Yazarlar deneysel cerrahi ve tıp Koleji, Kral Suud Üniversitesi, hayvan Laboratuvarı personeli onların işbirliği ve destek-hüseyin Almukhayzim ve özellikle Hisham Aloudah için teşekkür etmek istiyorum. Yazarlar ayrıca medya ekibinin King Saud bin Abdul-Aziz Üniversitesi'nde görsel hazırlanması için kabul etmek istiyorum malzeme özellikle Hüseyin bin Ghannam ve Abdulwahab Alsulami.

Protokol Kral Suud Üniversitesi'nin etik kurul hayvan bakımı kuralları izler. Cerrahi, anestezi ve hayvanlar üzerinde kullanılan diğer ilaçlar Kral Suud Üniversitesi'nin etik kurul tarafından onaylanır. Tüm deneysel çalışma uygun eğitimli personel tarafından gerçekleştirilir.
1. hücre sac İnşaat
<ol><li>Kültür yemekleri (3.5 cm sıcaklık duyarlı kültür yemekleri) su katılmamış fetal Sığır serum (FBS) ile ceket ve çanak tüm yüzeyi kapsayacak şekilde sağlamak. Not: Bu bir monolayer hücrelerinin büyümesini destekler ve hücre toplama engeller bu adımı hücre sayfası dekolmanı için önemlidir.</li>
	<li>Yemekleri için % 5 CO2 ve % 95 hava içeren bir oksijen atmosferde 37 ° C'de 24 saat kuluçkaya.</li>
	<li>Aşırı FBS kaplamalı yemeklerini kaldırın ve yemekleri için en az 1 h (RT) Oda sıcaklığında kurumasını sağlar.</li>
	<li>Üç hücre süspansiyonlar hazırlamak: 1 x 106 HepG2 hücreleri, 1 x 106 HepG2 hücreleri ile BMMSCs ve 1 x 106 HepG2 hücreleri ile UCMSCs (BMMSCs ve UCMSCs için 4:1 oranında).</li>
	<li>Tohum uygun hücre süspansiyon etiketli her yemeğin için.</li>
	<li>DMEM kültür % 10 FBS ve % 1 penisilin/streptomisin ile desteklenmiş orta bütün hücrelerdeki askıya alma.</li>
	<li>Hücreleri oksijen bir atmosferde % 5 CO2 ve % 95 hava içeren 37 ° C'de kuluçkaya.</li>
</ol>2. hücre sayfası dekolmanı
<ol><li>% 100 hücre izdiham bulaşıkları da 37 ° C kuluçka dışarı al.</li>
	<li>HepG2 hücre sayfası çanak RT, 1 h için HepG2/BMMSC ve HepG2/UCMSC hücre sayfası yemekler %5 CO2 ve % 95 hava içeren bir oksijen atmosferde 20 ° C'de 30-40 dk için kuluçka sırasında kuluçkaya. Not: sadece HepG2 yapılan hücre sayfası kırılgandır; sıcaklık 20 ° C-azaltarak sayfanın yapısına zarar neden.</li>
</ol>3. cerrahi işlem performansını
Not: hücre sayfası dekolmanı için kuluçka süre boyunca, hayvan yordam başlatın.
<ol><li>
Cerrahi alan hazırlanması için yönergeler
	<ol>
<li>Laboratuvar, bir başlık, laminar akış kabine veya aseptik cerrahi oda gibi bir steril ayrı alanındaki tüm cerrahi prosedürlerini yürütecektir. Cerrahi alan tüm yüzeyleri gözenekli olmayan, mühürlü, dayanıklı ve sanitizable olduğundan emin olun.</li>
		<li>Önlük, eldiven, facemasks ve baş ve ayakkabı kapakları da dahil olmak üzere uygun koruyucu giysiler giymek.</li>
		<li>Kullanım cerrahi alet are temiz ve steril (yolu ile ısıyla; yüksek basınç altında doymuş kök) başında cerrahinin.</li>
		<li>Eldiven fareler arasında değiştirin.</li>
	</ol>
</li>
	<li>
Ameliyat için hayvan hazırlanması
	<ol>
<li>8 - için 12-hafta-yaşlı çıplak fareleri kullanın.</li>
		<li>Fareler için enjekte edilebilir anestezi oluşturun.<ol>
<li>Anestezi çözüm hazırlamak için ketamin-xylazine, ketamin 2 mL, xylazine (bir konsantrasyon 20 mg/mL), 1 mL ve 1 mL steril fizyolojik serum (Örneğin, % 0,9 sodyum klorür) steril 10 mL serum koleksiyonu şişe içinde karıştırın ve çok önce sallamak kullanın.</li>
			<li>Anestezi uygulamak için intraperitoneally 0.2 mL farenin vücut ağırlığının 100 gr başına ketamin-xylazine çözeltisi enjekte et. Not: Çözüm toplam doz 100 mg/kg ketamin bas ve 10 mg/kg xylazine değil.</li>
		</ol>
</li>
		<li>Görünür kir/enkaz cerrahi sitesinden kaldırın.</li>
		<li>Anestezi derinliği pedal çekilme refleks (ayak pedi tutam her iki arka ayakları üzerinde) test ederek kontrol edin. Not: Ayak pedi tutam bir yanıt neden oluyorsa, bir doz, 0.05 mL/100 g (yaklaşık her 30 dakika) yineleyin ve sonra anestezik derinlik yeniden denetleyin.</li>
		<li>Bir iki aşamalı bodur ile cerrahi alan povidone-iyot/isopropanol tarafından dezenfekte.</li>
		<li>Belgili tanımlık kesme kirlenmesini önlemek için steril bir örtüsü fareyle kapsar.</li>
		<li>Fareler subkutan buprenorfin (0.01 - 0.05 mg/kg) ile enjekte kesi yapmadan önce8 .</li>
		<li>Steril neşter kullanmadan, bir 7 - için 10-cm derileri ve karaciğer ortaya çıkarmak için temel kaslar arasında kesin olun.<ol>
<li>Karın kas deri neşteri düz arka kenarı ile ayırın.</li>
			<li>Dikkatli bir şekilde bıçakla linea alba neşter kullanarak ve keskin makas ile kesme genişletmek.</li>
		</ol>
</li>
	</ol>
</li>
</ol>4. hücre sac ekimi
Not: Üzerinde karaciğer hücre sac ekimi gerçekleştirilir.
<ol><li>Hücre sayfası kültür çanak toplamak.<ol>
<li>Yavaşça kültür çanak yüzeyi sacdan ayırmak için tezgah üzerine dokunun.</li>
		<li>Hücre sayfası çanak'ın yüzeyinden bir yarım daire bir steril pipet ucu kullanarak sayfanın kenarına kazıma ayırın.</li>
		<li>Yavaşça tüm Orta kültür çanak çıkarın.</li>
		<li>Sayfanın herhangi bir zarar vermeden sağlam olduğundan emin olun; Aksi takdirde, olamaz (Şekil 1) kullanılır. Not: Elde edilen sayfaları doğrudan göz tarafından tespit edilebilir.</li>
	</ol>
</li>
	<li>Yemeğin hücre sacdan hasat için steril bir membran (filtre kağıdı) hazırlayın.<ol>
<li>İlk olarak, membran normal salin ile ıslatın. Sonra aşırı tuz ile steril pamuk yastık kaldırın.</li>
		<li>Islak membran hücre sayfası, kırışıklıklar ve hava kabarcıkları membran ve hücre sayfası arasındaki kaçınırken yerleştirin.</li>
		<li>Membran ve onları daha kolay toplamak için hücre sayfası normal salin birkaç damla ekleyin. Not: hücre sayfası bozulmaması için değil soğuk serum kullanın ve membran ve hücre sayfasını forseps ile kaldırın.</li>
		<li>Membran hücre sayfası membran için düzgün takıldığından emin olmak birkaç saniye için bırakın ve sonra onu toplamak.</li>
	</ol>
</li>
	<li>Karaciğer hücre sac ekimi için hazırlayın.<ol>
<li>Karaciğer yüzey hafifçe steril pamuk yastık ile dokunarak kuru olduğundan emin olun.</li>
		<li>Steril forseps kullanarak, membran hücre levha ile sıçan karaciğer tek bir lob yerleştirin.</li>
		<li>Steril serum fizyolojik birkaç damla ekleyin ve hücre sacdan ayırmak için membran için hafif bir basınç uygulayın.</li>
		<li>Dikkatle membran kaldırmadan önce 5 min için bekleyin. Not: Bu hücre sayfası karaciğere düzgün takıldığından emin olmak için yapılır.</li>
		<li>Son olarak, temel kasları kapatın ve insizyon ile 5-0 naylon dikiş cilt.</li>
		<li>Cerrahi alan povidone-iyot ile dezenfekte. Not: Resim 2 çıplak bir sıçan karaciğer üzerinde bir diyagram hücre sac ekimi yordam gösterir.</li>
	</ol>
</li>
</ol>5. ameliyat sonrası bakım
<ol><li>Ameliyattan hemen sonra tek tek yamyamlık veya boğulma önlemek için fareyi ev ve onu düzenli olarak (en azından her 15 dk) izlemek kadar bilinçli ve tam olarak ayakta.</li>
	<li>Sonra günlük 5-14 d, en az 5 d için fareler post-operatively, herhangi bir komplikasyon sağlamak için değerlendirmek. Günlük değerlendirme sırasında şunlardan emin olun.<ol>
<li>Yara kapalı ve bir dikiş aşırı sıkı olmadan arada.</li>
		<li>Aşırı ısı gibi kesi site enfeksiyon belirtisi yok şişme veya pürülan akıntı.</li>
		<li>Azalan yiyecek ve su tüketimi, kilo kaybı, dehidratasyon, cilt çadır veya hızlı ve açık ağız nefes gibi ağrı ile ilişkili belirti yok. Şiddetli ameliyat sonrası ağrı için ılımlı denetlemek için varsa, fareler subkutan buprenorfin (0.01 - 0.05 mg/kg) ile en az 48-72 saat için her 6-8 h9 postoperatively enjekte et.</li>
	</ol>
</li>
</ol>6. transplante çevrenin Analizi
<ol><li>Bir ay sonra nakli, scarifice fare ve transplante alan için toplamak histolojik ve immunohistokimyasal analizi.</li>
</ol>

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Vitro kanser çalışmaları hala büyük sınırlamalar ile ilişkilidir. Gerçeğini kanser hücrelerinin kültürü muhafaza ne zaman onların in vivo özelliklerinin çoğunu kaybetmek nedeni bu. Bütün doku Fizyoloji bir ex vivo ortamda olmamasından hücreleri vitro sonucu oluşan değişiklikleri. Kanser hücre hücre etkileşim (stromal, bağışıklık, damarlara, epitel, vb) tümör microenvironment içinde büyük ölçüde kanser hücre özellikleri2üzerinde yansıtır. Tümör microenvironment kanser hücre gen/protein ifade ve ek olarak angiogenetic ve metastatik potansiyeli fenotipik özellikleri değiştirmek. İki boyutlu (2B) vitro kültür sistemi de tümör ilerleme düzenlemek gerekli olan uygun doku matris yoksun. Böylece, bu sınırlamaları nedeniyle vivo içinde modelleri her zaman vitro modelleri ön bulguları desteklemek için kullanılması gereken. Bu çalışmada, HCC yatan tam biyolojik süreci elucidates in vivo hayvan modeli geliştirmek için hücre sayfası teknolojisini kullanır.
Daha--dan a on yıl önce Okano'nın laboratuvar doku mühendisliği hücre sayfa teknoloji3dayalı bir roman yöntemi kurdu. Bir termo-yanıt veren Kültür plastik geri dönüşümlü hücre adezyon/dekolmanı yüzey hydrophobicity kontrol ederek izin vermek için bu tekniği kullanır. Bu yöntem nazik bir biçimde olduğu gibi üç boyutlu (3-D) (i.e.,cell sayfa), kültürlü hücre hasat bakımlı hücre dışı matriks (ECM) ve hücre-hücre etkileşimleri ile sağlar. Hücre sayfası teknik ticari olarak kullanılabilir ve kullanıma hazır olan kültür yemekleri ile bir sıcaklık duyarlı polimer poly(N-isopropylacrylamide) (PIPA değilim), precoating gerektirir. Bir sıcaklık 20 ° C'den polimerler PIPA Am sulu olmak ve (37 ° C) daha yüksek bir sıcaklık, polimerler susuz haline ve bulanık bir çökelti dönüşümü ise sulu çözümler içinde çözülür. Polimer hidrofilik Amid zincirleri ve hidrofobik yan zincirleri (izopropil gruplar) içerir. Sulu yapısı dökümü izopropil grupları ve hidrofobik izopropil grupları çevreleyen su moleküllerinin düşük sıcaklıkta Toplam ise, yüksek sıcaklıklarda su molekülleri Albert hareketi, yoğun hidrofobik etkileşimler nedeniyle. Bu nedenle, tüm polimer zincirini toplar ve4precipitates.
Sunulan çalışmada, üç farklı hücre yaprak kullanarak bir HCC hayvan modeli geliştirmek için bu tekniği istihdam. Diğer iki yaprak HCC hücre satır hücreleri ve MSCs iki farklı kaynaklardan oluşur, ancak istihdam ilk sayfayı HCC hücre satır hücreleri yalnızca, oluşur: kemik iliği MSCs (BMMSCs) ve göbek kordonu MSCs (UCMSCs). MSCs intocell türevleri adipositler, osteocytes, kondrosit ve miyositler5gibi mezenkimal soyundan ayırt yeteneğine hematopoetik stromal hücreler vardır. Biz bu hücrelerin kanser hücre sayfası oluştururken istihdam MSCs etkisi kanser tutarsız raporlama nedenidir. MSCs iki ayrı fenotipleri olabilir sürülmüştür: "MSC1", proinflamatuar fenotip ve "MSC2", bir immünsüpresif fenotip6. MSCs toll benzeri reseptörler (TLR) hızlı. TLR3 astar immünsüpresif faktörler6onların salgılanmasını artırır, ancak TLR4 astar MSCs, proinflamatuar faktörler, onların salgılanmasını artırır. Bu iki fenotipleri vitro incelenmesi MSC2 ortak kültür ters etkisi7varken MSC1 ortak bir kültür kanser hücre hatları ile kanser hücre büyümesi, zayıflatılmış bildirdi. Bu MSCs yanlısı kanser veya bağlı olarak onların fenotip antikanser olabilir implicates. Böylece, hücre sayfası teknolojisi kullanarak HCC hayvan modeli geliştirmeye ek olarak, tümör gelişimi, MSC nakli etkisi keşfetmek istiyoruz ve bu hücreler kullanarak geliştirmek olup bu model geliştirilmesi azaltmak.

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		<tr height="22">
			<td height="22" style="height:22px;width:501px;">Reagents&#160;</td>
			<td></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:309px;">FBS&#160;</td>
			<td style="width:192px;">Gibco/Invitrogen</td>
			<td style="width:119px;">10270106</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:309px;">DMEM high glucose&#160;</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:119px;">D5671-500ML</td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:309px;">Penicillin/streptomycin&#160;</td>
			<td style="width:192px;">Life Technology</td>
			<td style="width:119px;">15070063</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:309px;">Sterile physiologic saline</td>
			<td style="width:192px;">Sigma&#160;</td>
			<td style="width:119px;">S0817-1GA</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:309px;">Human HepG2 cell line</td>
			<td style="width:192px;">ATCC, USA</td>
			<td style="width:119px;">HB-8065</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Human bone marrow MSCs cell line</td>
			<td style="width:192px;">PromoCell, USA</td>
			<td>C-12974</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:309px;">human umbilical cord tissue MSCs</td>
			<td style="width:192px;">PromoCell, USA&#160;</td>
			<td style="width:119px;">C-12971</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:309px;">Ketamine 50%</td>
			<td style="width:192px;">Rompun, Bayer</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:309px;">Xylazine 2%</td>
			<td style="width:192px;">Rompun</td>
			<td style="width:119px;">23076-35-9</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Alphadine&#174;&#160;solution.</td>
			<td style="width:192px;">Riyadh Pharma</td>
			<td style="width:119px;">LBL0816</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:501px;">Disposables:&#160;</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:309px;">15mL Polypropylene High Clarity PP Centrifuge Tube</td>
			<td style="width:192px;">Falcon&#160;</td>
			<td style="width:119px;">352097</td>
			<td></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:309px;">3.5 cm sterile UpCell culture dishes with the filter paper (membrane)</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:119px;">174904-1CS</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:309px;">100-1000 &#181;l &#160;Pipette Tips&#160;</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:119px;">CLS4868-1000EA&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Basic Procedure Drape</td>
			<td style="width:192px;">Thermofisher</td>
			<td style="width:119px;">PMD5293.0</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:501px;">Equipment&#160;</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:309px;">Plus pipette, variable volume</td>
			<td style="width:192px;">Eppendorf&#174;&#160;Research&#174;</td>
			<td style="width:119px;">Z683779-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Tissue culture incubator 37 &#176;C, 5% CO2</td>
			<td style="width:192px;">Any brand</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Biological safety cabinet</td>
			<td style="width:192px;">Any brand</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:309px;">Tissue culture incubator 20 &#176;C, 5% CO2</td>
			<td style="width:192px;">Any brand</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:501px;">Sterile surgical tools and nude rats:&#160;</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:501px;">Forceps</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:501px;">Scissors</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:501px;">scalpel&#160;</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:309px;">&#160;Nylon Suture&#160; 5-0</td>
			<td style="width:192px;">Accutome</td>
			<td style="width:119px;">AB-3854S</td>
			<td>Monofilament, Lancet</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:309px;">1 ml Tuberculin Syringes</td>
			<td style="width:192px;">Fisher Scientific</td>
			<td style="width:119px;">14-826-88</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:309px;">Nude rats&#160;</td>
			<td style="width:192px;">Charles river</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

exploring the potential of mesenchymal stem cell sheet on the development of hepatocellular carcinoma in vivo

İnsan kanseri taklit eden bir in vivo hayvan modeli önemli klinik bilgi sunar çeşitli uygulamalar olabilir. Şu anda kullanılan teknikleri vivo içinde kanser modelleri geliştirilmesi için önemli kısıtlamalar bulunmaktadır. Bu nedenle, bu çalışmada, bir vivo içinde kanser modeli geliştirmek için hücre sayfası teknolojiyi uygulamak için amacımız. Hepatosellüler karsinom (HCC) başarıyla çıplak fareleri HCC hücre satırı hücrelerinden oluşturulan hücre sayfalarını kullanarak geliştirilmiştir. Kanser hücre yaprak hücre içi adezyon ve hücre dışı matriks tarafından kontrollü bir tabakalı yapısı oluşumu aracılığıyla oluşturulur. Bu HCC sac ekimi karaciğer ve bir ay içinde bir tümörü taşıyan hayvan modeli oluşturulması için izin verir. Buna ek olarak, mezenkimal kök hücre (MSC), bu kanser modeli geliştirilmesinde rol araştırıldı. HCC hücre satırı sayfa ek olarak başka bir iki hücre sayfaları oluşturulur: HCC hücreleri ve kemik iliği MSCs (BMMSCs) ve bir levha HCC hücrelerin ve göbek kordonu MSCs (UCMSCs) bir sayfası. HCC hücreleri ve MSCs kombinasyon var sayfaları da bir tümörü taşıyan hayvanı üretme yeteneğine sahiptirler. Ancak, MSCs eklenmesiyle oluşan tümör boyutunu küçültür ve bu tümör gelişimi üzerinde olumsuz etkisi kullanılan MSCs kaynağına bağlı olarak değişir. Bu hücre sabıkası belirli MSC alt türlerinden yapılan tümör yönetimi ve denetimi kullanıldığında olabilir gösterir.

Transplante hücre yaprak sıçanlarda tumorigenicity:
Bir ay sonra nakli, tüm transplante hücre çarşaflarda rats karaciğer tümörleri (Şekil 3) geliştirdik. Gelişmiş tümör HepG2, HepG2/BMMSC ve HepG2/UCMSC hücre sayfaları üzerinden ortalama büyüklüğü 4,5 cm, 4 cm ve 2,5 cm, sırasıyla10vardı.
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Watch this Scientific Journal Video about Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo at JoVE.com

Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo

Burada, hücre sayfası teknolojisi kullanarak bir vivo içinde kanser modeli geliştirmek için bir protokol mevcut. Böyle bir model antikanser therapeutics değerlendirme için çok yararlı olabilir.

Hepatosellüler karsinom Vivo içinde geliştirilmesi üzerinde mezenkimal kök hücre sayfası potansiyelini keşfetmek

An in vivo animal model that mimics human cancer could have various applications that deliver significant clinical information. The currently used ...

exploring-potential-mesenchymal-stem-cell-sheet-on-development

Research

JoVE Journal

Bioengineering

Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo

5.8K Görüntüleme.  Ministry of National Guard Health Affairs. Burada, hücre sayfası teknolojisi kullanarak bir vivo içinde kanser modeli geliştirmek için bir protokol mevcut. Böyle bir model antikanser therapeutics değerlendirme için çok yararlı olabilir.

Video: Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

Farklı soy İnsan Embriyonik Kök Hücreleri (hESC) Yayılması ve Farklılaşma için 3D Platformu olarak aljinat Mikrokapsül

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Biyomühendislik

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

Geliştirme, Genişleme ve<em&gt; In vivo</em&gt; İnsan Embriyonik Kök Hücreleri (hESC) den İnsan NK Hücrelerinin İzleme ve ve Bağlı Pluripotent Kök Hücre (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to maintain cellular homeostasis. Puncture injury with glass needles provides a direct method to trigger a rapid epidermal wound response that activates wound transcriptional reporters, which can be visualized by a localized reporter signal in living embryos or larvae. Puncture or laser injury also provides signals that promote the recruitment of hemocytes to the wound site. Surprisingly, severe (through and through) puncture injury in late stage embryos only rarely disrupts normal embryonic development, as greater than 90% of such wounded embryos survive to adulthood when embryos are injected in an oil medium that minimizes immediate leakage of hemolymph from puncture sites. The wound procedure does require micromanipulation of the Drosophila embryos, including manual alignment of the embryos on agar plates and transfer of the aligned embryos to microscope slides. The Drosophila epidermal wound response assay provides a quick system to test the genetic requirements of a variety of biological functions that promote wound healing, as well as a way to screen for potential chemical compounds that promote wound healing. The short life cycle and easy culturing routine make Drosophila a powerful model organism. Drosophila clean wound healing appears to coordinate the epidermal regenerative response, with the innate immune response, in ways that are still under investigation, which provides an excellent system to find conserved regulatory mechanisms common to Drosophila and mammalian epidermal wounding.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The embryonic epidermis of very late stage Drosophila embryos provides an in vivo system for rapid puncture wound response analysis and can be combined with genetic manipulations or chemical microinjection treatments to advance studies in wound healing for translation into mammalian models.

Mikroenjeksiyon Yara Deneyi ve<em&gt; In vivo</em&gt; Epidermal lokalizasyonu Tepki Gazeteciler Yara<em&gt; Drosophila</em&gt; Embriyolar.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, and observation and quantification are very challenging. However, conventional in vitro microvessel assays have limitations when representing in vivo microvessels with respect to three-dimensional (3D) geometry and providing continuous fluid flow. Using a combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics, we have developed a multi-depth circular cross-sectional endothelialized microchannels-on-a-chip, which mimics the 3D geometry of in vivo microvessels and runs under controlled continuous perfusion flow. A positive reflowable photoresist was used to fabricate a master mold with a semicircular cross-sectional microchannel network. By the alignment and bonding of the two polydimethylsiloxane (PDMS) microchannels replicated from the master mold, a cylindrical microchannel network was created. The diameters of the microchannels can be well controlled. In addition, primary human umbilical vein endothelial cells (HUVECs) seeded inside the chip showed that the cells lined the inner surface of the microchannels under controlled perfusion lasting for a time period between 4 days to 2 weeks.

A microchannels-on-a-chip platform was developed by the combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics. The endothelialized microchannels platform mimics the three-dimensional (3D) geometry of in vivo microvessels, runs under controlled continuous perfusion flow, allows for high-quality and real-time imaging&#160;and can be applied for microvascular research.

Çok derinlemesine Dairesel kesit Endothelialized mikro kanallar-on-a-chip Geliştirme Prosedürü

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

Poli (ε-kaprolakton) electrospun İplik İnsan Mezenkimal Kök Hücre Ek Optimize

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

Tanımlı İnsan Engineered Kalp Dokuların inşaatı Kardiyak Hücre Tedavisinin Mekanizmaları Eğitim için

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

Yamultma stres gelişmekte olan zebra balığı kalp oransal etkileri 4 boyutlu görüntülerini yakalamak için ışık sayfalık floresans mikroskobu

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

3D dinamik kültür sistemi ile bir çok katmanlı mezenkimal kök hücre levha inşaatı

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

Doku Seviyesi Mekaniğinin Kontrol Altına Almak İçin Uyumlu Yüzeylerde İnsan Embriyonik Kök Hücre Kolonilerinin Geometrisinin Desenlemi

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Isolation, Expansion, and Differentiation of Mesenchymal Stem Cells from the Infrapatellar Fat Pad of the Goat Stifle Joint

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded during arthroscopic procedures and knee replacement surgeries. Additionally, its removal is associated with minimal donor site morbidity. Recent studies have demonstrated that IFP-MSCs do not lose their proliferation capacity during in vitro expansion and have age-independent osteogenic differentiation potential. IFP-MSCs possess superior chondrogenic differentiation potential compared to bone marrow-derived MSCs (BMSCs) and adipose-derived stem cells (ADSCs). Although these cells are easily obtainable from aged and diseased patients, their effectiveness is limited. Hence, using IFP-MSCs from healthy donors is important to determine their efficacy in biomedical applications. As access to a healthy human donor is challenging, animal models could be a better alternative to enable fundamental understanding. Large animals such as dogs, horses, sheep, and goats play a crucial role in translational research. Amongst these, the goat could be a preferred model since the stifle joint of the goat has the closest anatomy to the human knee joint. Moreover, goat-IFP can fulfill the higher MSC numbers needed for tissue regeneration applications. Furthermore, low cost, availability, and compliance with the 3R principles for animal research make them an attractive model. This study demonstrates a simple protocol for isolating IFP-MSCs from the stifle joint of goats and in vitro culture conditions for their expansion and differentiation. The aseptically isolated IFP from the goat was washed, minced, and digested enzymatically. After filtration and centrifugation, the collected cells were cultured. These cells were adherent, had MSCs-like morphology, and demonstrated remarkable clonogenic ability. Further, they differentiated into adipogenic, chondrogenic, and osteogenic lineages, demonstrating their multipotency. In conclusion, the study demonstrates the isolation and expansion of MSCs, which show potential in tissue engineering and regenerative medicine applications.

Infrapatellar fat pad mesenchymal&#160;stem cells (IFP-MSCs) can be isolated easily from the infrapatellar fat pad of the knee joint. They proliferate well in vitro, form CFU-F colonies, and differentiate into adipogenic, chondrogenic, and osteogenic lineages. Herein, the methodology for the isolation, expansion, and differentiation of IFP-MSCs from goat stifle joint is provided.

Mezenkimal Kök Hücrelerin Keçi Stifle Ekleminin İnfrapatellar Yağ Pedinden İzolasyonu, Genleşmesi ve Farklılaşması

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded ...

Hepatosellüler karsinom Vivo içinde geliştirilmesi üzerinde mezenkimal kök hücre sayfası potansiyelini keşfetmek

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