NIH kurumsal doktora sonrası eğitim desteği, NIH-HL-007260

Tüm hayvan cerrahi işlemler aşağıda açıklanan kurumsal hayvan bakım ve Komitesi (IACUC), tıbbi University of South Carolina herhangi bir hayvan ameliyat öncesinde kullanın veya herhangi bir hayvan doku kullanımı tarafından kabul edildi.
1. fare nefrektomi
<ol><li>Don bir ameliyat maskesi ve kabarık kapağı bulaşma riskini en aza indirmek için. Kısırlık cerrahi alan kurulum sırasında korumak.<ol>
<li>Delikli sigara cerrahi perdeler ameliyat masasında yerleştirin.</li>
		<li>Autoclaved aletleri steril örtüler üzerine Çantayı aç. Nefrektomi için gerekli aletleri 3 küçük hemostats dahil, iyi Forseps ile dişler, diş ve düz Iris makas olmadan iyi forseps.</li>
		<li>Başka bir delikli sigara cerrahi örtüsü ameliyat masasının ortasına yerleştirin.</li>
		<li>5 mL, Dulbecco'nın fosfat tamponlu tuz (DPBS) kalsiyum ve magnezyum, % 1 penisilin/streptomisin bir 15 mL steril konik tüp ile takıma hazırlayın. Bu çözüm buz ameliyat masasının yanındaki yerleştirin.</li>
	</ol>
</li>
	<li>Bir 8-12 haftalık C57BL/6 fare (erkek veya kadın) hayvan konut kompleksi edinin.</li>
	<li>Bir anestezi indüksiyon odası içinde fareyi getirin ve inhale % 3,5 isoflurane ile teşvik. Bakım anestezi için % 2 isoflurane kullanarak fare, bir burun konisi yerleştirin.<ol>
<li>Anestezi pedal refleks (firma ayak tutam) değerlendirilmesi tarafından düzenli olarak cerrahi işlem boyunca yeterli derinliği için monitör.</li>
	</ol>
</li>
	<li>Tüm saç saç dökücü krem kullanarak ventral gövde kaldırmak. Bu alan tüm saç karından kaldırıldığından emin olmak için distile su ile durulayın.</li>
	<li>Fare steril ameliyat masasının en iyi sigara delikli örtü altında sırtüstü pozisyonda aktarın. Sadece fare karın örten örtüsü içinde 2 x 2 cm2 fenestrasyon kesip.</li>
	<li>Povidone-iyot steril gazlı bez veya pamuk topları kullanarak karın % 70 etanol tarafından takip uygulanır.</li>
	<li>Diş ve iris ile iyi forseps kullanarak makas, 3-4 cm dikey karın cilt kesi orta hat, orta hat solundaki 0.5 cm paralel yapmak Not: Sağ böbrek kaldırmaya çalışan, bu bir kesik orta hat sağındaki 0.5 cm olacaktır.<ol>
<li>Diş olmadan iyi Forseps ile karın zarını kavramak ve karın boşluğuna girmek için Iris makasla deşmek. Hemostats üst ve AST yan kenarları deri ve gerginlik yanal yerleştirin ve pozlama geliştirmek için karın zarını için geçerlidir.</li>
		<li>Bağırsak içeriği ince sol böbrek ortaya çıkarmak için karın sağ tarafına kayması. Yavaşça dışarı karın yükseltmek için böbrek çekiş yerleştirin.</li>
		<li>Bir hemostat sol böbrek hilusu arasında yerleştirin. Iris makas Üreter ve böbrek damarlarının transect için kullanın.</li>
		<li>Sol böbrek % 1'yer penisilin/streptomisin DPBS çözüm buz üzerinde.</li>
		<li>Steril doku kültürü hood için böbrek içeren tüp aktarın. Böbrek 15 mL konik tüp sizden bir 60 mm petri kabına (şekil 1) aktarın. Durulama iki kez 5 mL DPBS kalsiyum ve magnezyum ile kullanma. 5 ml DPBS 60 mm tabak içinde askıya böbrek tutun.<ol>
<li>Bir ek 60 mm petri kabına 5 mL mikrodiseksiyon iletişim kuralı sırasında kullanılmak üzere DPBS ile hazırlayın.</li>
		</ol>
</li>
		<li>Fareyi Torakotomi ve böbrek, ihale takip kan kaybı tarafından onaylanmış bir IACUC hayvan protokole göre ötenazi.</li>
	</ol>
</li>
</ol>2. fare böbrek mikrodiseksiyon böbrek Segment yalıtım için
<ol><li>Steril eldiven, cerrahi maske, dahil olmak üzere iletişim kuralı, bu kısmı için steril tekniği kullanmaya devam etmek ve kabarık bulaşma riskini en aza indirmek için.<ol>
<li>De sadece sol alanları ve aletler için steril bir alanınız için mikroskop sağ stereo mikroskop platformda yer steril perdeler. Mikroskop odak kolları plastik yapışkan levhalar yerleştirin ve % 70 etanol ile sprey. Sahne ışığı çift boynu ışığı açın.</li>
		<li>(İyi forseps olmadan diş, neşter kolu, #15 neşter bıçak, iki düz hemostats, iki 30½ içi boş-geçişli iğneler ölçmek) açık steril aletler steril örtüler üzerine. Yer #15 bıçak neşteri üzerine baş ve bir hemostat her iğne adaptör/iğne diseksiyon aletleri daha sonra kullanmak, şekil 1' de gösterildiği gibi oluşturmak için hub ekleyin.</li>
	</ol>
</li>
	<li>60 mm petri yemekler, bir böbrek DPBS ve doku kültürü hood mikroskop alanına gelen diğer tek DPBS içeren taşımak.<ol>
<li>Amaç altında 60 mm petri kabına yerleştirin ve böbrek ortaya çıkarmak için kapağı kaldırın. DPBS içeren çanak tarafına daha sonra kullanmak için steril alanına bırakın.</li>
		<li>Sadece ön veya arka yüzey böbrek görünümünde (Yani, böbrek, büyük düz yüz çanak alt dokunmadan diğer yüzü) çanak taşımak. Yordamı bu kısmı için 3.2-4 x zoom. Sigara dominant el kullanarak, diş olmadan iyi forseps işlemek ve böbrek böbrek inferior ve superior yakın çanak karşı pin için kullanın.</li>
		<li>Sigara dominant el böbrek yıkmaya tutmak. Dominant el ile yarı saydam fibröz kapsül katman maruz yüzeyden kaldırmak için #15 bıçak kullanın. Böbrek kapsülü kalanı kaldırmak için maruz yüzeyinden en yüzeysel 0,5 mm tıraş.</li>
		<li>#15 bıçak doku decapsulated bölgeden yaklaşık 2 mm3 ' te boyutu bir ters piramit incelemek için kullanın. Sadece DPBS içeren 60 mm yemek almak ve doku piramit temiz tabak yerleştirin. Böbrek kalanını atmak.</li>
		<li>1.5-2.0 x diseksiyon kalanında büyütmeyi azaltma. Doku parçaları veya iğne ipuçları çapı eşit olana iki hemostat iğneli aracı kesme aletleri kullanarak, doku parçası giderek daha küçük parçalar halinde dilim. Bir böbrek doku 2 mm3 ' te boyutu 50'den fazla segmentleri bir kez tamamen disseke üretecektir.</li>
		<li>Böbrek kesimleri ile 60 mm çanak için doku kültürü hood taşıyın. DPBS 1000 µL pipet ile kaldırın. 5 mL DMEM, % 10 Fetal sığır Serum (FBS) ve % 1 penisilin/streptomisin ile desteklenmiş ekleyin.</li>
		<li>Kültür için bir kuluçka makinesi veya cep içine implant 37 ° C'de 3 gün hemen oluşturur.</li>
	</ol>
</li>
</ol>3. böbrek Segment hücresel yapı imalat
<ol><li>Kültür normal insan dermal fibroblastlar (NHDF) ve insan yağ mikrovasküler endotel hücreleri (HAMEC) 7.2 x 105 fibroblastlar ve 1.8 x 10 istenen yapı başına5 endotel hücre elde etmek birkaç hafta için. Doku kültürü başlık, fibroblastlar endotel hücreleri (4: çubuk şeklinde kuyu 1) daha önce Czajka tarafından açıklandığı gibi önceden iskele-Alerjik damar endotel fibroblast yapıları (SPEC) oluşturmak için özel kalıplar uygun oranını tohum ve ark 19.</li>
	<li>Steril bir hemostat, 30½ G iğne ve yassı ucuyla steril bir spatula elde edilir.<ol>
<li>Medya çoğunluğu renal kesimleri değil Aspire edin ve böbrek parçalarını kaldırmak için dikkatli olmak, içeren 60 mm plaka kaldırmak.</li>
		<li>Segmentleri implantasyonu görselleştirmek için cerrahi büyüteçler donatmak.</li>
		<li>Spatula yavaşça boyunca 10-15 parçaları elde etmek için spatula çok uç eşit olarak yayılmış için 60 mm çanak alt sonu kazı. Spatula ucu nerede SPEC seribaşı iyi dudak mümkün olduğunca yakın yerleştirin.</li>
		<li>Bir hemostat-30½ G iğne aleti diğer elinde her bireysel segmenti spatula ucundan kuyu kenarına taşımak için kullanın. Yavaşça her kesimi de biraz daha önce numaralı seribaşı hücresel süspansiyon batık kadar iğne ucu kullanarak içine aşağı taşıyın.</li>
		<li>Kültür böbrek segment-SPEC 2:1:1 oran FGM-2/EGM-2/DMEM orta (2.5 mL cilt toplam). Kültür medya her 24 h 3 gün boyunca değiştirin. 72 h kalıpları yapıları kaldırmak ve tamir veya flash-işleme için dondurma.</li>
	</ol>
</li>
</ol>

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Yazarlar ifşa gerek yok.

Böbrek doku yapıları hücrelerin ve Biyomalzeme kullanılan ve birçok durumda, her iki türü ile ilgili olarak değişkenlik yaşayan mühendis için kullanılan bir yöntem modası geçmiş ya da iyi edebiyat7karakterize. Çok kök hücre yaklaşımlar kullanıyorsanız veya böbrek mimarisinin izolasyon bireysel bileşenlerine recapitulating, yapay olarak hücresel kullanılamaz hale gelen 26 farklı farklı hücre tipleri ile tüm organ yeniden olasılığı sırasında 2...

Kronik böbrek hastalığı (CKD) geçerli büyük halk sağlığı sorunları dünya çapında1biridir. Amerika Birleşik Devletleri'nde CKD yaygınlığı % 14'den fazla 600.000 Amerikalılar en şiddetli, son aşama böbrek hastalığı (ESRD)2acı ile toplam nüfusun bitti. Mevcut tedavi seçenekleri bu ESRD ile diyaliz ve böbrek nakli dahil. Her ne kadar yaklaşık 25.000 hastalar böbrek nakli her yıl geçmesi, her yıl o bir hayat kurtarıcı organ ve o alıcı nakli3bekleyen arasında büyük bir eşitsizlik önde gelen hastaların önemli bir sayı eklenir. Uzun ömürlü ve yaşam kalitesini ciddi olumsuz etkileri yanı sıra, diyaliz şaşırtıcı bir mali yük ile ilişkilidir. 2014 yılında Medicare iddiaları ödenen üzerinde 30 milyar dolar ESRD hastalar2için buldu. Bir sınırlı organ tedarik ve hastaların diyaliz gerektiren hiçbir belirgin düşüş trendi, diyaliz için alternatif çözümler belirleme araştırma çabalarına yönelik ve nakli şimdiye kadar önemlidir. Diyaliz ihtiyacını bile nispeten kısa bir gecikme diyaliz ile ilgili maliyeti4,5,6ertelenmesi ise kalite-düzeltilir ömrü yıl ve verimlilik bir hastanın sayısını önemli ölçüde artırır.
Böyle ESRD, fonksiyonel doku kaybı için çözümler şu anda doku Mühendisliği ve rejeneratif tıp laboratuvarları, iskele tabanlı organoid imalat bütün organ mühendislik kullanmaya kadar yaygın olarak çeşitli yaklaşımlarla incelendiği organ yapıları hücresel implantasyon7,8,9,10,11decellularized. Marjinal veya gözardı edilen böbrek böbrek karmaşık yapıları recapitulating kısmen soruşturma. Aslında, neredeyse böbrek nakli için tedarik % 20'si çeşitli nedenlerle12,13için atılır. Bu sözde Greftler fonksiyonel böbrek dokusundan kullanılmaktadır ve bir veya daha fazla doku mühendislik yapıları dahil. Önceki çalışmalar atılan bu organlar, böbrekler doku mühendislik amaçlı14,15ekstra hücresel matris için kullanan çalışma fizibilite göstermiştir. Ancak, az sağlıklı böbrekler birincil nephronal dokudan doku Mühendisliği amacıyla16,17,18için kullandık.
Bir yöntem daha önce Kim vd tarafından açıklanan dan sonra üzerinde Poliglikolik asit (PGA) İskele yapı imalat16numaralı seribaşı sağlıklı sıçan böbrek, böbrek "parçaları" yalıtım içerir. Ancak, tam diseksiyon metodoloji ile ilgili çok az bilgi verilir ve kesimleri iyi kıyma ve filtrasyon bileşiminden elde edilmiştir. Benzer şekilde sağlam nephronal mimarisi ile ayrık böbrek segmentleri oluşturur, ancak yerine mikrodiseksiyon teknikleri üzerinde dayanır bu iletişim kuralı bir değişiklik açıklar. Nephrectomies sonra böbrekler nerede renal kapsül kaldırılır ve doku daha fazla disseke diseksiyon mikroskop aktarıldığı yaşam yetişkin fareler üzerinde gerçekleşir. Küçük 30½ G iğneler kesme aletleri kullanılır ve ayrıca diseksiyon içinde yardım kılavuzları gibi iğne çapı böbrek kesimleri hedef çapı eşittir. İzole, bu durumda fare, böbrek kesimleri canlılığı kültür korumak ve İskele-Alerjik endotel-fibroblast Hücresel yapıları19ile birleştirmek. Bu yapıları daha önce bir biyo-yapay pankreas20de dahil olmak üzere diğer organlara Mühendisi için kullanılmaktadır.

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		<col />
		<col />
		<col />
	</colgroup>
	<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

Böbrek nakli şimdi son aşama böbrek hastalığı için genel kullanıma uygun bir tedavi yöntemidir. Ancak, bekleme listesi ve sadece dörtte biri ekimi ulaşmak bu hastaların yaklaşık 96.000 kişi ile alternatifler organları başarısız olanlar için korkunç bir ihtiyaç yoktur. Diyaliz bu maruz kalırsa genel olarak sağlık maliyetleri ile birlikte zararlı sonuçlarını azaltmak için etkin soruşturma organ nakli için alternatif çözümler arayışı içinde devam ediyor. İmplante edilebilir doku Mühendisliği böbrek hücresel kayıp böbrek işlevleri yerine bir tür uygun yaklaşım yapılarıdır. İlk kez açıklanan, burada yaşayan corticomedullary yalıtım için fare böbrekler mikrodiseksiyon böbrek kesimleri. Bu kesimler bir kez implante ana damarlara ile hızlı bağlantı sağlayan iskele-Alerjik endotel-fibroblast yapıları içinde hızlı birleşme yeteneğine sahiptirler. Yetişkin fare böbrek böbrek kesimleri çapı 200-300 µm elde etmek için stereoscope mikrodiseksiyon ardından yaşam bağışçılardan tedarik. Birden fazla böbrek yapıları kullanarak tek bir böbrek hasat birincil böbrek kesimleri fabrikasyon. Bu yöntem aksi takdirde iptal edilecek organların fonksiyonel böbrek dokusundan kurtarmak bir işlem gösterilmektedir.

Açıklanan protokol piramit 2 mm3 bölüm böbrek dokusunun başına yaklaşık 50 böbrek kesimleri üretir. İşlenmiş ve yansıma böbrek kesimleri (bkz. Şekil 2) farklı oranlarda borulu ve glomerular bileşenleri var. Sağlam kesimleri bir tahlil için üç gün boyunca farklı kesimleri bir kez her 24 h canlılığı belirlemede tabi tutuldu. Yeşil-floresan calcein-AM ile hücre içi Esteraz etkinliğini, canlı hücreler gösterge mevcut...

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

Böbrek yapıları doku Mühendisliği zararlı etkileri diyaliz ve organ sıkıntısı için bir çözüm sağlar. Burada, bir protokol mikro tarif yalıtım kortiko-medüller segmentleri için fare böbrekler incelemek. Bu segmentler oluşturan böbrek organoids iskele-Alerjik Hücresel yapıları implante edilir.

Mikrodiseksiyon birincil böbrek doku parçaları ve roman iskele-Alerjik yapı teknolojisi ile birleşme

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

microdissection-primary-renal-tissue-segments-incorporation-with

Research

JoVE Journal

Bioengineering

7.3K Görüntüleme.  Medical University of South Carolina. Böbrek yapıları doku Mühendisliği zararlı etkileri diyaliz ve organ sıkıntısı için bir çözüm sağlar. Burada, bir protokol mikro tarif yalıtım kortiko-medüller segmentleri için fare böbrekler incelemek. Bu segmentler oluşturan böbrek organoids iskele-Alerjik Hücresel yapıları implante edilir.

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

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

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

Doku Değerlendirilmesinde Manyetik Rezonans Elastography Metodoloji Construct Büyüme Tasarlandı

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

Biyomühendislik

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

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, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Raman Spektroskopisi kullanarak Hücreler ve Ekstrasellüler Matriks Temassız, Etiket ücretsiz izleme

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

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Üç boyutlu Mürekkep Püskürtmeli Baskı Teknolojisi Kullanımı İnsan Kıkırdak Doku Fabrikasyon

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

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

Hücre ve Doku mechanobiology Applications A Novel Germe Platformu

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

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

Epitel Hücre Repopulation ve Böbrek Doku Kalkınma Kemirgen Ekstrasellüler Matriks iskelelerinin hazırlanması

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

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material.

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

Üç boyutlu Biyomimetik Teknoloji: Roman Biorubber Kollajen Hidrojeller içinde Mikro ve Makro ölçekli Mimarileri Tanımlı oluşturur

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

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

Üç boyutlu Poly(ε-caprolactone) İskele kontrol edilebilir türleri morfoloji ile Electrospinning yazma doku mühendisliği uygulamaları için erime

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 is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting kıkırdak ve cilt doku analogları bir roman pasif birimi teknik Bioink Precellularization için karıştırma kullanmak

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

Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all variations are based on the 20 canonical amino acids (cAAs). As a way to introduce additional physicochemical properties into polypeptides, the incorporation of non-canonical amino acids (ncAAs) is increasingly used in protein engineering. Due to their relatively short length, the modification of ribosomally synthesized and post-translationally modified peptides by ncAAs is particularly attractive. New functionalities and chemical handles can be generated by specific modifications of individual residues. The selective pressure incorporation (SPI) method utilizes auxotrophic host strains that are deprived of an essential amino acid in chemically defined growth media. Several structurally and chemically similar amino acid analogs can then be activated by the corresponding aminoacyl-tRNA synthetase and provide residue-specific cAA(s) &#8594; ncAA(s) substitutions in the target peptide or protein sequence. Although, in the context of the SPI method, ncAAs are also incorporated into the host proteome during the phase of recombinant gene expression, the majority of the cell&#39;s resources are assigned to the expression of the target gene. This enables efficient residue-specific incorporation of ncAAs often accompanied with high amounts of modified target. The presented work describes the in vivo incorporation of six proline analogs into the antimicrobial peptide nisin, a lantibiotic naturally produced by Lactococcus lactis. Antimicrobial properties of nisin can be changed and further expanded during its fermentation and expression in auxotrophic Escherichia coli strains in defined growth media. Thereby, the effects of residue-specific replacement of cAAs with ncAAs can deliver changes in antimicrobial activity and specificity. Antimicrobial activity assays and fluorescence microscopy are used to test the new nisin variants for growth inhibition of a Gram-positive Lactococcus lactis indicator strain. Mass spectroscopy is used to confirm ncAA incorporation in bioactive nisin variants.

The protocol presents the Escherichia coli-based selective pressure incorporation of non-canonical amino acids (ncAAs) into the lactococcal antimicrobial peptide nisin. Its properties can be changed during recombinant expression via substitution with desired ncAAs in defined growth media. Resulting changes in bioactivity are mapped by growth inhibition assays and fluorescence microscopy.

Antimikrobiyal peptidler kanonik olmayan Amino asitler ve seçici basınç eklenmesi tarafından üretilen

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all ...

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

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.