The University of Alabama at Birmingham Center for Metabolic Bone Disease performed the histologic processing and sectioning of surrogates. Southern Research (Birmingham, AL) provided support for the manufacture of the bioreactor system. Funding was provided by the United States Department of Defense Breast Cancer Research Program (BC121367).

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The authors declare that they have no competing financial interests.

Burada 3D kültür için bir yöntem daha yakın modeller, insan meme kanseri rekapitülasyon 3D morfoloji gelişimine izin vermek için bir hacim içinde, hücre dışı matrisin (ECM) ile, insan da dahil olmak üzere stromal fibroblastlann doku çevresinde bileşenlerinin içerdiğini tarif edilmiştir . tarif edilen 3D kültür yöntemi ECM&#39;nin 3B hacmi içine dahil edilen birden çok hücre tipinde geleneksel 2B hücre kültürü daha insan hastalığının daha iyi temsil eder. Mimari dokusu, mikroçevresinin v...

Daha doğru bir şekilde in vivo olarak tümör mimarisi ve mikro-taklit üç boyutlu (3D) kültür modelleri hücreler ile bunların mikro çevreleri arasındaki karmaşık etkileşimler incelemek için ve aday terapilerinin etkinliğini test etmek için amaçlanmıştır çalışmaları için önemlidir. Tümör boyut etkiler oksijen ve besin eğimleri, ilaç maruziyeti, interstisyel basınç / kan akımı ve 3D mimarisi 1-4 tekdüzelik. Uygun bir stromal mikroçevresinin varlığı tümör boyutluluğu ve etkileri, hücre-ECM sinyalizasyon ve stromal hücreleri ve malign epitel hücreleri arasındaki parakrin sinyalizasyon katkıda bulunur. Tümör boyutluluk ve hücresel fonksiyonu üzerindeki mikro-etkileri de ilaç yanıtı 1,3,5-8 değiştiren her iki faktörün birlikte, kurulur. Buna ek olarak, hücresel büyüme kinetiği, metabolik oranları ve hücre sinyal affe bu faktörler ile, 3D, iki boyutlu (2D) kültür ve kültür arasındaki farklılıkhücresel yanıtı 1,3,8-10 cting. İn vitro olarak, tümör taşıyıcı mikro-Örnek ECM bileşenleri ve stromal hücre popülasyonları da dahil olmak üzere modüle edilebilir. Malign epitel hücreleri tümörün daha fazla yayılma 5,6,10 inhibe ECM ve kanserle ilişkili stromal hücreler tarafından ya da tümör progresyonunu teşvik etmek için sinerjik / koruyucu bir şekilde ya da bastırıcı bir şekilde etkilenmektedir. Her iki çerçevede, stroma ilaç dağıtım 1,6 azalma parakrin sinyalizasyonu ile ve / veya elde edilen tümör geçiş basıncı artırarak terapötik cevabı ve ilaç olarak etkileyebilir. Bu nedenle, klinik öncesi modellerin içine ECM ve stromal hücrelerin eklenmesi 2B kültüründe de model olamaz tümörün yönlerini özetlemek yardımcı olacaktır. Burada bir yöntem olup, ECM bileşenleri ve s de dahil olmak üzere, bir mikro-ortam rekapitülasyon dahil meme kanseri temsilciler, kurma3B hacimde tromal hücreler tarif edilir. Meme kanserinde, stromal hücre popülasyonu baskın kanserle ilişkili fibroblastlar (CAF), stromal ECM oluşan büyük ölçüde laminin ve kolajen tip IV dahil olmak üzere, bazal membran bulunan matris bileşenlerinin küçük bir oran ile kollajen tip I oluşmaktadır 1,4,11-13. Bu nedenle, göğüs karsinoması mikro bu bileşenlerin (yani, CAF, kolajen I ve bazal membran) suretler halinde dahil edilmiştir. Bu yöntem, bir katı, un perfüze 3D suretler (Şekil 1A) oluşturmak için ya da biyoreaktör sisteminde (Şekil 1B) ile vekil ile orta perfüzyon, uyarlanabilir kullanılabilir. Her iki yaklaşım da burada açıklanmıştır. Bu yöntem, aynı zamanda, tümör ile ilişkili makrofaj gibi stromal elemanları içerecek şekilde ya da uygun olduğu şekilde, hücresel ve ECM bileşenleri ayarlayarak diğer katı tümörler modellemek için modifiye edilebilir. <pMeme karsinoması vekil için sınıf = &quot;jove_content&quot;&gt; Burada tarif edilen, MDA-MB-231 (231), meme kanseri hücre soyu, CAF daha önce insan göğüs karsinoma 14 izole kullanmışlardır ve bir ECM (% 90 kolajen I oluşan 6 mg / ml) ve% 10 büyüme faktörü indirgenmiş bazal membran malzemesi (BM). taşıyıcı ya da 8 oyuklu oda slayt (katı vekil) yetiştirilir veya biyoreaktör sistemi sürekli bir besin perfüzyon (perfüze vekil) temin etmek üzere kullanılır. ECM içeren bir hücre hacmine uygun herhangi bir perfüzyon biyoreaktör sistemi 15 kullanılabilir. Örnek olarak, bizim biyoreaktör sistemi doku suretler hazırlanmasını tarif etmektedir. Bu sistem, in-house geliştirilmiş ve ticari olarak mevcut değildir edildi. bizim odak burada 3D doku suretler hazırlanması ve analizi olduğundan, bizim biyoreaktör sistemi imalatı ve montajı özelliklerine ilişkin geniş detaya gitmiş değil. Ancak, ayrıntılı bir açıklamasıBu sistem ve gelişimi 16 yayımlanmıştır. Bu biyoreaktör sistemi içinde, bir polidimetilsiloksan (PDMS) akış kanalı PDMS köpük tarafından desteklenen vekil, ev için kullanılan (Calcagnile ve ark., 17 tarafından tarif edilenlere benzer yöntemler kullanılarak oluşturulan). Bu hacmi sürekli vekil oksijen ve besin kaynağı bir microphysiologic pompası vasıtasıyla ortamı ile perfüze 4 Mikrokanallarda (çapı her 400 mikron) tarafından nüfuz edilir. suretler uygun analiz tedavisi veya diğer manipülasyonlar yanıt olarak hücresel fonksiyonun ilgili önemli bilgileri elde etmek çok önemlidir. Suretler fiksasyon ve işlendikten sonra salgılanan ürünleri veya histolojik bölümlerde analiz için, konfokal mikroskopi veya non-invaziv görüntüleme diğer araçlar, koşullu ortam, ya da perfüzyon tahlil dolaylı hücresel analizi kullanılarak sağlam suretler doğrudan görüntüleme de dahil olmak üzere çeşitli yöntemlerle analiz edilebilir içinparafin. histolojik bölümlerde değerlendirilebilir Bu tür bir parametre, hücre yoğunluğu. Bu hücre yoğunluğu ölçmek için bir yaklaşım sunmaktayız (yani, kesit alanı başına çekirdekli hücrelerin sayısı) hematoksilin ve eozin (H &amp; E) ile boyandı vekil histolojik kesitlerinin fotomikrograflarının uygulanan yarı-otomatik bir görüntü işleme teknikleri kullanılarak. Hücre yoğunluğu zamanla hücre sayısı nispi değişimin bir göstergesi olarak kullanılabilir ya da farklı büyüme koşulları ve tedavi kaynaklanır. <img alt="Şekil 1" src="/files/ftp_upload/54004/54004fig1.jpg" /> Şekil 1. 3D ses ve biyoreaktör sistemi. Sürecin A) şematik katı 3D suretlerimi oluşturmak için. En: ECM (pembe), (sarı) epitel karsinoma hücreleri ve CAF (turuncu) içeren katı 3D hacmi karikatür; Alt:. 8-iyi odasının slayt içeren suretler üstten görünümü B) şematik sürecinin 3D suretlerimi perfüze üretir. En: cakanallı 3D hacmin rtoon orta perfüzyon ve içeren ECM (pembe), epitel karsinoma hücreleri (sarı) ve CAF (turuncu) için izin vermek; Orta: PDMS köpük (siyah ok) içeren PDMS akış kanalının görüntü hücresi + ECM enjekte ve çapı 400 mikron ölçme polimer kaplı paslanmaz çelik tel (pembe ok) tarafından nüfuz edilecek; Alt:., Suret ihtiva eden ve sürekli ortam perfüzyon için izin vermek için biyoreaktör sistemine bağlı PDMS akış kanalının görüntüsü (peristaltik pompa ve ortam haznesi gösterilmemiştir) C) kültürden sonra, katı ve perfüze suretler hem de işlem adımları görüntüleri. Sol: jel ve vekil işlem örneği içeren cryomold görüntü; Orta: Sabit ve işlenmiş vekil içeren bir parafin blok görüntü; Sağ:. Bir vekil bir H &amp; E boyalı histolojik kesiti olan bir cam slayt görüntüsü <a href="https://www.jove.com/files/ftp_upload/54004/54004fig1large.jpg" target="_blank">bir görmek için buraya tıklayınız</a>Bu rakamın daha büyük bir versiyonu.

<table border="0" cellpadding="0" cellspacing="0" width="465">
	<tbody>
		<tr height="94">
			<td height="94" style="height:94px;width:131px;">Dulbecco&#39;s Modified Eagel Medium 1x (DMEM)</td>
			<td style="width:107px;">Corning CellGro</td>
			<td style="width:107px;">10-014-CV</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:131px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:107px;">Atlanta Biologicals</td>
			<td style="width:107px;">S11150</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:131px;">0.25% Trypsin + 2.21 mM EDTA 1x</td>
			<td style="width:107px;">Corning</td>
			<td style="width:107px;">25-053-CI</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:131px;">Tissue Culture plates, 100mm</td>
			<td style="width:107px;">CellTreat Scientific Products</td>
			<td style="width:107px;">229620</td>
			<td style="width:121px;">Sterile</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:131px;">Tissue Culture plates, 35mm</td>
			<td style="width:107px;">CellTreat Scientific Products</td>
			<td style="width:107px;">229638</td>
			<td style="width:121px;">For PDMS foam formation&#160;</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:131px;">9&#34; Glass pipette</td>
			<td style="width:107px;">Fisher&#160;</td>
			<td style="width:107px;">13-678-20D</td>
			<td style="width:121px;">Sterile</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:131px;">10 ml pipette</td>
			<td style="width:107px;">CellTreat Scientific Products</td>
			<td style="width:107px;">229210B</td>
			<td style="width:121px;">Sterile</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:131px;">1000 &#181;l piptette tips</td>
			<td style="width:107px;">FisherBrand</td>
			<td style="width:107px;">02-717-166</td>
			<td style="width:121px;">Sterile Filtered</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:131px;">200 &#181;l pipette tips</td>
			<td style="width:107px;">FisherBrand</td>
			<td style="width:107px;">02-717-141</td>
			<td style="width:121px;">Sterile Filtered</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:131px;">10 &#181;l pipette tips</td>
			<td style="width:107px;">FisherBrand</td>
			<td style="width:107px;">02-717-158</td>
			<td style="width:121px;">Sterile Filtered</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:131px;">15 ml conical tubes</td>
			<td style="width:107px;">CellTreat Scientific Products</td>
			<td style="width:107px;">229410</td>
			<td style="width:121px;">Sterile</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:131px;">50 ml conical tubes</td>
			<td style="width:107px;">CellTreat Scientific Products</td>
			<td style="width:107px;">229422</td>
			<td style="width:121px;">Sterile</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:131px;">1.5 ml microcentrifuge tubes</td>
			<td style="width:107px;">FisherBrand</td>
			<td style="width:107px;">05-408-129</td>
			<td style="width:121px;">Sterile</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:131px;">Trypan blue</td>
			<td style="width:107px;">Corning Cellgro</td>
			<td style="width:107px;">25-900-CI</td>
			<td style="width:121px;">Sterile</td>
		</tr>
		<tr height="125">
			<td height="125" style="height:125px;width:131px;">Sylgard 184</td>
			<td style="width:107px;">Electron Microscopy Sciences</td>
			<td style="width:107px;">24236-10</td>
			<td style="width:121px;">PDMS elastomer and curing agent. Used for our in-house bioreactor.</td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;width:131px;">PDMS Foam</td>
			<td style="width:107px;"></td>
			<td style="width:107px;"></td>
			<td style="width:121px;">Made in-house for use in our in-house bioreactor.</td>
		</tr>
		<tr height="86">
			<td height="86" style="height:86px;width:131px;">High Concentration Bovine Collagen Type I</td>
			<td style="width:107px;">Advanced Biomatrix</td>
			<td style="width:107px;">5133-A</td>
			<td style="width:121px;">FibriCol ~10 mg/mL</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:131px;">Growth Factor Reduced Matrigel (Basement Membrane)</td>
			<td style="width:107px;">Corning</td>
			<td style="width:107px;">354230</td>
			<td style="width:121px;">Basement membrane material</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:131px;">Sodium Bicarbonate</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:107px;">S8761</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:131px;">Molecular Biology Grade Water</td>
			<td style="width:107px;">Fisher</td>
			<td style="width:107px;">BP2819-1</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:131px;">DMEM 10x&#160;</td>
			<td style="width:107px;">Sigma-Aldrich</td>
			<td style="width:107px;">D2429</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:131px;">Nunc Lab-Tek Chamber Slide System&#160;</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td style="width:107px;">177402</td>
			<td style="width:121px;">8-well</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:131px;">Bioreactor</td>
			<td style="width:107px;"></td>
			<td style="width:107px;"></td>
			<td style="width:121px;">Made in-house.</td>
		</tr>
		<tr height="185">
			<td height="185" style="height:185px;width:131px;">Spring-Back 304 Stainless Steel&#8212;Coated with PTFE polymer</td>
			<td style="width:107px;">McMaster-Carr</td>
			<td style="width:107px;">1749T19</td>
			<td style="width:121px;">Stainless steel wires to generate microchannels in our in-house&#160; bioreactor system. 0.016&#34; Diameter</td>
		</tr>
		<tr height="154">
			<td height="154" style="height:154px;width:131px;">BioPharm Plus platinum-cured silicone pump tubing, L/S 14</td>
			<td style="width:107px;">Masterflex&#160;</td>
			<td style="width:107px;">EW-96440-14</td>
			<td style="width:121px;">For use in our in-house bioreactor system. Tubing ID: 1.6 mm, Hose barb size: 1/16 in.</td>
		</tr>
		<tr height="158">
			<td height="158" style="height:158px;width:131px;">2-Stop Tubing Sets, non-flared PVC, 1.52 mm ID</td>
			<td style="width:107px;">Cole-Parmer&#160;</td>
			<td style="width:107px;">EW-74906-36</td>
			<td style="width:121px;">For use in our in-house bioreactor system (with microperistalitic pump).&#160;</td>
		</tr>
		<tr height="158">
			<td height="158" style="height:158px;width:131px;">Six Channel precision micro peristaltic pump</td>
			<td style="width:107px;">Cole-Parmer</td>
			<td style="width:107px;">EW-74906-04</td>
			<td style="width:121px;">For use with our in-house bioreactor system</td>
		</tr>
		<tr height="166">
			<td height="166" style="height:166px;width:131px;">Labtainer BPC Bag &#8211; 2 Ports, Luer Lock 
			50mL</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td style="width:107px;">&#8203;SH3065711</td>
			<td style="width:121px;">Example Media Reservoir&#160;</td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:131px;">Tuberculin Syringes</td>
			<td style="width:107px;">BD Medical</td>
			<td style="width:107px;">309625</td>
			<td style="width:121px;">26 gauge 3/8 in. needle; Sterile</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:131px;">Dissecting Tissue Forceps</td>
			<td style="width:107px;">FisherBrand</td>
			<td style="width:107px;">13-812-36</td>
			<td style="width:121px;">5.5 inch</td>
		</tr>
		<tr height="180">
			<td height="180" style="height:180px;width:131px;">Mini Tube Rotator</td>
			<td style="width:107px;">Boekel Scientific</td>
			<td style="width:107px;">260750</td>
			<td style="width:121px;">Equipment option for surrogate rotation. Used with carousel for 50 ml tubes (model number 260753)</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:131px;">50 ml tube carousel</td>
			<td style="width:107px;">Boekel Scientific</td>
			<td style="width:107px;">260753</td>
			<td style="width:121px;">Used with mini tube rotator</td>
		</tr>
		<tr height="88">
			<td height="88" style="height:88px;width:131px;">Bambino&#160; Hybridization Oven</td>
			<td style="width:107px;">Boekel Scientific</td>
			<td style="width:107px;">230301</td>
			<td style="width:121px;">Equipment option for surrogate rotation</td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;width:131px;">HistoGel Specimen Processing Gel</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td style="width:107px;">HG-4000-012</td>
			<td style="width:121px;">Specimen Processing Gel described in Step 5.2</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:131px;">Cryomold</td>
			<td style="width:107px;">Andwin Scientific</td>
			<td style="width:107px;">4566</td>
			<td style="width:121px;">15 mm x 15 mm x 5 mm</td>
		</tr>
		<tr height="192">
			<td height="192" style="height:192px;width:131px;">Tissue Marking Dye</td>
			<td style="width:107px;">Cancer Diagnostics, inc.&#160;</td>
			<td style="width:107px;">03000P</td>
			<td style="width:121px;">Can be used to mark surrogates,&#160; allowing multiple samples to be included in one tissue cassette&#160;</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:131px;">Hinged tissue cassettes&#160;</td>
			<td style="width:107px;">FisherBrand</td>
			<td style="width:107px;">22-272-416</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:131px;">Formalin</td>
			<td style="width:107px;">Fisher</td>
			<td style="width:107px;">23-245-685</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:131px;">GoldSeal Plain Glass Slides</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td style="width:107px;">3048-002</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:131px;">Xylene</td>
			<td style="width:107px;">Fisher</td>
			<td style="width:107px;">X3P-1GAL</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:131px;">Ethanol, 200 proof (100%), USP</td>
			<td style="width:107px;">Decon Laboratories, Inc.</td>
			<td style="width:107px;">2805M</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;width:131px;">Hematoxylin</td>
			<td style="width:107px;">Thermo Scientific Richard-Allan Scientific</td>
			<td style="width:107px;">7211</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;width:131px;">Clarifier</td>
			<td style="width:107px;">Thermo Scientific Richard-Allan Scientific</td>
			<td style="width:107px;">7401</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;width:131px;">Bluing Solution</td>
			<td style="width:107px;">Thermo Scientific Richard-Allan Scientific</td>
			<td style="width:107px;">7301</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;width:131px;">Eosin Y</td>
			<td style="width:107px;">Thermo Scientific Richard-Allan Scientific</td>
			<td style="width:107px;">7111</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;width:131px;">Cytoseal XYL mounting media</td>
			<td style="width:107px;">Thermo Scientific Richard-Allan Scientific</td>
			<td style="width:107px;">83124</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:131px;">Coverslips</td>
			<td style="width:107px;">Fisher Scientific</td>
			<td style="width:107px;">12-548-5G</td>
			<td style="width:121px;"></td>
		</tr>
	</tbody>
</table>

preparation and analysis of in vitro three dimensional breast carcinoma surrogates

Üç boyutlu (3D) bir kültür, iki boyutlu kültüründen in vitro hücre davranışını model fizyolojik olarak daha uygun bir yöntemdir. Meme karsinomu kapsayan karsinomları, fibroblastlar ve hücre dışı matrisin (ECM) de dahil olmak üzere kanser epitelyal hücreleri ve stromal bileşenleri içeren karmaşık 3D dokulardır. Ancak, meme kanserinin en in vitro modeller nedenle stroma atlama sadece kanser epitelyal hücrelerden oluşur ve in vivo olarak tümör 3D mimari. karsinom uygun 3D modelleme tümör biyolojisi, davranış ve tedaviye yanıtın doğru anlaşılması için önemlidir. Ancak, kültür ve 3D modelleri hacmi süresi kültürü içinde oksijen ve besin mevcudiyeti ile sınırlıdır. Burada, meme kanseri, epitelyal hücreleri ve stromal fibroblastlann stroma içeren bir şekilde kültüre edilebilir 3B meme kanseri vekil üretmek üzere ECM&#39;nin dahil edildiği bir yöntem ortaya koymaktadırKatı 3D yapı veya oksijen ve besin sağlamak için bir perfüzyon biyoreaktör sistemi kullanılarak. kurulum ve ilk büyüme döneminden sonra, suretler klinik öncesi ilaç testleri için de kullanılabilir. Seçenek olarak ise, bir vekil hücresel ve matris bileşenleri, biyolojik çeşitli sorular gidermek için modifiye edilebilir. kültürden sonra, suretler sabit ve ilgi parametrelerinin değerlendirilmesi için klinik göğüs karsinoma örneklerinin işleme benzer bir şekilde, parafin için işlenir. ImageJ ve CellProfiler görüntü analiz yazılım sistemleri suretler histolojik kesitlerinin fotomikrograflarının uygulanır, açıklanmıştır Bu tür bir parametre, bu hücre yoğunluğu değerlendirmesi alan başına çekirdekli hücrelerin sayısını belirlemek için. Bu, zaman içinde hücre sayısı değişikliği veya değişen büyüme koşulları ve tedavi sonucunda, hücre sayısındaki değişim bir göstergesi olarak da kullanılabilir.

7 gün yukarıda tarif yetiştirilen hem katı hem de perfüze 3D meme kanseri suretler hazırlanmıştır. Daha sonra, suretler, tespit edilmiş parafine işlenmiş, kesit ve yukarıda tarif edildiği gibi, hematoksilin ve eosin ile boyandı. çekirdekli alan başına hücreleri (her ikisi de 231 hücre ve CAF) her vekil sayısı ölçüldü. Şekil 12&#39;de görülebileceği gibi, H &amp; E ile boyanmış bölümlerin temsil edici fotomikrografikler hücre yoğunluğu...

Watch this Scientific Journal Video about Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates at JoVE.com

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, ...

preparation-analysis-vitro-three-dimensional-breast-carcinoma

Research

JoVE Journal

Bioengineering

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

10.6K Görüntüleme.  University of Alabama at Birmingham. 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.

Video: Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

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 ...

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 ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

3-Boyutlu Hücre Kültürü için Self-raporlama iskeleleri

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

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 ...

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.

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 ...

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 ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Elektrospinning Süreci Uyarlama bir Tri-katmanlı için üç Benzersiz ortamlar sağlamak için<em&gt; In Vitro</em&gt; Havayolu Wall Modeli

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

Ekstraselüler Matrix içinde Gömülü Mühendislik Üç boyutlu Epitel Dokular

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.