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).

NOTA: Questo protocollo descrive l&#39;uso di una contropressione assistita sistema di erogazione robotico (Figura 1A) come incisione della superficie (Figura 1B) e bioprinter estrusione-based (Figura 1C) 10. 1. Modifica di una superficie polistirolo <ol><li> Utilizzare 1 fogli mm di polistirene da piastre di coltura dei tessuti in polistirene tendono a piegarsi verso l&#39;alto al centro, rovinando la consistenza altezza sia incisione e la stampa. NOTA: i fogli di polistirolo non sono modificati per l&#39;adesione cellulare, viene eseguito il trattamento al plasma. </li><li> trattamento al plasma di ossigeno. <ol><li> Innanzitutto, selezionare una pressione di 2 bar sul regolatore del cilindro di ossigeno collegato alla macchina plasma. Poi accendere la macchina del plasma e l&#39;ingresso alle seguenti condizioni nel pannello di controllo della macchina: 150 W, 30 SSCM di ossigeno, 10 min (per il potere, il flusso del gas e del tempo, rispettivamente). </li><li> Posizionare il Substrat polistirolovia e nella camera di plasma, sigillare la porta e premere il pulsante di avvio sul pannello di controllo. Bagnare il plasma di ossigeno polistirene trattata substrato in siero bovino fetale e incubare a 37 ° C per 2 ore prima del lavaggio tre volte 1x tampone fosfato salino (PBS). </li></ol></li></ol> 2. I modelli di programmazione di stampa <ol><li> Utilizzare il programma per primo Etch la superficie con uno stilo o bisturi. <ol><li> Quando si utilizza uno stilo, inserire un ago tessili 1,5 millimetri di diametro (dall&#39;interno con grande cura) nell&#39;ugello di una siringa di erogazione (5 o 10 ml) fino a quando non viene incastrato e fissato. Se si utilizza un bisturi, scegliere un bisturi rotondo maneggiati in modo che possa essere fissato nel meccanismo di fissaggio del braccio di stampa. NOTA: Lo stilo incide modelli curvi meglio del bisturi. </li><li> Quando si tenta prima di creare una disposizione bioprinted, disegnare il modello desiderato su carta millimetrata con assi numerati per generare le coordinate XY. Quindi ionput le coordinate del modello inciso / bioprinted nel software foglio di calcolo (Figura 2). NOTA: Il &quot;modello desiderato&quot; può essere in molte forme come lineari, a forma di S, o circolare. La scelta dipende dal modello sperimentale richiesta, come ad esempio le linee lineari parallele per la differenziazione delle cellule staminali mesenchimali per cardiomiociti come dimostrato qui. La funzione di grafico a dispersione permette la visualizzazione del pattern grafico proposto. </li><li> Aprire la stampa / software di erogazione. Selezionare &quot;Programma&gt; Aggiungi programma&quot;, seguito da &quot;Modifica&gt; Aggiungi punto&quot; per impostare il programma. Esportare il xey coordinate valori ottenuti dal foglio di calcolo nella stampa / erogazione software utilizzando il &quot;copia e incolla&quot; la funzione. </li><li> Calibrare il &quot;z&quot; altezza del robot prima di ogni corsa in modo da mettere l&#39;ugello stilo / stampa sulla superficie. <ol><li> Nella stampa / software di erogazione, selezionare l&#39;opzione &quot;Robot&quot;, cliccare su &quot;Modifica della modalità &amp;# 34; e attivare l&#39;opzione &quot;modalità di insegnamento&quot;. Una volta selezionato, il software consente la funzione &quot;Jog&quot; del robot. </li><li> Fare jogging, prima inizializzare il robot nella posizione predefinita selezionando i seguenti comandi dalla barra dei menu; &quot;Robot&gt; Meca inizializzazione&quot;, quindi selezionare &quot;Robot&gt; Jog&quot;. Input i valori numerici (in mm) nelle &quot;slot X e Y&quot; necessari per posizionare lo stilo esattamente sulla origine del pattern. </li><li> Quindi, immettere un valore numerico (in mm) nella &quot;fessura Z&quot; per posizionare l&#39;ugello stilo o la stampa a contatto con la superficie, ma non flex o rientro della superficie. Questo punto è designato come valore di partenza &quot;Z&quot;. NOTA: La profondità di ogni scanalatura può essere variata facilmente usando la Z-altezza del sistema. Scanalature di 40, 80 e 170 micron sono dimostrati per tagliare nella superficie di 1 mm di spessore fogli di polistirene (Figura 4 e Tabella 1). </li></ol></li><li> Selezionare il pistruzioni Rint per ciascuno dei punti di coordinate, vale a dire, &quot;Avvio della linea di erogazione&quot; per definire il primo punto e l&#39;iniziazione di stampa, &quot;linea che passa&quot; per designare i punti intermedi e &quot;End of Line Dispense&quot; per segnalare al robot di risolvere il tiratura. </li><li> Comunicare il programma per il robot selezionando i comandi successivi dalla barra dei menu in alto: &quot;Robot&gt; Invia C &amp; T dati&quot;. </li><li> Avviare l&#39;incisione corsa / stampa, modificando il robot in modalità &quot;Run&quot;. A tale scopo, selezionando &quot;Robot&gt; Modifica della modalità&gt; Switch Mode Run&quot; dalla barra dei menu in alto. </li><li> Avviare la procedura di stampa, premendo il tasto &quot;Start&quot; verde sulla console dispenser robot. </li></ol></li></ol> 3. Preparazione e stampa di Cell contenenti gelatina Bioink <ol><li> Sciogliere 2% di gelatina in Minimum Essential Media Alpha Media (αMEM) (supplementato con 10% FBS e 2% antibiotico / antimycotic) a 60 ° C per 2 ore per preparare le soluzioni bioink. </li><li> Pre-cultura della Proteina Fluorescente Rosso esprimendo cellule staminali mesenchimali (MSC-RFP) al 70% di confluenza in 10 cm piatti di coltura utilizzando αMEM / 10% FBS. Rilasciare le cellule attaccate in sospensione rimuovendo il mezzo e rivestimento con una soluzione di tripsina-EDTA 1x per 5 min a 37 ° C. </li><li> Agglomerare le cellule per centrifugazione a 1.000 xg per 5 minuti e rimuovere il surnatante. Risospendere il pellet di cellule in 0,5 ml di PBS 1x e contare la densità cellulare utilizzando un emocitometro. </li><li> Dopo aver lasciato la bioink raffreddare a temperatura ambiente, mescolare delicatamente in un volume sufficiente per la sospensione di RFP-MSC nel bioink per ottenere una concentrazione finale di 5 x 10 6 cellule ml -1. </li><li> Versare il bioink cuscinetto cella in una siringa di stampa con l&#39;ugello sigillato. Raffreddare la siringa caricata a 4 ° C per raggiungere una viscosità stampabile. </li><li> Posizionare la siringa caricata sul automatizzatosistema di distribuzione robotizzato e fissare le linee di pressione dell&#39;aria. Rimuovere la siringa tenuta Luer e attaccare l&#39;ugello di stampa. </li><li> Estrudere la bioinks cellularized in linee sottili utilizzando 0,05 MPa contropressione, a 5 mm / sec velocità di scrittura da una siringa da 10 ml con una 27 ago G / ugello (stretti consigliato sopra cilindrica) su una superficie del film di polistirene, a seguito della deposizione di pre-programmati modello descritto al punto 2.1 per posizionare il bioink cellularized sulle scanalature pre-incise. </li><li> Dopo 1 ora di incubazione a temperatura ambiente, si aggiungono 10 ml mezzi di crescita (supplementato con 10% FBS e antibiotici) e incubare le cellule per 24 ore (per permettere alle cellule di senso e reagiscono alle caratteristiche incise) prima di visualizzare utilizzando un microscopio a fluorescenza invertito a 10X di ingrandimento. </li></ol>

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The authors declare they have no competing financial interest.

La fase critica di questa procedura è la consegna bioprinting reale delle cellule staminali come il processo deve consentire di sedimentazione delle cellule alle caratteristiche, di stampa senza la diffusione bioink / sanguinamento, fornire cellule senza morte cellulare sforzo di taglio e non innescare la differenziazione verso lignaggio indesiderati. Se l&#39;allineamento delle cellule previsto non si verifica, quindi la viscosità bioink dovrebbe essere valutato per la sua idoneità per l...

Il patterning deliberata di posizionamento cellulare consente la formazione di culture che imitano in vivo organizzazione cellulare 1. In effetti, la ricerca l&#39;interazione tra più tipi di cellule può essere assistito da organizzare la loro collocazione spaziale 2,3. La maggior parte dei sistemi di patterning si basano su procedure di modifica della superficie per favorire o impedire l&#39;adesione delle cellule con successiva deposizione delle cellule passiva. Bioprinting offre un controllo spaziale e temporale su distribuzioni cella 1. Oltre a queste funzioni, bioprinting è stato descritto come un metodo tecnicamente semplice, rapida ed economica per generare scaffold geometricamente complesse 4. Utilizza software di progettazione e consente l&#39;introduzione di cellule nel processo di fabbricazione 4. Sistemi Bioprinting sono stati classificati in base alle loro principi di funzionamento il laser a base, a getto d&#39;inchiostro a base o di estrusione a base di 4. Estrusione bioprinting è stato descritto come il più promettente in quanto permette la realizzazione di costrutti organizzati di dimensioni clinicamente rilevanti entro un lasso di tempo realistico 4-6. E &#39;eseguita da una pressione meccanica o posteriore di estrusione assistito di un idrogel bioink cuscinetto cellule. Nel metodo qui presentato, è stato impiegato contropressione. Come accennato, le cellule vengono consegnati in un bioink cytocompatible. Tale bioink dovrebbe sostenere la fornitura di cellule senza produrre deleteria sollecitazione di taglio, e di essere di una viscosità sufficiente a mantenere l&#39;integrità della traccia stampata, senza collassare o diffusione (denominato &quot;spurgo inchiostro&quot;) 7-10. L&#39;interazione delle cellule con la loro superficie di adesione è noto per influenzare il comportamento cellulare. La topografia della superficie può controllare la forma delle cellule, l&#39;orientamento 11, e anche il fenotipo. In particolare, la realizzazione di scanalature e canali sono stati dimostrati per indurreun allungato, morfologia allungata su diversi tipi di cellule. L&#39;adozione di questa morfologia è stato trovato per influenzare il fenotipo di cellule multipotenti e pluripotenti. Ad esempio, quando allineato scanalature, cellule staminali mesenchimali (MSC) mostrano segni di differenziamento verso cardiomiociti 12,13 e cellule muscolari lisce vascolari adottare il fenotipo contrattile sul sintetico 10,14-17. La cella allineando canali o scanalature può essere generato su una superficie polimerica tramite un certo numero di metodi, per esempio, profonda attacco con ioni reattivi, litografia a fascio elettronico, stampa laser diretta, laser a femtosecondi, fotolitografia e attacco a secco plasma 18. Questi approcci sono spesso lunghi, richiedono apparecchiatura complessa e può essere limitante nella forma del modello generato. Inoltre, essi non si sincronizzano con patterning bioprinting e non consentono cellularization immediato. Il movimento coordinato controllato di un sistema automatizzatosistema di erogazione può seguire schemi complessi per la deposizione di soluzioni. Qui mostriamo come il movimento microscala controllata può essere sfruttata per creare canali per l&#39;orientamento delle cellule. Uno stilo o bisturi affilato è collegata alla testina di stampa al posto della siringa estrusione e le attrezzature possono incidere la superficie del polimero sotto la guida del software di tracciatura. Il metodo offre versatilità nella progettazione del modello ed è applicabile ai materiali polimerici comunemente utilizzati in bioingegneria come polistirolo, PTFE, e policaprolattone. Come fase successiva alla incisione, le cellule possono essere bioprinted direttamente alle scanalature graffiati. Il bioink gelatina utilizzato qui è stato in grado sia di mantenere la traccia e consentire alle cellule depositate per rilevare la funzionalità incise. Le cellule staminali mesenchimali bioprinted alle scanalature incise sono state dimostrate per allungare lungo loro in linee distinte.

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			<td height="21" style="height:21px;width:151px;">USB Microscope</td>
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			<td height="41" style="height:41px;width:151px;">Printing Co-ordinate Software</td>
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			<td height="21" style="height:21px;">Name</td>
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			<td height="21" style="height:21px;width:151px;">5ml printing syringe</td>
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			<td height="21" style="height:21px;width:151px;">&#945;MEM</td>
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			<td height="61" style="height:61px;width:151px;">Red Fluorescent Protein Mesenchymal Stem Cells (RFP-MSCs)</td>
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			<td style="width:104px;">RASMX-01201</td>
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automated robotic dispensing technique for surface guidance and bioprinting of cells

Questo manoscritto descrive l&#39;introduzione di elementi di orientamento cellule seguita dalla consegna diretta delle cellule di queste caratteristiche in un bioink idrogel utilizzando un sistema di erogazione automatico robotizzato. La particolare bioink stata selezionata in quanto consente alle cellule di sedimentare verso e rilevano le caratteristiche. Il sistema di erogazione bioprints cellule vitali in bioinks idrogel usando una contropressione assistito testina di stampa. Tuttavia, sostituendo la testina di stampa con uno stilo o bisturi affilata, il sistema di erogazione può anche essere impiegato per creare segnali topografici attraverso incisione superficiale. Il movimento dello stilo può essere programmata in passi di 10 micron in X, Y e Z. Le scanalature modellate erano in grado di orientare le cellule staminali mesenchimali, influenzando ad adottare una morfologia allungata in allineamento con la direzione dei solchi. Il patterning potrebbe essere progettato utilizzando il software tracciare linee rette, cerchi concentrici, e onde sinusoidali. In una procedura successiva, fibroesplosioni e le cellule staminali mesenchimali sono stati sospesi in una gelatina bioink 2%, per bioprinting in una contropressione guidato di estrusione della testina di stampa. Il bioink cuscinetto cella è stata poi stampata utilizzando le stesse coordinate programmate utilizzate per l&#39;incisione. Le cellule bioprinted erano in grado di rilevare e reagire alle caratteristiche incise come dimostrato dal loro orientamento allungata lungo la direzione delle scanalature incise.

I risultati rappresentativi dimostrano che il sistema di applicazione robotizzata contropressione assistita può essere utilizzato come bioprinter estrusione-based per effettuare sia l&#39;incisione superficiale e la stampa bioink (Figura 1 A). Può essere usato per la generazione di scanalature incise in superfici polimeriche, e per stampare successivamente un bioink cuscinetto cellule direttamente alle caratteristiche (Figura 1 B e C). <p class="jo...

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.

Tecnica Automated robotica erogazione di orientamento della superficie e Bioprinting di celle

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

6.4K Views.  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

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Ponteggi self-reporting per coltura cellulare 3-Dimensional

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Ricerca

Bioingegneria

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 Costruisce Utilizzando un tessuto-specifici idrogel Bioink

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

Robotic Mirror Therapy System for Functional Recovery of Hemiplegic Arms

Mirror therapy has been performed as effective occupational therapy in a clinical setting for functional recovery of a hemiplegic arm after stroke. It is conducted by eliciting an illusion through use of a mirror as if the hemiplegic arm is moving in real-time while moving the healthy arm. It can facilitate brain neuroplasticity through activation of the sensorimotor cortex. However, conventional mirror therapy has a critical limitation in that the hemiplegic arm is not actually moving. Thus, we developed a real-time 2-axis mirror robot system as a simple add-on module for conventional mirror therapy using a closed feedback mechanism, which enables real-time movement of the hemiplegic arm. We used 3 Attitude and Heading Reference System sensors, 2 brushless DC motors for elbow and wrist joints, and exoskeletal frames. In a feasibility study on 6 healthy subjects, robotic mirror therapy was safe and feasible. We further selected tasks useful for activities of daily living training through feedback from rehabilitation doctors. A chronic stroke patient showed improvement in the Fugl-Meyer assessment scale and elbow flexor spasticity after a 2-week application of the mirror robot system. Robotic mirror therapy may enhance proprioceptive input to the sensory cortex, which is considered to be important in neuroplasticity and functional recovery of hemiplegic arms. The mirror robot system presented herein can be easily developed and utilized effectively to advance occupational therapy.

We developed a real-time mirror robot system for functional recovery of hemiplegic arms using automatic control technology, conducted a clinical study on healthy subjects, and determined tasks through feedback from rehabilitation doctors. This simple mirror robot can be applied effectively to occupational therapy in stroke patients with a hemiplegic arm.

Sistema di terapia robotica Specchio per il recupero funzionale di emiplegici Arms

Mirror therapy has been performed as effective occupational therapy in a clinical setting for functional recovery of a hemiplegic arm after stroke. It is ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Automatizzato robot Liquid Handling Assembly di dispositivi modulari del DNA

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

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.

Vascularized microfluidic multimateriali per l'ingegneria dei tessuti e Organoids

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.

Multimateriali di cartilagine e pelle tessuto analoghi utilizzando un romanzo passivo unità tecnica di miscelazione per Bioink Precellularization

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

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

Uso di High-Throughput Microbioreactor sistema automatizzato per la produzione del modello IgG1 in cellule CHO

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, manipulate, and follow single cells in the brain tissue with high resolution over time. This task is extremely challenging due to the complexity of tissues in the brain. We have recently developed a robot, that guide a microinjection needle into brain tissue upon utilizing images acquired from a microscope to deliver femtoliter volumes of solution into single cells. The robotic operation increases resulting an overall yield that is an order of magnitude greater than manual microinjection and allows for precise labeling and flexible manipulation of single cells in living tissue. With this, one can microinject hundreds of cells within a single organotypic slice. This article demonstrates the use of the microinjection robot for automated microinjection of neural progenitor cells and neurons in the brain tissue slices. More broadly, it can be used on any epithelial tissue featuring a surface that can be reached by the pipette. Once set up, the microinjection robot can execute 15 or more microinjections per minute. The microinjection robot because of its throughput and versality will make microinjection a broadly straightforward high-performance cell manipulation technique to be used in bioengineering, biotechnology, and biophysics for performing single-cell analyses in organotypic brain slices.

This protocol demonstrates the use of a robotic platform for microinjection into single neural stem cells and neurons in brain slices. This technique is versatile and offers a method of tracking cells in tissue with high spatial resolution.

Manipolazione di singole cellule staminali neurali e neuroni nelle fette cerebrali utilizzando la microiniezione robotica

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, ...

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.

Bioprinting 3D di astrociti corticali murini per l'ingegneria del tessuto neurale

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

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.