Name: automated robotic dispensing technique for surface guidance and bioprinting of cells
Uploaded: 2016-11-18T13:00:00.000+00:00
Duration: 10 min 14 s
Description: Watch this Scientific Journal Video about Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells at JoVE.com

The work presented here is supported by the Singapore National Research Foundation under CREATE program (NRF-Technion): The Regenerative Medicine Initiative in Cardiac Restoration Therapy Research Program and by the Public Sector Funding (PSF) 2012 from the Science and Engineering Research Council (SERC) under the Agency for Science, Technology and Research (A*STAR).

NOT: Bu protokol, yüzey pürüzlülük (Şekil 1B) ve ekstrüzyon bazlı bioprinter (Şekil 1C) 10 olarak bir karşı basınç destekli robotik sevk sistemi (Şekil 1A) kullanımını tarif etmektedir. Bir Polistiren Yüzey 1. Modifikasyon <ol><li> polistiren doku kültürü plakaları aşındırma ve baskı hem de yükseklik tutarlılığını bozma, merkezde yukarı yay eğilimi beri 1 mm polistren yapraklarını kullanın. NOT: polistiren levhalar hücre yapışması için modifiye edilmemiş gibi, plazma tedavisi yapılır. </li><li> Oksijen plazma tedavisi. <ol><li> İlk olarak, plazma sistemi bağlı oksijen tüpünün regülatör 2 bar&#39;lık bir basınç seçin. Sonra makinenin kontrol panelinden aşağıdaki koşulları ve giriş plazma makineyi açıp: 150 W, oksijen 30 sscm, 10 dakika (güç, gaz akımı ve zaman sırasıyla). </li><li> polistiren substrat yerleştirinPlazma odasına e kapıyı mühür ve kontrol panelindeki başlangıç ​​düğmesine basın. Fetal sığır serumu içinde oksijen plazma muamele edilmiş polistiren substrat ıslatın ve üç kez 1 x fosfat tamponlu tuzlu su (PBS) yıkamadan önce 2 saat boyunca 37 ° C&#39;de inkübe edin. </li></ol></li></ol> 2. Programlama Baskı Kalıpları <ol><li> Bir Stylus veya Neşter Birinci Etch için Yüzey Programı kullanın. <ol><li> Bir iğne kullanan zaman sıkıştırılmış ve güvenli hale gelene kadar, bir dağıtma şırınga (5 ya da 10 mi) namlunun (özenle içinden), 1.5 mm çapında bir tekstil iğneyi. bu baskı kolu kelepçe mekanizması tespit edilebilir ve böylece bir neşter kullanılarak ise, bir yuvarlak ele neşter seçin. NOT: Stylus iyi neşter bıçağı daha kavisli desen etches. </li><li> ilk bioprinted düzenleme oluşturmaya çalışırken, XY koordinatlarını oluşturmak için numaralandırılmış eksenli grafik kağıt üzerinde istenilen desen kroki. Sonra benelektronik tablo yazılımı (Şekil 2) içine kazınmış / bioprinted desen koordinatları Temel giriş. Not: &quot;istenen bir desen&quot; lineer, S-şeklinde veya dairesel olarak birçok şekillerde olabilir. Burada gösterildiği gibi seçim gibi kardiyomiyositlerinin için MKH ayrımında paralel doğrusal çizgiler olarak gerekli deneysel model bağlıdır. dağılım grafiği fonksiyonu önerilen arsa desen görselleştirme sağlar. </li><li> Baskı / dağıtım yazılımı açın. &quot;&gt; Program Program Ekle&quot; yi seçin program kurmak için &quot;Noktası Ekle Düzenle&gt;&quot; izledi. x İhracat ve y fonksiyonunu kullanarak yazılımı dağıtma / baskı içine e-tablodan elde edilen koordinat değerleri &quot;Kopyala ve yapıştır&quot;. </li><li> yüzeye kalemi / baskı meme yerleştirmek için her çalıştırmadan önce robotun &quot;z&quot; yüksekliği ayarlayın. <ol><li> Baskı / dağıtım yazılımında, &quot;Robot&quot; seçeneğini değiştirme Mode &amp; &quot;üzerine tıklayın# 34; ve &quot;Öğretim modu&quot; seçeneğini etkinleştirin. Seçildikten sonra, yazılım robot &quot;Jog&quot; işlevini sağlar. </li><li> Jog için önce menü çubuğunda aşağıdaki komutları seçerek varsayılan konumuna robot başlatmak; &quot;Robot&gt; Meca başlatma&quot;, ardından &quot;Robot&gt; Jog&quot; seçeneğini seçin. Girdi desen kökeni tam olarak kalemi yerleştirmek için gerekli &quot;X ve Y yuvaları&quot; olarak (mm) sayısal değerler. </li><li> Sonra, &quot;Z yuvasına&quot; giriş (mm) bir sayısal değer yüzeyi ile temas kalemini veya baskı meme koyun ama esnek değil ya çukurlaştırılır. Bu nokta &quot;Z&quot; başlangıç ​​değeri olarak belirlenmiştir. NOT: Her oluğun derinliği sisteminin Z yüksekliği ile kolayca değiştirilebilir. 40, 80 ve 170 um oluklarının 1 mm kalınlığında polistiren levha (Şekil 4 ve Tablo 1) yüzeyine oyulmuş gösterilmiştir. </li></ol></li><li> p seçinkoordinat noktalarının her biri için rint talimat, yani &quot;Çizgi dağıtmak Başlat&quot; sona erdirmek için robota sinyal &quot;Satır dağıtmak Sonu&quot; ara noktaları tayin ve &quot;Geçiş Hattı&quot;, ilk noktasını ve baskı başlatılması tanımlamak için baskı çalıştırın. </li><li> Üst menü çubuğundan takip komutları seçerek robot programı iletişim: &quot;Robot&gt; C &amp; T Veri Gönder&quot;. </li><li> &quot;Çalıştır&quot; moduna robot değiştirerek, gravür / yazdırma işlemini başlatın. Üst menü çubuğundan &quot;&gt; Çalıştır Switch Mode Modunu Değiştirme&gt; Robot&quot; seçerek bu etmeyin. </li><li> Robot dağıtıcı konsolunda yeşil &quot;başlat düğmesine&quot; basarak yazdırma işlemine başlayın. </li></ol></li></ol> 3. hazırlama ve baskı hücre içeren Jelatin Bioink <ol><li> Minimum Essential Medium alfa ortamında (αMEM) içinde% 2 jelatin çözülür (% 10 FBS ve% 2 antibiyotik / antimycot ile desteklenmiş2 saat boyunca 60 ° C&#39;de IC) bioink çözüm hazırlanması için. </li><li> Ön kültür αMEM /% 10 FBS ile 10 cm doku kültür tabaklarında% 70 ortak akışa kadar mezenkimal kök hücreleri tanımlayan kırmızı flöresanlı protein (RFP MSC&#39;ler). 37 ° C&#39;de 5 dakika boyunca 1 x tripsin-EDTA çözeltisi ile orta kaplama ve kaldırarak süspansiyona eklenen hücre salgısıdır. </li><li> 5 dakika boyunca 1000 x g&#39;de santrifüj ile hücreler pelet ve supernatant çıkarın. 1x PBS 0.5 ml hücre pelletini ve bir hemasitometre kullanılarak hücre yoğunluğu sayısı. </li><li> Bioink oda sıcaklığına soğumaya bırakıldıktan sonra, yavaşça 5 x 10 6 hücre mL-1 bir son konsantrasyon elde etmek için bioink RFP-MSC nin süspansiyonuna yeterli hacimde karıştırılır. </li><li> mühürlü başlığı ile bir baskı enjektöre hücre taşıyan bioink dökün. yazdırılabilir bir viskoziteye ulaşmak için 4 ° C&#39;ye yüklenen şırınga Chill. </li><li> otomatik yüklenen şırınga yerleştirinRobotik dağıtma sistemi hava basınç hatları takın ve. Şırınga Luer mührünü çıkarın ve baskı ağzına takılır. </li><li> 5 mm, 0,05 MPa geri basınç kullanarak ince çizgiler halinde cellularized bioinks a&#39;ya / 27 G iğne / meme aracılığıyla 10 ml şırınga sn yazma hızı önceden programlanmış birikimi sonra bir polistiren filmi yüzeyine (silindirik üzerinde tavsiye konik) Adım 2.1 açıklanan model öncesi kazınmış oluklar üzerine cellularized bioink yerleştirmek için. </li><li> Oda sıcaklığında 1 saat inkübe edildikten sonra, bir ters flüoresan mikroskop kullanılarak görüntülemeden önce 24 saat (hücreler algılar ve kazınmış özelliklerine tepki sağlamak için) için hücreler (% 10 FBS ve antibiyotikler ile takviye edilmiş), 10 ml büyüme ortamı ilave ve inkübe 10X bir büyütmede gösterilmiştir. </li></ol>

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The authors declare they have no competing financial interest.

Bu prosedürün kritik bir adım süreci, özellikleri, hücre sedimantasyon izin kanama / bioink yayma olmadan yazdırmak, kayma gerilmesi hücre ölümü olmadan hücreleri teslim ve istenmeyen soy doğru farklılaşma tetiklemek olmamalıdır olarak kök hücrelerin gerçek bioprinting doğumdur. beklenen hücre hizalama meydana başarısız olursa, o zaman bioink viskozite baskı için uygunluğu açısından değerlendirilmelidir. Bioink hücreleri desenli polimer yüzeyine tortu sağla...

Hücre yerleştirme kasıtlı desenlendirme in vivo hücresel organizasyonun 1 taklit kültürlerin oluşumunu sağlar. Nitekim, birden fazla hücre tipleri arasındaki etkileşim içine araştırmalar mekansal yerleştirme 2,3 düzenleyerek yardım edilebilir. Çoğu desenlendirme sistemleri teşvik veya sonraki pasif hücre birikimi ile hücre yapışmasını önlemek için yüzey modifikasyon prosedürleri güveniyor. Bioprinting hücre dağılımları üzerinde 1 mekansal ve zamansal kontrol sunuyor. Bu fonksiyonlara ek olarak, bioprinting geometrik olarak kompleks yapı iskelesi 4 üretilmesi için bir teknik, basit bir hızlı ve düşük maliyetli bir yöntem olarak tarif edilmiştir. Bu bilgisayar tasarım yazılımı kullanan ve üretim süreci 4 içine hücrelerin girişine izin verir. Bioprinting sistemleri lazer tabanlı olarak çalışma prensiplerine göre sınıflandırılır edilmiştir, mürekkep püskürtmeli tabanlı veya 4 ekstrüzyon tabanlı. Gerçekçi bir süre 4-6 içinde klinik olarak önemli boyutta organize yapıların imalatı izin verdiği Ekstrüzyon bioprinting en umut verici olarak tarif edilmiştir. Bir hücrenin dayanma hidrojel bioink mekanik ya da geri basınç destekli ekstrüzyon biri tarafından gerçekleştirilir. Burada yer alan yöntemde, geri dönüş basıncı kullanılmıştır. belirtildiği gibi, hücreler, bir cytocompatible bioink teslim edilir. Böyle bir bioink zararlı kayma gerilimi üretmeden hücrelerin teslim destek ve çöken ya da 7-10 ( &quot;baskı kanaması&quot; olarak anılacaktır) yayılma olmadan basılı izidir bütünlüğünü korumak için yeterli bir viskozite olması gerekmektedir. kendi yapışan yüzeyi ile hücrelerin etkileşim hücre davranışını etkilemek için bilinmektedir. Yüzey topoğrafyası hücre şekli, yönü 11, ve hatta fenotip kontrol edebilirsiniz. Özel olarak, oluklar ve kanallar imalat indüklediği gösterilmiştirçok sayıda hücre türleri üzerinde gerilmiş, ince uzun morfolojisi. Bu morfoloji kabul multipotent ve pluripotent hücreler fenotipini belirler bulunmuştur. Örneğin, oluklar hizaya zaman, mezenkimal kök hücreler (MSC) sentetik 10,14-17 üzerinde kasılma fenotip kabul kardiyomiyositlerinin 12,13 ve damar düz kas hücrelerine karşı farklılaşma kanıt gösterebilir. Kanallar veya oluklar hizalayarak hücre, örneğin, derin reaktif iyon dağlama, elektron demeti, doğrudan lazer baskı, femtosaniye laser, fotolitografi ve plazma kuru aşındırma 18 için, bir dizi yöntem vasıtasıyla bir polimerik yüzey üzerinde oluşturulabilir. Bu yaklaşımlar, çoğu zaman alıcıdır karmaşık bir düzeneği gerekli ve oluşturulan desen şeklinde sınırlayıcı olabilir. Buna ek olarak, bioprinting olan model vermenin senkronize olmayan ve anında Hücreselleştirmeden izin vermemektedir. otomatik bir bir koordineli kontrollü hareketdağıtım sistem çözümleri birikimi için karmaşık desenleri takip edebilirsiniz. Burada mikro kontrollü hareket hücre yönlendirme için kanalları oluşturmak için kullanılabilir gösterilmektedir. Bir sivriltilmiş kalem veya neşter ekstrüzyon şırınga yerine baskı kafası takılır ve ekipmanları daha sonra çizim yazılımı gözetiminde polimer yüzeyi aşındırma edebilirsiniz. yöntemi, desen tasarım esnekliği sağlar ve genel olarak, polistiren, PTFE ve polikaprolakton gibi Biomühendislik kullanılan polimerik materyallere uygulanabilir. Çatlatma, bir sonraki aşamada olduğu gibi, hücreler, çizik oluklar, doğrudan bioprinted edilebilir. Burada kullanılan jelatin bioink iz korumak ve biriken hücreler kazınmış özellikleri hissetmeye izin hem başardı. kazınmış oluklarına bioprinted mezenkimal kök hücrelerin farklı hatlarda kendilerine boyunca uzamaya gösterildi.

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Robotic Dispensing System</td><td>Janome</td><td>2300N</td><td></td></tr><tr><td>Plasma Machine</td><td>Femto Science</td><td>Covance</td><td></td></tr><tr><td>USB Microscope</td><td></td><td></td><td></td></tr><tr><td>Optical Microscope</td><td>Olympus</td><td>IX71</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Spreadsheet</td><td>Excel</td><td>Excel</td><td></td></tr><tr><td>Printing Co-ordinate Software</td><td>Janome</td><td>JR C-Points</td><td></td></tr><tr><td>Imaging Software</td><td>National Institutes of Health (NIH)</td><td>ImageJ</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Stylus (Blade)</td><td>OLFA</td><td>AK-5</td><td></td></tr><tr><td>5 ml printing syringe</td><td>San-ei Tech</td><td>SH10LL-B</td><td></td></tr><tr><td>30 G printing needle</td><td>San-ei Tech</td><td>SH30-0.25-B</td><td></td></tr><tr><td>1 mm polystyrene sheets</td><td></td><td>Purchased locally</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Invitrogen&amp;nbsp;</td><td>10270-098</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>Gelatin from porcine skin, Gel strength 300, Type A</td><td>Sigma Aldrich</td><td>9000-70-8</td><td></td></tr><tr><td>&amp;alpha;MEM</td><td>Invitrogen</td><td>41061-029</td><td></td></tr><tr><td>Antibiotc antimycotic</td><td>Sigma Aldrich</td><td>A5955-100ML</td><td></td></tr><tr><td>Red Fluorescent Protein Mesenchymal Stem Cells (RFP-MSCs)</td><td>Cyagen Biosciences Incorporation</td><td>RASMX-01201</td><td></td></tr></tbody></table>

automated robotic dispensing technique for surface guidance and bioprinting of cells

Bu el yazması bir otomatik robot dağıtım sistemini kullanarak bir hidrojel bioink bu özelliklere hücrelerin doğrudan teslim takiben hücre rehberlik özelliklerinin tanıtım açıklar. hücrelerin doğru tortu ve özellikleri anlamda verir gibi belirli bioink seçildi. dağıtma sistemi bir geri basınç destekli yazıcı kafasını kullanarak hidrojel bioinks içinde canlı hücreleri bioprints. Ancak, bilenmiş kalemle veya neşter ile yazıcı kafasını değiştirerek, dağıtım sistemi de yüzey aşındırma yoluyla topografik ipuçları oluşturmak için kullanılabilir. stylus hareketi X, Y 10 mikron adımları ve Z yönlerinde programlanabilir. desenli oluk oluklar &#39;yönü ile uyum içinde uzunlamasına bir morfolojisi benimsemeye bunları etkileyen, mezenkimal kök hücreleri yönlendirmek başardık. desenlendirme düz çizgiler, iç içe daireler ve sinüzoidal dalgalar halinde komplo yazılımı kullanılarak dizayn edilebilir. Bir sonraki işlem, fibro içindepatlamalar ve mezenkimal kök hücreleri, bir karşı basınç tahrik ekstrüzyon yazıcı kafasının bioprinting için,% 2 jelatin bioink içinde süspansiyon haline getirildi. Hücre taşıyan bioink sonra gravürü için kullanılan aynı programlanmış koordinatları kullanılarak basılmıştır. bioprinted hücreler duyu ve kazınmış oluklar doğrultusu boyunca onların uzatılmış yönelim gösterdiği gibi kazınmış özelliklere tepki başardık.

Örnek sonuçlar karşı basınç destekli robot dağıtma sistemi yüzey pürüzlülüğü ve bioink baskı (Şekil 1 A), hem de gerçekleştirmek için bir çekme-bazlı bioprinter olarak kullanılabileceğini göstermektedir. Bu polimer yüzeylerinin kazınmış olukların oluşturulması için kullanılabilir, ve daha sonra özellikleri (Şekil 1 B ve C) için doğrudan bir cep taşıyıcı bioink baskı. <p class="jove_content" fo:keep-together....

Watch this Scientific Journal Video about Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells at JoVE.com

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

Hücre Yüzey Rehberlik ve Bioprinting için Otomatik Robotik Dağıtım Tekniği

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using ...

automated-robotic-dispensing-technique-for-surface-guidance

Research

JoVE Journal

Bioengineering

6.5K Görüntüleme.  Nanyang Technological University. This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

Video: Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

Prescribed 3-D Direct Writing of Suspended Micron/Sub-micron Scale Fiber Structures via a Robotic Dispensing System

A 3-axis dispensing system is utilized to control the initiating and terminating fiber positions and trajectory via the dispensing software. The polymer fiber length and orientation is defined by the spatial positioning of the dispensing system 3-axis stages. The fiber diameter is defined by the prescribed dispense time of the dispensing system valve, the feed rate (the speed at which the stage traverses from an initiating to a terminating position), the gauge diameter of the dispensing tip, the viscosity and surface tension of the polymer solution, and the programmed drawing length. The stage feed rate affects the polymer solution&#8217;s evaporation rate and capillary breakup of the filaments. The dispensing system consists of a pneumatic valve controller, a droplet-dispensing valve and a dispensing tip. Characterization of the direct write process to determine the optimum combination of factors leads to repeatedly acquiring the desired range of fiber diameters. The advantage of this robotic dispensing system is the ease of obtaining a precise range of micron/sub-micron fibers onto a desired, programmed location via automated process control. Here, the discussed self-assembled micron/sub-micron scale 3D structures have been employed to fabricate suspended structures to create micron/sub-micron fluidic devices and bioengineered scaffolds.

Here, we present a protocol to fabricate freely-suspended, micron/sub-micron scale polymer fibers and &#8220;web-like&#8221; structures generated via automated direct writing procedure by means of a 3-axis dispensing system.

Asma Micron / Sub-mikron Ölçeği Fiber Yapıların 3-D Doğrudan Yazma Robotik Dağıtım Sistemi aracılığıyla Prescribed

A 3-axis dispensing system is utilized to control the initiating and terminating fiber positions and trajectory via the dispensing software. The polymer ...

Mühendislik

The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.

This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

Sürekli Akış Microspotter kullanma Modifiye yüzeye hücrelerin Batık Baskı

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell ...

Biyomühendislik

Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing both mono-culture and co-culture spheroids in a high throughput 96-well plate format is described in this work. This approach utilizes an aqueous two-phase system to confine cells into a drop of the denser aqueous phase immersed within the second aqueous phase. The drop rests on the well surface and keeps cells in close proximity to form a single spheroid. This technology has been adapted to a robotic liquid handler to produce size-controlled spheroids and expedite the process of spheroid production for compound screening applications. Spheroids treated with a clinically-used drug show reduced cell viability with increase in the drug dose. The use of a standard micro-well plate for spheroid generation makes it straightforward to analyze viability of cancer cells of drug-treated spheroids with a micro-plate reader. This technology is straightforward to implement both robotically and with other liquid handling tools such as manual pipettes.

A protocol for robotic printing of cancer cell spheroids in a high throughput 96-well plate format using an aqueous two-phase system is presented.

İlaç test için bir sulu iki-fazlı bir sistem ile Cancer Celi sferoitlerin Robotik üretimi

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing ...

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.

A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced.

Üç Verici Kartezyen Yazıcı Kullanımı Bioprinted Hücresel Yapıların Canlılık

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of ...

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

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and fibroblasts are first isolated, counted and mixed at desired cell ratios. They are co-cultured in individual wells in ultra-low attachment 96-well plates. Within 3 days, beating spheroids form. These spheroids are then picked up by a nozzle using vacuum suction and assembled on a needle array using a 3D bioprinter. The spheroids are then allowed to fuse on the needle array. Three days after 3D bioprinting, the spheroids are removed as an intact patch, which is already spontaneously beating. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

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 ...

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 ...

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 ...

Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue assembling units for biofabrication. While the main power of the spheroid model is in mimicking physical-chemical gradients at the tissue microscale, the real physiological environment (including dynamics of metabolic activity, oxygenation, cell death, and proliferation) inside the spheroids is generally ignored. At the same time, the effects of the growth medium composition and the formation method on the resulting spheroid phenotype are well documented. Thus, characterization and standardization of spheroid phenotype are required to ensure the reproducibility and transparency of the research results. The analysis of average spheroid oxygenation and the value of O2 gradients in three dimensions (3D) can be a simple and universal way for spheroid phenotype characterization, pointing at their metabolic activity, overall viability, and potential to recapitulate in vivo tissue microenvironment. The visualization of 3D oxygenation can be easily combined with multiparametric analysis of additional physiological parameters (such as cell death, proliferation, and cell composition) and applied for continuous oxygenation monitoring and/or end-point measurements. The loading of the O2 probe is performed during the stage of spheroid formation and is compatible with various protocols of spheroid generation. The protocol includes a high-throughput method of spheroid generation with introduced red and near-infrared emitting ratiometric fluorescent O2 nanosensors and the description of multi-parameter assessment of spheroid oxygenation and cell death before and after bioprinting. The experimental examples show comparative O2 gradients analysis in homo- and hetero-cellular spheroids as well as spheroid-based bioprinted constructs. The protocol is compatible with a conventional fluorescence microscope having multiple fluorescence filters and a light-emitting diode as a light source.

The protocol describes high-throughput spheroid generation for bioprinting using the multi-parametric analysis of their oxygenation and cell death on a standard fluorescence microscope. This approach can be applied to control the spheroids viability and perform standardization, which is important in modeling 3D tissue, tumor microenvironment, and successful (micro)tissue biofabrication.

Çok Hücreli Sferoidlerin Uygun Fiyatlı Oksijen Mikroskobu Destekli Biyofabrikasyonu

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

3D Biyobaskı için Adipoz Türevi Kök Hücre Sferoidlerinin Büyük Ölçekli, Otomatik Üretimi

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

Agarose Fluid Gels Formed by Shear Processing During Gelation for Suspended 3D Bioprinting

The use of granular matrices to support parts during the bioprinting process was first reported by Bhattacharjee et al. in 2015, and since then, several approaches have been developed for the preparation and use of supporting gel beds in 3D bioprinting. This paper describes a process to manufacture microgel suspensions using agarose (known as fluid gels), wherein particle formation is governed by the application of shear during gelation. Such processing produces carefully defined microstructures, with subsequent material properties that impart distinct advantages as embedding print media, both chemically and mechanically. These include behaving as viscoelastic solid-like materials at zero shear, limiting long-range diffusion, and demonstrating the characteristic shear-thinning behavior of flocculated systems. 
On the removal of shear stress, however, fluid gels have the capacity to rapidly recover their elastic properties. This lack of hysteresis is directly linked to the defined microstructures previously alluded to; because of the processing, reactive, non-gelled polymer chains at the particle interface facilitate interparticle interactions-similar to a Velcro effect. This rapid recovery of elastic properties enables bioprinting high-resolution parts from low-viscosity biomaterials, as rapid reformation of the support bed traps the bioink in situ, maintaining its shape. Furthermore, an advantage of agarose fluid gels is the asymmetric gelling/melting transitions (gelation temperature of ~30 &#176;C and melting temperature of ~90 &#176;C). This thermal hysteresis of agarose makes it possible to print and culture the bioprinted part in situ without the supporting fluid gel melting. This protocol shows how to manufacture agarose fluid gels and demonstrates their use to support the production of a range of complex hydrogel parts within suspended-layer additive manufacture (SLAM).

Shear processing during hydrogel formation results in the production of microgel suspensions that shear-thin but rapidly restructure following the removal of shear forces. Such materials have been used as a supporting matrix for bioprinting complex, cell-laden structures. Here, methods used to manufacture the supporting bed and compatible bioinks are described.

Askıda 3D biyobaskı için jelasyon sırasında kesme işlemi ile oluşturulan agaroz sıvı jelleri

The use of granular matrices to support parts during the bioprinting process was first reported by Bhattacharjee et al. in 2015, and since then, several ...