NIH hibe HD052670 tarafından desteklenir.

1. ECM proteinleri ile plakaların Kaplama <ol><li> Steril koşullar altında, plaka başına soğuk steril 1X PBS 12 ml ile laminin 120 mikrogram karıştırın. </li><li> Bioflex tedavi edilmemiş levhalar alın ve her kuyuya çözüm 2ml ekleyin (laminin son konsantrasyonu 2 mg / cm 2 &#39;dir). Alternatif olarak, diğer proteinler gibi ECM&#39;yi kolajen-1 olarak, kullanılan olabilir [10 ug / cm 2], fibronektin [5 ug / cm 2], vitronektin [0,5 ug / cm 2] veya elastin [10 ug / cm 2]. Kaplama tekniği bunun laminin-kaplanması için tarif edilene benzer. Kuyular tamamen çözümü kapsamında olduğundan emin olun. </li><li> Plastik tabak sarın ve kuyu dibine tutunmasını laminin izin gecede düz bir yüzey üzerinde 4 ° C de koydu. Hala iyi ECM işlevini koruyarak Bu koşullarda, tabaklar en az bir hafta saklanabilir. </li><li> Ertesi gün, steril koşullar altında, 1X kuyular PBS ile 3 defa yıkanır. </li><li&gt; her oyuğa 1X PBS içinde% 1 BSA 1 ml ekleyin ve 37 &#39;de inkübe edilir zarları üzerindeki spesifik olmayan bağlanma yerleri bloke etmek için 1H ° C. </li><li> Plakalar adzorbe edilmeyen proteinleri kaldırmak için 1X PBS ile 3 defa çalkalayın. </li><li> Hücreler adım 2.14 izole edilene kadar 37 ° C&#39;de DMEM içinde plaka tutmak. </li></ol> 2. Fetal kemirgen Akciğer tip II epitel hücrelerinin izolasyonu Izolasyon önceki gün otoklavlanmış ve hazır farklı boyut naylon kafesleri ile ekran bardak var. <ol><li> Gebeliğin E17-19 fare / sıçan zamanlı gebe fetal akciğer edinin. DMEM ortamı içeren 50 ml konik tüpe doku aktarın ve buz üzerine yerleştirin. </li><li> 50 ml&#39;lik bir konik tüpe sindirim tampon hale: (10 ml). Bu birim için, fare / sıçan yaklaşık 20 fetusa ait akciğer dokusu kullanılır. 8.5 ml DMEM 0.5 ml 500mM Hepes pH 7.4 5 mg Kollajenaz 1 5 mg Kollajenaz 1A 1 ml Tavuk serum, inaktif ısı </li><li>Tüm bileşenleri steril kaput içinde 0.2 mikron şırınga filtre ile çözünmüş ve filtre kadar iyice karıştırınız. 37 su banyosunda sindirim tampon içeren konik tüp ° C yerleştirin </li><li> Adım 2.1 &#39;den konik tüp DMEM ortamı çıkarın ve steril Petri kabına doku aktarın. </li><li> 1 mm&#39;den daha az doku parçaları yapmak ve adım 2.3 &#39;önceden ısıtılmış sindirim tampon içeren konik tüp içine doku aktarımı için iyi steril makas veya jilet ile akciğerlere kıyma. </li><li> Aşağı pipetleme tarafından doku sindirmek: 10 ml pipet ile 100 defa 5 ml pipet ile 100 defa Pasteur pipeti ile 100 defa </li><li> Oda sıcaklığında 5 dakika boyunca 1300 rpm&#39;de santrifüje Homojenat. </li><li> Dikkatle aspirasyon ile süpernatantı kaldırmak ve pelet 15 ml DMEM artı% 20 FBS içeren hücreler yeniden askıya. </li><li> , - 30 - 100 ekran bardak çıkarın ve 15 mikron naylon kafesleri ve yer thSteril 150 ml bardak üzerinde em. </li><li> Adım 2.8 hücre Homojenat ekleyin ve sırasıyla 100 süzülmeye -, 30 - ve 15-mikron örgü ekran bardak. </li><li> Non-epitelyal hücrelerin filtrasyon kolaylaştırmak için DMEM artı% 10 FBS ile çok yıkamadan sonra ve 15 mikron kafesleri - clumped 30 olmayan filtre hücreleri toplayın. </li><li> Çoğunlukla fibroblastlar içeren 15 mikronluk bir elekten süzüntü atılır. </li><li> 30 min (şişeye başına hücre süspansiyonu approximatelly10 ml) 2-75 cm şişeler içerisinde aşama 2.11 hücreleri tarafından daha kuluçkaya tip II hücreleri saflaştırmak. </li><li> Pelet hücreleri için oda sıcaklığında 5 dakika boyunca 1300 rpm&#39;de süpernatan toplamak ve santrifüj. </li><li> Dikkatle aspirasyon ile süpernatantı kaldırmak ve pelet serumsuz DMEM hücre içeren yeniden askıya. Yeniden süspanse hacmi işlenmiş doku miktarına bağlı olarak değişir. Yaklaşık olarak, bu plaka hücreler denk Achi etmek için (2 ml) de, her biri fetus elde edilenErtesi gün eve% 60-70 arasında birbirine karışmış. </li><li> Bioflex altı iyi tabaklarda Plaka hücre süspansiyonu laminin ve fibronektin ile önkaplamalı. </li></ol> 3. Fetal kemirgen akciğer fibroblast İzolasyonu <ol><li> 2.11 kadar tip II hücrelerin izolasyonu için yukarıda açıklanan aynı adımları izleyin. </li><li> Fibroblastlar yapışma sağlamak için 30-60 dakika boyunca 37 ° C&#39;de 75 cm-2 üzerine ve plaka, 15 mikron meş (yaklaşık 10-15 ml hacim olduğu önceki yıkama olmadan) gelen süzüntü al. </li><li> Süpernatantından aspire ve geceleme serumsuz DMEM ve inkübe ile değiştirin. </li><li> Ertesi gün,% 0.25 (ağırlık / hacim) 0.4 mM EDTA ve fibronektine ile önkaplamalı Bioflex plakalar üzerine plakası bunları tripsin ile hücreler hasat. </li></ol> 4. Akciğer hücreleri için mekanik uyarılması için deneysel sistem <ol><li> Serumsuz DMEM (2 ml / kuyu) ve bunların ekleyin ve spre içeri Bioflex kültür plakaları üzerine tohum hücreleri (yaklaşık% 50 konfluent)deneyler önce en az 6 saat reklam. Genel olarak laboratuvar mekanik zorlama uygulamadan önce yaklaşık 24 saat için kültür hücreleri korur. Hücreler belirli bir sayıda deneyler için gerekli olduğu durumda, izolasyon sonra, tip II hücreler 75 cm 2 matara ve ertesi gün, hücreleri, tripsinize sayılır ve sonra tohumlanmıştır serumsuz DMEM tutulur. </li><li> Ertesi gün, taze ortam serumsuz DMEM (2 ml / çukur) ve levhalar bioflex ile değiştirilir, bir Flexcell FX-5000 Suş birimi içinde monte edilmiştir. Monotabaka deneyler başlamadan önce hiçbir büyük 80 den% birleşik olmalıdır. </li><li> Equibiaxial suşu membranları uygulanır. Gerilme rejimi (bakınız tartışma) in vivo mekanik güçlerin simülasyon bağlı olarak değişir. </li><li> Paralel kültüre olmayan gergin zarlar üzerinde yetiştirilen hücreler kontrol olarak kullanılır. </li><li> Deneyler sonucunda, mono tabakalar gen ifadesi üzerine değişiklik (örneğin yüzey aktif protein C) analiz etmek için işlenebilirWestern Blot, vb, zaman-PCR gerçek bir protein bolluğu Ayrıca, mono tabakaları immünsitokimya deneylerde tespit edilebilir. Bu teknik için, tespit sonrasında, silastik membranlar plakalardan kesilir ve antikorlar ile permeabilization ve inkübasyon önce montaj ajan olarak su 10-20 ul kullanılarak cam slaytlar üzerine monte edilmiş. Süpernatant da büyüme faktörleri, sitokinler, vs varlığının belirlenmesi için de kullanılabilir </li></ol> 5.. Temsilcisi sonuçları Şekil 1 ve E18 fetal fare tip II hücrelerin Şekil 2 gösterisi temsilcisi faz-kontrast fotoğrafları bu makalede açıklanan yöntemi kullanarak izole. Şekil 3 bu mekanik zorlama bir belirteç olarak surfaktan protein-C kullanarak fetal tip II epitel hücrelerin farklılaşmasını uyarır göstermektedir. <img src="/files/ftp_upload/3543/3543fig1.jpg" /> Şekil 1. Tecrüberesentative faz-kontrast fotoğrafın sağ fetal tip II hücrelerin clumped görünümünü gösteren izolasyon sonra alınır. <img src="/files/ftp_upload/3543/3543fig2.jpg" /> Laminin ile kaplanmış bioflex plakaları üzerine kaplanmış ve burada tarif edildiği gibi Şekil 2. E18 fetal tip II epitel hücreleri izole edildi. Olmayan gergin hücreleri paraformaldehid tespit edildi sonra Fotoğraf ertesi gün çekildi. Hücre 90 saflıkta olduğu tespit edildi ± SP-C için epitel hücre morfoloji ve immun mikroskopik analizi ile% 5 idi. <img src="/files/ftp_upload/3543/3543fig3.jpg" /> Şekil 3. Fetal tip II epitel hücreleri 40 devir / dk için 16h azından% 5 siklik suşu maruz bırakıldı. Yüzey aktif protein C (SP-C) o suşu farklı ECM ​​substrat kullanılarak tip II hücre farklılaşması indüklemektedir gösteren mRNA ekspresyonu A) Northern blot . + / - Işaretler s maruz kalma ya da temsilsırasıyla, eğitmek. Veriler ortalama olarak sunulmaktadır + / - SEM, n = 3; * p &lt;0.05 B) mekanik zorlamalara SP-C proteini maruz fetal tip II hücrelerinde düzeyleri (yeşil) ya da (kontrol) gösteren Floresans immünsitokimya görüntüler.. Çekirdekler DAPI (mavi) ile zıt edildi. Bar, bu mekanik gerilmenin gösteren üç deneyler 10 mikron. C) Western Blot sonuçları SP-C proteini artırır. N = 3; * p &lt;0.05.

<ol>
	<li>Sanchez-Esteban, J., Tsai, S. W., Sang, J., Qin, J., Torday, J. S., Rubin, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+mechanical+forces+on+lung-specific+gene+expression.">Effects of mechanical forces on lung-specific gene expression.</a> Am. J. Med. Sci. 316, 200-204 (1998).</li><li>Sanchez-Esteban, J., Cicchiello, L. A., Wang, Y., Tsai, S. W., Williams, L. K., Torday, J. S., Rubin, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+stretch+promotes+alveolar+epithelial+type+II+cell+differentiation.">Mechanical stretch promotes alveolar epithelial type II cell differentiation.</a> J. Appl. Physiol. 91, 589-595 (2001).</li><li>Sanchez-Esteban, J., Wang, Y., Cicchiello, L. A., Rubin, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+mechanical+stretch+inhibits+cell+proliferation+and+induces+apoptosis+in+fetal+rat+lung+fibroblasts.">Cyclic mechanical stretch inhibits cell proliferation and induces apoptosis in fetal rat lung fibroblasts.</a> Am. J. Physiol. Lung. Cell. Mol. Physiol. 282, L448-L456 (2002).</li><li>Sanchez-Esteban, J., Wang, Y., Cicchiello, L. A., Rubin, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+mechanical+stretch+inhibits+cell+proliferation+and+induces+apoptosis+in+fetal+rat+lung+fibroblasts.">Cyclic mechanical stretch inhibits cell proliferation and induces apoptosis in fetal rat lung fibroblasts.</a> Am. J. Physiol. Lung. Cell. Mol. Physiol. 282, L448-L456 (2002).</li><li>Sanchez-Esteban, J., Wang, Y., Gruppuso, P. A., Rubin, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+stretch+induces+fetal+type+II+cell+differentiation+via+an+epidermal+growth+factor+receptor-extracellular-regulated+protein+kinase+signaling+pathway.">Mechanical stretch induces fetal type II cell differentiation via an epidermal growth factor receptor-extracellular-regulated protein kinase signaling pathway.</a> Am. J. Respir. Cell. Mol. Biol. 30, 76-83 (2004).</li><li>Wang, Y., Maciejewski, B. S., Lee, N., Silbert, O., McKnight, N. L., Frangos, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-induced+fetal+type+II+epithelial+cell+differentiation+is+mediated+via+cAMP-PKA-dependent+signaling+pathway.">Strain-induced fetal type II epithelial cell differentiation is mediated via cAMP-PKA-dependent signaling pathway.</a> Am. J. Physiol. Lung. Cell. Mol. Physiol. 291, L820-L827 (2006).</li><li>Sanchez-Esteban, J., Wang, Y., Filardo, E. J., Rubin, L. P., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrins+{beta}1,+{alpha}6,+and+{alpha}3+contribute+to+mechanical+strain-induced+differentiation+of+fetal+lung+type+II+epithelial+cells+via+distinct+mechanisms.">Integrins {beta}1, {alpha}6, and {alpha}3 contribute to mechanical strain-induced differentiation of fetal lung type II epithelial cells via distinct mechanisms.</a> Am. J. Physiol. Lung. Cell. Mol. Physiol. 290, L343-L350 (2006).</li><li>Wang, Y., Maciejewski, B. S., Soto-Reyes, D., Lee, H. S., Warburton, D., Sanchez-Esteban, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+stretch+promotes+fetal+type+II+epithelial+cell+differentiation+via+shedding+of+HB-EGF+and+TGF-alpha.">Mechanical stretch promotes fetal type II epithelial cell differentiation via shedding of HB-EGF and TGF-alpha.</a> J. Physiol. 587, 1739-1753 (2009).</li><li>Wang, Y., Maciejewski, B. S., Drouillard, D., Santos, M., Hokenson, M. A., Hawwa, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+caveolin-1+in+mechanotransduction+of+fetal+type+II+epithelial+cells.">A role for caveolin-1 in mechanotransduction of fetal type II epithelial cells.</a> Am. J. Physiol. Lung. Cell. Mol. Physiol. 298, L775-L783 (2010).</li><li>Lee, H. S., Wang, Y., Maciejewski, B. S., Esho, K., Fulton, C., Sharma, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-10+protects+cultured+fetal+rat+type+II+epithelial+cells+from+injury+induced+by+mechanical+stretch.">Interleukin-10 protects cultured fetal rat type II epithelial cells from injury induced by mechanical stretch.</a> Am. J. Physiol. Lung. Cell. Mol. Physiol. 294, L225-L232 (2008).</li><li>Hawwa, R. L., Hokenson, M. A., Wang, Y., Huang, Z., Sharma, S., Sanchez-Esteban, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-10+inhibits+inflammatory+cytokines+released+by+fetal+mouse+lung+fibroblasts+exposed+to+mechanical+stretch.">IL-10 inhibits inflammatory cytokines released by fetal mouse lung fibroblasts exposed to mechanical stretch.</a> Pediatric Pulmonology. , (2011).</li><li>Kitterman, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+mechanical+forces+on+fetal+lung+growth.">The effects of mechanical forces on fetal lung growth.</a> Clin. Perinatol. 23, 727-740 (1996).</li><li>Harding, R., Crystal, R. G., West, J. B., Banes, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+breathing+movements.">Fetal breathing movements.</a> The Lung: Scientific Fountations. , 2093-2104 (1997).</li><li>Harding, R., Liggins, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+thoracic+dimensions+induced+by+breathing+movements+in+fetal+sheep.">Changes in thoracic dimensions induced by breathing movements in fetal sheep.</a> Reprod. Fertil. Dev. 8, 117-124 (1996).</li></ol>

Çıkar çatışması ilan etti.

Bu yazıda, fetal tip II epitel hücreleri ve fibroblastlar yalıtmak ve Flexcell Gerinim Aparatı kullanarak mekanik stimülasyonu almaları için bir metodu tanımlar. Biz epitel hücrelerinin farklılaşması 1,2 değerlendirmek ve reseptör ve streç 3-9 aktive sinyal yolları incelemek için bu tekniği kullandık. Buna ek olarak, bu yöntem ayrıca mekanik yaralanması 10,11 uyarılan hücre cevabını araştırılması için de kullanılabilir. Prosedürü kollajenaz ile akciğer ...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma</td>
			<td>D5648</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Collagenase 1</td>
			<td>Sigma</td>
			<td>C0130</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Collagenase 1A</td>
			<td>Sigma</td>
			<td>C9891</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chicken serum</td>
			<td>Sigma</td>
			<td>C5405</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Screen cups</td>
			<td>Sigma</td>
			<td>CD1-1KT</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Syringe filters</td>
			<td>Fisher Scientific</td>
			<td>09-754-25</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>100 micron nylon mesh</td>
			<td>Small Parts, INC</td>
			<td>CMN-100-D</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>30 micron mesh</td>
			<td>Small Parts, INC</td>
			<td>CMN-30-D</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>15 micron mesh</td>
			<td>Dynamic Aqua-Supply Ltd.</td>
			<td>NTX15</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma</td>
			<td>L2020</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Collagen-1</td>
			<td>Collagen Biomed</td>
			<td>PC0701</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma</td>
			<td>F1141</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vitronectin</td>
			<td>Sigma</td>
			<td>V-0132</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Elastin</td>
			<td>Sigma</td>
			<td>E-6402</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bioflex plate</td>
			<td>Flexcell International</td>
			<td>BF-3001U</td>
			<td>Uncoated</td>
		</tr>
		<tr>
			<td>Flexcell Strain Unit</td>
			<td>Flexcell International</td>
			<td>FX-5000</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

an experimental system to study mechanotransduction in fetal lung cells

Tekrarlayan solunum benzeri hareketleri ve sıvı distansiyonu ile utero üretilen mekanik güçlerin normal akciğer gelişimi için kritik öneme sahiptir. Akciğer gelişimi temel bileşeni, alveolar tip II epitel hücreleri, pulmoner surfaktan en önemli kaynağı bölünme. Bu hücreler aynı zamanda alveolar lümen, konak savunma ve yaralanma tamir sıvı dengesinin katılırlar. Ayrıca, distal akciğer parankima hücreleri doğrudan doğum sonrası mekanik ventilasyon sırasında abartılı streç maruz kalabilirler. Ancak, akciğer hücreleri akciğer gelişimini etkilemek için ve akciğer hasarı teşvik mekanik uyarıcılar hissedebiliyorum hangi hassas moleküler ve hücresel mekanizmaları tam olarak anlaşılamamıştır. Burada, kemirgen fetal akciğerlerden tip II hücreleri ve fibroblastlar yalıtmak için basit ve yüksek saflıkta bir yöntem sunar. Sonra, mekanik kuvvetleri taklit, fetal hücreler mekanik uyarılması için, in vitro sistemi, Flexcell Zorlanma Birimi bir tariffetal akciğer gelişimini veya akciğer hasarı. Bu deneysel sistem germek maruz fetal akciğer hücrelerinde moleküler ve hücresel mekanizmaları araştırmak için mükemmel bir araç sunmaktadır. Bu yaklaşımı kullanarak, laboratuvar fetal akciğer gelişimi ve akciğer hasarında mechanotransduction katılmak çeşitli reseptörler ve sinyal proteinlerini tespit etti.

Watch this Scientific Journal Video about An Experimental System to Study Mechanotransduction in Fetal Lung Cells at JoVE.com

An Experimental System to Study Mechanotransduction in Fetal Lung Cells

Mekanik kuvvetler akciğer gelişimi ve akciğer hasarı önemli bir rol oynamaktadır. Burada, kemirgen fetal akciğer tip II epitel hücreleri ve fibroblastlar izole etmek ve bir kullanarak mekanik stimülasyonu almaları için bir yöntem tarif<em&gt; In vitro</em&gt; Sistemi.

Fetal Akciğer Hücrelerinde Mechanotransduction Okumak Deneysel Bir Sistem

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key ...

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12.9K Görüntüleme.  Alpert Medical School of Brown University. Mekanik kuvvetler akciğer gelişimi ve akciğer hasarı önemli bir rol oynamaktadır. Burada, kemirgen fetal akciğer tip II epitel hücreleri ve fibroblastlar izole etmek ve bir kullanarak mekanik stimülasyonu almaları için bir yöntem tarif In vitro Sistemi.

Video: An Experimental System to Study Mechanotransduction in Fetal Lung Cells

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

Akson Streç Büyüme: Nöronal Büyüme Mechanotransduction

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

Biyomühendislik

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Devirli Basınç Biyoreaktör Tasarım Ex vivo</emAort Kalp Vanalar> Eğitim

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans1. The main difficulty of this assay is the perceived cost ($25-100K) and technical expertise required for implementing a laser ablation system2,3. However, solid-state pulse lasers of modest costs (&lt;$10K) can provide robust performance for laser ablation in transparent preparations where target axons are "close" to the tissue surface. Construction and alignment of a system can be accomplished in a day. The optical path provided by light from the focused condenser to the ablation laser provides a convenient alignment guide. An intermediate module with all optics removed can be dedicated to the ablation laser and assures that no optical elements need be moved during a laser ablation session. A dichroic in the intermediate module allows simultaneous imaging and laser ablation. Centering the laser beam to the outgoing beam from the focused microscope condenser lens guides the initial alignment of the system. A variety of lenses are used to condition and expand the laser beam to fill the back aperture of the chosen objective lens. Final alignment and testing is performed with a front surface mirrored glass slide target. Laser power is adjusted to give a minimum size ablation spot (&lt;1um). The ablation spot is centered with fine adjustments of the last kinematically mounted mirror to cross hairs fixed in the imaging window. Laser power for axotomy will be approximately 10X higher than needed for the minimum ablation spot on the target slide (this may vary with the target you use). Worms can be immobilized for laser axotomy and time-lapse imaging by mounting on agarose pads (or in microfluidic chambers4). Agarose pads are easily made with 10% agarose in balanced saline melted in a microwave. A drop of molten agarose is placed on a glass slide and flattened with another glass slide into a pad approximately 200 um thick (a single layer of time tape on adjacent slides is used as a spacer). A "Sharpie" cap is used to cut out a uniformed diameter circular pad of 13mm. Anesthetic (1ul Muscimol 20mM) and Microspheres (Chris Fang-Yen personal communication) (1ul 2.65% Polystyrene 0.1 um in water) are added to the center of the pad followed by 3-5 worms oriented so they are lying on their left sides. A glass coverslip is applied and then Vaseline is used to seal the coverslip and prevent evaporation of the sample.

Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

Laser axotomy followed by time-lapse imaging is a sensitive way to assay the effects of mutations in C. elegans on axon regeneration. A high quality, but inexpensive, laser ablation system can be easily added to most microscopes. Time lapse imaging over 15 hours requires careful immobilization of the worm.

Axon Rejenerasyon Eğitim Düşük bütçeli Lazer Axotomy Sistemi oluşturulması C. elegans</em

Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans1.  The main difficulty of this assay ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

Bir Çok Kanallı Çift elektrik alan mikroakışkan Chip kullanarak Akciğer Kanseri Hücreleri Electrotaxis Çalışmaları

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Decellularized Akciğer Yapı iskeleleri kullanarak ESC türetilmiş Fare Havayolu Epitel Hücreleri Üretimi

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

DNA nanotüpler Semiflexible polimerler eğitim için çok yönlü bir araç olarak

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based method is one of the simplest techniques for the mass production of cellular aggregates, but it does not protect the cultures against over-aggregation, which occurs when they gather at the bottom center of the culture vessel. To solve this problem, we developed an O-shaped dish and an O-shaped bag, neither of which contains a central region. Aggregates grown in either O-shaped culture vessel were noticeably more uniform in size than aggregates grown in conventional vessels. Histological analyses showed that aggregates in conventional culture dishes contained necrotic cores most likely caused by a poor oxygen supply. In contrast, aggregates that were grown in the O-shaped bag, even those with similar diameters to aggregates in conventional culture dishes, did not show necrotic cores. These results suggest that the O-shaped bag provides sufficient oxygen to the aggregates due to the oxygen permeability of the bag material. We, therefore, propose that this novel gas-permeable O-shaped culture bag is suitable for the mass production of uniform aggregates that are necessary in various biotechnological fields.

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

Memeli hücre kültür Tekdüzen toplamları üretmek için O şeklindeki damarlarının sallayarak bir Orbital

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Sıçan Vivo içinde kısmi karaciğer Dairesi'nde için bir temel olarak yeni bir cerrahi teknik

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Experimental Methods to Study Human Postural Control

Many components of the nervous and musculoskeletal systems act in concert to achieve the stable, upright human posture. Controlled experiments accompanied by appropriate mathematical methods are needed to understand the role of the different sub-systems involved in human postural control. This article describes a protocol for performing perturbed standing experiments, acquiring experimental data, and carrying out the subsequent mathematical analysis, with the aim of understanding the role of musculoskeletal system and central control in human upright posture. The results generated by these methods are important, because they provide insight into the healthy balance control, form the basis for understanding the etiology of impaired balance in patients and the elderly, and aid in the design of interventions to improve postural control and stability. These methods can be used to study the role of somatosensory system, intrinsic stiffness of ankle joint, and visual system in postural control, and may also be extended to investigate the role of vestibular system. The methods are to be used in the case of an ankle strategy, where the body moves primarily about the ankle joint and is considered a single-link inverted pendulum.

This article presents an experimental/analytic framework to study human postural control. The protocol provides step-by-step procedures for performing standing experiments, measuring body kinematics and kinetics signals, and analyzing the results to provide insight into the mechanisms underlying human postural control.

İnsan Postural Kontrolünü İncelemek İçin Deneysel Yöntemler

Many components of the nervous and musculoskeletal systems act in concert to achieve the stable, upright human posture. Controlled experiments accompanied ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

İnsan kaynaklı Pluripotent Kök Hücre Türevli Kardiyomiyositlerde Tek Hücreli Optik Eylem Potansiyeli Ölçümü

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...