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 институциональных докторантура Грант, низ HL-007260

Все животные хирургических процедур, описанных ниже были утверждены институциональный уход животных и использовать Комитет (IACUC) в медицинский университет Южной Каролины до любых животных операций или использование любых животных тканей.
1. мышиных нефрэктомия
<ol><li>До?...

<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>

Методы, используемые для инженер почечной ткани жизни конструкции различаются в отношении как тип клеток и биоматериалов использованы и во многих случаях являются устаревшими или не хорошо изученных в литературе7. Хотя многие используют подходы стволовых клеток или пере?...

Хроническая болезнь почек (CKD) является одним из текущих общественного здравоохранения проблемы во всем мире1. Распространенность CKD в Соединенных Штатах составляет более 14% от общей численности населения, с более чем 600 000 американцев страдают от наиболее тяжелой формой, терминальная стадия почечной недостаточности (ТПН)2. Текущие варианты лечения доступны для тех, кто с ТПН включают диализа и почечной трансплантации. Хотя приблизительно 25 000 пациентов проходят почечной трансплантации каждый год, значительное количество пациентов добавляются ежегодно приводит к большой разрыв между теми ожидающих спасения жизни органа и тех принимающих трансплантации3. В дополнение к его серьезное негативное воздействие на продолжительность жизни и качество жизни диализ ассоциируется с удивительной финансового бремени. В 2014 году Medicare заплатил претензии составил более 30 миллиардов долларов для ТПН больных2. С ограниченной орган и без очевидной нисходящий тренд в пациентов, нуждающихся в диализе исследовательские усилия, направленные на выявление альтернативных решений для диализа и трансплантации всегда важны. Даже относительно небольшой задержки в необходимости диализа увеличивает количество пациента лет жизни с поправкой на качество и производительность существенно отложив расходов, связанных с диализа4,5,6.
Решения для потери функциональных тканей, как в ТПН, в настоящее время изучаются в тканевой инженерии и регенеративной медицины лаборатории, с широко разнообразных подходов, начиная от изготовления на основе эшафот органоид инженерных с помощью весь орган decellularized орган структур для сотовых имплантации7,8,9,10,11. Лишь частично исследован сводный комплекс почечной структур от маргинальных или брошенных почек. В самом деле почти 20% почек для пересадки удаляются для различных причин12,13. Функциональной почечной ткани из этих предполагаемых графтов может использоваться и включены в одной или нескольких тканей инженерных конструкций. Предыдущие исследования показали возможность работы с этими органами отбрасываются, используя почки внеклеточных матрицы для ткани инженерных целей14,15. Однако мало использовали первичной nephronal ткани от здоровой почки для тканевой инженерии целей16,17,18.
Один из методов описано ранее, Ким et al. включает изоляции почечной «сегменты» от здоровых крыс почки, которые затем были посеяны на Полигликолидная кислота (PGA) подмости для изготовления конструкции16. Однако мало информации дается относительно методологии точного рассечение, и сегменты были получены из комбинации тонкой мясорубки и фильтрации. Мы описываем изменения настоящего Протокола, который аналогичным образом производит дискретных почечной сегментов с нетронутыми nephronal архитектуры, но вместо этого опирается на методы microdissection. Nephrectomies выполняются на жизни взрослых мышей, после чего почки передаются рассечение Микроскоп где почечной капсула удаляется, и далее рассекается ткани. Малокалиберная 30½ G иглы используются как инструменты для резки и также как направляющие, пособничество в рассечение, как иглы диаметром равным диаметру целевых сегментов почек. Изолированные, в этом случае мышиных, почечной сегменты сохранять жизнеспособность в культуре и соединяются с сотовой конструкции бесплатно эшафот эндотелия фибробластов19. Ранее были использованы эти конструкции инженера других органов, включая био Искусственная поджелудочная железа20.

<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

Трансплантация почки в настоящее время основной терапии для терминальной стадии почечной болезни. Однако с примерно 96 000 человек на лист ожидания и только четверть из этих пациентов, достижение трансплантации, существует острая потребность в альтернативных вариантов для тех, кто не в состоянии органов. Для уменьшения вредных последствий диализа наряду с общей медицинских расходов, которую он несет, активное расследование продолжается в поисках альтернативных решений для трансплантации. Имплантируемые почечной сотовой конструкции, ткани инженерии, один такой реальный подход к замене потерянных почечной функции. Описанные в первый раз, вот microdissection мышиных почек для изоляции жизни corticomedullary почечной сегментов. Эти сегменты способны быстрого включения в течение бесплатно эшафот эндотелия фибробластов конструкций, которые могут включить быстрое соединение с принимающей сосудистую после имплантации. Взрослый мыши почки были закуплены от живых доноров, следуют стереоскоп microdissection для получения почечной сегментов 200-300 мкм в диаметре. Несколько почечной конструкции были изготовлены с использованием первичных почек сегментов заготавливаемым от только одной почки. Этот метод демонстрируется процедура, которая может спасти функциональной почечной ткани от органов, которые в противном случае будет игнорироваться.

Протокол, в описанный производит около 50 почечной сегментов на пирамидальную 2мм3 часть почечной ткани. Почечная сегментов, которые были обработаны и образы имеют трубчатых и клубочковых компоненты в различных пропорциях (см. Рисунок 2). Нетронуты...

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

Ткани инженерных конструкций почечной предоставляют решение для органа нехватка и пагубные последствия диализа. Здесь мы описываем протокол к микро вскрыть мышиных почки для изоляции cortico Медуллярная сегментов. Эти сегменты имплантируется в эшафот бесплатный сотовых конструкций, образуя почечной organoids.

Microdissection первичной почечной ткани сегментов и интеграция с технологией Роман эшафот свободной конструкции

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

Магнитно резонансная Эластография методологии по оценке тканей Разработан Construct роста

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

Бесконтактная, этикетки без мониторинга клеток и внеклеточного матрикса использованием спектроскопии комбинационного

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

Человека хрящевой ткани Изготовление с использованием трехмерных струйных технологии печати

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

Роман Растяжка Платформа для приложений в клеточной и тканевой 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

Эпителиальные Репопуляции сотовый и подготовка грызунов внеклеточный матрикс строительные леса для почечной ткани развития

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

Трехмерная Биомиметический Технология: Роман Biorubber Создает Определено микро- и макроуровне архитектур в коллагене гидрогелей

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

Расплава Electrospinning написания трехмерных Poly(ε-caprolactone) леса с контролируемым морфологии тканей инженерных приложений

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

Подложке хряща и аналогов ткани кожи, используя Роман пассивного смешивания единицы техники для 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

Новый метод наложения швов теноррафи с тканевым коллагеновым трансплантатом для восстановления больших дефектов сухожилий

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

Чистый метод, основанный на плесень, создания эшафот бесплатный трехмерный сердечной ткани

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