Yazarlar kültürleme Şekil 2&#39;de sunulmuştur dokular için Yabin Wu teşekkür etmek istiyorum, resmi Bart van Overbeeke tarafından çekildi. Iş mali SenterNovem, hibe ISO 42022 tarafından desteklenmiştir.

1. Mürin myoblast Progenitör Hücreleri veya C2C12 Hücre Kültürü <ol><li> Başlangıçta Shefer ve arkadaşları 10 tarafından yayımlanan ve Collins et al. 11 ve Boonen ve ark. 12 göre adapte protokolüne göre hücreler izole ve sıvı azot içinde bu saklayın. Bu fareler, örneğin, C57BL / 6 gerektirir. Alternatif yöntemler örneğin Li, Y ve arkadaşları ile görselleştirilmiş Deney yayınlanan bir yöntemi, diğer laboratuarlarda kullanılmaktadır. 13. Reaktifler ve cihazlar için, sayfa 7 ve 8&#39;de verilen tabloda verilir. Tek bir fare kas itibaren genellikle sıvı azot içinde yaklaşık 20 şişeleri cryopreserve yeterli hücre elde edecektir. Sonra, sıvı azot depolama hücrelerinin çözülme için protokol başlar. </li><li> 25 cm 2 Matrigel (1 mg / ml) kaplanmış doku kültürü şişesi içine 1 şişeden hücrelerin yerleştirin ve oluşan büyüme ortamı (GM) (335 ml DMEM gelişmiş, 100 ml cenin bovin eklemeke serum (FBS), 50 ml HS, 5 ml penisilin / streptomisin, 5 ml L-glutamin, 5 ml tavuk embriyo özü (OA)). </li></ol> Not: Oda sıcaklığı az 2 saat süreyle ceket ve aspirasyon ile Matrigel çıkarın. <ol start="3"><li> Hücrelerin yaklaşık olarak 3 gün boyunca her 01:03 alt kültür. Bu şu anlama gelir: 3. gün: biri 75 cm 2 balon iki 150 cm 2 matara (kaplamasız şişeler preplating 30 dk) geçişi hücreleri: günde 6, 75 cm 2 balon bir 25 cm 2, gelen Passage hücreler. Günde 9 tek transfer 150 cm 2 balon üç 150 cm 2 matara (gerekirse preplate tekrar hücrelerin fenotipi bağlı olarak) kullanılamayabilir 1 üçlü 150 cm 2 balon veya eğer. Günde 12 hücreleri kas yapı içine tohum hazır bulunmaktadır. </li></ol> Notlar: - Üçlü 150 cm 2 başına hücre sayısı yaklaşık 4,5 x 10 6 hücre olacak. - Via kullanınfarklı izolasyonların gelen ls ekimi sırasında hücre karışık bir nüfus elde etmek karıştırın. Not: birincil piller ile çalışmak istemezseniz, C2C12 miyoblastları iyi bir alternatiftir. 2. Mühendislik İskelet Kası Dokular <ol><li> &quot;Sertleştirici&quot; (10:1) ile &quot;elastomer&quot; karıştırılarak silikon yapıştırıcı hazırlayın. Tek üçgen tarafı (; Şekil 1 şekil ev gibi) ile Velcro 5 mm kare bölümler + / - Kes. Kare arasında yaklaşık olarak 12 mm arasında bir boşluk bir 6-kuyulu kültür plakasının kuyu içine Tutkal Velcro. </li></ol> Notlar: - Sadece Velcro yumuşak tarafı kullanın ve bu yan yukarıya dönük. - Çatıları birbirine baktığından emin olun. - Sadece silikon yapıştırıcı ile Velcro kapak, iyi boyunca tutkal yayılır yok. - Daha sonra elektriksel stimülasyon için bu iyi bir maç dikey yönde yapıları hizalamak alakalı(uzun ekseni boyunca) yedik. <ol start="2"><li> Vakumlu fırın içinde gece boyunca kurumaya bırakın, ısıtmalı değil, öncelikle hava kabarcıklarını çıkarmak için. 15 dakika için inkübe kuyuları ve% 70 EtOH ilave edilerek sterilize edin. PBS ile 3x durulayın ve 15 dakika UV altında koymak. Kuyulardan tüm PBS ve kullanıma kadar kuvöz Velcro ve yer çıkarın. </li><li> Buzdolabında Matrigel çözüm çözülme ve kısa süre 3D yapıları yapmadan önce istenilen konsantrasyona kollajen solüsyon hazırlanır. Steril% 0.02 asetik asit (nihai konsantrasyon 3.2 mg / ml) ile stok kollajen seyreltilir. </li></ol> Not: buz üzerinde her şeyi bırakın. <ol start="4"><li> GM ve sayısında tekrar süspansiyon hücreleri, Trypsinize. Inkübatör santrifüj tüpündeki hücreler bırakın. </li><li> Bu kollajen çözüm 0.5M NaOH ekleyin, bir tüp (Tablo 1 &#39;e göre) yapmak istediğiniz yapıları numaraları için Kollajen stok solüsyonu istenilen miktarda Ekle açık pembe Colo kadarr 7,5 arasında bir pH değerine işaret etmektedir. Yukarı ve aşağı pipetleme tarafından yavaşça karıştırın ve kabarcık oluşumunu önlemek. Sonra Matrigel ekleyin ve yavaşça ama iyice çok karıştırın. Nihayet, Kollajen / Matrigel karışımı (uygun miktarlarda için Tablo 1&#39;e bakınız) için GM ekleyin. </li></ol> Notlar: - buz üzerinde her adımı gerçekleştirin! Matrigel ve kollajen artan sıcaklıkta kolayca jel olacaktır. - Rengi aslında pembe olduğunu dikkat! PH seviyesindeki artış da hızlı jelleşme neden olacaktır. Kollajen - nihai konsantrasyon: 1.6 mg / ml. <ol start="6"><li> 5 dakika için 1000 rpm&#39;de hücrelerin istenen miktarda santrifüjlenir ve süpernatant çıkarın. </li></ol> Not: - yapı başına hücre sayısı ayarlanması gerektiğinde hücre faaliyeti bağlı olarak değişir. Tipik olarak, hücre sayısını yapı başına 750,000 ila 1,250,000 hücreler yer alır. <ol start="7"><li> Hücre pelet tekrar süspansiyon jel karışımı bazı kullanınve kalan karışımı içine hücreleri aktarmak ve iyice karıştırın, ama hava kabarcıkları tanıtan olmadan. </li><li> Kuvöz ve pipet Velcro ilk içine hücre jel karışımı 0.35 -0.4 ml ısıtılmış kuyucuğu çıkarın. Ardından, ek siteler arasında merkezine karışımı Pipeti ve Velcro uzatmak başlar. Son olarak, Velcro parçaları etrafında iki Velcro çapa ve pipet arasındaki boşluğu doldurmak için jel kalanını da kullanabilirsiniz. </li><li> Jeller aktarılacak yeterince sağlam olup olmadığını dikkatlice 5-10 dakika sonra kontrol edin, yani hafifçe yemekleri dokunun ve jel katı ise görsel inceleyin. Eğer öyleyse, bir inkübatörde yemekleri dikkatle yerleştirin. Genellikle, onlar on dakika sonra alınabilir. Daha sonra, 1-2 saat sonra, yavaşça sıcak GM 4 ml her biri jel üzerinde görüntülenir. </li></ol> Not:-Do tabak tutarken güçlü hareketleri yapamaz. <ol start="10"><li> (Büyüme ortamında farklılaşma, orta GM değiştirincivciv embriyo özü olmadan) 24 saat sonra, ve taze orta 2-3 günde bir ile değiştirin. Gün 7 sitesinde sigortalı kas liflerinin çapraz çizgili gelişimi için boyama ile değerlendirilebilir gibi, olgun odaklı kas liflerinin elde olmalıdır. </li></ol> 3. Elektriksel Stimülasyon <ol><li> Bir güvenlik kabininde UV altında% 70 etanol ve sonra kuru kullanmadan önce ionoptix plaka elektrotlar (reaktifler ve ekipman tabloya bakınız) sterilize edin. Yapıları ile kültür plaka üzerine elektrotlar ile plaka yerleştirin ve kültür plaka ve bir inkübatöre transfer kapağı ile kapatın. Kablolar ile uygun Ionoptix C-pacer bağlayın. </li></ol> Not: Elektrotlar kas yapı (Şekil 2) ile birlikte paralel olarak yerleştirilir. <ol start="2"><li> Beklendiği gibi stimülasyon protokolü uygulayın. </li></ol> Not: Biz genellikle, 4 V / CM kullanın2Hz bir frekansta 6ms bakliyat. Kültür ortamı uyarılma sırasında her 24 saat değiştirin.

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Yazarlar ifşa etmek başka bir şey var.

Kas dokularının mühendislik rejeneratif tıpta ilaç tarama için bir hastalık modeli olarak ve et üretimi için kullanılmak üzere büyük bir potansiyele sahiptir. Bununla birlikte, bu tür uygulamalar için gereksinimleri değişir. Kollajen hücre hizalama sağlar çünkü önceki 2B çalışmaları 12 yılında belirlenen miyoblast progenitör hücreler bazal membran kaynaklı proteinlerin varlığını gerektirir çünkü, kollajen ve Matrigel kombinasyonu ile çalışmayı seçti. Ayrıca, fibrin...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> Matrigel-büyüme faktörü azalır </td><td> Beckton ve Dickinson </td><td></td><td></td></tr><tr><td> DMEM (yüksek glukoz) * </td><td> Gibco </td><td> 42430 </td><td></td></tr><tr><td> Gelişmiş DMEM </td><td> Gibco </td><td> 12491 </td><td></td></tr><tr><td> At serumu </td><td> Gibco </td><td> 65050-122 </td><td></td></tr><tr><td> Fetal bovin serumu </td><td> Greiner </td><td> 758075 </td><td></td></tr><tr><td> 0.45 ve 0.22 mikron şırınga filtresi * </td><td> Whatmann (Schleicher ve Scheull) </td><td> 10462100 </td><td></td></tr><tr><td> L-glutamin </td><td> Gibco </td><td> 25030024 </td><td></td></tr><tr&gt; <td> Penisilin / streptomisin </td><td> Gibco </td><td> 10378016 </td><td></td></tr><tr><td> Amfoterisin </td><td> Gibco </td><td> 15290-018 </td><td></td></tr><tr><td> Kültür plastik </td><td> Greiner </td><td></td><td> Kültür şişeleri ve pipetler içerir </td></tr><tr><td> Civciv embriyo özü </td><td> Amerika Birleşik Devletleri Biyolojik </td><td> C3999 </td><td></td></tr><tr><td> Pasteur pipeti * </td><td> Hilgenberg </td><td></td><td> Pamuk, açık ucu L daralma ile Pasteur pipetler,: 0,9 ucu çapı 230 mm - 1,1 mm </td></tr><tr><td> Pasteur pipeti * </td><td> Hilgenberg </td><td></td><td> Pamuk, açık ucu L daralma ile Pasteur pipetler,: 1,4 ucu çapı 230 mm - 1,6 mm </td></tr><tr><td> Pasteur pipeti </td><td> VWR </td><td> 612-1702 </td><td></td></tr><tr><td> Kollajenaz tip I* </td><td> Sigma </td><td> C0130-16 </td><td></td></tr><tr><td> 40 mikron hücre süzgecinden * </td><td> BD Falcon </td><td> 352340 </td><td></td></tr><tr><td> 19G iğne </td><td></td><td></td><td></td></tr><tr><td> Elastomer </td><td> Dow Corning şirketi </td><td> 3097358-1004 </td><td> Silastik MDX 4-4210 # </td></tr><tr><td> Ajan Kür </td><td> Dow Corning şirketi </td><td></td><td> Silastik MDX 4-4210 # </td></tr><tr><td> Velcro </td><td> Düzenli mağaza </td><td></td><td> Siz düzenli bir mağazasında bu satın alabilirsiniz, sadece yumuşak tarafı kullanın </td></tr><tr><td> Kollajen tip I, sıçan kuyruğu </td><td> BD Biosciences </td><td> 3544236 </td><td></td></tr><tr><td> C-Pace EP Kültür Pacer </td><td> Ionoptix </td><td></td><td></td></tr><tr><td> Elektriksel uyarım için 6-kuyulu kültür tabaklarında </td><td> Beckton Dickinson-Falcon </td><td> BD Falcon # 353846 </td><td></td></tr><tr><td> C-Bulaşık kültür çanak elektrotlar </td><td> Ionoptix </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> * Hücrelerin izolasyonu (puan 1.1) için ihtiyaç duyulan # Birlikte tek bir kit içerisinde </td></tr></tbody></table>

engineering skeletal muscle tissues from murine myoblast progenitor cells and application of electrical stimulation

Engineered kas dokularının ve rejeneratif ilaç için bir alternatif et 1. basınç ülserleri çalışmak için in vitro olarak örneğin, bir hastalığın örnek olarak kullanım için dokuların üretimi, dahil çeşitli farklı amaçlar için kullanılabilir. İlk rapor 3D kas yapıları yıllar önce yapılan ve alanında öncü Vandenburgh ve arkadaşları 2,3 olan oylandı. Kas doku mühendisliği alanındaki ilerlemeler biyokimyasal faktörler, kök hücre ve progenitör hücrelerin bilgisi engin kazanç gelen sonuç değil sadece, ama fiziksel faktörlerin hücre davranış kontrolünde önemli rol oynadığı araştırmacılar tarafından kazanılan öngörüleri özellikle tabanlı olan ve doku gelişimi. State-of-the-art mühendislik kas anda hücre nüfuslu hidrojel yapıları oluşur oluşturur. Bizim laboratuvarımızda bu genellikle C2C12, mil murin arka bacak kasları izole sıçan miyoblast progenitör hücreleri, ya da bir fare miyoblast hücre hattı oluşurkollajen / Matrigel oluşan bir karışım ile Duran ve iki ankraj noktası arasındaki kaplama, kas bağları taklit. Diğer hücrelerin yanı gibi L6 sıçan miyoblastları 4, yenidoğan kas kaynaklı progenitör hücreleri 5, insan 6 hatta indüklenen pluripotent kök hücreler (iPS hücreleri) 7 gibi diğer türlerden erişkin kas dokularının türetilen hücreler olarak örneğin alternatif hücre hatları kabul edilebilir . Hücre kontraktilite yapı 8,9 ve kültür yaklaşık bir hafta sonra kas progenitör hücrelerin farklılaşma uzun ekseni boyunca hücre hizalama yapar. Dahası, elektrikli uyarım uygulama ölçüde 8 farklılaşma sürecini geliştirmek olabilir. Çünkü sınırlı boyutu (8 x 2 x 0,5 mm) tam doku örneğin canlılığı, farklılaşma ve hücre hizalama izlemek için konfokal mikroskopi kullanılarak analiz edilebilir. Söz konusu uygulamaya enginee için gereksinimleri bağlı olarakkırmızı kas dokusu değişecektir; diğer türler için bir et alternatif çeviri gerekli olarak hizmet ederken rejeneratif tıp örneğin kullanımı, doku boyutu ve vaskülarizasyon yukarı ölçeklendirme gerektirir.

Son ürün olarak Şekil 3 &#39;de gösterilen kas yapı olacaktır. Doku büyüklüğü yaklaşık 8 mm uzunluğunda, 2 mm genişliğinde ve 0,5 mm kalınlığında olacaktır. Farklılaşması sırasında elektrik stimülasyonu miyozin ağır zincir izoform ifadesi değişecektir, ancak farklılaşma orta 8 ile uyarılan olarak büyük ölçüde farklılık sürecini geliştirmek değil, aynı zamanda elektrik stimülasyonu kas işlevselliğinin kontrol etmek için işlem sonunda uygulanabilmektedir ?...

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Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Üretimli kas dokusu hastalığı modeli olarak hem de eti için alternatif bir kaynak olarak, rejeneratif tıpta büyük bir potansiyele sahiptir. Burada fare miyoblast projenitör hücreleri ve elektrik sinyalleri tarafından uyarılması, bu durumda, bir kas yapısının mühendislik açıklanmaktadır.

Elektrik Stimülasyonu murin myoblast Progenitör Hücreler ve Uygulama Mühendislik İskelet Kası Dokular

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

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Research

JoVE Journal

Bioengineering

20.8K Görüntüleme.  Eindhoven University of Technology, The Netherlands. Üretimli kas dokusu hastalığı modeli olarak hem de eti için alternatif bir kaynak olarak, rejeneratif tıpta büyük bir potansiyele sahiptir. Burada fare miyoblast projenitör hücreleri ve elektrik sinyalleri tarafından uyarılması, bu durumda, bir kas yapısının mühendislik açıklanmaktadır.

Video: Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

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

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized tissues.  The modules can be made of collagen as well as other gelable or crosslinkable materials.  They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells.  The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable.  These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues.  There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.

Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.

Kollajen, Modüller kullanarak Micro-dokuların Fabrikasyon İçeren Hücreleri Jel

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

Biyomühendislik

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim to use these principles to create tissues with predefined collagen architectures. A full understanding of the exact underlying processes of collagen remodeling to control the final tissue architecture, however, is lacking. In particular, little is known about the (re)orientation of collagen fibers in response to changes in tissue mechanical loading conditions. We developed an in&#160;vitro model system, consisting of biaxially-constrained myofibroblast-seeded fibrin constructs, to further elucidate collagen (re)orientation in response to i) reverting biaxial to uniaxial static loading conditions and ii) cyclic uniaxial loading of the biaxially-constrained constructs before and after a change in loading direction, with use of the Flexcell FX4000T loading device. Time-lapse confocal imaging is used to visualize collagen (re)orientation in a nondestructive manner.
Cell and collagen organization in the constructs can be visualized in real-time, and an internal reference system allows us to relocate cells and collagen structures for time-lapse analysis. Various aspects of the model system can be adjusted, like cell source or use of healthy and diseased cells. Additives can be used to further elucidate mechanisms underlying collagen remodeling, by for example adding MMPs or blocking integrins. Shape and size of the construct can be easily adapted to specific needs, resulting in a highly tunable model system to study cell and collagen (re)organization.

This model system starts from a myofibroblast-populated fibrin gel that can be used to study endogenous collagen (re)organization real-time in a nondestructive manner. The model system is very tunable, as it can be used with different cell sources, medium additives, and can be adapted easily to specific needs.

Mühendislik Fibrin tabanlı Doku miyofibroblastlar ve Kısıtlar Uygulamasından Yapıları ve Hücre ve Kollajen (Re) Organizasyon tetikleyebilecek süzün

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim ...

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named&#160;poly(glycerol-dodecanoate) (PGD)1 by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

Fare Embriyonik kök hücrelerden Mühendislik Sinir Dokular için Poli electrospun fibröz iskeleleri (gliserol-dodecanedioate)

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. In this article we outline a valid and reliable technique to prepare and test permeabilized skeletal muscle fibers in vitro.

Permeabilize İskelet Kası Fibers tarafından oluşturulan Maksimum İzometrik Kuvvet Ölçümü

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

Ekstraselüler Matrix içinde Gömülü Mühendislik Üç boyutlu Epitel Dokular

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Cardiac Muscle-cell Based Actuator and Self-stabilizing Biorobot - PART 1

Biological machines often referred to as biorobots, are living cell- or tissue-based devices that are powered solely by the contractile activity of living components. Due to their inherent advantages, biorobots are gaining interest as alternatives to traditional fully artificial robots. Various studies have focused on harnessing the power of biological actuators, but only recently studies have quantitatively characterized the performance of biorobots and studied their geometry to enhance functionality and efficiency. Here, we demonstrate the development of a self-stabilizing swimming biorobot that can maintain its pitch, depth, and roll without external intervention. The design and fabrication of the PDMS scaffold for the biological actuator and biorobot followed by the functionalization with fibronectin is described in this first part. In the second part of this two-part article, we detail the incorporation of cardiomyocytes and characterize the biological actuator and biorobot function. Both incorporate a base and tail (cantilever) which produce fin-based propulsion. The tail is constructed with soft lithography techniques using PDMS and laser engraving. After incorporating the tail with the device base, it is functionalized with a cell adhesive protein and seeded confluently with cardiomyocytes. The base of the biological actuator consists of a solid PDMS block with a central glass bead (acts as a weight). The base of the biorobot consists of two composite PDMS materials, Ni-PDMS and microballoon-PDMS (MB-PDMS). The nickel powder (in Ni-PDMS) allows magnetic control of the biorobot during cells seeding and stability during locomotion. Microballoons (in MB-PDMS) decrease the density of MB-PDMS, and enable the biorobot to float and swim steadily. The use of these two materials with different mass densities, enabled precise control over the weight distribution to ensure a positive restoration force at any angle of the biorobot. This technique produces a magnetically controlled self-stabilizing swimming biorobot.

In this two-part study, a biological actuator was developed using highly flexible polydimethylsiloxane (PDMS) cantilevers and living muscle cells (cardiomyocytes), and characterized. The biological actuator was incorporated with a base made of modified PDMS materials to build a self-stabilizing, swimming biorobot.

Kardiyak Kas hücresine Dayalı Aktüatör ve Kendiliğinden Sertleşen Biorobot - BÖLÜM 1

Biological machines often referred to as biorobots, are living cell- or tissue-based devices that are powered solely by the contractile activity of living ...

Cardiac Muscle Cell-based Actuator and Self-stabilizing Biorobot - Part 2

In recent years, hybrid devices that consist of a living cell or tissue component integrated with a synthetic mechanical backbone have been developed. These devices, called biorobots, are powered solely by the force generated from the contractile activity of the living component and, due to their many inherent advantages, could be an alternative to conventional fully artificial robots. Here, we describe the methods to seed and characterize a biological actuator and a biorobot that was designed, fabricated, and functionalized in the first part of this two-part article. Fabricated biological actuator and biorobot devices composed of a polydimethylsiloxane (PDMS) base and a thin film cantilever were functionalized for cell attachment with fibronectin. Following functionalization, neonatal rat cardiomyocytes were seeded onto the PDMS cantilever arm at a high density, resulting in a confluent cell sheet. The devices were imaged every day and the movement of the cantilever arms was analyzed. On the second day after seeding, we observed the bending of the cantilever arms due to the forces exerted by the cells during spontaneous contractions. Upon quantitative analysis of the cantilever bending, a gradual increase in the surface stress exerted by the cells as they matured over time was observed. Likewise, we observed movement of the biorobot due to the actuation of the PDMS cantilever arm, which acted as a fin. Upon quantification of the swimming profiles of the devices, various propulsion modes were observed, which were influenced by the resting angle of the fin. The direction of motion and the beating frequency were also determined by the resting angle of the fin, and a maximum swim velocity of 142 &#181;m/s was observed. In this manuscript, we describe the procedure for populating the fabricated devices with cardiomyocytes, as well as for the assessment of the biological actuator and biorobot activity.

In this study, a biological actuator and a self-stabilizing, swimming biorobot with functionalized elastomeric cantilever arms are seeded with cardiomyocytes, cultured, and characterized for their biochemical and biomechanical properties over time.

Kardiyak Kas Hücresine Dayalı Aktüatör ve Kendiliğinden Sertleşen Biorobot - Bölüm 2

In recent years, hybrid devices that consist of a living cell or tissue component integrated with a synthetic mechanical backbone have been developed. ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

Mikrosıvısal Bioprinting mühendisliği için doku ve Organoids skarların

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

Doku mühendisliği uygulamaları için bitki dokuların Decellularization için iki yöntem

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated researchers in the stem cell and bioengineering fields to develop in vitro high-fidelity human myocardium for both basic research and clinical applications. However, the immature phenotype of cardiac cells is a limitation on obtaining tissues that functionally mimic the adult myocardium, which is mainly characterized by mechanical and electrical signals. Thus, the purpose of this protocol is to prepare and mature the target cell population through electromechanical stimulation, recapitulating physiological parameters. Cardiac tissue engineering is evolving toward more biological approaches, and strategies based on biophysical stimuli, thus, are gaining momentum. The device developed for this purpose is unique and allows individual or simultaneous electrical and mechanical stimulation, carefully characterized and validated. In addition, although the methodology has been optimized for this stimulator and a specific cell population, it can easily be adapted to other devices and cell lines. The results here offer evidence of the increased cardiac commitment of the cell population after electromechanical stimulation. Electromechanically stimulated cells show an increased expression of main cardiac markers, including early, structural, and calcium-regulating genes. This cell conditioning could be useful for further regenerative cell therapy, disease modeling, and high-throughput drug screening.

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells&#039; Cardiomyogenic Potential

Here we present a protocol for training a cell population using electrical and mechanical stimuli emulating cardiac physiology. This electromechanical stimulation enhances the cardiomyogenic potential of the treated cells and is a promising strategy for further cell therapy, disease modeling, and drug screening.

Hücrelerin Cardiomyogenic potansiyeli geliştirmek için aynı anda elektrik ve mekanik stimülasyon

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated ...