The authors thank the Fraunhofer ISC for the collaboration concerning the construction of the automated wounding device. The project was founded by Fraunhofer internal project &#8220;M&#228;rkte von &#220;bermorgen&#8221; (SkinHeal).

NOT: protokol 24 tam kalınlıkta deri eşdeğerleri üretimi için tasarlanmıştır. Dermal fibroblastlar ve epidermal keratinositler İnsan 21,22 Aydınlatılmış onam önceden elde edildi. Daha önce yayınlanmış protokole göre deri biyopsi izole edilmiş ve çalışma Julius-Maximilians-Üniversitesi Würzburg insan araştırma kurumsal etik komitesi tarafından onaylanmıştır (182 oy / 10). Dermal Bileşeni 1. Üretimi <ol><li> 6 mg / ml&#39;lik nihai bir konsantrasyona kadar% 0.1 asetik asit ile kollajen çözülür. Jel nötrleştirme çözeltisi için, 232,5 mi 2x DMEM, 7.5 ml cenin buzağı serumu, 7.5 ml 3 M HEPES, 2.5 ml kondroitin sülfat (5 mg / ml) karıştırın. Buz üzerinde jel nötrleştirme çözeltisi ve kolajen çözeltisi tutun. Insert / deri eşdeğeri başına jel 500 ul hesaplayın. NOT: jel nötrleştirme çözeltisi kolajen çözeltisi Prepa iki parça ile karıştırılacaktır Adıkolajen çift miktarı yeniden. </li><li> 24 plaka içine 24 ekler yerleştirin. </li><li> 10 ml DMEM, 270 x g&#39;de 5 dakika boyunca santrifüj insan dermal fibroblastları (HDF) yeniden süspanse edin ve hücreleri sayın. X g, 270 ° C&#39;de 5 dakika için gerekli HDF bir miktar (500 ul kollajen jel başına 5 x 10 4 hücrelerinde) ve santrifüj hücreleri ekstrakte edin. </li><li> 4 ml hava kabarcıkları üretmeden jel nötralizasyon çözümü soğutulmuş HDF tekrar süspansiyon dikkatlice Süpernatantı ve. Bu aşamada itibaren kollajen jeller erken katılaşmasını önlemek için mümkün olduğunca hızlı çalışması için emin olun. </li><li> Kolajen çözeltisi, 8 ml çözelti yeniden süspanse edin. </li><li> Çok aşamalı-pipet ile kollajen-hücre-karışımı alın ve her insert hızlı karışımın 500 ul doldurun. Bir kuluçka makinesi içinde 20 dakika süreyle inkübe edin jeller (37 ° C,% 5 CO2) jelleri jöle için. </li><li> Oyuk başına 2 ml&#39;lik Dulbecco&#39;nun Modifiye Edilmiş Eagle Ortamı (DMEM)% 10 fetal dana serumu (FCS) içinde jeller daldırınve bir kuluçka makinesi içinde 24 saat (37 ° C,% 5 CO2) için kuluçkalayın. </li></ol> İnsan epidermal Keratinositler 2. eklenmesi (HEK) <ol><li> 24 saat sonra, orta çıkarın. 50 ug / ml&#39;lik nihai bir konsantrasyona kadar ultra-saf su ile fibronektin insan proteinini çözülür. Fibronektin çözeltisi 25 ul eşdeğer her dermal örtün. Bir kuluçka makinesi içinde, 30 dakika süre ile inkübe jeller (37 ° C,% 5 CO2). </li><li> Bu arada, 10 mi KGM-2&#39;de HEK tekrar süspansiyon ve KGM-2 5 + uyumlu% FCS ile 1 x 10 6 hücre / ml hücre konsantrasyonu ayarlanır. Her jel üzerinde Tohum 1 x 10 5 HEK. Inkübatörde 45 dakika boyunca (37 ° C,% 5 CO2) hücre yapışmasının sağlanması için jeller inkübe edin. </li><li> Kuluçka makinesi içinde, KGM-2 + uyumlu,% 5 FCS ve kültür olması (37 ° C,% 5 CO2) ile jeller batırın. </li></ol> Tam Kalınlıkta Deri Benzerleri 3. Kültür (FTSE) <ol><li> 6-7 gün boyunca kültür ftseFCS konsantrasyonu (% 5,% 2, ve% 0 FCS) inen. Bir sonraki alt FCS konsantrasyonu ile 2-3 günde orta değiştirin. Bunun için, tam orta kaldırmak ve taze ortam içinde 1.6 ml ilave ediniz. </li><li> 7. günde, tam ortam maddesinin ayrılması. NOT: Bunu yaparken pipet ucu ile FTSE yüzeyine dokunmayın. </li><li> Her steril forseps ile 6 plaka 1 kuyuya eklemek yerleştirin. FTSE yüzeyini ıslak değil sağlanabil- iyi başına 1.5 ml hava ikmal orta ekleyin. Ilave 14 gün boyunca 2-3 günde bir değiştirilen ortam içinde. NOT: orta dolum seviyesi FTSE menisküsüne kadar. </li></ol> 4. Histolojik analiz <ol><li> Tespit ve FTSE parafin-gömme <ol><li> Oda sıcaklığında 2 saat boyunca dokular taze 24 yuvalı plakalar içinde uçlar aktarmak ve de 1.6 mi, her ila% paraformaldehid 4 eklenerek dokular tamir ve inkübe, kültür ortamı çıkarın. Dikkat! Paraformaldehyd toksik, davlumbaz altında manipüle. TransferBir doku gömme kaset FTSE. Su ile yıkama ile geri kalan fiksatif çıkarın ve artan etanol konsantrasyonu kullanılarak doku dihidrat. </li><li> Orta dikey bir kesik yoluyla deri eşdeğer bölün. Aşağı kesilmiş yüzeyleri ile bir parafin dolu metal taban kalıp iki adet yerleştirin. Bir destek olarak, kalıbın üst doku kaseti ekleyin. </li><li> Uygun mikroskop lamı üzerine slaytlar monte, 3-5 mikron dilim kesin ve straitening için 40 ° C su banyosunda onları yüzer. Iyice kuru dilim. </li></ol></li><li> Hematoksilen &amp; Eosin (H &amp; E) ve immunohistokimyasal (İHK) boyama <ol><li> Talep rafa kayar ve 60 ° C de 1 saat süreyle inkübe edin. Parafin erimiş olduğundan emin olun. Leke standart bakış boyama gibi H &amp; E ile dilimleri rehidre. </li><li> H &amp; E boyama için etanol% 50, 4x deiyonize su, 1x Hematoksilin, 1x HCI-e 1x, etanol ile 2x, etanol ile 3x, ksilolde ile% 70% 96 4x cam boyama cuvettes hazırlamak-metanol (13.7 mi, 1 M HCI reklam 200 ml% 50 etanol), 1x kullanma suyu, 1x eozin ve 2 x 2-propanol (100 ml deiyonize su içinde 1 g eosin). </li><li> Yer ksilol I 10 dakika ve deparaffinize ve slaytlar rehydrate xylol II 10 dakika slaytlar. Daldırma etanol,% 50 etanol% 70, 3 x 3x, etanol ile% 96 I, 3 x 96% II 3x kayar. Deiyonize su slaytlar döndürün. Hematoksilen boyası, HCI-etanol daldırma 2x ayırt etmek. Iyonu giderilmiş su içinde durulayın. Blueing için, yer sekme suda 5 dakika slaytlar. Eozin boyama, yer eozin 1 dakika kayar ve deiyonize su içinde daha sonra yıkanır. Dehidrasyon için, daldırma slaytlar etanol,% 96 etanol içinde% 70, 2 x 2 x ve 2-propanol II, 5 dakika için, 2-propanol I, 5 dakika için koyun, ksilol içinde 5 dakika I ve ksilol II 5 dak. H &amp; E boyama sonra, hücre çekirdekleri kırmızı lekeli mavi, sitoplazma ve hücre dışı matriks içinde lekeli. </li><li> Antijen alımı için, ksilen ile parafin kaldırmak ve boyama için dilimleri rehydrate. Yer dilimleriDüdüklü tencereyi önceden ısıtılmış 10 mM, 20 dakika sitrat tamponu (pH 6) için deparafinize ve rehidre dilimleri yemek ve. </li><li> Bir sıvı itici slayt işaretleyici kalem veya elmas kalemle yıkama tamponu yerleştirin slaytlar (PBS tampon +% 0.5 Polisorbat 20) ve daire dilimleri. </li><li> Bir nem odasında slaytlar yerleştirin. Her bir dilim üzerine 100 ul% 3 hidrojen peroksit çözeltisi ilave edilerek, endojenez peroksidazı bloke eder. Dikkat! Cilt ve göz temasında Çok tehlikeli. Kişisel koruyucu ekipman ile taşıyınız. Yıkama yıkama tamponu slaytlar. </li><li> Birincil antikor uygulayın ve oda sıcaklığında 1 saat boyunca inkübe edilir. Yıkama Yıkama tamponu ile üç kez kayar. Burada, numuneler karanlıkta tutulmalıdır. </li><li> Bağlayıcı spesifik bir antijen-antikor tespiti için biyotin-streptavidin algılama sistemi kullanın. </li><li> 1 dakika boyunca Hematoksilen çekirdekler Counterstain. Kurutmak ve slaytlar monte edin. </li><li> Bir ters mikroskop kullanılarak görüntü örnekleri. </li></ol></li></ol> &lt;p class = &quot;jove_title&quot;&gt; 5. Otomatik Yaralama Cihazı Kullanımı yaralanma (AWD) <ol><li> % 70 etanol ve ultraviyole ışık ile çalışma alanını dezenfekte ile AWD hazırlayın. Istenilen büyüklükte sondaj kafası emin olun (örneğin, 1 mm) eklenmiştir. </li><li> Steril forseps kullanarak otoklava numune taşıyıcı plakanın belirlenen alanlara cilt eşdeğerleri yerleştirin. Sondaj başının altındaki sokete örnek taşıyıcı plaka yerleştirin. </li><li> Spin hızı: aşağıdaki gibi yaralama parametrelerini ayarlamak için kontrol yazılımını kullanın 15,000 rpm; penetrasyon: 1,5 mm; tahrik: 100 Hz. Bu parametreler, her bir numune tipi için tanımlanması gerekir. Transferi AWD parametreleri yaralandı. </li><li> Prosedürü yaralama başlayın. Soket örnek taşıyıcı plakasını çıkarın. Aktarım steril forseps kullanarak kültürü için kullanılan ekler geri FTSE yaralandı. </li><li> Rejenerasyon araştırılması için kuluçka makinesi içinde yaralı ftse kültürü (37 ° C,% 5 CO2) devam edin. Alternatively, herhangi bir zamanda, histolojik analiz için modeller kullanmaktadır. </li></ol>

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The authors have nothing to disclose.

Nitro hücrelerde genellikle hücreler plastik yüzeylere yapışan iki boyutlu hücre kültürlerinde, yayılır. Bununla birlikte, bu kültür koşulları hücrelerinin in vivo büyümek olan fizyolojik üç boyutlu koşulları yansıtmamaktadır. Üç boyutlu koşulları altında, hücreler, doğal hücre-hücre ve hücre-matris bağlanmalarının ve üç boyutlu olarak yer değiştirmektedir. Hücre göçü ve matriks üretimi yara iyileşmesinin temel unsurları olarak özellikle in vivo ...

Deri vücudun en büyük organıdır. Bu dış çevre ve iç organlar arasında bir bariyer oluşturur. Ayrıca, cilt sıvı kaybı, çevresel etkilere, yaralanma ve enfeksiyonlara karşı vücudu korur ve vücut ısısını 1 düzenlemeye yardımcı olur. Nedeniyle açıkta bir konumda, cilt genellikle mekanik, termal veya kimyasal travma etkilenir. Cilt kendini tamir genellikle yetenekli olmasına rağmen, böyle bir enfeksiyon, oksijenlenme ve venöz yeterlilik gibi birden fazla yerel faktörler bozulmuş yara iyileşmesi yol açabilir. Yara iyileşmesi de obezite, alkolizm, sigara, ilaç, beslenme ve diyabet gibi hastalıklar gibi sistemik faktörler tarafından müdahale edilebilir. 2 (i) iltihaplı faz, (ii) proliferatif ve (iii) yeniden modelleme aşaması: yara iyileşmesi süreci 3 evreye ayrılabilir. Cilde hasar üzerine, karmaşık bir sinyal kademeli yara kapanmasına neden başlar. 3Yaralanma sonrası yara kanama ve bir kan pıhtısı meydana gelir. Fibroblastlar kan pıhtısı içine taşımak ve daha sonra yılda yenilenmiş yeni doku ile değiştirin. Biyolojik süreçler altta yatan deri onarım mevcut anlayış sınırlıdır. Küçük hayvan ve domuz modelleri yara iyileşmesini incelemek için kullanılmıştır. Bununla birlikte, bu sonuçlar, doğrudan türe özgü farklılıklar nedeniyle insanlarda transfer edilemez. Bu in vivo modellerde ek olarak, yara iyileşmesi bazı yönlerini 4 Bu çizilmeye modeller oldukça standardize edilir. Ölümsüzleştirdi hücre hatları veya primer hücrelerde dayalı in vitro tek tabaka kültürlerinin çizilmemesi yoluyla bir yara durumu taklit ederek ele alınabilir, ancak yeterince yansıtmıyor kompleks in vivo olarak fizyoloji. 5, iki-boyutlu bir model yanı sıra, üç-boyutlu bir insan derisi eşdeğer dermatolojik araştırmaları için geliştirilmiştir. Bu modellerin dermal kısmı ge vardırnerated decellularized dermis, 6 kollajen hidrojeller, 7,8 glikosaminoglikanlar 9 veya sentetik malzemelerden dahil olmak üzere çeşitli iskele kullanarak. 10 bu deri eşdeğerleri istihdam, epitelyal-mezenkimal etkileşimleri 11 rolünü yeniden epitelizasyon, fibroblast ve keratinositler arasındaki hücresel karışma Farklı büyüme faktörlerinin etkisi incelenebilir. Ayrıca, bu modeller fibroblastlar yaralı bölgeye göç ve nasıl kemotaktik faktörler doku yenilenmesini nasıl etkilediği hakkında yeni bilgiler elde etmek için yararlıdır. 12 Yara iyileşmesi modelinin kendisinde değil, sadece üretim zor olmakla birlikte, aynı zamanda, bir modelde yüksek ölçüde standardize edilmiş yara sorunludur kurmak. Ortak teknikler yaralar çizik testleri, 13 yanıklara, 14 teyp aşınma, 15 termal yaralanma, 16 emme kabarcıklar, 17 sıvı azot, 18 lazerler, 19 olan oluşturmak için </sup&gt; neşter, 18 meshers 6 ve biyopsi yumruklar. Bu yöntemlerin çoğu 20 aynı tuzaklar var. Elle uygulanan yaralanmalar standartlaştırmak ve çoklu testler arasında yeniden zordur. Boyut, şekil ve yaranın derinliği çalışmalar arasında değişir ve böylece araştırma verilerinin kalitesini bozar. tanımlı cilt yaralanması için lazer kullanımı nispeten kolay standardize ancak yanık yaraları taklit bir duruma yol açar edilebilir. Lazer tarafından uygulanan ısı nekrotik dokunun yol açabilir protein denatürasyonu, trombosit agregasyonu veya damarların daralmasına neden olabilir. Alternatif bir yaklaşımda, bize steril koşullar altında tanımlanmış ve hassas deri yaraları oluşturmak için izin veren bir otomatik yaralama cihazı (AWD) geliştirdi. Derinlik ve hız penetrasyonu olarak matkap ucu devir gibi yaralanması parametreleri ayarlanabilir. Bu çalışmada, biz evde geliştirilen tam kalınlıkta deri eşdeğerleri ile AWD kombine (ftGangatirkar ve arkadaşları tarafından yayınlanmış bir protokol ile karşılaştırılabilir SE). 8 deri eşdeğerinde dermal katman, hidrojel bir kollajen gömülü, insan dermal fibroblastları (HDF), oluşmaktadır. Dermal katmanda, insan epidermal keratinositler (HEK) numaralı seribaşı bulunmaktadır. Hava-sıvı arayüzü iki hafta içinde HEK birçok hayati hücre katmanları ve stratum corneum oluşan bir epidermis kurmak. Bu modelin oluşturmasının yanı sıra, bu çalışma FTSE tanımlanmış ve hassas yaralar oluşturmak üzere AWD kullanımını göstermektedir.

<table border="0" cellpadding="0" cellspacing="0" width="750">
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		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Name of Material/ Equipment</td>
			<td style="width:135px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:329px;"></td>
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			<td style="width:119px;"></td>
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			<td height="21" style="height:21px;width:329px;">Trypsin EDTA (1:250) 0.5 % in DPBS&#160;</td>
			<td style="width:135px;">PAA</td>
			<td style="width:119px;">L11-003</td>
			<td align="right">0.05%</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">D8537</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Collagen (6 mg/ml in 0,1 % acetic acid)</td>
			<td style="width:135px;">Produced in house</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:329px;">Fibronectin Human Protein, Plasma (50 &#181;g/ml)</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:119px;">33016-015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Inserts</td>
			<td style="width:135px;">Nunc</td>
			<td align="right" style="width:119px;">140627</td>
			<td></td>
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			<td height="21" style="height:21px;width:329px;">6 well plate&#160;</td>
			<td style="width:135px;">Nunc</td>
			<td align="right" style="width:119px;">140685</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">24 well plate</td>
			<td style="width:135px;">Nunc</td>
			<td align="right" style="width:119px;">142485</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Microscope slides</td>
			<td style="width:135px;">R. Langenbrinck</td>
			<td style="width:119px;">03-0070</td>
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			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:329px;">Fibroblasts culturing (500 ml):</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:329px;">DMEM, high glucose</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:119px;">11965-092</td>
			<td>89% (445 ml)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Fetal bovine serum</td>
			<td style="width:135px;">Bio &#38; Sell</td>
			<td style="width:119px;">FCS.ADD.0500</td>
			<td>10% (50 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td>1% (5 ml)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:329px;">Keratinozyten culture medium (500 ml):&#160;</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Keratinocyte Growth Medium 2</td>
			<td style="width:135px;">Promocell&#160;</td>
			<td style="width:119px;">C-20111</td>
			<td>89% (445 ml)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Keratinocyte Growth Medium 2 SupplementPack</td>
			<td style="width:135px;">Promocell&#160;</td>
			<td style="width:119px;">C-39011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td>1% (5 ml)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Gel neutralization solution (250 ml):</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Dulbecco&#8217;s Modified Eagle Medium, high Glucose Powder with L-Glutamine</td>
			<td style="width:135px;">PAA</td>
			<td style="width:119px;">G0001,3010</td>
			<td>93% (232,5 ml)</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Chondroitin sulfate sodium salt from shark cartilage</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">C4384-1g</td>
			<td>1% (2,5 ml)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Fetal bovine serum</td>
			<td style="width:135px;">Bio &#38; Sell</td>
			<td style="width:119px;">FCS.ADD.0500</td>
			<td>3% (7,5 ml)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:329px;">HEPES</td>
			<td style="width:135px;">Sigma&#160;</td>
			<td style="width:119px;">H3375-1kg</td>
			<td>3% (7,5 ml)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
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			<td height="21" style="height:21px;width:329px;">Skin model submers medium (500ml):&#160;</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Keratinocyte Growth Medium 2</td>
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			<td style="width:119px;">C-20111</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Keratinocyte Growth Medium 2 SupplementPack</td>
			<td style="width:135px;">Promocell&#160;</td>
			<td style="width:119px;">C-39011</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Fetal calf serum</td>
			<td style="width:135px;">Bio &#38; Sell</td>
			<td style="width:119px;">FCS.ADD.0500</td>
			<td>5%-2% (25 ml-10 ml)&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td>1% (5 ml)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Skin model air-liquid interface medium (500 ml):&#160;</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Keratinocyte Growth Medium 2</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Keratinocyte Growth Medium 2 Supplement Pack</td>
			<td style="width:135px;">Promocell&#160;</td>
			<td style="width:119px;">C-39011</td>
			<td>adding only supplements: Insulin, Hydrocortisone, Epinephrine, Transferrin, CaCl2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td>1% (5 ml)</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:329px;">CaCl2 (300 mM)</td>
			<td style="width:135px;">Sigma&#160;</td>
			<td style="width:119px;">C7902-500g</td>
			<td>&#160;0,62% (3,1 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Histology:</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">IHC-Kit DCS SuperVision 2 HRP</td>
			<td style="width:135px;">DCS</td>
			<td style="width:119px;">PD000KIT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Vimentin antibody</td>
			<td style="width:135px;">Abcam</td>
			<td>ab92547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">CK14 antibody</td>
			<td style="width:135px;">Sigma&#160;</td>
			<td>HPA023040-100&#181;l</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">CK10 antibody</td>
			<td style="width:135px;">Dako</td>
			<td style="width:119px;">M7002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Filaggrin antibody</td>
			<td style="width:135px;">Abcam</td>
			<td>ab81468</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">H&#38;E staining</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Mayer&#180;s Haemalaun</td>
			<td style="width:135px;">AppliChem</td>
			<td style="width:119px;">A0884,2500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Xylol</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td>296325-4X2L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Ethanol</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td>32205-4X2.5L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">HCl</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td>H1758-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Eosin</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td>E4009-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">2-Propanolol</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td>I9516-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Mounting Medium</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td>M1289-10ML</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of a three-dimensional full thickness skin equivalent and automated wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

izole edilmiş bir HDF ve HEK morfolojisinde ve tipik markerlerin ekspresyonu bakımından farklılık gösterir. Hek morfolojisi bir Arnavut kaldırımı morfolojisi ile tarif edilebilir ise Hdf, tipik mil-şekil morfolojisi gösterdi. Hücreler FTSE için bunları kullanmadan önce immünohistokimyasal boyama (Şekil 1) ile karakterize edilmiştir. Hdf vimentin (Şekil 1A), fibroblastlar için bir marker için olumludur. İlköğretim Hek derece erken farklılaşma proteini sitokerati...

Watch this Scientific Journal Video about Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding at JoVE.com

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Üç-boyutlu Tam Kalınlığı Deri Üretimi Eşdeğer ve Otomatik Yaralama

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

generation-three-dimensional-full-thickness-skin-equivalent-automated

Research

JoVE Journal

Bioengineering

18.6K Görüntüleme.  University Hospital Würzburg. The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility. 

Video: Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Üç boyutlu optik çözünürlük foto akustik Mikroskopi

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Biyomühendislik

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Yumurtalıklar ve fallop borularında Üç Boyutlu Organ Kültür Aljinatta Hidrojeller

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

Üç Boyutlu İnsan Cilt Model Yeniden İnşa: Aracı Normal Cilt ve Melanom İlerleme Eğitim

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

İletken Mürekkepler düzlemsel ve Üç Boyutlu Baskı

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

Model Vocal Fold Polip'in Neden Olduğu Üç Boyutlu Akış Ayrımının Araştırılması

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

A Tam Cilt Hata Modeli Biyomateryallerin vaskülarizasyonunu değerlendirin<em&gt; In Vivo</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

Bir Üç boyutlu Kollajen Matrix içindeki Hücre Göç Analizi

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

Hazırlama ve Analizler<em&gt; İn Vitro</em&gt; Üç Boyutlu Meme Kanseri Suretler

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

Three-dimensional Printing of Thermoplastic Materials to Create Automated Syringe Pumps with Feedback Control for Microfluidic Applications

Microfluidics has become a critical tool in research across the biological, chemical, and physical sciences. One important component of microfluidic experimentation is a stable fluid handling system capable of accurately providing an inlet flow rate or inlet pressure. Here, we have developed a syringe pump system capable of controlling and regulating the inlet fluid pressure delivered to a microfluidic device. This system was designed using low-cost materials and additive manufacturing principles, leveraging three-dimensional (3D) printing of thermoplastic materials and off-the-shelf components whenever possible. This system is composed of three main components: a syringe pump, a pressure transducer, and a programmable microcontroller. Within this paper, we detail a set of protocols for fabricating, assembling, and programming this syringe pump system. Furthermore, we have included representative results that demonstrate high-fidelity, feedback control of inlet pressure using this system. We expect this protocol will allow researchers to fabricate low-cost syringe pump systems, lowering the entry barrier for the use of microfluidics in biomedical, chemical, and materials research.

Here we present a protocol to construct a pressure-controlled syringe pump to be used in microfluidic applications. This syringe pump is made from an&#160;additively manufactured body, off-the-shelf hardware, and open-source electronics. The resulting system is low-cost, straightforward to build, and delivers well-regulated fluid flow to enable rapid microfluidic research.

Üç boyutlu termoplastik malzemelerin oluşturmak için yazdırma şırınga pompalar geribildirim denetimiyle mikrosıvısal uygulamalar için otomatik

Microfluidics has become a critical tool in research across the biological, chemical, and physical sciences. One important component of microfluidic ...

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated. Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.

This protocol describes a straightforward method to cultivate three-dimensional reconstituted human epidermis in a reproducible and robust manner. Additionally, it characterizes the structure-function relationship of the epidermal barrier model. The biological responses of the reconstituted human epidermis upon proinflammatory stimuli are also presented.

Büyük Ölçekte Üç Boyutlu Yeniden Yapılandırılmış İnsan Epidermisi Yetiştirmek

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process ...