Yazarlar, aşağıdaki finansman kaynaklarını kabul etmektedir: Kardiyovasküler Araştırmalar İçin Önemli olan Sihir ve Maryland Kök Hücre Araştırma Fonu (2016-MSCRFI-2735).

1. Kardiyomiyositlerin hazırlanması <ol><li> Tanımlanan temel zar matrisi ile kaplanmış 6 yuvalı plakalar üzerinde insan kaynaklı pluripotent kök hücreleri (hiPSC&#39;ler) üretin ve kültürleyin. </li><li> Daha önce tarif edilen yöntemler 11 , 12 kullanarak hiPSC&#39;leri hiPSC türevi kardiyomiyositlere (hiPSC-CM&#39;ler) ayırt edin. </li><li> Farklılaştıktan 19 gün sonra, oda sıcaklığında 5 dakika süreyle her oyuğa 2 mL Tripsin / EDTA% 0.05&#39;lik kardiyomiyosit sostantivoek izole edin. </li><li> Hücrenin ayrışmasını izlemek için kardiyomiyositleri optik bir mikroskop altında izleyin. </li><li> 2 mL Tripsin inhibitörü 0.0125% kullanarak tripsin nötralize edin. </li><li> Izole edilmiş kardiyomiyositleri boşaltın ve bir motorize pipet dolgu kullanarak bir 50 mL konik tüp içine aktarın. </li><li> Bir pellet elde etmek için hücre süspansiyonunu oda sıcaklığında 250 xg&#39;de 5 dakika santrifüjleyin. </li><li> Pelleti 5 mL Roswell Park&#39;a tekrar süspanse edinB-27 (RPMI / B-27 hücre ortamı) ile takviye edilmiş Memorial Institute (RPMI) hücre ortamı. </li><li> Hücre ortamındaki hücrelerin 20 μL&#39;sini pipetleyin ve eşit miktarda% 0.4 Trypan Mavi çözeltiyle leke uygulayın. </li><li> Hücre süspansiyonunun konsantrasyonunu ve hücre canlılığını saymak ve elde etmek için otomatik bir hücre sayacı veya manuel hemositometre kullanın. </li></ol> 2. Fibroblastların Hazırlanması <ol><li> İnsan kalp fibroblast (HCF) (yetişkin ventriküler tip) hücre hattını 13 başlatınız. </li><li> HCF kültürü protokollerine uygun olarak geçiş yapın ve izole edin 13 . </li><li> Hücre ortamındaki hücrelerin 20 μL&#39;sini pipetleyin ve Trypan Mavisi çözeltisinin% 0.4&#39;lük eşit miktarda lekelenmesini sağlayın. </li><li> Yeni hücre süspansiyonunun konsantrasyonunu ve hücre yaşayabilirliğini saymak ve elde etmek için otomatik bir hücre sayacı veya manuel hemositometre kullanın. </li></ol> 3. Endotel Hücrelerinin Hazırlanması <ol><li> Insan umn başlatmakAçıklandığı gibi ilik ven endotel hücresi (HUVEC) hattı. Geçiş yapın ve tarif edildiği gibi HUVEC kültürleme protokollerine uygun olarak izole edin. </li><li> Hücre ortamındaki hücrelerin 20 μL&#39;sini pipetleyin ve eşit miktarda% 0.4 Trypan Mavi çözeltiyle leke uygulayın. </li><li> Hücre süspansiyonunun konsantrasyonunu ve hücre canlılığını saymak ve elde etmek için otomatik bir hücre sayacı veya manuel hemositometre kullanın. </li></ol> 4. Ortak kültür: <ol><li> Bir hiPSC-CM&#39;de 50 mL&#39;lik bir konik tüp içinde karışık hücre çözeltisi stoğu oluşturmak için RPMI / B-27 hücre ortamında üç hücre tipini (hiPSC-CM, HCFler ve HUVEC&#39;ler) karıştırın: HCF: HUVEC hücre oranı 70:15:15 ve mL başına 165.000 hücre konsantrasyonu. </li><li> Karışık hücre çözeltisini, çok kanallı bir pipet kullanarak, oyuk başına 200 μL ya da oyuk başına 33.000 hücre ile ultra-düşük ek 96-iyi U-alt plakalara dağıtın. </li><li> 96 oyuklu plakaları 3 gün inkübe edin (37 ° C,% 5 karbon dioksIde,% 95 nem). </li><li> Işık mikroskopisi altında bireysel kuyuların merkezinde ve altındaki karışık hücre çok hücreli sferoitlerin varlığını kontrol edin. NOT: İki boyutlu bir mikroskobik projeksiyonda, bu sfero şekli dairesel görünür ( Şekil 1 ). Sferoitler, ultra-düşük bağlanma 96 delikli plakalara hücre tohumlamasından sonra 24 saat içinde oluşmalıdır. Sferoidler, 96 gözlü plakalara hücre tohumlamasından sonra 48 saat içinde atmaya başlamalıdır. Dövülmeyen sferoitler, son 3D biyoprintili kalp yamasının işlevsel olmasını sağlamak için 3D biyoprinting için kullanılmamalıdır. Connexin 43 (Cx43) ve troponin için spermlerin immünfloresansı, sfero hücresel bileşimi ve kalitesini değerlendirmek için kullanılabilir 15 . </li></ol> 5. İskele-Özlü Kardiyak Dokuların 3D Biyoprinti <ol><li> Sferoidleri içeren plakları, iskelesiz sferoid tabanlı 3D bioprinter dergilerine yerleştirin. </li><li> 3D bioprinter&#39;ı açın ve 3D bioprinting yazılımını çalıştırın. <ol><li> &quot;Auxillary&quot;, &quot;Operation Preparation&quot;, &quot;Inspect Mode&quot; ve &quot;Home Return&quot; tuşlarına tıklayın. </li><li> Sfero kontrolü başlatmak için &quot;Başla&quot; altındaki yeşil simgeyi tıklayın. </li><li> Ekrandaki &quot;(2) Dia (μm)&quot; sütununun altında sferoid çaplarının görünmesini bekleyin. </li></ol></li><li> Ortalama sfero çapını en yakın 100 μm&#39;ye çıkarmak için &quot;Başlangıç ​​ayarları&quot; nı tıklayın ve yığın Z aralığına (μm) ortalama küresel dönüş çapını girin. </li><li> &quot;Toplam&quot; ı tıklayın, &quot;Katmanlar&quot; altındaki sayı otomatik olarak yeniden ayarlanmalıdır. Ayarları onaylamak için &quot;Uygula&quot; yı tıklayın. </li><li> &quot;İğne Dizisi Haritası&quot; nı tıklayın, &quot;Katman No&quot; na gidin, 3B biyoprint için istenen katmanı seçin ve bireysel küre biçimlerini seçerek ekranın solundaki haritada harita üzerinde istediğiniz 3D biyoprint tasarımını çizin. ClicK &quot;3D bioprinting tasarımını onaylamak için&quot; Uygula &quot;yı tıklayın. </li><li> İğne dizisi incelemesine başlamak için &quot;İğne Dizi Kontrolü&quot; nü tıklayın. İğne dizisi odak dışındaysa, kamera odağı ayarlamak için 0 ila 500 μm arasında değişen değerleri &quot;OffsetZ (μm)&quot; içine girin, 500 μm&#39;den başlayan ve 100 μm ila 0 μm&#39;lik bir azalma. </li><li> &quot;Muayene Parametreleri&quot;, &quot;Tip 1&quot; i tıklayın ve &quot;Çap (μm)&quot; değerini istediğiniz çap aralığına ayarlayın. &quot;Yuvarlaklık (%)&quot; ve &quot;Düzgünlük (%)&quot; değerlerini istediğiniz yüzdelere ayarlayın. Ayarları onaylamak için &quot;Uygula&quot; yı tıklayın. NOT: Burada sfero çapları 450 μm ila 550 μm arasında ayarlanmıştır. &quot;Yuvarlaklık (%)&quot; ve &quot;Düzgünlük (%)&quot;, her bir kürecik için bilgisayar tarafından hesaplanan değerlendirmelerdir. Burada &quot;Yuvarlaklık (%)&quot; için kullanılan ayarlar% 60 ve &quot;Düzgünlük (%)&quot; için% 70&#39;tir. </li><li> &quot;Durum&quot; u tıklayın ve &quot;Çap (μm)&quot; değerini aynı ayara güncelleyinS önceki adımda. Ayarları onaylamak için &quot;Uygula&quot; yı tıklayın. </li><li> 3D biyoprinterin memesini vakumla emerek ve bir iğne dizisindeki belirli yerlere sferoid yerleştirerek sfero başlamak için &quot;Yığın Modu&quot; nu ve ardından &quot;Başlat&quot; altındaki yeşil simgeyi tıklayın. NOT: 81 sfero (9 küremsi x 9 küremsi) içeren bir kalp yaması için, 3B biyoprodizyonu yaklaşık 20 ila 30 dakika sürmelidir. </li><li> Biyoprintten sonra, steril forseps kullanarak, 3 boyutlu biyoprote edilmiş yama içeren iğne dizisini topla ve 150 mL RPMI / B-27 hücre ortamı içeren 250 mL steril behergreye aktar. </li><li> 3D biyoprote edilmiş yamayı iğne dizisi ile 3 gün inkübe edin (37 ° C,% 5 karbon dioksit,% 95 nem). </li></ol> 6. İğne Dizi ve Doku Matürasyonundan 3D Biyopetilen Düzeltme Bırakma <ol><li> Üç gün bekletildikten sonra bir eliniz iğne dizisinin tabanında, diğeri plastik kapakta tYama, ipliğin dizisinden 3D biyoprote edilmiş yamayı çıkarmak için plastik kapağı hafifçe kaydırın. NOT: Fosfat tamponlu tuzlu su (PBS) ile yağlama işlemi, bu adımdan hemen önce iğnelere gerektiğinde uygulanabilir ve daha düzgün ve kolay bir şekilde çıkarılabilir. </li><li> Bir çift steril forseps kullanarak, üzerine 3D biyoprote edilmiş yama ile plastik kapağı kaldırın ve yama ve plastik kapağı RPMI / B-27 hücre ortamı içeren 35 mm çanağa aktarın. </li><li> Yamayı medyaya bırakmak için forseps hala plastik kapağı tutarak, RPMI / B-27 hücre medyasındaki yama ile plastik kapağı hafifçe sallayın. </li><li> 3D biyopetilen yamanın işlevsel olduğundan emin olmak için dayak mı edip etmediğini görselleştirmek için ışık mikroskopisi altında 3D biyoprote edilmiş yamayı inceleyin. </li><li> 3 boyutlu biyoprote edilmiş yamayı 3 gün veya daha uzun süre 35 mm çanağında inkübe edin (37 ° C,% 5 karbon dioksit,% 95 nem). NOT: 3 gün sonra sferoidler birbirine kaynaşır ve içindeki deliklerIğnelerin bulunduğu yama artık görünür olmamalıdır. </li></ol>

<ol>
	<li>Murphy, S. V., Atala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+bioprinting+of+tissues+and+organs.">3D bioprinting of tissues and organs.</a> Nat Biotech. 32, 773-785 (2014).</li><li>Bajaj, P., Schweller, R. M., Khademhosseini, A., West, J. L., Bashir, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+biofabrication+strategies+for+tissue+engineering+and+regenerative+medicine.">3D biofabrication strategies for tissue engineering and regenerative medicine.</a> Ann Rev Biomed Eng. 16, 247-276 (2014).</li><li>Patra, S., Young, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Review+of+3D+Printing+Techniques+and+the+Future+in+Biofabrication+of+Bioprinted+Tissue.">A Review of 3D Printing Techniques and the Future in Biofabrication of Bioprinted Tissue.</a> Cell Biochem Biophy. 74, 93-98 (2016).</li><li>Moldovan, N. I., Hibino, N., Nakayama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+the+Kenzan+Method+for+Robotic+Cell+Spheroid-Based+Three-Dimensional+Bioprinting.">Principles of the Kenzan Method for Robotic Cell Spheroid-Based Three-Dimensional Bioprinting.</a> Tissue Eng Part B Rev. , (2017).</li><li>Cui, X., Boland, T., D'Lima, D. D., Lotz, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+inkjet+printing+in+tissue+engineering+and+regenerative+medicine.">Thermal inkjet printing in tissue engineering and regenerative medicine.</a> Recent Pat Drug Deliv Formul. 6, 149-155 (2012).</li><li>Murphy, C. M., Haugh, M. G., O'Brien, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+mean+pore+size+on+cell+attachment,+proliferation+and+migration+in+collagen-glycosaminoglycan+scaffolds+for+bone+tissue+engineering.">The effect of mean pore size on cell attachment, proliferation and migration in collagen-glycosaminoglycan scaffolds for bone tissue engineering.</a> Biomater. 31, 461-466 (2010).</li><li>Noguchi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+three-dimensional+pre-vascularized+scaffold-free+contractile+cardiac+patch+for+treating+heart+disease.">Development of a three-dimensional pre-vascularized scaffold-free contractile cardiac patch for treating heart disease.</a> J Heart Lung Transpl. 35, 137-145 (2016).</li><li>Mironov, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+printing:+tissue+spheroids+as+building+blocks.">Organ printing: tissue spheroids as building blocks.</a> Biomater. 30, 2164-2174 (2009).</li><li>Itoh, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffold-Free+Tubular+Tissues+Created+by+a+Bio-3D+Printer+Undergo+Remodeling+and+Endothelialization+when+Implanted+in+Rat+Aortae.">Scaffold-Free Tubular Tissues Created by a Bio-3D Printer Undergo Remodeling and Endothelialization when Implanted in Rat Aortae.</a> PloS One. 10, e0136681 (2015).</li><li>Lieu, P. T., Fontes, A., Vemuri, M. C., Macarthur, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+induced+pluripotent+stem+cells+with+CytoTune,+a+non-integrating+Sendai+virus.">Generation of induced pluripotent stem cells with CytoTune, a non-integrating Sendai virus.</a> Methods Mol Biol. 997, 45-56 (2013).</li><li>Li, S., Cheng, H., Tomaselli, G. F., Li, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+basis+of+excitation-contraction+coupling+in+human+pluripotent+stem+cell-derived+ventricular+cardiomyocytes+revealed+by+Ca2++spark+characteristics:+direct+evidence+of+functional+Ca2+-induced+Ca2++release.">Mechanistic basis of excitation-contraction coupling in human pluripotent stem cell-derived ventricular cardiomyocytes revealed by Ca2+ spark characteristics: direct evidence of functional Ca2+-induced Ca2+ release.</a> Heart Rhythm. 11, 133-140 (2014).</li><li>Boheler, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Human+Pluripotent+Stem+Cell+Surface+N-Glycoproteome+Resource+Reveals+Markers,+Extracellular+Epitopes,+and+Drug+Targets.">A Human Pluripotent Stem Cell Surface N-Glycoproteome Resource Reveals Markers, Extracellular Epitopes, and Drug Targets.</a> Stem Cell Rep. 3, 185-203 (2014).</li><li>ScienCell Research Laboratories. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human Cardiac Fibroblasts (adult ventricular) Product Sheet. , (2017).</li><li>Lonza Walkersville, Inc. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Endothelial Cell Systems - Technical Information &amp; Instructions.,. , (2015).</li><li>Thermo Fisher Scientific, Inc. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Immunofluorescence Method for IHC Detection. , (2017).</li><li>Murata, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+preliminary+study+of+osteochondral+regeneration+using+a+scaffold-free+three-dimensional+construct+of+porcine+adipose+tissue-derived+mesenchymal+stem+cells.">A preliminary study of osteochondral regeneration using a scaffold-free three-dimensional construct of porcine adipose tissue-derived mesenchymal stem cells.</a> J Orthop Surg Res. 10, (2015).</li><li>Mosadegh, B., Xiong, G., Dunham, S., Min, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+progress+in+3D+printing+for+cardiovascular+tissue+engineering.">Current progress in 3D printing for cardiovascular tissue engineering.</a> Biomed Mater. 10, 034002 (2015).</li><li>Chang, C. C., Boland, E. D., Williams, S. K., Hoying, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct-write+Bioprinting+Three-Dimensional+Biohybrid+Systems+for+Future+Regenerative+Therapies.">Direct-write Bioprinting Three-Dimensional Biohybrid Systems for Future Regenerative Therapies.</a> J Biomed Mater Res. Part B, Appl Biomater. 98, 160-170 (2011).</li><li>Lee, J. M., Sing, S. L., Tan, E. Y. S., Yeong, W. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioprinting+in+cardiovascular+tissue+engineering:+a+review.">Bioprinting in cardiovascular tissue engineering: a review.</a> International J Bioprinting. 2 (2016), (2016).</li></ol>

Yazarların açıklayacak bir şeyi yok.

It is important to use beating, functional spheroids for 3D bioprinting. If spheroids are not beating, continuing to use them will invariably result in a non-functional 3D bioprinted patch.
One benefit of this approach is the ability to manipulate the cell content of the patch by varying the total number of cells and the percentage of cardiomyocytes, endothelial cells, and fibroblasts in the spheroids. This allows for many different types of cardiac patches to be printed, with varying histological and mechanical properties.
This approach also allows different types of spheroids to be used to make a patch, as well as the 3D bioprinting of cardiac patches with complex 3D designs. This complexity of the final 3D construct is achievable by 3D bioprinting with precise spheroid positioning, but may be difficult to achieve by gentle rotation of floating spheroids7 or simple molding of spheroids into a cylindrical 3D construct16.
The limitations of this approach are the relatively fixed size of the spheroids between 450 to 550 &#181;m due to the fixed distance of 400 &#181;m between the needles in the needle array, as well as possibly reduced cell viability if the spheroids are too large in diameter. The patches are mechanically fragile immediately after removal from the need array; however, the strength and durability are improved with culture for longer durations of time.
Finally, 3D bioprinting of cardiac patches has promising clinical applications17, in cardiac regeneration18,19 and as 3D models of heart disease. These 3D models can be used for applications in predictive pharmacology/toxicology and development of cell and genome directed therapies. 
In conclusion, it is feasible to 3D bioprint cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids. 3D bioprinting of cardiac tissue has promising clinical applications in cardiac tissue regeneration and as 3D models of heart disease.

3D biyoprintleme için birçok farklı yöntem vardır 1 , 2 , 3 . 3D biyoprinting, mürekkep püskürtmeli biyoprint, mikro ekstrüzyon biyoprint, lazer yardımlı biyoprint, metotların bir kombinasyonu veya daha yeni yaklaşımlar gibi örneklerle baskı teknolojisi 1 ile sık sık sınıflandırılır. 3D biyoprinting, iskeleye bağlı olmayan veya iskeleye bağlı yöntemler olarak sınıflandırılabilir. 3D biyoprintinin çoğu metodu, biyo-materyallere, örn., Biyometrik bağlantılar 5 veya iskele 6&#39;ya ihtiyaç duyulduğunda, iskeleye bağlıdır. Bununla birlikte, iskele bağımlı 3D biyoprinting, iskele materyalinin immünojenisitesi, patentli bioink maliyeti, bozunma ürünlerinin yavaş hızı ve toksisitesi gibi birçok konuyu ve sınırlamaları 4 , 7 ile karşı karşıya bırakıyor. ScafKüresel olmayan kardiyak doku mühendisliği, iskeleye bağlı doku mühendisliğinin bu dezavantajlarının üstesinden gelebilecek potansiyele sahip 8 olarak denenmiştir. Bununla birlikte, yazarların bu makalede belirttiği gibi, biofabrikasyon sürecinde sferoidleri sabit yerlerde sağlam bir şekilde tutmak ve konumlandırmak zordu. 3D biyoprint ve sferoya dayalı doku mühendisliğinin birlikte kullanımı bu zorlukların üstesinden gelme potansiyeline sahiptir. Bu protokolde, yalnızca sfero formundaki hücreleri kullanarak, diğer biyomalzemeler olmadan kardiyak dokunun 3D biyoprintini açıklıyoruz. İskeletsiz sferoid tabanlı 3B biyoeneratörler 9 , vakumlu emici kullanarak tekli sferoidleri alıp iğne dizisine yerleştirme özelliğine sahiptir. 3D biyoprintide bir iğne dizisine küre biçimlendirme konsepti , eski Japonya&#39;da iğne dizilerinin (&quot; kenzan &quot; olarak bilinir) kullanılmasından esinlenmiştirÇiçek düzenleme düzenlemeleri, ikebana. Bu sistem, sferoidlerin herhangi bir konfigürasyonda tam olarak konumlandırılmasını sağlar ve bireysel sferoitler, bir 3D biyoprote edilmiş doku oluşturmak için kısa bir süre boyunca kaynaşarak sonuçlanır. Bu yöntem, bu sayede, sferoidlerin kolaylıkla manipüle edilmesine ve iskelesiz organ biyofabrasyonunun geleceği için potansiyel etkilere sahip olmasına izin verir.

<table border="0" cellpadding="0" cellspacing="0" width="587">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">Geltrex</td>
			<td style="width:157px;">Invitrogen&#160;</td>
			<td style="width:88px;">A1413202</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">Trypsin/EDTA 0.05%</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">15400054</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">Defined Trypsin inhibitor 0.0125%</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">R007100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:176px;">RPMI Cell Media</td>
			<td style="width:157px;">Invitrogen</td>
			<td style="width:88px;">11875-093</td>
			<td style="width:167px;">RPMI supplemented with B27 constitutes HIPSC-CM culture media</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:176px;">B-27 Supplement</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">17504044</td>
			<td style="width:167px;">RPMI supplemented with B27 constitutes HIPSC-CM culture media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">Countess Automated Cell Counter</td>
			<td style="width:157px;">Invitrogen</td>
			<td style="width:88px;">C10227</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:176px;">Human cardiac fibroblasts (adult ventricular type)</td>
			<td style="width:157px;">Sciencell</td>
			<td style="width:88px;">6310</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">Human umbilical vein endothelial cells</td>
			<td style="width:157px;">Lonza</td>
			<td style="width:88px;">CC-2935</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:176px;">PrimeSurface ultra-low attachment 96-well U-bottom plates&#160;</td>
			<td style="width:157px;">Akita Sumitomo Bakelite Co.</td>
			<td style="width:88px;">MS-9096UZ</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">Regenova Bio 3D Printer</td>
			<td style="width:157px;">Cyfuse Biomedical K.K.</td>
			<td style="width:88px;">N/A</td>
			<td>www.cyfusebio.com/en/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">15250061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">Troponin T Antibody</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">701620</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">Connexin 43 (Cx43) Antibody</td>
			<td style="width:157px;">Chemicon</td>
			<td>MAB3068</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">ProLong Gold Antifade Mountant with DAPI</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">P36935</td>
			<td></td>
		</tr>
	</tbody>
</table>

creation of cardiac tissue exhibiting mechanical integration of spheroids using 3d bioprinting

Bu protokol, sadece hücreler kullanılarak, biyomalzemeler kullanılmadan kalp dokusunun 3D biyoprintini açıklamaktadır. Kardiyomiyositler, endotel hücreleri ve fibroblastlar önce izole edilir, sayılır ve istenen hücre oranlarında karıştırılır. Bunlar, ultra-düşük ek 96 delikli plakalar içinde bireysel kuyularda birlikte kültürlenir. 3 gün içinde sopoidler atılır. Bu sfero daha sonra vakum emiş kullanarak bir meme tarafından toplanır ve bir 3D bioprinter kullanarak bir iğne dizisi üzerine monte edilir. Daha sonra sferoidlerin iğne dizisi üzerinde sigortalanmasına izin verilir. 3D biyoprintten üç gün sonra, sferoidler, bozulmamış bir yama olarak çıkarılır; bu zaten kendiliğinden dayaktır. 3D biyoprote edilmiş kalp yamaları bileşen küremsiklerin mekanik entegrasyonunu sergiler ve kalp dokusunun yenilenmesinde ve kalp hastalığının 3D modellerinde son derece umut vericidir.

Adım 4.4&#39;ün (ortak kültür) sonunda, yerçekimi ile küre biçimlendirmek için her kuyucuktaki hücreler ultra-düşük ek 96-kuyucuk U-alt plakalarının tabanında birikmelidir. Bu sferoitler, hiPSC-CM, HCF&#39;ler ve HUVEC&#39;leri içerir ve iki boyutlu projeksiyon ile dairesel görünmeleri gereken ışık mikroskopisi altında görsel olarak incelenebilir ( Şekil 1 ). 6.3. Adımın sonunda, 3D biyoprote edilmiş kardiyak yama, iğne dizisi tarafından oluşturulan iğne deliklerine bağlı olarak doku boşluklarını içermelidir ( Şekil 2 , solda). Bu noktada, sferoitler arasındaki sınırlar belirsizleşmeli ve yamaların mekanik bütünleşmesi sergileyerek kendiliğinden atılmaya başlamış olması gerekirdi. 6. adımın sonunda doku boşlukları çevreleyen doku tarafından doldurulmalıdır ( Şekil 2 , sağ), 3D biyoprote edilmiş kalp yaması bea devam etmelidirKendiliğinden. <img alt="Şekil 1" class="xfigimg" src="/files/ftp_upload/55438/55438fig1.jpg" /> Şekil 1 : Karışık hücre çok hücreli sferoidlerin oluşturulması. HiPSC-CM, HCFler ve HUVEC&#39;ler, 70:15:15 hiPSC-CM: HCF: HUVEC hücre oranı ile karıştırıldı ve hücre karışımı, ultra-düşük ekli 96 gözlü U-alt plakalara dağıtıldı. 24 saat içinde, karışık hücre çok hücreli sferoitler (sol, sağ) her kuyuda bir tane meydana geldi. Ölçek çubuğu = 500 μm. <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55438/55438fig1large.jpg" target="_blank">Bu rakamın daha büyük bir versiyonunu görmek için lütfen tıklayınız.</a> <img alt="şekil 2" class="xfigimg" src="/files/ftp_upload/55438/55438fig2.jpg" /> Şekil 2 : Sferoidlerin mekanik bütünleşmesini gösteren kalp dokusunun oluşturulması . 3D biyoGörülebilir iğne delikleri olan iğne dizisinden çıkarıldıktan hemen sonra, basılı kalp pili (solda). Şu anda küremsiler arasındaki sınırlar belirsizleşti ve yama kendiliğinden dayanıyordu, bu nedenle küremsikler mekanik olarak bütünleşmişti. İğne dizisinden çıkarıldıktan üç gün sonra, iğne dizisinden kaynaklanan doku boşlukları çevreleyen doku (sağda) ile dolduruldu ve yama spontan atmaya devam etti. Ölçek çubuğu = 1 mm. <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55438/55438fig2large.jpg" target="_blank">Bu rakamın daha büyük bir versiyonunu görmek için lütfen tıklayınız.</a>

Watch this Scientific Journal Video about Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting at JoVE.com

Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

Bu protokol, biyomalzemeler kullanılmadan kalp dokusunun 3D biyoprintini açıklamaktadır. 3D biyoprote edilmiş kalp yamaları bileşen küremsiklerin mekanik entegrasyonunu sergiler ve kalp dokusunun yenilenmesinde ve kalp hastalığının 3D modellerinde son derece umut vericidir.

3D Bioprinting&#39;i Kullanan Sferoidlerin Mekanik Entegrasyonu Sergileyen Kalp Doku Oluşturma

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and ...

creation-cardiac-tissue-exhibiting-mechanical-integration-spheroids

Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.9K Görüntüleme.  Johns Hopkins Hospital.  Bu protokol, biyomalzemeler kullanılmadan kalp dokusunun 3D biyoprintini açıklamaktadır. 3D biyoprote edilmiş kalp yamaları bileşen küremsiklerin mekanik entegrasyonunu sergiler ve kalp dokusunun yenilenmesinde ve kalp hastalığının 3D modellerinde son derece umut vericidir. 

Video: Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

Plazma Litografi Yüzey Desenlendirme Hücre Ağları Oluşturma

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

Biyomühendislik

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

Bioprinting cellularized dokuya özel Hidrojel Bioink kullanılarak dahil edilmesi

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting kıkırdak ve cilt doku analogları bir roman pasif birimi teknik Bioink Precellularization için karıştırma kullanmak

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

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

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

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. ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

Jelatin Metacryloyl Hidrojel Bazlı Bioinks 3D Biyobaskı Protokolleri

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by shear stress and improve the survival of the encapsulated cells. However, rapid gravity-driven cell sedimentation in the reservoir could lead to an inhomogeneous cell distribution in bioprinted structures and therefore hinder the application of liquid-like bioinks. Here, we developed a novel multilayered modified strategy for liquid-like bioinks (e.g., gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells. Multiple liquid interfaces were manipulated in the multilayered bioink to provide interfacial retention. Consequently, the cell sedimentation action going across adjacent layers in the multilayered system was retarded in the bioink reservoir. It was found that the interfacial retention was much higher than the sedimental pull of cells, demonstrating a critical role of the interfacial retention in preventing cell sedimentation and promoting a more homogeneous dispersion of cells in the multilayered bioink.

Here, we developed a novel multilayered modified strategy for liquid-like bioinks (gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells.

Homojen Hücre Dağılımı için Üç Boyutlu Biyobaskıda Çok Katmanlı Hidrojel Bioink'in Kullanılması

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by ...

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue architecture. In current layer-by-layer extrusion bioprinting, individual cells are extruded in a bioink together with complex spatial and temporal cues to promote hierarchical tissue self-assembly. However, this biofabrication technique relies on complex interactions among cells, bioinks and biochemical and biophysical cues. Thus, self-assembly may take days or even weeks, may require specific bioinks, and may not always occur when there is more than one cell type involved. We therefore developed a technique to directly bioprint pre-formed 3D breast epithelial spheroids in a variety of bioinks. Bioprinted pre-formed 3D breast epithelial spheroids sustained their viability and polarized architecture after printing. We additionally printed the 3D spheroids onto vascular endothelial cell networks to create a co-culture model. Thus, the novel bioprinting technique rapidly creates a more physiologically relevant 3D human breast model at lower cost and with higher flexibility than traditional bioprinting techniques. This versatile bioprinting technique can be extrapolated to create 3D models of other tissues in additional bioinks.

The goal of this protocol is to directly bioprint breast epithelial cells as multicellular spheroids onto pre-formed endothelial networks to rapidly create 3D breast-endothelial co-culture models which can be used for drug screening studies.

3D Çok Hücreli Meme Sferoidlerinin Endotel Ağlarına Doğrudan Biyobaskı

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue ...

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

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

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 ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

Mikroakışkan Bir Platformda 3D Organize İnsan Kardiyak Dokusu Geliştirme

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

Mühendislik Nöral Benzeri Dokular için Murine Kortikal Astrositlerin 3D Biyobaskı

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...