Yazarlar Prof Hendrik Tevaearai ve çalışmanın ilk kısmı için mali destek için Kalp Damar Cerrahisi Bölümü teşekkür ederim.

NOT: Lewis Kadın ve Erkek sıçanlar, 200-220 gr standart laboratuar koşullarında (12 saat aydınlık ve karanlık döngüsü, istenildiği kadar su ve gıda, IVC kafes) altında tutuldular. Tüm hayvanlar hayvan koruma FELASA ve İsviçre Kanun tavsiyelerine uygun olarak tedavi edildi. 1. Hücre Hazırlanışı: Kemik İliği dan Mezenkimal Kök Hücre İzolasyonu <ol><li> 5 dakika boyunca bir indüksiyon odasında izofluran% 5 ve O 2 5 L / dak, sıçanları anestezisi. Anestezi sistemine bağlı bir burun konisi içine hayvanın burun yerleştirin. Sedasyon onaylamak için bir ayak veya kuyruk tutam gerçekleştirin. </li><li> Kalça ayak bileği arasında cildi keserek makasla bacaktan deri kaldırmak. Kasları çıkarın ve hayvan eksanguinasyonla için femoral arter kesti. Kalça eklemi Açığa ve femur başını yerinden. Bu femur başını zarar vermemek önemlidir. 50 ml&#39;lik plastik bir tüp içinde, steril PBS içinde kemikleri yerleştirin. </li><li> Tüm mu kaldıronları kırmadan kemiklerden scles ve ligament. Not: Bu, kemik, etanol ile yıkama çözeltisi ya da kirlenme bağlantı bilmek bozulmadan kalması çok önemlidir. 5 dakika boyunca% 70 etanol içinde soyulmuş kemikleri durulayın. </li><li> Steril bir laminer akış kaputu altında, makas ile kemiklerin hem ipuçları kesti. Kemik ekstremite steril PBS enjekte edilerek kemik iliği yıkayın. 15 ml tüp içine kemik iliği toplayın. 300 x g ile santrifüj 7 Min. </li><li> Süpernatantı. Kırmızı kan Celi Lysis tamponunda 3 ml pelet askıya alınması. 300 x g, 7 dakika boyunca tekrar iplik önce, oda sıcaklığında 1 dakika boyunca süspansiyonu inkübe edin. 150 ml kültür, bir şişe içinde, steril, hücre kültür ortamı (395 ml IMDM ortamı, 5 ml pen / strep, 100 ml FBS) ekleyin ve tohum hücreleri. </li><li> Nonadherent hücreleri çıkarmak için ikinci gün orta değiştirin. <ol><li> Istenilen hücre miktarı elde edilinceye kadar 2-3 hafta boyunca her ikinci gün orta değişiklikleri gerçekleştirin. </li><li> Alternatif olarak, tohum tarafından biograft hazırlamakgerekli ve 3 başka bir yerde tarif edildiği gibi bir katı matris üzerine hücreleri ing. </li></ol></li><li> Kalp implantasyonu günü, hücreleri hazırlamak ya da sadece epikardiyal uygulamadan önce biograft hasat. <ol><li> Accutase hücre dekolmanı çözüm kullanarak hücreleri toplamak. Hücreleri saymak. Istenilen hücre miktarı elde etmek için hesaplanır hücre süspansiyonu hacmi, 1.5 ml santrifüj tüpü doldurun. 300 xg&#39;de 7 dakika santrifüj ve süpernatant kaldırmak. </li><li> Seçenek olarak ise, steril PBS içerisinde çalkala ve hücre kültür ortamından hasat biograft ve serumsuz taze kültür ortamı içinde tutar. </li></ol></li></ol> 2. İlk Torakotomi ve LAD ligasyon <ol><li> Sıçan tartılır. 37 ° C&#39;de ısıtma yastığı açın. 5-7 dakika indüksiyon odasında% 5 izofluran ve 5 L / dk% 100 O 2 ile sıçan anestezisi. Sedasyon onaylamak için bir ayak veya kuyruk tutam gerçekleştirin. Isıtma yastığı sıçan yerleştirin. </li><li> 14 ile hayvan entübeG iv kateter. 2.5 L / dk oksijen,% 2.5 izofluran, 2 ml tidal hacim ve / dak 90 nefeslerin bir nefes frekans için programlanmış bir kemirgen ventilatöre entübasyon kateteri bağlayın. </li><li> 0.1 mg / kg bir buprenorfin çözelti hazırlayın. Deri altına çözeltinin üçte biri enjekte edilir. Toraks sol kısmını tıraş. % 1 Betadine solüsyonu ile dezenfekte. </li><li> Dördüncü interkostal boşlukta sternum dik cilt İnsizyon. 3 göğüs kasının katmanları (pektoralis majör, artan pektoralis ve dış eğik pektoralis) ayırın. (Kaburga arasında 4 ve 5), dördüncü interkostal boşluk açın. </li><li> Kaburga yaymak ve kalp maruz küçük bir ekartörünü kullanın. Dikkatli perikard açın. Bulun ve atrium altında 7.0 dikiş 4 mm ile koroner arter (LAD) sol ön inen Arter. </li><li> 3,0 suturlerle iki dikiş ile interkostal alanı kapatın. Sternum proksimal ve distalde iki sütür yerleştirin. İlk olarak, tDistal dikiş ighten. Göğüs şişirmek ve herhangi bir pnömotoraks önlemek için 2 saniye boyunca ventilatör egzoz boru kelepçe. İkinci dikiş sıkın. (Hayır dikişler ihtiyaç vardır) geri yerine kasları katmanları yerleştirin. 5,0 dikiş ile cilt kapatın. </li><li> % 1 Betadine solüsyonu ile dezenfekte dikiş. Buprenorfin, solüsyonun geri kalanı enjekte edilir. Anestezi sistemi kapatın. Entübasyon kateteri çıkarın. </li><li> 1-2 saat ılık bir Lamp altında kafeste sıçan tutun. Kafes içinde bir termometre tutun ve aşırı ısınmaya önlemek için sıcak lamba ve kafesin arasındaki mesafeyi kontrol eder. VKİ ünitesinde sıçan dön. Deri altından Buprenorfin 0.05-0.1 mg / kg enjekte Gönder -operatively 48 saat boyunca 6-12 saat. </li></ol> İkinci Torakotomi Tedavi Via 3. Epikardiyal İdaresi <ol><li> Tekrarlayın 2,1-2,3 adımları. </li><li> Beşinci interkostal boşlukta sternum dik cilt İnsizyon. 3 tabakanın ayrılmasıTorasik kas s (pektoralis majör, artan pektoralis ve dış eğik pektoralis). (Kaburgalar 5 ve 6 arasındaki) beşinci interkostal aralık açın. </li><li> Kaburga yaymak ve kalp maruz küçük bir ekartörünü kullanın. Bazı bağlılık varsa, dikkatle ince forseps ile bunları kaldırmak. </li><li> Dikilen altında bir soluk alanı olarak görünen enfarktüs alanı bulun. Not: Gerektiğinde daha sol karıncığa görselleştirmek için tepe eklenir ameliyat dikiş ipliğinin bir 10 cm&#39;lik bir parça (7,0) kullanır. Kalp maruz bir kelepçe ile gevşek tutulan sütür hafifçe çekin. </li><li> Aşağıdaki tedavilerden birini uygulayın: <ol><li> Kalp yüzeyinde biograft yerleştirin. 10 dakika boyunca, oda sıcaklığında önceden ısıtılmış doldurulmuş fibrin yapıştırıcı şırınga kullanın. 25 G Luer iğne kullanılarak biograft altında fibrin yapıştırıcı bir damla (50-100 ul) uygulayın. Biograft tamamen sızdırmaz olduğundan emin olun. </li><li> Seçenek olarak ise, yüzey o bir pipet ile toplanmış hücre pelletini geçerlidirdikilen altında soluk alan üzerine kalp f. Hücre pelet üzerine fibrin yapıştırıcı bir damla (50-100 ul) uygulayın. </li></ol></li><li> Apeksinden sütür parça çıkarın. </li><li> Tekrarlayın 2,6-2,8 adımları. </li></ol>

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The authors have nothing to disclose.

LAD kalıcı ligasyonu geri dönüşümsüz miyokard yaralanmasına neden olmaktadır. İlk hayvan modeli, o günden beri kronik MI için standart ve uygun model olarak kabul edilmiştir. 1960 4 tarif edilmiştir. Onun kararlılığı ve uyarlık MI 5 için tedavilerinin deneysel değerlendirilmesini sağladı. İlk açıklama aşağıdaki Geliştirilmiş prosedürler 35-13% 6 bir operatif mortalite oranı bildirdi. Beklendiği gibi, LAD ligasyon 2 hafta içi...

19. yüzyılın sonundan bu yana, kardiyovasküler hastalık sanayileşmiş ülkelerde bir numaralı seri katili kalmıştır. Bunlar arasında, koroner arter hastalığı, ana etiyolojisi temsil eder. Miyokard infarktüsü (Mİ) ve akut faz sonuçları giderek kronik fazda ve ciddi kalp yetmezliği doğru gelişir uyumsuz biçimlenme tarafından takip edilmektedir. Önemli en son teknolojik ve terapötik ilerlemelere rağmen, bağlı kalp yetmezliğinin ilerlemesine morbidite ve mortalitesi hala 1 büyüyor. Bu bağlamda, hücre tedavisi kalp yetmezliği doğru hastalığın ilerlemesini durdurmak ve miyokardın son zamanlarda belirlenen rejeneratif kapasitesini harekete geçirmek için yeni bir tedavi seçeneği olarak artan bir ilgi kazanmıştır. Deneysel ve klinik araştırmalar çeşitli hücre tiplerinin kalp naklinden sonra elde yararlı etkileri ikna edici bir kanıt sağladı. Önemli sonuçlar geliştirilmiş cardi dahilac kasılma fonksiyonu, sol ventrikül remodeling azaldı infarkt azalır ve infarkt alanında damar yoğunluğu artmıştır. Fakat, hücre enjeksiyonundan sonra düşük hücre sayısı tutma önemli bir dezavantajı kalmıştır. Hücre teslim 2 iyileştirmek için bir alçı ile hücrelerin Derneği son zamanlarda araştırmacı ve klinik çıkarlarını teşvik etmişlerdir. Koroner arter (LAD) sol ön inen ligasyonu transmural infarktüsü ve olgun bir yara sonuçlanan küçük bir hayvan modeli MI için bir referans yöntemidir. MI, kronik fazda uygulanan hücre tedavisi ikinci bir cerrahi müdahale gerektirir. Bir medyan sternotomi genellikle hücrelerin veya biografts bir epikardiyal implantasyon intramiyokardiyal enjeksiyonu sağlamak için gerçekleştirilir. Bu tür invaziv cerrahi işlemler ölüm oranı, ameliyat sonrası iyileşme süresi, ağrı ve enfeksiyon riskini arttırır. Burada sunulan minimal invaziv yaklaşım değil sadece bu önyargı engeller ama aynı zamanda o sağlarTedavi uygulaması için kalbin ptimal erişilebilirlik. MI ve jel tipi biyolojik matris ile ilişkili hücrelerin epikardiyal implantasyon sol interkostal torakotomilerde üzeri kalpte yapılır.

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			<td height="21" style="height:21px;width:216px;">POLYSORB 3-0 suture</td>
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			<td height="21" style="height:21px;width:216px;">POLYSORB 5-0 suture</td>
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			<td height="21" style="height:21px;width:216px;">SURGIPRO II 7-0 Suture</td>
			<td style="width:104px;">COVIDIEN</td>
			<td style="width:119px;">VP904X</td>
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			<td height="21" style="height:21px;width:216px;">CATHETER INSYTE 14G</td>
			<td style="width:104px;">BD</td>
			<td style="width:119px;">381267</td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">INSPIRA ADVANCED VOLUME CONTROLLED VENTILATOR</td>
			<td style="width:104px;">HARVARD APPARATUS</td>
			<td style="width:119px;">557058</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">DUMONT #7 Forceps</td>
			<td style="width:104px;">FST Germany</td>
			<td style="width:119px;">11274-20</td>
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			<td height="21" style="height:21px;width:216px;">Fibrin sealant</td>
			<td style="width:104px;">BAXTER</td>
			<td style="width:119px;">1501441</td>
			<td>TISSEEL Glue, Frozen -20&#176;C</td>
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			<td height="42" style="height:42px;width:216px;">CASTROVIEJO EYE SPECULA</td>
			<td style="width:104px;">HARVARD APPARATUS</td>
			<td style="width:119px;">72-8925</td>
			<td>use as retractor for the ribs</td>
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			<td height="21" style="height:21px;">IMDM Glutamax</td>
			<td>Gibco</td>
			<td>31980</td>
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			<td height="21" style="height:21px;">Pen/Strep</td>
			<td>Gibco</td>
			<td>15140</td>
			<td></td>
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			<td height="21" style="height:21px;">FBS</td>
			<td>PAA Clone</td>
			<td>&#160;&#160;&#160;&#160;&#160; A15-101</td>
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			<td height="42" style="height:42px;">bone scissors</td>
			<td style="width:104px;">Fine Science Tols</td>
			<td style="width:119px;">16044-10</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Red Blood Cell Lysis Solution</td>
			<td>Gentra Systems</td>
			<td>D-50k1</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Accutase cell detachment solution</td>
			<td style="width:104px;">Stem cell technology</td>
			<td style="width:119px;">7920</td>
			<td></td>
		</tr>
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cell-based therapy for heart failure in rat: double thoracotomy for myocardial infarction and epicardial implantation of cells and biomatrix

Kardiyak hücre tedavisi hücreleri ile ilişkili biyomateryallerin ilgi ve implantasyonu artan kazanmış miyokart hücre teslim optimize etmek için önemli bir sorun haline gelmiştir. Miyokard infarktüsü kemirgen modeli (MI) arter (LAD) ligasyonu yaygın bir torakotomi yapılmıştır sol ön inen oluşan; bir sternotomi ile bir ikinci açık kalp cerrahisi, geleneksel olarak tedavi epikardiyal uygulanması için gerçekleştirilmiştir. LAD ligasyon modeli açıklaması yana, ameliyat sonrası ölüm oranı ancak ikinci ameliyat kritik kalmıştır, 35-13% düşmüştür. Ağrı ve ameliyat sonrası enfeksiyon kazanımını arttırmak ve azaltmak için, minimal invaziv cerrahi işlemler sunulmaktadır. İki torakotomi, LAD ligasyon için ilk biri ve tedavi epikardiyal yönetimi için ikinci bir yapıldı. Bir katı ya da jel matrisler özellikleri ile bağlantılı hücrelerden oluşan Biografts enfarktüslü ar üzerine uygulanmıştırad. 2 ve 6 hafta sonra gerçekleştirilmiştir ekokardiyografi ile teyit edildiği gibi, LAD ligasyon kalp fonksiyonlarının kaybı ile sonuçlandı. Goldner kalp bölümlerinde gerçekleştirilen trikrom boyama Transmural skar oluşumunu doğruladı. Birinci ve ikinci ameliyatlar daha az olduğunu,% 10 post-operatif mortalite sonuçlandı.

Tüm hayvanlar torakotomilerde sonra 1 saat içinde iyileşti. Yara iyileşmesi hızlı oldu. Enfeksiyon veya ödemi gözlendi. Çift sol torakotomi kalbe iyi erişim (Şekil 1) izin verdi. Ağrı ve ameliyat sonrası mortalite düşüktür. Hayvan cerrahi hızla iyileşti ve (Şekil 2) ağırlık kazandı. Kaplan Meir sağkalım yüzde ilk torakotomi için% 96 oldu. 104 üzerinde dört sıçan ikinci torakotomi ile 24 saat aşağıdaki tedavi uygul...

Watch this Scientific Journal Video about Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix at JoVE.com

Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix

Bir kemirgen modelinde, LAD ligasyonu ile uyarılmış miyokardiyal enfarktüsü tedavi etmeye yönelik bir biograft implantasyonu, geleneksel olarak iki açık kalp ameliyatı gerektirmiştir. Ölümlerini azaltmak ve hücreleri ile ilişkili solid ve jelatinli biyolojik matrisler tespiti için en uygun koşulları sağlamak amacıyla, minimal invaziv prosedürler geliştirilmiştir.

Rat Kalp Yetmezliği Hücre Terapisi: Miyokard İnfarktüsü için çift Torakotomi ve Hücreleri ile BioMatrix arasında Epikardiyal implantasyonu

Cardiac cell therapy has gained increasing interest and implantation of biomaterials associated with cells has become a major issue to optimize myocardial ...

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Research

JoVE Journal

Bioengineering

11.8K Görüntüleme.  University of Fribourg.  Bir kemirgen modelinde, LAD ligasyonu ile uyarılmış miyokardiyal enfarktüsü tedavi etmeye yönelik bir biograft implantasyonu, geleneksel olarak iki açık kalp ameliyatı gerektirmiştir. Ölümlerini azaltmak ve hücreleri ile ilişkili solid ve jelatinli biyolojik matrisler tespiti için en uygun koşulları sağlamak amacıyla, minimal invaziv prosedürler geliştirilmiştir. 

Video: Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

Engineered Kalp Doku İmplantasyonu takiben İmmün Yanıtların Değerlendirilmesi (EHT) Biyoparlaklık Görüntüleme

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

Biyomühendislik

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Kas Yenilenmesinin Fare Modellerinde İndüklenmiş Pluripotent Kök Hücre türevi Mezoangioblast benzeri Miyojenik Progenitörlerin Nakli

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+ release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.

In cardiac myocytes, tubular membrane structures form intracellular networks. We describe optimized protocols for i) isolation of myocytes from mouse heart including quality control, ii) live cell staining for state-of-the-art fluorescence microscopy, and iii) direct image analysis to quantify the component complexity and the plasticity of intracellular membrane networks.

Kulakçık ve karıncığa gelen kardiyak miyozitlerde Boru Membran Ağlarının Analizi

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

Tohum ve bir biyosentetik doku Mühendisliği trakeal greft bir fare modeli implantasyonu

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

Keratinosit yayılması üzerine iki ve üç boyutlu değerlendirilmesi tip ı kollajen yüzeylerde

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has been challenged. Here, we fabricate iron oxide nanoparticle shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform. Iron oxide nanoparticles serve as both magnetic and photothermal agents. Once intravenously injected, NSMs can be magnetically guided to the tumor site. Ultrasound triggers the release of iron oxide nanoparticles, facilitating the penetration of nanoparticles deep into the tumor due to the cavitation effect of microbubbles. Thereafter, magnetic hyperthermia and photothermal therapy can be performed on the tumor for combinational cancer therapy, a solution for cancer resistance due to the tumor heterogeneity. In this protocol, the synthesis and characterization of NSMs including structural, chemical, magnetic and acoustic properties were performed. In addition, the anti-cancer efficacy by thermal therapy was investigated using in vitro cell cultures. The proposed delivery strategy and combination therapy holds great promise in cancer treatment to improve both delivery and anticancer efficacies.

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

Manyetik Hipertermi ve Pothototermal Kombinasyon Kanser Tedavisi için Manyetik, Akustik ve Optik-Üçlü Duyarlı Mikrobubbles

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has ...

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

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from human induced pluripotent stem cells (hiPSCs), which are developed towards the goal of clinical use. HiPSC-derived cardiomyocytes, endothelial cells, and vascular mural cells are mixed with gel matrix and then poured into a polydimethylsiloxane (PDMS) tissue mold with rectangular internal staggered posts. By culture day 14 ECTs mature into a 1.5 cm x 1.5 cm mesh structure with 0.5 mm diameter myofiber bundles. Cardiomyocytes align to the long-axis of each bundle and spontaneously beat synchronously. This approach can be scaled up to a larger (3.0 cm x 3.0 cm) mesh ECT while preserving construct maturation and function. Thus, mesh-shaped ECTs generated from hiPSC-derived cardiac cells may be feasible for cardiac regeneration paradigms.

The present protocol generates mesh-shaped engineered cardiac tissues containing cardiovascular cells derived from human induced pluripotent stem cells to allow the investigation of cell implantation therapy for heart diseases.

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 ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

Miyokard Enfarktüsü'nün Murine Modelinde 3Boyutlu Biyobaskılı Yama Transplantasyonu

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

Quantification of Mouse Heart Left Ventricular Function, Myocardial Strain, and Hemodynamic Forces by Cardiovascular Magnetic Resonance Imaging

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations result in pathology, and how myocardial diseases may be treated. Cardiovascular magnetic resonance imaging (CMR) has become an indispensable tool for a comprehensive in vivo assessment of cardiac anatomy and function. This protocol shows detailed measurements of mouse heart left ventricular function, myocardial strain, and hemodynamic forces using 7-Tesla CMR. First, animal preparation and positioning in the scanner are demonstrated. Survey scans are performed for planning imaging slices in various short- and long-axis views. A series of prospective ECG-triggered short-axis (SA) movies (or CINE images) are acquired covering the heart from apex to base, capturing end-systolic and end-diastolic phases. Subsequently, single-slice, retrospectively gated CINE images are acquired in a midventricular SA view, and in 2-, 3-, and 4-chamber views, to be reconstructed into high-temporal resolution CINE images using custom-built and open-source software. CINE images are subsequently analyzed using dedicated CMR image analysis software.
Delineating endomyocardial and epicardial borders in SA end-systolic and end-diastolic CINE images allows for the calculation of end-systolic and end-diastolic volumes, ejection fraction, and cardiac output. The midventricular SA CINE images are delineated for all cardiac time frames to extract a detailed volume-time curve. Its time derivative allows for the calculation of the diastolic function as the ratio of the early filling and atrial contraction waves. Finally, left ventricular endocardial walls in the 2-, 3-, and 4-chamber views are delineated using feature-tracking, from which longitudinal myocardial strain parameters and left ventricular hemodynamic forces are calculated. In conclusion, this protocol provides detailed in vivo quantification of the mouse cardiac parameters, which can be used to study temporal alterations in cardiac function in various mouse models of heart disease.

This study describes a comprehensive cardiovascular magnetic resonance imaging (CMR) protocol to quantify the left ventricular functional parameters of the mouse heart. The protocol describes the acquisition, post-processing, and analysis of the CMR images as well as assessment of different cardiac functional parameters.

Kardiyovasküler Manyetik Rezonans Görüntüleme ile Fare Kalbi Sol Ventrikül fonksiyonu, Miyokard Zorlanması ve Hemodinamik Kuvvetlerin Nicelemesi

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations ...

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format.
The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Here, we demonstrate a non-invasive cardiac medical device contractility evaluation method using 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, plated on a flexible substrate, coupled with video-based microscopy. This tool will be useful for the in vitro evaluation of the contractile properties of cardiac electrophysiology devices.

2D İnsan Kök Hücre Kaynaklı Kardiyomiyositlerde Kardiyak Kontraktilite Modülasyon Tedavisinin Değerlendirilmesi

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used ...