E. F., Rabelink, T. J., van den Hout, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+the+Cost-Effectiveness+of+Delaying+End-Stage+Renal+Disease.">Modelling the Cost-Effectiveness of Delaying End-Stage Renal Disease.</a> Nephron. 133 (2), 89-97 (2016).</li><li>Lefebvre, P., Duh, M. S., Mody, S. H., Bookhart, B., Piech, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+economic+impact+of+epoetin+alfa+therapy+on+delaying+time+to+dialysis+in+elderly+patients+with+chronic+kidney+disease.">The economic impact of epoetin alfa therapy on delaying time to dialysis in elderly patients with chronic kidney disease.</a> Disease management : DM. 10 (1), 37-45 (2007).</li><li>Mennini, F. S., Russo, S., Marcellusi, A., Quintaliani, G., Fouque, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Economic+effects+of+treatment+of+chronic+kidney+disease+with+low-protein+diet.">Economic effects of treatment of chronic kidney disease with low-protein diet.</a> Journal of renal nutrition : the official journal of the Council on Renal Nutrition of the National Kidney Foundation. 24 (5), 313-321 (2014).</li><li>Moon, K. H., Ko, I. K., Yoo, J. J., Atala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney+diseases+and+tissue+engineering.">Kidney diseases and tissue engineering.</a> Methods. 99, 112-119 (2016).</li><li>Wobma, H., Vunjak-Novakovic, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Engineering+and+Regenerative+Medicine+2015:+A+Year+in+Review.">Tissue Engineering and Regenerative Medicine 2015: A Year in Review.</a> Tissue engineering. Part B, Reviews. 22 (2), 101-113 (2016).</li><li>Langer, R., Vacanti, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+tissue+engineering.">Advances in tissue engineering.</a> Journal of pediatric surgery. 51 (1), 8-12 (2016).</li><li>Jakab, 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=Tissue+engineering+by+self-assembly+and+bio-printing+of+living+cells.">Tissue engineering by self-assembly and bio-printing of living cells.</a> Biofabrication. 2 (2), 022001 (2010).</li><li>Fisher, M. B., Mauck, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+and+regenerative+medicine:+recent+innovations+and+the+transition+to+translation.">Tissue engineering and regenerative medicine: recent innovations and the transition to translation.</a> Tissue engineering. Part B, Reviews. 19 (1), 1-13 (2013).</li><li>Stewart, D. E., Garcia, V. C., Rosendale, J. D., Klassen, D. K., Carrico, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosing+the+Decades-Long+Rise+in+the+Deceased+Donor+Kidney+Discard+Rate+in+the+United+States.">Diagnosing the Decades-Long Rise in the Deceased Donor Kidney Discard Rate in the United States.</a> Transplantation. 101 (3), 575-587 (2017).</li><li>Mohan, S., 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+weekend+effect+alters+the+procurement+and+discard+rates+of+deceased+donor+kidneys+in+the+United+States.">The weekend effect alters the procurement and discard rates of deceased donor kidneys in the United States.</a> Kidney international. 90 (1), 157-163 (2016).</li><li>Orlando, 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=Discarded+human+kidneys+as+a+source+of+ECM+scaffold+for+kidney+regeneration+technologies.">Discarded human kidneys as a source of ECM scaffold for kidney regeneration technologies.</a> Biomaterials. 34 (24), 5915-5925 (2013).</li><li>Katari, R., 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=Renal+bioengineering+with+scaffolds+generated+from+human+kidneys.">Renal bioengineering with scaffolds generated from human kidneys.</a> Nephron. Experimental nephrology. 126 (2), 119 (2014).</li><li>Kim, S. S., Park, H. J., Han, J., Choi, C. Y., Kim, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+tissue+reconstitution+by+the+implantation+of+renal+segments+on+biodegradable+polymer+scaffolds.">Renal tissue reconstitution by the implantation of renal segments on biodegradable polymer scaffolds.</a> Biotechnology letters. 25 (18), 1505-1508 (2003).</li><li>Guimaraes-Souza, N. K., Yamaleyeva, L. M., AbouShwareb, T., Atala, A., Yoo, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+reconstitution+of+human+kidney+structures+for+renal+cell+therapy.">In vitro reconstitution of human kidney structures for renal cell therapy.</a> Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association. 27 (8), 3082-3090 (2012).</li><li>Kelley, R., 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=Tubular+cell-enriched+subpopulation+of+primary+renal+cells+improves+survival+and+augments+kidney+function+in+rodent+model+of+chronic+kidney+disease.">Tubular cell-enriched subpopulation of primary renal cells improves survival and augments kidney function in rodent model of chronic kidney disease.</a> American journal of physiology. Renal physiology. 299 (5), 1026-1039 (2010).</li><li>Czajka, C. A., Drake, 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=Self-assembly+of+prevascular+tissues+from+endothelial+and+fibroblast+cells+under+scaffold-free,+nonadherent+conditions.">Self-assembly of prevascular tissues from endothelial and fibroblast cells under scaffold-free, nonadherent conditions.</a> Tissue engineering. Part A. 21 (1-2), 277-287 (2015).</li><li>Rhett, J. M., Wang, H., Bainbridge, H., Song, L., Yost, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexin-Based+Therapeutics+and+Tissue+Engineering+Approaches+to+the+Amelioration+of+Chronic+Pancreatitis+and+Type+I+Diabetes:+Construction+and+Characterization+of+a+Novel+Prevascularized+Bioartificial+Pancreas.">Connexin-Based Therapeutics and Tissue Engineering Approaches to the Amelioration of Chronic Pancreatitis and Type I Diabetes: Construction and Characterization of a Novel Prevascularized Bioartificial Pancreas.</a> Journal of diabetes research. 2016, 7262680 (2016).</li><li>Al-Awqati, Q., Oliver, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+in+the+kidney.">Stem cells in the kidney.</a> Kidney international. 61 (2), 387-395 (2002).</li><li>Aboushwareb, T., 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=Erythropoietin+producing+cells+for+potential+cell+therapy.">Erythropoietin producing cells for potential cell therapy.</a> World journal of urology. 26 (4), 295-300 (2008).</li></ol>

Methoden die worden gebruikt om ingenieur levend renale weefsel constructies sterk uiteen ten aanzien van zowel het type van cellen en biomaterialen gebruikt, en in veel gevallen lopen zijn verouderd of niet goed gekarakteriseerd in de literatuur7. Terwijl velen maken gebruik van benaderingen van de cel van de stam of Recapitulerend afzonderlijke onderdelen van de renale architectuur in isolement, het vooruitzicht van kunstmatig het herscheppen van een gehele orgaan met meer dan 26 verschillende g...

Chronische nierziekten (CKD) is één van de huidige grote volksgezondheid uitdagingen wereldwijd1. De prevalentie van Cementovenstof in de Verenigde Staten is meer dan 14% van de totale bevolking, met meer dan 600.000 Amerikanen lijden aan de meest ernstige vorm, eind-fase nierziekte (ESRD)2. De huidige behandelingsopties beschikbaar voor mensen met ESRD omvatten dialyse en de nieren transplantatie. Hoewel ongeveer 25.000 patiënten ondergaan niertransplantatie elk jaar, zijn een groot aantal patiënten toegevoegd jaarlijks leidt tot een grote ongelijkheid tussen degenen die wachten op een levensreddende orgel en die ontvangende transplantatie3. Naast haar ernstige negatieve gevolgen op de lange levensduur en kwaliteit van leven wordt dialyse geassocieerd met een verbazingwekkende financiële lasten. In 2014 bedroeg Medicare betaald vorderingen meer dan $30 miljard voor ESRD patiënten2. Met de levering van een beperkt orgel en geen duidelijk dalende bij patiënten waarbij dialyse, onderzoek gericht op het identificeren van alternatieve oplossingen voor dialyse en transplantatie zijn ooit belangrijk. Zelfs een relatief korte vertraging in de noodzaak van dialyse stijgt van een patiënt aantal kwaliteit-aangepast levensjaren en productiviteit aanzienlijk terwijl het uitstel van de dialyse-gerelateerde kosten4,5,6.
Oplossingen voor functionele weefsel schade, zoals die in ESRD, worden momenteel bestudeerd in de weefselkweek en regeneratieve geneeskunde laboratoria, met gevarieerd benaderingen variërend van steiger gebaseerde organoid fabricage tot hele orgel engineering met behulp van decellularized orgel structuren voor cellulaire implantatie7,8,9,10,11. Recapitulerend complexe renale structuren van marginale of afgedankte nieren is slechts gedeeltelijk onderzocht. In feite, is bijna 20% van de nieren voor transplantatie aangeschaft teruggegooid voor verschillende redenen12,13. De functionele nier weefsel van deze vermeende protheses kan worden gebruikt en opgenomen in één of meerdere weefsel-engineered constructies. Voorafgaande studies hebben aangetoond dat de haalbaarheid van het werken met deze afgedankte organen, gebruik makend van de nieren voor de extra-cellulaire matrix voor weefsel engineering doeleinden14,15. Weinigen hebben echter de primaire nephronal weefsel van gezonde nieren gebruikt voor weefselengineering doeleinden16,17,18.
Een methode die hierboven worden beschreven door Kim et al. omvat isolatie van renale "segmenten" vanaf gezonde rat nieren, die vervolgens werden zaadjes op polyglycolic zuur (PGA) steigers voor construct fabricage16. Echter weinig informatie wordt gegeven met betrekking tot de exacte dissectie methodologie en segmenten werden verkregen uit een combinatie van fijn hakken en filtratie. We beschrijven een wijziging van dit protocol, dat ook produceert discrete renale segmenten met intact nephronal architectuur, maar in plaats daarvan vertrouwt op microdissection technieken. Nephrectomies worden uitgevoerd op levende volwassen muizen, waarna de nieren worden overgedragen aan de dissectie Microscoop waar de renale capsule wordt verwijderd en het weefsel is verder ontleed. Lijst 30½ G naalden worden gebruikt als snijden instrumenten en ook als gidsen helpend in dissectie, als de naald diameter is gelijk aan de diameter van de doelstelling van de renale segmenten. De geïsoleerde, in dit geval lymfkliertest, renale segmenten behouden levensvatbaarheid in cultuur en nemen met steiger-vrije endotheel-fibroblast cellulaire constructies19. Deze constructies zijn eerder gebruikt om andere organen, met inbegrip van een bio-artificiële pancreas20ingenieur.

<table border="0" cellpadding="0" cellspacing="0" style="width:707px;" width="706">
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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Non-fenestrated Sterile Field</td>
			<td style="width:181px;">Busse Hospital Disposables</td>
			<td style="width:153px;">696</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Fenestrated Sterile Field</td>
			<td style="width:181px;">Busse Hospital Disposables</td>
			<td style="width:153px;">697</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Halsted Mosquito Forceps 5 Curved</td>
			<td style="width:181px;">Miltex</td>
			<td style="width:153px;">Mil-7-4</td>
			<td>&#34;Hemostat&#34; in manuscript</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:173px;">Extra Fine Graefe Forceps, Curved with teeth</td>
			<td style="width:181px;">Fine Science Tools</td>
			<td style="width:153px;">11155-10</td>
			<td>Fine forceps with teeth</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:173px;">Extra Fine Graefe Forceps, Serrated (without teeth)</td>
			<td style="width:181px;">Fine Science Tools</td>
			<td style="width:153px;">11152-10</td>
			<td>Fine forceps without teeth</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Fine Scissors - Tungsten Carbide</td>
			<td style="width:181px;">Fine Science Tools</td>
			<td style="width:153px;">14568-09</td>
			<td>Iris Scissors</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:173px;">Betadine Surgical Scrub with Pump, Povidone-iodine 7.5%</td>
			<td style="width:181px;">Purdue Products L.P.</td>
			<td style="width:153px;">67618-151-17</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Sterile Cotton Gauze Pad (4&#34; x 4&#34;)</td>
			<td style="width:181px;">Fisher Healthcare</td>
			<td style="width:153px;">22-415-469</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Dulbecco&#39;s Phosphate Buffered Solution</td>
			<td style="width:181px;">Corning</td>
			<td style="width:153px;">21-030-CV</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Penicillin/Streptomycin Solution, 100X</td>
			<td style="width:181px;">Corning</td>
			<td style="width:153px;">30-002-Cl</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Isoflurane, USP</td>
			<td style="width:181px;">Manufacturer: Piramal, Distributor: McKesson</td>
			<td style="width:153px;">2254845</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Nair Hair Remover</td>
			<td style="width:181px;">Nair</td>
			<td style="width:153px;">22600-23307</td>
			<td>Hair Removal Cream in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">200 Proof Ethanol</td>
			<td style="width:181px;">Decon Laboratories</td>
			<td style="width:153px;">2705</td>
			<td>Diluted to 70% Ethanol Solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">BioLite 60mm Tissue Culture Dish</td>
			<td style="width:181px;">Themo-Scientific</td>
			<td style="width:153px;">130181</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Press&#39;n Seal</td>
			<td style="width:181px;">Glad</td>
			<td style="width:153px;">12587-70441</td>
			<td>Applied to Stereoscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">SZX16 Stereo Microscope</td>
			<td style="width:181px;">Olympus</td>
			<td style="width:153px;">SZX16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Fiber Optic Illuminator</td>
			<td style="width:181px;">Cole Parmer</td>
			<td style="width:153px;">41720-20</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:173px;">Self-Supporting Dual-Light Pipe, 23&#34; L Gooseneck</td>
			<td style="width:181px;">Cole Parmer</td>
			<td style="width:153px;">EW-41720-60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Scalpel Handle #3</td>
			<td style="width:181px;">Miltex</td>
			<td style="width:153px;">Mil-4-7</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:173px;">Sterile Rib-Back Carbon Steel Blade, Blade Size 15</td>
			<td style="width:181px;">Bard-Parker</td>
			<td style="width:153px;">371115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">31 1/2 Gauge Needle</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:153px;">14-826F</td>
			<td style="width:199px;">Becton Dickinson 305106</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td style="width:181px;">Corning</td>
			<td style="width:153px;">10-017-CV</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Fetal Select 100% Bovine Serum</td>
			<td style="width:181px;">Atlas Biologicals</td>
			<td style="width:153px;">FS-0500-AD</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Normal Human Dermal Fibroblasts</td>
			<td style="width:181px;">Lonza</td>
			<td style="width:153px;">CC-2511</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:173px;">Human Adipose Microvascular Endothelial Cells</td>
			<td style="width:181px;">Sciencell Research Laboratories</td>
			<td style="width:153px;">7200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Surgical Loupes (2.5x)</td>
			<td style="width:181px;">Orascoptic</td>
			<td style="width:153px;">(N/A) Custom Order</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:173px;">FGM-2 (Fibroblast Basal Medium with FGM-2 SingleQuots Added)</td>
			<td style="width:181px;">Lonza</td>
			<td style="width:153px;">CC-3131, CC-4126</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:173px;">EGM-2 (Endothelial Basal Medium with EGM-2 SingleQuots Added)</td>
			<td style="width:181px;">Lonza</td>
			<td style="width:153px;">CC-3156, CC-4176</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:173px;">Live/Dead Viability/Cytotoxicity Kit for Mammalian Cells</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:153px;">L3224</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Anti-Cytokeratin-18 Antibody</td>
			<td style="width:181px;">Abcam</td>
			<td style="width:153px;">ab668</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Goat anti-Mouse IgG, Alexa Fluor 633</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:153px;">A-21052</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Goat anti-Rabbit IgG, Alexa Fluor 546</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:153px;">A-11010</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Anti-Von Willebrand Factor Antibody</td>
			<td style="width:181px;">Abcam</td>
			<td style="width:153px;">ab6994</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:173px;">Albumin, Fluorescein isothiocyanate Conjugate</td>
			<td style="width:181px;">Sigma Aldrich</td>
			<td style="width:153px;">A9771-50MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Hoescht 33342</td>
			<td style="width:181px;">BD Pharmingen</td>
			<td style="width:153px;">561908</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Background Buster</td>
			<td style="width:181px;">Innovex Biosciences</td>
			<td style="width:153px;">NB306</td>
			<td></td>
		</tr>
	</tbody>
</table>

microdissection of primary renal tissue segments and incorporation with novel scaffold-free construct technology

Niertransplantatie is nu een reguliere therapie voor einde-fase nierziekte. Met ongeveer 96.000 mensen op de wachtlijst en slechts een kwart van deze patiënten bereiken van transplantatie, is er echter een grote behoefte voor alternatieven voor degenen met falende organen. Ter vermindering van de schadelijke gevolgen van dialyse samen met de totale gezondheidskosten die het oploopt, is actief onderzoek voortdurend op zoek naar alternatieve oplossingen voor orgaantransplantatie. Implanteerbare weefsel-engineered renale cellulaire constructies zijn een dergelijke haalbare benadering ter vervanging van verloren renale functionaliteit. Beschreven voor de eerste keer, hier is de microdissection van lymfkliertest nieren voor isolatie van levende corticomedullary renale segmenten. Deze segmenten zijn geschikt voor snelle integratie binnen steiger-vrije endotheel-fibroblast constructies waarmee snelle verbinding met host therapieën eenmaal geïmplanteerd kan. Volwassen muis nieren waren verkregen van levende donoren, gevolgd door de stereoscoop microdissection verkrijgen van renale segmenten 200-300 µm in diameter. Meerdere renale constructies werden vervaardigd gebruikend primaire renale segmenten geoogst van slechts één nier. Deze methode geeft aan een procedure die functionele nier weefsel van organen die anders zouden worden weggegooid kon redden.

Het protocol beschreven produceert ongeveer 50 renale segmenten per piramidale 2 mm3 sectie van renale weefsel. De renale segmenten die zijn verwerkt en beeld hebben buisvormige en glomerulaire componenten in verschillende verhoudingen (Zie Figuur 2). De intact segmenten werden onderworpen aan een test om te bepalen van de levensvatbaarheid van de verschillende segmenten eenmaal elke 24 uur per dag voor drie dagen. Groen-fluorescerende calceïne-AM...

Watch this Scientific Journal Video about Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology at JoVE.com

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

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

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

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

The overall goal of this microscopic dissection procedure is to isolate discrete segments of living renal tissue with intact renal architecture for tissue engineering applications. This method can help answer key questions in the field of tissue engineering such as can whole segments of isolated primary organ tissue be harvested sterilely and be kept alive in culture? The main advantage of this technique is the segments contain intact renal architecture as opposed to using cell lines to recreate a complex organ with over 26 different cell types.The implications of this technique extend towards therapy of end-stage renal disease because these constructs will improve filtration and may help patients stay off dialysis. During the surgery, wear proper surgical gear including a mask and a cap to minimize the risk of contamination. Likewise, make every effort to maintain sterility throughout.Start preparations by draping the operating table. Next, open the pack of autoclaved instruments onto the sterile drapes. They must include three small hemostats, fine forceps with teeth, fine forceps without teeth, and straight iris scissors.Cover the center of the operating table with a second non-fenestrated surgical drape. Prior to setting up the operating room, prepare 50 milliliters of antibiotic supplemented DBPS and aliquot five milliliters into a 15 milliliter tube. Now anesthetize an eight to 12-week-old C57 black six mouse and periodically monitor the level of anesthesia by assessing its pedal reflex with a firm toe pinch.Next, use a hair removal cream to remove all of the hair from the ventral torso area and rinse off the cream and hair with distilled water. Then transfer the mouse to the operating table and place it supine under the non-fenestrated drape. Now cut out a two centimeter square fenestration in the drape over the mouse's abdomen.Then scrub the exposed skin with povidone-iodine followed by 70%ethanol three times to sterilize the skin and complete the preparations for the surgery. Begin the surgery by using fine forceps with teeth and iris scissors. Make a three to four centimeter skin incision parallel to the midline one-half centimeter to the left of the midline.Next, grasp the peritoneum using fine forceps without teeth and incise with iris scissors to enter the abdominal cavity. Then apply hemostats to the superior and inferior lateral edges of skin and peritoneum to place tension laterally and enhance the exposure. Now delicately shift the intestinal contents to the right of the abdomen to expose the left kidney.Then gently place traction on the kidney to elevate it out of the abdomen. Now place a hemostat across the hilum of the kidney and use iris scissors to transect the ureter and renal vessels. Then transfer the kidney to the tube of 1%pen-strep DBPS on ice.Take this tube to a sterile culture hood for processing. There transfer the kidney to a 60 millimeter Petri dish and rinse it twice with fresh five milliliters of DPBS containing calcium and magnesium. After the second wash, suspend the kidney in fresh five milliliters of DBPS in a new 60 millimeter dish.Then prepare an additional Petri dish containing only five milliliters of DBPS for later use. Continue using sterile technique including sterile gloves, surgical mask, and bouffant to minimize contamination risks. Then drape the stage area and area surrounding the stereo microscope.Next, place plastic adhesive sheets on the microscope focus knobs and spray them with 70%ethanol. Then turn on the dual gooseneck illuminator to light the stage. Now onto the sterile drapes, open a package of sterilized instruments containing fine forceps without teeth, a scalpel handle, a number 15 scalpel blade, two straight hemostats, and two 30.5 gauge hollow-bore needles.Place the number 15 blade onto the scalpel handle and attach one hemostat to each needle adapter hub to create needle dissection instruments. Now transfer the two dishes, one with the kidney in DPBS and the other only DPBS, to the microscope area. Remove the lid on the kidney dish and focus the microscope on the anterior or posterior surface of the kidney at 3.2 to 4X magnification.Next, with the non-dominant hand, use fine forceps without teeth to pierce and pin the kidney against the dish at the inferior and superior poles of the kidney. Then with the dominant hand, use the number 15 blade to remove the translucent fibrous capsular layer from the exposed surface. Shave the most superficial 0.5 millimeters from the exposed surface of the kidney surface to remove the remainder of the capsule.Continue by using the number 15 blade to dissect an inverted pyramid of tissue from the de-capsulated area that is approximately two square millimeters. Then transfer the tissue into the clean dish of DBPS and discard the remainder of the kidney. Now focus on the dissected tissue and decrease the magnification to 1.5 to 2.0X.Using the two hemostat needle instruments as cutting tools, slice the tissue fragment into progressively smaller pieces until the tissue segments are the size of the needle tips or smaller. About 50 segments should be produced. This is a critical portion of the procedure.Each segment needs to closely approximate the diameter of the 30-1/2 gauge needle tip to avoid large segments which could hinder diffusion. Return the tissue to the tissue culture hood and remove the DPBS using a P1000 micropipette. Then add back five millimeters of DMEM supplemented with 10%fetal bovine serum and 1%pen-strep.Then culture the tissue or implant it within cellular constructs immediately. The viability of the intact renal segments produced by the described procedure was examined over three days in culture using a viability assay. Green fluorescent calcium AM is present with intracellular esterase activity indicative of living cells.Red fluorescent ethidium homodimer-1 is seen with loss of integrity of the plasma membrane. When the renal segments were embedded in scaffold-free endothelial fibroblast constructs, by day three they were incorporated with the cellular constructs to form intact structures. The constructs maintained their pre-vascular endothelial network shown by labeling with von Willebrand factor.Renal epithelial cells can be seen in green labeled by cytokertain-18. To test in-vitro renal functionality, the constructs were incubated with FITC-labeled albumin. While there was residual albumin found away from the segments of embedded renal tissue, there were also hotspots in the renal tubular epithelial cells.These hotspots are albumin that traverses intraluminally which is thought to represent albumin reuptake in the renal segment cellular construct. After watching this video, you should have a good understanding of how to isolate renal segments from murine kidneys for tissue engineering applications. Once mastered, this procedure can be done in one to 1-1/2 hours.Following this procedure, other methods including seeding cellularized constructs can be performed in order to answer other questions regarding renal construct fabrication and characterization. Don't forget that working with surgical instruments and pressurized oxygen cylinders can be extremely hazardous. Be careful when working with sharp objects and remember to anchor oxygen tanks to a lab bench.

Research

JoVE Journal

Bioengineering

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Magnetic Resonance elastografie methodologie voor de evaluatie van Tissue Engineered Construct groei

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-contact, Label-free monitoring van cellen en extracellulaire matrix met behulp van Raman spectroscopie

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Human kraakbeenweefsel Fabrication behulp van drie-dimensionale Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

A Novel Stretching platform voor toepassingen in de Cel en Weefsel Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

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

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Driedimensionale Biomimetic Technologie: Novel Biorubber Creëert Defined micro- en macro-schaal Architectures in Collageen Hydrogels

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

Electrospinning schrijven van driedimensionale Poly(ε-caprolactone) steigers met controleerbare Morphologies voor Tissue Engineering toepassingen smelten

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting van kraakbeen en huid weefsel analogen met behulp van een roman passief mengen eenheid techniek voor Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Een nieuwe tenorrfaphy hechttechniek met weefselmanipulatie collageentransplantaat om grote peesdefecten te repareren

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

Een netto schimmel gebaseerde methode van de steiger-free driedimensionale cardiale weefsel schepping

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...