Finansman Ulusal Sağlık Enstitüsü (EB 001.013), Fen Bilimleri ve Kanada&#39;nın Mühendislik Araştırma Konseyi ve Kanada Sağlık Araştırma Enstitüleri tarafından sağlanmıştır. Biz onun uzmanlık için Dr. AP McGuigan&#39;ın teşekkür ve modüllerin geliştirilmesi yardımcı.

1) Boru hazırlanması <ol><li> Polietilen borular 2 ila 3 m uzunluğunda (0,76 mm ID x 1.22 mm OD) kesin. </li><li> Hortumun bir ucunu 20 G1 İğneyi. </li><li> Dönüştürücüler kendini kapatmalı (7 1 / 2 &quot;x 13&quot;) Bobin boru ve yer. Hortumun iki ucu çanta kaplanmadan sonunda yerleştirilir ve yerde gaz sterilizasyon bandı ile bantlanmış olmalıdır. </li><li> Mühür çanta ve gaz sterilize edin. </li></ol> 2) Nötralizasyon Kollajen Not: 1 ml kollajen, yaklaşık 2 metre polietilen boru doldurur. <ol><li> 15 ml konik bir tüp içine boru istenilen uzunlukta için gerekli kollajen gerekli miktarda koyun ve buz üzerinde tutmak. </li><li> 128 uL 10x α-MEM başına mL tüp ve art arda çözüm pipetleme karıştırın kollajen. Not: (1) çözüm asitlendirilmiş kollajen α-MEM sarı karışık açmalısınız ve (2) karıştırma kabarcıkları en aza indirmeye çalışın. </li><li> Kollajen 7.4 pH kadar 0.8 M sodyum bikarbonat ekleyerek kollajen nötralize. PH eklemek sodyum bikarbonat genel bir tahmin için sarı kollajen çözümü açık pembe renk olana kadar. Not: Bu genellikle kollajen her ml başına 30 ila 60 mcL alır. </li><li> Nötralize kolajen olarak kullanmak için hazır olana kadar jel 4 tutulması değilse etkisiz hale kollajen buz üzerinde tutun ° C </li></ol> 3) Hücre Gömme (isteğe bağlı) Not: hücreler, hücre tipine bağlı olarak bir konsantrasyon ml başına 20 milyon hücre ml başına 1 milyon hücre arasında gömülü olabilir. Gömmek istediğiniz hücreleri için gerekli ortamı kullanın. <ol><li> Hücrelerden ortamı çıkarın ve 5 ml PBS ile yıkayın. </li><li> PBS çıkarın ve 3 ml tripsin-EDTA ekleyin. </li><li> 37 °, 5 dakika boyunca tripsin-EDTA hücrelerin inkübe ° C </li><li> 7 ml medya hücrelerin tekrar </li><li> Hücreleri saymak. </li><li> 15 ml konik tüp gömmek için gerekli sayıda hücre aktarın. </li><li> Pelet 5 dakika boyunca 300 xg&#39;de hücreleri. </li><li> Kapalı hücre pelletini medya aspire. </li><li> Nötralize kolajen hücrelerinin yeniden süspanse edin ve buz üzerinde tutmak. </li></ol> 4) Jelleşme Kollajen <ol><li> Biyolojik güvenlik kabini polietilen boru içeren torba mühürlü ucunu kesti. </li><li> Bir ucunu sterilize cımbız kullanarak boru için 3 ml şırınga 20G iğne takın. Tüp torba içinde saklayın. </li><li> Hortumun diğer ucunu steril cımbız kullanarak konik tüp alt ucu kadar nötralize kollajen içine yerleştirin. Konik tüp buz üzerinde tutun. </li><li> Polietilen boru içine yavaş yavaş şırınga pistonu geri çekilmeden kollajen çekin. Not: kollajen hızlı kollajen sonra kabarcıklar oluşturacak boru içine çekilir. </li><li> Tüp yoluyla kollajen çekilmesinden sonra, boru 20G iğne çekin ve her iki ucunda çantaya geri koymak. Teyp çanta kapattı. </li><li> 60 dakika için 37 ° C çanta inkübe edin. Kollajen hücreler içeriyorsa, her 15 dakika içinde çanta çevirin. böylece hücrelerin modülün bir tarafında razı olmayın. </li></ol> 5) Boru kesimi jel Kollajen İçeren <ol><li> Otoklav başlıkları ve bıçak kesme cihazı ve tüp tutmak için tepsi. </li><li> Kesme cihazı birleştirin. </li><li> Kesme aleti önünde steril bir tepsiye yerleştirin boru ve boru kesme cihazı yerleştirin. </li><li> 50 ml konik boru kesme cihazı çıkışında kesme modülleri toplamak için 25 ml medya içeren bir ayarlayın. Not: modülleri medya FBS olmadan boru ayrı olarak sadece kolajen modüller için bile FBS içeren medya kullanın. Eğer modülleri içeren hücrelerin gömülü hücreler için gerekli olan medya kullanımı. Modüller hiçbir gömülü hücreler endotel hücreleri için ortam varsa kullanın. </li><li> 2 mm uzunlukta boru kesme ve boru kesmek için kesme cihazı ayarlayın. </li><li> 37 ° kesme modülleri inkübe ° C 50 ml konik tüp içinde 60 dk. </li></ol> 6) Boru Kollajen Modüllerini Çıkarma <ol><li> 10 sn için tüp modülleri içeren Vortex 50 ml konik boru. Not: kırılmaları önlemek için yüksek hücre yoğunluğu ile gömülü modüller ayıran zaman nazik olun. </li><li> Modülleri tüpün dibine dinlendiriniz. Yaklaşık 5 dakika sürer. </li><li> Geniş bir ağız 10 ml serolojik pipet kullanarak yerleşik modülleri 15 ml konik tüp transferi. </li><li> 6,1-6,3 adımları iki kez tekrarlayın. </li><li> Olmayan bir doku kültürü tedavi plaka endotel hücreleri ya da transfer modülleri ile kat modülleri için 7. adıma gidin. </li></ol> 7) Kaplama endotel hücreleri ile modülleri <ol><li> Th için medya 5 ml, 15 ml konik tüp dibine modülleri Settlee gömülü hücreler. </li><li> Endotel hücreleri medya çıkarın ve 5 ml PBS ile yıkayın. </li><li> PBS çıkarın ve 3 ml tripsin-EDTA ekleyin. </li><li> 37 °, 5 dakika boyunca tripsin-EDTA hücrelerin inkübe ° C </li><li> 7 ml medya hücrelerin tekrar </li><li> Hücreleri saymak. Konik tüpün dibinde paketlenmiş modülleri mL başına 5x10 6 endotel hücreleri ihtiyacınız var. </li><li> Pelet 300 xg&#39;de 5 dakika ve 5 ml endotel hücre medya tekrar süspansiyon hücreleri. </li><li> Modülleri endotel hücreleri içeren medyanın 5 ml ekleyin. Not: Bu, biz, her iki hücre tipinin sağkalım ve işlevi için çalışmak bulduk iki medya türleri 50:50 oranı verir. Kullanmadan önce, iki hücre tiplerinin fenotipleri uygun bakım sağlamak için formüle edilmiş bir ko-kültür ortamında test edin. </li><li> Endotel hücreleri ile hem statik ve dinamik modülleri inkübe edin. </li><li> Tersine çevirerek statik inkübasyon karışımı endotel hücreleri ile modülleri ve 37 ° dik tüp seti ° C de 15 dakika. Tekrarlayın. </li><li> 37 ° tüp hafifçe sallanan tarafından dinamik tohum endotel hücreleri ° C de 60 dakika. </li><li> Statik inkübasyon iki kez tekrarlayın. </li><li> Olmayan doku kültürü tedavi plaka modülleri yerleştirin ve 37 ° gece inkübe ° C </li><li> Taze medya ile yeni bir non-doku kültürü tedavi plaka ertesi gün yerine modülleri (50:50 karışımı kullanmadan ko-kültür sistemi) unattached endotel hücreleri çıkarmak için. </li><li> Modülleri yüzeyinin% 100 kapağı kadar endotel hücreleri ile kaplı modüllerin inkübe edin. Bu genellikle 7 gün sürer. </li></ol> 8) Temsilcisi Sonuçlar: Ilk tüp çıkarılır modülleri silindirik bakacağız. Modülleri gömülü hücreleri içeren ve / veya endotel hücreleri ile kaplı hacim olarak% 50 kadar sözleşme ve oval bir şekil (Şekil 1) geliştirmek. Modülleri de ışık mikroskobu (Şekil 1) bakıldığında daha yoğun ve daha opak hale gelecektir. Ayrıca endotel hücreleri, modülleri yüzeyinde konfluent zaman VE-Cadherin immünofloresan boyama (Şekil 2) tarafından görülebilir sıkı bağlantıları bir oluşum var. Kütle transferi analizi modülleri, yetersiz oksijen taşıma, (örneğin büyük çaplı modülleri, D = 1.4, büyük dokular genellikle sorunlu nedeniyle nekrotik çekirdek gelişmekte olmadan yüksek hücre yoğunluğu (8x10 7 hücre / cm 3) destekleme yeteneğine sahip olduğunu göstermiştir mm) (Şekil 3). <img src="/files/ftp_upload/2177/2177fig1.jpg" alt="Şekil 1" /> Şekil 1: Modül imalat ve daralma. Modül daralma tohumlama daha küçük modül çapı ve uzunluğu (p &lt;0,001) (A) sonuçları HUVEC-C (endotelyal hücre hattı) takip eden üç gün boyunca oluştu. Gömülü HepG2 hücreleri düzgün uydurmalarla zamanda modülleri içinde dağıtılmış ve (B) yüksek canlılığını korudu. [Hücreleri yeşil Live ölü hücreleri kırmızı], [2010 Corstorphine adapte] <img src="/files/ftp_upload/2177/2177fig2.jpg" alt="Şekil 2" /> Şekil 2: bir modül yüzeyi kaplı AT RAEC 10 gün sonra sıkı hücre kavşak VE Cadherin immunofluorescent boyama. Ölçek çubuğu = 50 mm. <img src="/files/ftp_upload/2177/2177fig3.jpg" alt="Şekil 3" /> Şekil 3: Masson tipik modülleri trikrom boyama (0.76 mm başlangıç ​​çapı) ve büyük modüller (1.4 mm başlangıç ​​çapı), oksijen difüzyon sınırlamaları etkisi göstermektedir . Imalat aşağıdaki yedi gün, ölü hücrelerden çok sayıda canlı hücreler sadece ince bir jant (~ 200μm kalın) bırakarak, büyük modülleri (sağ alt panel) çekirdek içinde kurmuştu. Tersine, küçük modülleri canlı hücreler bir üniforma ve yüksek dağıtım korudu. [Modülleri, HepG2 hücreleri ile gömülü ve endotel hücreleri ile kaplı.] [Corstorphine 2010 tarihinden itibaren Çekildiği] <img src="/files/ftp_upload/2177/2177fig4.jpg" alt="Şekil 4" /> Şekil 4: (A) poloxamine HMEC-1 numaralı seribaşı kollajen modülleri. Poloxamine-kollajen modülleri, silindir şeklinde korumak ve sınırlı hücre eki özellikleri kollajen sadece modülleri göre ekim 1 Gün sonra. Ölçeği bar = 200 mm. PLGA tabanlı biyobozunur mikroküreler içeren bir kollajen modülü (B) Işık mikroskop görüntüsü. Ölçeği bar = 500 mm. <img src="/files/ftp_upload/2177/2177fig5.jpg" alt="Şekil 5" /> Şekil 5: in vitro tayinlerde örnekleri. (A) Anjiyogenez tahlil: kapiller benzeri oluşumlar endotel hücreleri ile seribaşı modül yağ elde edilen kök hücreleri ile kolayca tespit edilir ve inkübasyon 5 gün sonra sayısal olabilir. (B) s modülleri canlı / ölü testinin mikroskopisi görüntülericanlı hücreler için yerleştirme yazılımı yeşil ve ölü hücreleri için kırmızı. <img src="/files/ftp_upload/2177/2177fig6.jpg" alt="Şekil 6" /> Şekil 6: Kollajen gömülü mezenkimal kök hücreleri ve endotel hücreleri bir yüzey tabakası içeren modüller. Modüller mikroakışkan odasında 7 gün kayma gerilmesi (~ 0.64 dyn / cm 2) maruz kalan ve temas noktalarında füzyon göstermeye başlar. Endotel hücreleri vWF (kahverengi) ile boyandı ve mezenkimal kök hücre çekirdekleri mavi (H &amp; E) görünür. Ölçek çubuğu = 100μm. <img src="/files/ftp_upload/2177/2177fig7.jpg" alt="Şekil 7" /> Şekil 7: Yağ elde edilen kök hücreler (ASC) ve İnsan mikrovasküler endotel hücreleri ile kaplı içeren kollajen modülleri yüzlerce (HMEC) ASC / HMEC yağ rejenerasyon uygulamalar için in vivo etkileşim çalışmasında fare cilt altına yerleştirilir.

<ol>
	<li>Chamberlain, M. D., Gupta, R., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+vessel+tissue+engineering+driven+by+endothelialized+modules.">Chimeric vessel tissue engineering driven by endothelialized modules.</a> , (2010).</li><li>Corstorphine, L. E., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+factor+and+diffusion+limitations+in+collagen+gel+modules+containing+HepG2+cells.">Effectiveness factor and diffusion limitations in collagen gel modules containing HepG2 cells.</a> J Tissue Eng Regen Med. , (2010).</li><li>Gupta, R., Van Rooijen, N., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of+endothelialized+modular+constructs+implanted+in+an+omental+pouch+in+nude+rats.">Fate of endothelialized modular constructs implanted in an omental pouch in nude rats.</a> Tissue Eng. Part A 15, 2875-2887 (2009).</li><li>Leung, B. M., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modular+tissue+engineering+construct+containing+smooth+muscle+cells+and+endothelial+cells.">A modular tissue engineering construct containing smooth muscle cells and endothelial cells.</a> Ann Biomed Eng. 35, 2039-2049 (2007).</li><li>McGuigan, A. P., Leung, B., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+cell-containing+gel+modules+to+assemble+modular+tissue-engineered+constructs+[corrected].">Fabrication of cell-containing gel modules to assemble modular tissue-engineered constructs [corrected].</a> Nat Protoc. 1, 2963-2969 (2006).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularized+organoid+engineered+by+modular+assembly+enables+blood+perfusion.">Vascularized organoid engineered by modular assembly enables blood perfusion.</a> Proc Natl Acad Sci U S A. 103, 11461-11466 (2006).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+tissue+engineering:+fabrication+of+a+gelatin-based+construct.">Modular tissue engineering: fabrication of a gelatin-based construct.</a> J Tissue Eng Regen Med. 1, 136-145 (2007).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+fabrication+of+sub-mm-sized+modules+containing+encapsulated+cells+for+modular+tissue+engineering.">Design and fabrication of sub-mm-sized modules containing encapsulated cells for modular tissue engineering.</a> Tissue Eng. 13, 1069-1078 (2007).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+criteria+for+a+modular+tissue-engineered+construct.">Design criteria for a modular tissue-engineered construct.</a> Tissue Eng. 13, 1079-1089 (2007).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thrombogenicity+of+human+umbilical+vein+endothelial+cell+seeded+collagen+modules.">The thrombogenicity of human umbilical vein endothelial cell seeded collagen modules.</a> Biomaterials. 29, 2453-2463 (2008).</li><li>Sosnik, A., Leung, B., McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen/poloxamine+hydrogels:+cytocompatibility+of+embedded+HepG2+cells+and+surface-attached+endothelial+cells.">Collagen/poloxamine hydrogels: cytocompatibility of embedded HepG2 cells and surface-attached endothelial cells.</a> Tissue Eng. 11, 1807-1816 (2005).</li></ol>

Biz farklı hücre tipleri 1-11 gömülü modüllerini kullanarak birçok farklı mikro-dokulara fabrikasyon var. Biz başarıyla birincil kardiyomiyositlerde, adacıklar, yağ kök hücreler ve mezenkimal stromal hücrelerinin yanı sıra HepG2, NIH 3T3 ve klon 9 karaciğer hücreleri de dahil olmak üzere çok sayıda hücre hatları gömülü. Biz, sıçan aort endotel hücreleri, insan umbilikal ven endotel hücreleri ve insan mikrovasküler endotel hücreleri de dahil olmak üzere endotel hücreleri çe?...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Intramedic tubing PE60</td>
<td>&nbsp;</td>
<td>Becton Dickson</td>
<td>427416</td>
<td>Different diameter of tubing can be used to change the diameter of the modules</td>
</tr>
<tr>
<td>Phosphate-buffered saline (PBS)</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>20012-027</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25200-072</td>
<td>&nbsp;</td>
<tr>
<td>Purcol acidificed collagen, 3 mg/mL</td>
<td>&nbsp;</td>
<td>Cedarlane</td>
<td>5005-B</td>
<td>&nbsp;</td>
<tr>
<td>Sodium bicarbonate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S5761</td>
<td>&nbsp;</td>
<tr>
<td>20G needle</td>
<td>&nbsp;</td>
<td>Becton Dickson</td>
<td>305175</td>
<td>Diameter of the needle needs to be similar to the diameter of the tubing</td>
</tr>
<tr>
<td>3 mL syringe</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>309585</td>
<td>&nbsp;</td>
<tr>
<td>15 mL tube</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>352096</td>
<td>&nbsp;</td>
<tr>
<td>50 mL tube</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>352070</td>
<td>&nbsp;</td>
<tr>
<td>Convertors Self-Seal Pouch 7 1/2&#x201D; x 13&#x201D;</td>
<td>&nbsp;</td>
<td>Cardinal Health</td>
<td>92713</td>
<td>&nbsp;</td>
<tr>
<td>10 ml Wide Tip serological pipet</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>357504</td>
<td>&nbsp;</td>
<tr>
<td>10 ml serological pipet</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>357551</td>
<td>&nbsp;</td>
<tr>
<td>5 ml serological pipet</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>357543</td>
<td>&nbsp;</td>		</tbody>
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fabrication of micro-tissues using modules of collagen gel containing cells

Bu protokol, modüller olarak adlandırılan mikro-dokuların bir tür fabrikasyon açıklamaktadır. Modülü yaklaşım, üniforma, ölçeklenebilir ve vaskülarize doku üretir. Modülleri kollajen yanı sıra diğer gelable veya çapraz bağlanabilir malzemeler yapılabilir. Onlar imalat üzerine çapı yaklaşık 2 mm uzunluğunda ve 0.7 mm ama gömülü hücreleri veya modülleri endotel hücreleri ile kaplıdır boyutu küçültmek. Modüllerin ayrı ayrı gömülü hücreler oksijen ve diğer besinleri difüzyon sınırı içinde ama modülleri perfusable olan büyük dokular oluşturmak için birlikte paketlenmiş olabilir bu kadar küçük. Bu dokular, farklı hücre türleri, karmaşık dokuları oluşturmak için birlikte paketlenmiştir önce gömülü veya modülleri kaplı olabilir çünkü inşaat modüler. Modülleri yapmak için üç temel adım vardır: (1) kollajen nötralize ve bu hücreleri gömerek, tüp içinde (2) jelleşme kolajen ve endotel hücreleri ile modülleri ve (3) kaplama modülleri kesme.

Watch this Scientific Journal Video about Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells at JoVE.com

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

Gömülü hücreler ve yüzey içeren modüller olarak adlandırılan silindirik kollajen jeller, mikro-dokuların oluşturulması endotel hücreleri ile kaplanmıştır.

Kollajen, Modüller kullanarak Micro-dokuların Fabrikasyon İçeren Hücreleri Jel

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

fabrication-micro-tissues-using-modules-collagen-gel-containing

Research

JoVE Journal

Bioengineering

12.6K Görüntüleme.  University of Toronto. Gömülü hücreler ve yüzey içeren modüller olarak adlandırılan silindirik kollajen jeller, mikro-dokuların oluşturulması endotel hücreleri ile kaplanmıştır.

Video: Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Dinamik Maske Projeksiyon Fotolitografi kullanarak Sinir Kültür Sistemleri Micropatterned Hidrojeller Fabrikasyon

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Biyomühendislik

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

Doğallığı Kollajen Tellerinin Görüntüleme<em&gt; In vivo</em&gt; Ve<em&gt; Ex vivo</em&gt; Kafesli Kollajen Mimetik Peptidlerinin Fotoğraf-tetiklenen Hybridization yoluyla

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

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

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

Üç boyutlu Mürekkep Püskürtmeli Baskı Teknolojisi Kullanımı İnsan Kıkırdak Doku Fabrikasyon

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

Sıkıştırılmış Kolajen kıyılmış Doku: Tek aşamalı Rekonstrüktif Onarım için Cep içeren Biotransplant

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

Altın nanorods İçeren Hücresel Araçların Hazırlama ve foto akustik Analizi

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

Forming Giant-sized Polymersomes Using Gel-assisted Rehydration

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier properties, chemical versatility and tunable physical characteristics. Typical methods used to prepare giant-sized (&gt; 4 &#181;m) vesicles, however, are both time and labor intensive, yielding low numbers of intact polymersomes. Here, we present for the first time the use of gel-assisted rehydration for the rapid and high-yielding formation of giant (&gt;4 &#181;m) polymer vesicles (polymersomes). Using this method, polymersomes can be formed from a wide array of rehydration solutions including several different physiologically-compatible buffers and full cell culture media, making them readily useful for biomimicry studies. This technique is also capable of reliably producing polymersomes from different polymer compositions with far better yields and much less difficulty than traditional methods. Polymersome size is readily tunable by altering temperature during rehydration or adding membrane fluidizers to the polymer membrane, generating giant-sized polymersomes (&gt;100 &#181;m).

We present a protocol to rapidly form giant polymer vesicles (pGVs). Briefly, polymer solutions are dehydrated on dried agarose films adhered to coverslips. Rehydration of the polymer films results in rapid formation of pGVs. This method greatly advances the preparation of synthetic giant vesicles for direct applications in biomimetic studies.

Jel destekli rehidrasyon kullanma Dev boyutlu Polymersomes Şekillendirme

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

İmalat ve Macrostructure içeren optik doku hayaletler karakterizasyonu

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

Hazırlık, arıtma ve yağ asidi içeren lipozomlar kullanımı

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Falcarindiol tarafından insan mezenkimal kök hücre içeren yeni Lipid kaplı nano tanecikleri alımını

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading chemotherapeutic drugs, such as doxorubicin (DOX). To achieve this, an ethanolic lipid mixture and ammonium sulfate solution are injected into a staggered herringbone micromixer (SHM) microfluidic device. The solutions are rapidly mixed by the SHM, providing a homogeneous solvent environment for liposomes self-assembly. Collected liposomes are first annealed, then dialyzed to remove residual ethanol. An ammonium sulfate pH-gradient is established through buffer exchange of the external solution by using size exclusion chromatography. DOX is then remotely loaded into the liposomes with high encapsulation efficiency (&#62; 80%). The liposomes obtained are homogenous in size with Z-average diameter of 100 nm. They are capable of temperature-triggered burst release of encapsulated DOX in the presence of mild hyperthermia (42 &#176;C). Indocyanine green (ICG) can also be co-loaded into the liposomes for near-infrared laser-triggered DOX release. The microfluidic approach ensures high-throughput, reproducible and scalable preparation of LTSLs.

The protocol presents the optimized parameters for preparing thermosensitive liposomes using the staggered herringbone micromixer microfluidics device. This also allows co-encapsulation of doxorubicin and indocyanine green into the liposomes and the photothermal-triggered release of doxorubicin for controlled/triggered drug release.

Lysolipid İçeren Sıcaklığa Duyarlı Lipozomların Mikroakışkan Üretimi

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading ...