Name: construction of defined human engineered cardiac tissues to study mechanisms of cardiac cell therapy
Uploaded: 2016-03-01T13:00:00
Description: Watch this Scientific Journal Video about Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy at JoVE.com

Bu çalışma TJC, KDC, Amerikan BDG Hong Kong TRS T13-706 / 11 (KDC), NIH (R01 HL113499) araştırma hibe konseyi NIH / NHLBI PEN sözleşme HHSN268201000045C için NIH (1F30HL118923-01A1) tarafından desteklenmiştir Ek finansman rl NIH DRB 5T32GM008553-18 tarafından ve Sistemleri ve gelişimsel olarak NIDCR-Disiplinlerarası Eğitim bir staj olarak TJC sağlanmıştır kalp Derneği (12PRE12060254) RJ ve HKSAR Araştırma Hibe Konseyi (/ 11 T13-706 TBRS) Biyoloji ve Doğum Kusurları T32HD075735. Yazarlar ayrıca minnetle, teknik yardım için biyoreaktör ve Memduh Eldaly işleme ile yardım için New York City College of Zahn Merkezi&#39;nde Arthur Autz kabul etmek istiyoruz. Biz de cömertçe insan mezenkimal kök hücreleri sağlamak için kalp farklılaşma tavsiye Dr. Kenneth Boheler, Dr. Joshua Hare teşekkür ederim.

Not: Bir HEPA filtreli sınıf II biyolojik güvenlik kabini kullanılarak aseptik koşullarda tüm hücre manipülasyonlar gerçekleştirin ve 0.2 mikron filtre aracılığıyla filtre edilerek tüm çözümler sterilize edin. Aynı aseptik şartlarda ya da bir laminer akış kaputu ya doku yapım ve fonksiyon testlerini gerçekleştirmek. Kardiyak Farklılaşma için Hazırlık H7 hESC 1. Tohum <ol><li> (Gün 1) bazal membran Matrix hazırlanması <ol><li> 4 ° C&#39;de bir gece boyunca ...

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tanımlanmış insan mühendisliği kalp dokularında (Hect) İnşaatı insan kalp miyosit fonksiyonunun daha tutarlı ve güvenilir bir model sağlayabilir. Kritik olarak, sistemdeki tüm hücre ve hücre-dışı bileşenler bilinmektedir ve dolayısıyla farklılaşma işleminden kaynaklanan diğer bilinmeyen hücre tipleri karıştırıcı etki çıkarılması istendiği gibi işlenebilir. hızlı hücre büyümesi ve yüksek verim dengelemek için, farklılaşma ideal dört gün sonra kaplama HESC% 75 birleşme nokt...

Kardiyak doku mühendisliği daha yakın hem de fare kardiyomiyositlerinin 1-6 ve yapılan tam fonksiyonel, dayak dokular, insan kök hücresi kaynaklı kalp miyositlere 7-12 sonuçlarını yayınlayarak çoklu gruplar, son on yılda büyük ölçüde ilerlemiştir. Kalp doku mühendisliği alanında iki birincil ve esas bağımsız hedefler tarafından tahrik edilmektedir: 1) işlevini 4-6 iyileştirmek için kalpleri başarısız içine transplante edilebilir eksojen greft geliştirmek; ve 2) fizyoloji ve hastalık eğitimi için in vitro modellerinde geliştirmek, ya da terapötik gelişim 2,7 için tarama araçları olarak. Üç boyutlu (3-D) hücre kültürü 3-D matrisi geleneksel 2-D tek tabakalı hücre kültürü daha doğal kalp mikro yansıtır, yeni nesil tarama araçları geliştirmek için gerekli olarak kabul edilir; Gerçekten hücre biyolojisi bazı yönlerini 2-D vs 3-D kültürlerinde 13,14 temelde farklıdır </sus&gt;. hücre dışı bir matris, bir hücre popülasyonu: Buna ek olarak, işlenmiş kardiyak dokular tam olarak tanımlanmış bileşenlerin inşa edilir. Ekstraselüler matriks kompozisyonu (genellikle fibrin 9 veya kollajen 7,8,10) sıkı kontrol edilirken geleneksel mühendislik insan kalp dokuları için giriş hücre kompozisyon daha az bir yönlendirilmiş kalp farklılaşma hücrelerin tüm karışımı ile tanımlanır ya embriyonik kök hücreler (ESC 7,9) ya da uyarılmış pluripotent kök hücreler (iPSC 10,12) dokulara ilave edilir. Spesifik hücre hattı ve ikinci farklılaşma protokolünün verimliliğine bağlı olarak, kardiyomiyositlerin sonuçtaki yüzdesi üzerinden% 90 den az% 25, belirli kardiyomiyosit fenotipi arasında olabilir (yani, ventricular-, atrial-, kalp pili gibi ya da) Ayrıca bile olmayan kardiyomiyosit fraksiyon 15,16 derece heterojen ve farklılaşmış kardiyak m olgunluk değiştirebilir, değişir17 yocytes. Son kardiyak doku mühendisliği çalışmaları kalp reporter insan embriyonik kök hücre hattı 8 veya hücre yüzey işaretleyicileri 18 farklılaşma kardiyak miyosit bileşenini izole etmek için kullanılan biriyle, hücrelerin giriş nüfusunu kontrol etmek için çalıştı. Sadece kalp miyositler oluşan başlangıçta doku ideal gibi görünüyor olsa da, bu gerçeği değil durumda olduğunu; Kalp miyositler 1 oranında: fibroblastlar yüksek seğirme kuvveti 8 üreten sadece kardiyak miyositler oluşan hECTs bazı gruplar, 3 bulma, fonksiyonel dokulara sıkıştırmak için başarısız. Canlı hücre sıralama için yüzey belirteçleri de dahil olmak üzere çeşitli hücre seçim yöntemleri kullanılarak, tanımlanmış hücre populasyonlarına sahip hECTs oluşturmak mümkündür. Kardiyak olmayan stromal hücrelerin belirteçleri CD90 19,20 gibi, farazi fibroblast markeri olarak bir süre için uygun olduğu halde, kardiyak miyositler yüzey işaretleyicileri daha zor olmuşturtespit etmek. SIRPα insan kalp miyositlere 18 için belirlenen ilk kalp yüzey belirteçleri arasında yer aldı ve kalp soy için son derece seçici olduğu gösterilmiştir. Hücreler bir fibroblast-benzeri fenotip (Josowitz, yayınlanmamış gözlemler) sergileyen CD90 + nüfusu ile hemen hemen saf kardiyomiyositlerde verir - Son zamanlarda, iki sıralama SIRPα + ve CD90 için bulduk. Kardiyomiyositlerde ve CD90 + fibroblastlar - SIRPα + / CD90 1 kombinasyonu: Bu toplanan bulgulara dayanarak, burada biz bir 3 kullanan hECTs oluşturmak açıklar. tamamen tanımlanmış insan kalp dokusu mühendisi yeteneği güçlü tarama araçları oluşturmak için değil, aynı zamanda gelişmekte olan hücreye ve gen bazlı kardiyak tedaviler araştırmak için bir model sistemleri geliştirmek için değil, sadece esastır. Mezenkimal kök hücrelerin (MSC) 21 olmak üzere hücre tipleri kullanan kalp yetmezliği için özellikle çok sayıda hücre tedavileri, içinde </sup&gt;, kardiyak kök hücreler 22 ve kemik iliği mononükleer hücreleri 23-25, klinik çalışmalarda test edilmiştir. İlk sonuçların pek 21,23,25 umut verici olsa da, ilk fayda genellikle zaman 26-29 üzerinde azalır. Benzer bir eğilim nedeniyle MSC takviyesi önemli bir fonksiyonel fayda gösterir kemirgen mühendislik kardiyak dokularda rapor edilmiştir, ancak fayda uzun vadeli kültürü 1 sırasında sürekli değildir. sub-optimal performans altında yatan hücre tedavileri düzenleyen mekanizmaların bizim sınırlı bilgidir. onların olumlu etkisi yanı sıra, miyosit-nonmyocyte etkileşimlerin potansiyel olumsuz sonuçların uygulamak nasıl tedavi hücreler daha derin bir anlayış, kalp yetmezliği olan hastalarda minimal yan etkileri klinik olarak anlamlı ve sürekli faydalar veren gelişmiş tedavilerin gelişimini, sağlayacaktır. Burada, interrog tanımlanmış hECTs kullanımını tarifhücre tabanlı tedavinin mekanizmaları yedik. kontrollü doku kompozisyonu kardiyomiyosit performansını etkilemeden belirli faktörleri belirlemek önemlidir. Sıçan AKTS 1&#39;de gösterildiği gibi, doğrudan ilgi terapötik hücre tipi (örneğin, MKH) ile hECTs tamamlayan, kardiyak miyosit performansı üzerindeki etkileri ortaya çıkarabilir. Aşağıdaki çok adımlı protokol çoklu doku biyoreaktör imalatı izledi ve doku yapımı ve fonksiyonel analiz bir açıklama ile sonuçlandırılması, yönetmenliğini kardiyak kök hücre farklılaşması ile başlar. Bizim deneyler NIH onaylı H7 insan embriyonik kök hücre (hESC) hattı kullanılarak gerçekleştirilir. Bununla birlikte, aşağıdaki protokol ayrıca bir HESC hattı ve benzer sonuçlar ile üç uyarılmış pluripotent kök hücre (hiPSC) çizgileri kullanılarak test edilmiştir. Biz Hect üretiminde kardiyomiyosit farklılaşma ve başarı verimlilik, özellikle fo, hücre hattı bağımlı olabilir buldukBireysel hastalar türetilen R hiPSC hatları. Bu protokolü takip ederek, iki adet 6-kuyu yemekleri 20 gün boyunca ayırt ve yeterince altı tanımlanan dokuları yapmak için, sıraladıktan sonra yaklaşık 2,5 milyon miyositleri verir 1680000 hESC (oyuk başına 140,000 hücre), toplam kaplanmıştır.

<table border="0">
	<tbody>
		<tr>
			<td>Cell Culture</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma-Aldrich</td>
			<td>A2411</td>
			<td>Prepare a 2.5 mg/ml stock in DMSO and filter-sterilize</td>
		</tr>
		<tr>
			<td>B27 with Insulin</td>
			<td>Life Technologies</td>
			<td>17505055</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 without Insulin</td>
			<td>Life Technologies</td>
			<td>A1895601</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Stemgent</td>
			<td>04-0004</td>
			<td>Create 6 &#956;M stock, then aliquot and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Essential 8 Media</td>
			<td>Life Technologies</td>
			<td>A1517001</td>
			<td></td>
		</tr>
		<tr>
			<td>H7 Human Embryonic Stem Cells</td>
			<td>WiCell</td>
			<td>WA07</td>
			<td></td>
		</tr>
		<tr>
			<td>hESC Qualified Matrix, Corning Matrigel</td>
			<td>Corning</td>
			<td>354277</td>
			<td>Thaw on ice at 4 &#176;C overnight then aliquot 150 &#956;l into separate tubes and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>IWR-1</td>
			<td>Sigma-Aldrich</td>
			<td>I0161</td>
			<td>Create 10 mM stock and aliquot. Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Neonatal Calf Serum</td>
			<td>Life Technologies</td>
			<td>16010159</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-enzymatic Dissociation Reagent: Gentle Cell Dissociation Reagent</td>
			<td>Stem Cell Technologies</td>
			<td>7174</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Life Technologies</td>
			<td>11875-093</td>
			<td>Keep refrigerated</td>
		</tr>
		<tr>
			<td>Y-27632 (ROCK Inhibitor)</td>
			<td>Stemgent</td>
			<td>04-0012</td>
			<td>Resuspend to a 10 mM stock concentration, aliquot and store at -20 &#176;C.&#160; Avoid freeze thaw cycles.</td>
		</tr>
		<tr>
			<td>Cell Sorting</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>4&#8217;,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)</td>
			<td>Life Technologies</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>CD90-FITC</td>
			<td>BioLegend</td>
			<td>328107</td>
			<td></td>
		</tr>
		<tr>
			<td>Enzymatic Dissociation Reagent: Cell Detach Kit I (0.04 % Trypsin/ 0.03% EDTA, Trypsin neutralization solution and Hanks Buffered Salt Solution)&#160;</td>
			<td>PromoCell</td>
			<td>C-41200</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologics</td>
			<td>S11250</td>
			<td></td>
		</tr>
		<tr>
			<td>SIRP&#945;-PE/Cy7</td>
			<td>BioLegend</td>
			<td>323807</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Construction</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>0.25% Trypsin/0.1% EDTA</td>
			<td>Fisher Scientific</td>
			<td>25-053-CI</td>
			<td>Optional: For collection of supplemental cells of interest</td>
		</tr>
		<tr>
			<td>10x MEM</td>
			<td>Sigma-Aldrich</td>
			<td>M0275-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>10X PBS Packets</td>
			<td>Sigma-Aldrich</td>
			<td>P3813</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen, Bovine Type I</td>
			<td>Life Technologies</td>
			<td>A10644-01</td>
			<td>Keep on ice</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Life Technologies</td>
			<td>11330057</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagles Medium (DMEM), High Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>D5648</td>
			<td></td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3784</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>221465</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>1.5 ml microcentrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>NC0536757</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml polyproylene centrifuge tube</td>
			<td>Corning</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml Polystyrene Round-Bottom Tube</td>
			<td>Corning</td>
			<td>352235</td>
			<td>With integrated 35 &#956;m cell strainer</td>
		</tr>
		<tr>
			<td>50 ml polyproylene centrifuge tube</td>
			<td>Corning</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well flat bottom tissue-culture treated plate</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Scraper, Disposable</td>
			<td>Biologix</td>
			<td>70-2180</td>
			<td></td>
		</tr>
		<tr>
			<td>Polysulfone</td>
			<td>McMaster-Carr</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polytetrafluoroethylene (Teflon)</td>
			<td>McMaster-Carr</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>Olympus</td>
			<td>SZ-61</td>
			<td>Or similar, must have a mount for the high speed camera to attach</td>
		</tr>
		<tr>
			<td>Electrical Pacing System</td>
			<td>Astro-Med, Inc</td>
			<td>Grass S88X Stimulator</td>
			<td></td>
		</tr>
		<tr>
			<td>High Speed Camera</td>
			<td>Pixelink</td>
			<td>PL-B741U</td>
			<td>Or similar, but must be capable of 100 frames per second for accurate data acquisition</td>
		</tr>
		<tr>
			<td>Plate Temperature Control</td>
			<td></td>
			<td></td>
			<td>Used to maintain media temperature during data acqusition.</td>
		</tr>
		<tr>
			<td>Custom Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>LabView Post-tracking Program</td>
			<td></td>
			<td></td>
			<td>available upon request from the authors</td>
		</tr>
	</tbody>
</table>

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.

Kalp miyositleri elde etmek için, Boheler ve Lian farklılaşma yöntemleri biraz değiştirilmiş bir sürümü 30,31 kullanılır. Farklılaşma, hücre büyümesi log-faz esnasında başlar, ama aynı zamanda başlangıç ​​popülasyonu (yaklaşık% 75 en uygunudur) sıralama sonra bir hücre kullanılabilir sayısını elde etmek için yeterli sıvıyla zorunludur. Tipik haliyle, H7 hESC için, 37 ° C &#39;de muhafaza esas 8 ortam ve% 5 CO2 inkübatöründe 6 oyuklu çanak oyuk başına 1...

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

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 ...

Research

JoVE Journal

Bioengineering

Shock Wave Application to Cell Cultures

Kültürler Hücre Şok Dalga Uygulama

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to ...

Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

Minyatür Doku Kültürü Sistemde Fare Kalp Vanalar Kültürleme

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Kardiyak İlaç güvenlik Taraması için insan mühendisliği kalp dokusunun Otomatik Büzülme Analizi

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum

İnsan Serumda kardiyak biyolojik ölçmek için olağanüstü optik iletim kullanma

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

Yetişkin kemik iliği türevi hematopoetik kök hücre mühendislik manyetik hedefleme ile kombine etkinleştirmek için hücre içine mikroRNA Transfer için iletişim kuralı

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

Net kalıp tabanlı yöntemi iskele ücretsiz üç boyutlu kalp doku oluşturma

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

İnsan Organotipik Kardiyak Dilimlerinin Çift Voltaj ve Kalsiyum Optik Haritalaması ile Preklinik Kardiyak Elektrofizyoloji Değerlendirmesi

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...

Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair

In Vivo Miyokardiyal Onarım için İnsan iPS Hücrelerinden Elde Edilen Örgü Şekilli Mühendislik Kardiyak Dokuların Hazırlanması

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

İnsan iPSC Türevli Kardiyomiyositler, Kardiyak Fibroblastlar ve Endotel Hücreleri Kullanılarak 3D Kardiyak Mikrotissue Dizilerinin İmalatı

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...

Cardiac Spheroids as in vitro Bioengineered Heart Tissues to Study Human Heart Pathophysiology

İn vitro Biyomühendislik Kalp Dokuları Olarak Kardiyak Sferoidler İnsan Kalp Patofizyolojisi Çalışma

Despite several advances in cardiac tissue engineering, one of the major challenges to overcome remains the generation of a fully functional vascular ...