Yazarlar Gelişmiş Oftalmik Malzemelerin Geliştirilmesi için fon kaynağı NSERC 20/20 Ağ kabul etmek istiyorum.

Tüm deneyler Üniversitesi&#39;nin Waterloo hayvan araştırma etik komitesi tarafından belirtilen tüm ilgili kılavuzlara uygun ve uyumlu tamamlanmıştır. sığır gözler cömertçe yerel mezbahaya bağışlanan. 1. Göz Modeli <ol><li> Tasarım ve Kalıp 13 Üretimi <ol><li> İnsan yetişkin gözlerin ortalama fizyolojik boyutlarına göre göz modelleri tasarlar. 13 </li><li> göz küresi ve göz modelinin göz kapağı parçaları arasında 250 mikron bir boşluk bırakın. bilgisayar destekli tasarım (CAD) yazılımı kullanılarak ilgili kalıp tasarımı. </li><li> AutoCAD veya Solidworks ile yeni .cad dosyası veya .sldprt dosyası oluşturun. insan göz küresi / göz kapağı 3D modelleri oluşturun. modellerin kalıpları oluşturun ve stl dosyaları olarak kalıpları kaydedin. </li><li> 3D yazıcı yazılımı içine İthalat stl dosyaları (replicator2 örneğin, makeware). Baskı parametrelerini (yer, seyrekliği, ölçek, oryantasyon, pürüzsüzlük, vb belirtin </em&gt;) 13. </li><li> 3D yazıcılar okumak için G-kod dosyası olarak kaydedin. Bu PLA (polilaktik asit), ABS (akrilonitril butadien stiren), PC (polıkarbonat) veya bunların bir kombinasyonu gibi seçin malzeme, kalıp 13 baskı. </li><li> seçim malzemesi istenilen iplik takın. okumak için 3D yazıcının içine G-kod dosyasını içe aktarın. kalıp yazdırın. NOT: Göz modeline yumuşak bir yüzey isteniyorsa, alternatif olarak, (CNC) makine kontrollü bir bilgisayar nümerik kullanarak göz kalıpları üretmek. CNC kalıp üretimi için kalıp malzemeleri artık termal plastik sınırlıdır, ancak metal, seramik ve bu Politetrafloroetilen gibi kimyasal direnç polimerler uzatmak. </li><li> Bir kesim matkap bağlı olan CNC yazılım arayüzü açın. Ön, üst, yan ve kontrol yazılımı arayüzü, daha önce inşa göz küresi / gözkapağı modeli kalıp perspektif görünümleri göre 3D kalıpları oluşturun. için uygun parametreleri seçinMakinede (bit boyutu, alt tabaka malzemesi, malzeme kalınlığı) ve kalıp kesim geçin. </li></ol></li><li> PDMS kullanılması Oküler sentezi <ol><li> Bir şırınga kullanılarak, PDMS (polidimetilsiloksan) baz 10 mi hacmini ölçmek ve 15-50 ml santrifüj tüpüne doldurun. PDMS toplam ağırlığına göre, elastomer çözeltisi ağ / hac% 10 ekleyin. Bir karıştırma çubuğu kullanarak, iyi çözüm karıştırın. </li><li> göz küresi ve göz kapağı kalıpların içine PDMS çözüm dökün. PDMS RT O / N yerleşmek için izin verin (ya da en azından 12 saat süreyle) polimerizasyonu başlatmak ve kabarcıklar polimerin dışına çözünmesi için izin vermek. NOT: yükselecek ya da genişletmek olabilir PDMS sol hava kabarcığı olmadığından emin olun. </li><li> Daha sonra, 1 saat, ya da 5 dakika için 150 ° C (302 ° F) 75 ° C (167 ° F) fırına kalıplar koydu. daha yumuşak bir jel, PDMS tamamen polimerize için en az 48 saat boyunca oda sıcaklığında bekletin. </li><li> Bir kaç dakika dondurucuda örnekleri koymak; Bu PDMS küçültmek ve basitleştirecekkalıplardan numune alım. ince bir spatula kullanılarak kalıplardan oküler ekstrakte edin. </li><li> göz küresi ve göz kapağı parçaları arasındaki boşluğa bir çözüm teslimi için, 1/16 &quot;eşit bacak bağlantı borusu konnektörü ile 1/16&quot; x 1/8 &quot;politetrafloroetilen tüp bağlamak ve boru delik göz kapağı parça ekleyin . </li></ol></li><li> Agaroz kullanma Gözküresi Piece sentezi NOT: gözün parçası örneğin agaroz gibi polimerler kullanılarak sentezlenebilir. Aşağıdaki prosedür, aynı zamanda, PDA (patates dekstroz ağar) ya da SDA&#39;nın (Sabouraud Dextrose Agar) agar türleri, çeşitli göz parçaları üretmek için değiştirilebilir. <ol><li> % 2 (2 g / 100 mi) jel, ölçü agaroz 2 g üretim ve ultra saf su ve 100 ml karıştırın. Agaroz tamamen eriyene şekilde Çıban (100 ºC) çözüm getirin. Çözelti 5 dakika soğumaya bırakın. </li><li> göz küresi kalıba çözüm dökün ve çözüm 3 soğumasını bekleyinOda sıcaklığında 0 dak. bir spatula ile gözün parçalarını çıkarın. Bir de gözün agar Mağaza -20 daha sonra kullanmak üzere ° C dondurucu. Mikrobiyoloji çalışmaları için, otoklav ve / veya UV-ışıması ile gözün kalıpları sterilize edin. </li></ol></li><li> PDMS göz küresi üzerinde Sığır Kornea Ortaklığın Not:. Bu protokol Parekh ark adapte edilmiştir 14 <ol><li> bir laminar akış başlığı altında steril şartlarda sığır kornea diseksiyon ve birleşme gerçekleştirmek. gözleri kazanmak ve aynı gün onları teşrih. </li><li> Kullanmadan önce 10 dakika akış kaputu açın ve% 70 etanol alkol ile dezenfekte. Tüm malzemeler ve aletler 273 ° F / 45 dakika 133 ° C&#39;de otoklav ile steril ve akış kaputu girişinde az 4 inç konumlandırılmış emin olun. </li><li> 2 dakika için seyreltilmiş bir povidon-iyodin solüsyonu içeren bir cam kapta sığır göz bırakın. fosfat tamponlu tuzlu su ihtiva eden bir beher içinde göz yıkama (PBS) p-H 7.4. Forseps hafifçe cam petri, kornea yüzü yukarı göz yerleştirin. </li><li> künt uç diseksiyon makas ile skleral eki noktalarında kesim tarafından aşırı kas ve yağ dokusu çıkarın. hayvan atıkları için belirlenmiş bir steril beher içine aşırı doku atınız. </li><li> Mikro-makas kullanarak, gözün konjonktiva çıkarın. limbusta en az 1 cm&#39;lik bir mesafeyi koruyarak, steril gazlı bez ile göz sarın. </li><li> Altta yatan koroid ve vitreus penetrasyonunu önlemek ve yüzeysel şekilde bir neşter kullanılarak, yaklaşık 2 mm limbus bölgeden sklera insizyon. Dikkatle doğal eğrilik kornea deforme olmadan bir neşter veya diseksiyon makas kullanılarak 360 ° kesi uzatmak. </li><li> ince forseps ile gözün kornea çıkarın. forseps kullanarak, dikkatli herhangi yapışan uveal dokuyu çıkarmak ve PBS ile kornea durulayın. </li><li> Kültür steril bir kap içinde 31ºC kornea MağazaOrta (Medium 199 gibi), doku nem ve hücre besin sağlamak için% 3 fetal sığır serumu ihtiva eden. </li><li> deney öncesinde, PDMS göz küresi üzerinde eksize kornea dinlenme ve özel bir klipsli ile birlikte iki adet kelepçe. </li></ol></li></ol> 2. Blink platform <ol><li> Blink-platform Tasarımı ve Üretimi NOT: kırpma platform üç fonksiyonel bölümden oluşmaktadır: göz modeli (bölüm 1 &#39;de tarif edilmiştir), bir vites indirgeme sistemi, elektronik sistem. <ol><li> Tasarım göz modeli (bölüm 1.1) için tarif edilene benzer CAD ve 3D baskı kullanarak yanıp platformu üretim ve. O eyepieces yanal ve dönme hareketleri içine motorların basit dönüşünü çevirir şekilde vites sistemi tasarlayın. 15 </li><li> pinyon ve dişli mekanizmasını kullanarak, kapak parçalarına bağlı olan bir dişli, yatay hareketine bir kademeli motorun dönme hareketi çevirir. </li><li> Kullanmakkonjuge bir vites indirgeme sistemi, üç farklı küresi parçaları için üç (veya daha fazla) dönme hareketleri bir adım motoru ile ilgili bir dönme hareketi yükseltmek. </li><li> ikisi arasındaki mesafenin sabit böylece, iki dişli sistemleri, göz kapağı diğeri göz küresi için bir hizaya getirin. bir mikro motor kalkan ve iki motor ile elektronik sistem monte edin. NOT: yanıp sönen bir hareket içine dişli sistemi tarafından çevrilmiştir dönme motorları sağlamak için iki step motorlar kullanın. </li><li> mikrodenetleyici yığılmış bir motorlu kalkan oluşan bir sistem ile iki step motorlar bağlayın. Bağlayın ve açık kaynak kodlu yazılım ürünleri ile çalışmak için elektronik bileşenler yapılandırın. </li><li> Böyle dakikada mermi (RPM), ileri mermi sayısı, geriye mermi sayısı ve dönme tarzı olarak motor parametrelerini kontrol etmek için sistemin programlanmasına. NOT: Ayrıntılı bilgi için tamamlayıcı &quot;Arduino kod dosyası&quot; bölümüne bakın. </li><li> ma sistem yazılımını indirinnufacturers &#39;web sitesi. </li><li> yazılımı yükleyin ve açın. İstenilen yapılandırmada step motorları kontrol etmek için bir kod yazın. araştırmacı tarafından tanımlanan motorlar istenilen şekilde hareket böylece elektronik sistem güç bir kaynağı ile bağlantı sistemi. NOT: Ek &quot;Arduino kod dosyası&quot; bölümüne bakın. </li></ol></li><li> Mikroakiskan ile Meclisi (Yapay Gözyaşı Çözüm) <ol><li> Sentezlenen göz küresi ve göz kapağı parçaları alın ve göz modeli için bunlara karşılık gelen klip-ons üzerine onları kayma. gözkapağı parça (bölüm 1.2.5) ile mikroakışkan pompa üzerinde bir şırınga ile katıldı ve konumlandırılmış boru bağlayın. Test platformu çalıştırmak ve tutarlı hareket edin. </li><li> Başbakan boru ve suni gözyaşı solüsyonu (ATS) sürekli bir akış kontrol edin. ATS için reçete daha önce rapor edilmiştir. 16 </li><li> El ile, bir seviye uçakta birlikte göz modeli parçaları taşımak şekilde göz küresi ve gözKapak temas halindedir. istenen değerlere mikroakışkan pompa akış hızını ayarlayın. 1-1.5 ul / dk fizyolojik debileri ayarlayın. 17 </li><li> deney başlamak için pompayı ve aktüatörler başlayın. ilaç verme deneyleri için, göz küresi parçasına ilaç içeren kontakt lens yerleştirin. </li><li> akış sıvısı 12 plaka içine damla izin verin. İstenen belirlenen zaman aralıklarında, UV-Vis spektroskopisi veya floresan gibi ortak bulgulama yöntemleri kullanarak analit veya ilaç konsantrasyonunu ölçmek. 1,4,18 </li><li> kontakt lensler göz yaşartıcı bileşenlerinin birikimi değerlendirmek çalışmaları için, &quot;göz küresi&quot; parçasının üzerine kontakt lens yerleştirin. atılabilir akış sıvısı, toplayın. </li><li> istenilen zaman aralıklarında sonra, göz küresi parçasından kontakt lensi çıkarın ve konfokal mikroskopi olarak daha fazla analiz için lensi hazırlamak. </li></ol></li></ol>

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Yazarlar ifşa hiçbir şey yok.

Orada özel dikkat gerektiren protokolü içinde üç kritik adımlar şunlardır: tasarım ve kalıp üretimi (bölüm 1.1), platformu montajı (bölüm 2.2.1-2.2.3) ve deneysel koşmak izleme (bölüm 2.2.4-2.2.7 ). tasarımı ve kalıplar (Bölüm 1.1) üretimi açısından, gözün parçası, bir insan kornea boyutlarına göre tasarlanmış olmalıdır. Ancak, mükemmel bir ticari kontakt lens (CL) uyuyor oluşturulabilir bir göz küresi parçasının önce kalıbın birden prototipler gerektirebilir. göz küresi...

kontakt lens (KL) arena içinde ilgi iki önemli alanlar rahatsızlık ve yeni CL uygulamalarının geliştirilmesini içermektedir. CL rahatsızlık altında yatan mekanizmaların tanıtılması yıllardır alanını atlatmış olan bir konudur. 8 romanın gelişimi, bu tür ilaç iletim cihazları 1,3,9 ve biyosensörler gibi fonksiyonel ZL, 10-12 büyüyen bir ilgi alanıdır, önemli potansiyel pazarları ile. Her iki durumlarda, in vitro model sofistike bir gelişim aşamasında lens malzemeleri veya tasarım özellikleri uygun seçilmesi ile yardımcı olmak için ilgili bilgileri sağlayacaktır. Ne yazık ki, cls ve diğer göz ile ilgili uygulamalar değerlendirilmesi için in vitro modellerinde geçerli nispeten kaba ve deneyimsiz bulunmaktadır. Geleneksel olarak, gözyaşı filmi birikimi veya ilaç dağıtım değerlendiren in vitro CL çalışmalar sabit bir sıvı hacmi içeren statik, büyük hacimli şişeler içinde yapılır hangi greatly fizyolojik miktarlarda aşmaktadır. Ayrıca, bu basit model, doğal gözyaşı akışı bileşen ve göz çevresinin faktörleri tanımlayarak, her ikisi de yanıp sönen refleks, yoksun. Sofistike, fizyolojik ilgili göz &quot;model&quot; geliştirilmesi multi-disipliner bir yaklaşım gerektiren ve in vivo doğrulama önemli gerektirecektir. Bu nedenlerden dolayı, bizim in vitro göz modeli için temel çerçeve modeli sürekli gelecek yükseltmeleri ve modülasyon yoluyla geliştirilebilir şekilde, son derece çok yönlüdür. Bugüne kadar, bir model gözyaşı hacmini, yırtılma akışı mekanik aşınma ve hava maruz uyarabilmesi. Amaç vivo ve ex vivo gözlemlerinde için akıllı ve ücretsiz bir anlamlı sonuçlar verecektir in vitro bir model, yaratmaktır.

<table border="0" cellpadding="0" cellspacing="0" width="1129">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">Arduino Uno R3 (Atmega328 - assembled)</td>
			<td style="width:125px;">Adafruit</td>
			<td style="width:451px;">50</td>
			<td style="width:273px;">Board</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Stepper motor</td>
			<td style="width:125px;">Adafruit</td>
			<td style="width:451px;">324</td>
			<td>Motor and Motor shield</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Equal Leg Coupler 1.6mm 1/16&#34;</td>
			<td style="width:125px;">VWR</td>
			<td style="width:451px;">CA11009-280</td>
			<td>50 pcs of tube connector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Tubing PT/SIL 1/16&#34;x1/8&#34;</td>
			<td style="width:125px;">VWR</td>
			<td style="width:451px;">16211-316</td>
			<td>Case of 50feet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">PDMS</td>
			<td style="width:125px;">Dow Corning</td>
			<td style="width:451px;">Sylgard 184 Solar Cell Encapsulation</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Agarose, Type 1-A, low EEO</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:451px;">A0169-25G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">PHD UltraTM</td>
			<td style="width:125px;">Harvard Apparatus</td>
			<td style="width:451px;">703006</td>
			<td>MicroFluidic Pump</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">Bovine cornea</td>
			<td style="width:125px;">Cargill, Guelph/ON</td>
			<td style="width:451px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">Soldidworks</td>
			<td style="width:125px;">Dassault Systemes</td>
			<td style="width:451px;"></td>
			<td>Software</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:281px;">3-D printing</td>
			<td style="width:125px;">University of Waterloo - 3D Print Centre</td>
			<td style="width:451px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">Dissection tools</td>
			<td style="width:125px;">Fine Science Tools</td>
			<td style="width:451px;"></td>
			<td>General dissection tools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Medium 199</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:451px;"></td>
			<td>Culture medium storage for cornea</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Fetal bovine serum</td>
			<td style="width:125px;">Thermo Fisher</td>
			<td style="width:451px;"></td>
			<td>Add to culture medium, 3% total volume</td>
		</tr>
	</tbody>
</table>

development of an in vitro ocular platform to test contact lenses

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic physiological tear volumes.7 The traditional model also lacks the natural tear flow component and the blinking reflex, both of which are defining factors of the ocular environment. The development of a novel model is described in this study, which consists of a unique 2-piece design, eyeball and eyelid piece, capable of mimicking physiological tear volume. The models are created from 3-D printed molds (Polytetrafluoroethylene or Teflon molds), which can be used to generate eye models from various polymers, such as polydimethylsiloxane (PDMS) and agar. Further modifications to the eye pieces, such as the integration of an explanted human or animal cornea or human corneal construct, will permit for more complex in vitro ocular studies. A commercial microfluidic syringe pump is integrated with the platform to emulate physiological tear secretion. Air exposure and mechanical wear are achieved using two mechanical actuators, of which one moves the eyelid piece laterally, and the other moves the eyeballeyepiece circularly. The model has been used to evaluate CLs for drug delivery and deposition of tear components on CLs.

Makine dükkandan ve 3-D baskı elde edilen sentez göz kalıplar, Şekil 1 &#39;de gösterilmiştir. Bu kalıplar, istenen özelliklere sahip oküler üretilmesi için, örneğin agaroz PDMS ve benzeri gibi polimerler, çok çeşitli kullanılabilir. Mikroakışkan şırınga pompası göz modeli platformun işaret düzeneği, Şekil 2 &#39;de gösterilmiştir. Platformu ve kapak parçasının hareketi üzerinden yan boyunca gözün parçasının dönd?...

Watch this Scientific Journal Video about Development of an In Vitro Ocular Platform to Test Contact Lenses at JoVE.com

Development of an In Vitro Ocular Platform to Test Contact Lenses

Kontakt lens (ZL) ve diğer göz ile ilgili başvurularının değerlendirilmesi için mevcut in vitro modeller ciddi sınırlıdır. sunulan okular platformu fizyolojik gözyaşı akışı, gözyaşı hacmini, hava pozlama ve mekanik aşınma simüle. Bu sistem, çok yönlü ve CL analizler nitro çeşitli uygulanabilir.

Bir Gelişimi<em&gt; İn Vitro</em&gt; Göz Platformu Lensler Test

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic ...

development-of-an-in-vitro-ocular-platform-to-test-contact-lenses

Research

JoVE Journal

Bioengineering

Development of an In Vitro Ocular Platform to Test Contact Lenses

10.5K Görüntüleme.  University of Waterloo.  Kontakt lens (ZL) ve diğer göz ile ilgili başvurularının değerlendirilmesi için mevcut in vitro modeller ciddi sınırlıdır. sunulan okular platformu fizyolojik gözyaşı akışı, gözyaşı hacmini, hava pozlama ve mekanik aşınma simüle. Bu sistem, çok yönlü ve CL analizler nitro çeşitli uygulanabilir. 

Video: Development of an In Vitro Ocular Platform to Test Contact Lenses

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

İnsan 3D Doku Mühendisliği<em&gt; In vitro</em&gt; Tümör Test Sistemi

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Biyomühendislik

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or erosion. Amelogenin has been proven to be a critical protein for controlling the organized growth of apatite crystals. In this paper, we present a detailed protocol for superficial enamel reconstruction by using a novel amelogenin-chitosan hydrogel. Compared to other conventional treatments, such as topical fluoride and mouthwash, this method not only has the potential to prevent the development of dental caries but also promotes significant and durable enamel restoration. The organized enamel-like microstructure regulated by amelogenin assemblies can significantly improve the mechanical properties of etched enamel, while the dense enamel-restoration interface formed by an in situ regrowth of apatite crystals can improve the effectiveness and durability of restorations. Furthermore, chitosan hydrogel is easy to use and can suppress bacterial infection, which is the major risk factor for the occurrence of dental caries. Therefore, this biocompatible and biodegradable amelogenin-chitosan hydrogel shows promise as a biomaterial for the prevention, restoration, and treatment of defective enamel.

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

In this article, we describe a protocol for fabricating an amelogenin-chitosan hydrogel for superficial enamel reconstruction. Organized in situ growth of apatite crystals in the hydrogel formed a dense enamel-restoration interface, which will improve the effectiveness and durability of restorations.

Için Amelogenin-kitosan hidrojelin geliştirilmesi<em&gt; In Vitro</emBir Yoğun Arayüzü ile&gt; Emaye büyütme

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

Yeniden İnşa İnsan Kornea gibi Epitelyal (RHCE) Doku Modeli kullanılarak Göz tahriş Kimyasalların Tehlike Tanımlaması Göz tahrişi Testi (EIT)

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

Vasküler Hücre Remodeling Soruşturma Fizyolojik in vitro Modeli ve Uygulaması ile Patolojik Kan Akış

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

bir<em&gt; İn Vitro</emKemirgen İntervertebral Disk&gt; Organ Kültürü Modeli

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

Bir Mikro Kuyruklu Dizi Platformunda Mikrobik Topluluk Geliştirmenin Montajı ve Takibi

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

Gıda kaynaklı patojen tespit etmek için bir elektrokimyasal DNA biyoalgılayıcı gelişimi

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

Tek hücreli genom sıralama için bir üretilen iş Ultrahigh mikrosıvısal Platform

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

Rodent Behavioral Testing to Assess Functional Deficits Caused by Microelectrode Implantation in the Rat Motor Cortex

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes demonstrate the ability to record action potentials from individual or small groups of neurons. Such recorded signals have successfully been used to allow patients to interface with or control&#160;computers, robotic limbs, and their own limbs. However, previous animal studies have shown that a microelectrode implantation in the brain not only damages the surrounding tissue but can also result in functional deficits. Here, we discuss a series of behavioral tests to quantify potential motor impairments following the implantation of intracortical microelectrodes into the motor cortex of a rat. The methods for open field grid, ladder crossing, and grip strength testing provide valuable information regarding the potential complications resulting from a microelectrode implantation. The results of the behavioral testing are correlated with endpoint histology, providing additional information on the pathological outcomes and impacts of this procedure on the adjacent tissue.

We have shown that a microelectrode implantation in the motor cortex of rats causes immediate and lasting motor deficits. The methods proposed herein outline a microelectrode implantation surgery and three rodent behavioral tasks to elucidate potential changes in the fine or gross motor function due to implantation-caused damage to the motor cortex.

Kemirgen fonksiyonel açıkları sıçan Motor korteks elektrot implantasyon nedeniyle değerlendirmek için sınama davranış

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Uzun Süreli Nanomalzeme Maruziyetini Takiben İn vitro Genotoksisite Testi için Gelişmiş 3D Karaciğer Modelleri

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

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İnsan 3D Doku Mühendisliği

Için Amelogenin-kitosan hidrojelin geliştirilmesi