We thank the laboratory of Dr. Stephen Moss for providing embryonic rat brain tissues. M.D.T. designed the original protocol. This work was funded by National Institutes of Health P41 Tissue Engineering Resource Center Grant EB002520. K.C. was supported with Postdoctoral Fellowship from German Research Foundation (DFG) (CH 1400/2-1).

Das Hirngewebe Isolation Protokoll wurde von der Tufts University Institutional Animal Care und Verwenden Committee genehmigt und entspricht den National Institutes of Health Guide für die Pflege und Verwendung von Labortieren (Institutional Animal Care und Verwenden Committee B2011-45). 1. Silk Scaffold Vorbereitung <ol><li> Herstellung von Seidenlösung von Bombyx mori Kokons wie zuvor 19,20 beschrieben. <ol><li> Schneiden Sie die einzelnen Kokon in 8 gleiche Teile mit einer Schere. Verwenden Sie ca. 11 abhaspelbare 5 g fragmentierten Kokons. (15 min) </li><li> Bereiten 2 l 0,02 M Na 2 CO 3 -Lösung und bringen es zum Kochen unter Verwendung einer Heizplatte. (15 min) </li><li> Man wiegt 5 g fragmentierten Kokons und kochen Sie sie in Na 2 CO 3 Lösung für 30 min. Rühren Sie das kochende Seiden alle paar Minuten mit einem Spachtel. Dieser Schritt, auch als de-Gummierung, reinigt Seidenfibroin von hydrophilen Proteinen, sericins. (30 min) </li><li&gt; Wringen Sie das Fibroin von Hand und spülen Sie ihn in destilliertem Wasser mindestens dreimal zu waschen Sie das restliche Sericin und Chemikalien. (5 Min) </li><li> Setzen Sie den nassen Fibroin auf eine Petrischale und trocknen Sie das Fibroin-Extrakt in der Flow-Haube O / N. </li><li> Am nächsten Tag wiegen die Trockenmasse Fibroin und legen Sie das Fibroin in einem Becherglas. (15 min) </li><li> Um das Fibroin in 9,3 M LiBr-Lösung, berechnen die erforderliche Volumen (in ml) LiBr durch Multiplikation der Masse des trockenen Fibroin von 4. Gießen Sie langsam auflösen LiBr-Lösung über die Seidenfibroin und tauchen alle Fibroinfasern mit Spachtel. Decke den Becher, um Verdunstung zu vermeiden und ihn bei 60 ° C für 4 Stunden, damit die Fasern sich aufzulösen. (15 min) </li><li> Mit der Spritze, sammeln die Fibroinlösung aus dem Becherglas und laden Sie es in die MWCO 3.500 Dialyse-Kassetten. Zuführen Dialyse gegen destilliertes Wasser für 48 Stunden. Wechseln Sie das Wasser alle paar Stunden. </li><li> Mit der Spritze, sammeln die Fibroinlösung ausdie Kassetten in 50 ml konischen Röhrchen und zentrifugiert zweimal bei 9000 UpM (~ 12.700 xg) bei 4 ° C für 20 min. Gießen Sie den Überstand in ein frisches Röhrchen nach jedem Zentrifugationsschritt und entsorgen Sie das Pellet. (40 min) </li><li> Messung der Konzentration des Fibroin durch Abschätzen der Trockenmasse. Gießen Sie 1 ml Seidenlösung in einen wiegen Boot. Trocknen der Probe im Ofen bei 60 ° C für 2 Std. Das trockene Seidenfibroin und berechnet die Konzentration des Seidenfibroinlösung durch Multiplikation des Gewichts durch 100. Der erwartete Konzentration Seide Lösung 6-9% (w / v). </li><li> Stellen Sie die Seiden Konzentration auf 6% (w / v), indem sie in destilliertem Wasser verdünnt. Haltepunkt: Die Flüssigkeit Seidenfibroin kann bei 4 ° C für bis zu einem Monat in einem geschlossenen Behälter gelagert werden. </li></ol></li><li> Herstellung von porösen Gerüsten aus Seide Lösung. <ol><li> Sieb des körnigen NaCl zur Trennung der Granulate bemessen 500-600 &amp; mgr; m, die in späteren Schritten verwendet werden sollen. Entsorgen Sie das Granulats bemessen unterhalb 500 um und oberhalb von 600 &amp; mgr; m. (15 min) </li><li> Pour 30 ml 6% Seide Lösung in das Polytetrafluorethylen (PTFE) Form (Abbildung 1). Streuen Sie vorsichtig 60 g gesiebten NaCl über die Seide. Tippen Sie auf den Container, um eine gleichmäßige Schicht von Salz zu erhalten. Man inkubiert 48 Stunden bei Raumtemperatur, um die Seide zu polymerisieren. </li><li> Legen Sie das Gerüst haltigen PTFE-Form im Ofen bei 60 ° C für 1 Stunde, um die Polymerisation abzuschließen und zu verdampfen restliche Flüssigkeit. </li><li> Platzieren des Inhalts des PTFE-Form in ein Becherglas mit 2 l destilliertem Wasser für 48 Stunden zum Auslaugen des Salzes. Wechseln Sie das Wasser 2-3 mal pro Tag. Entfernen Sie die Schwamm Gerüste aus den Formen, wenn das Salz vollständig ausgelaugt Haltepunkt:. Die Schwämme können gespeichert unter Wasser bei 4 ° C in einem geschlossenen Behälter, um die Gerüste vor dem Austrocknen zu verhindern. </li><li> Wenn Sie fertig sind, schneiden Sie die Gerüste mit 5 mm Durchmesser Biopsie Punsch. Vorgeschnitten die Gerüste auf etwa 2 mm in der Höhe zu erreichen. PuNch die Mitte des Gerüstes mit 2 mm Durchmesser Biopsie Stempel (2A). (1 h) </li><li> Autoklaven werden die Gerüste in Wasser eingetaucht, um sie zu sterilisieren (Nasszyklus, 121 ° C, 20 min) Haltepunkt:. Die Schwämme können gespeichert unter Wasser bei 4 ° C in einem geschlossenen Behälter, um die Gerüste vor dem Austrocknen zu verhindern. </li><li> Vor dem geplanten Zellaussaat, tauchen die Gerüste in sterile 0,1 mg / ml Poly-D-Lysin (PDL) Lösung. Inkubation für 1 h bei 37 ° C. </li><li> Waschen Sie die Gerüste 3x mit phosphatgepufferter Salzlösung (PBS), um nicht gebundene PDL zu entfernen. (30 min) </li></ol></li></ol> 2. Isolierung kortikale Neuronen der Ratte <ol><li> Sezieren Rinden vom embryonalen Tag 18 (E18) Sprague-Dawley-Ratten, wie vorher durch Pacifici und Peruzzis 27 beschrieben nach bestätigten Tier-Protokoll erhalten wird. (2 h) </li><li> Für 20 min bei 10 inkubieren 3 cortices in 5 ml 0,25% Trypsin mit 0,3% DNase I (aus Rinderpankreas)7 ° C. </li><li> Inaktivierung des Trypsins durch Zugabe von gleichen Volumen von 1 mg / ml Sojabohnen-Protein. </li><li> Man reibt den Kortex mit 10 ml Pasteurpipette durch Auf- und Abpipettieren 20 Mal, bis eine Einzelzellsuspension erzeugt wird. Seien Sie sanft und Luftblasenbildung zu vermeiden. (10 min) </li><li> Zentrifugieren Sie die Zellsuspension bei 127 xg für 5 min. </li><li> Re-suspend das Zellpellet in 10 ml Kulturmedium (Neurobasalmedium, 1x B27 ergänzen, 1x Glutamax, 1% Penicillin / Streptomycin). Zählen Sie die Zellen. Die erwartete Zellkonzentration etwa 2x10 7 / ml. (20 min) </li></ol> 3. Konstruieren Montage und Kultur <ol><li> Scaffold Impfen mit Zellen. <ol><li> Verschieben Sie die sterile Gerüsten und alle notwendigen Utensilien in einer Zellkultur Kapuze. Mit einer sterilen Pinzette legen die Gerüste in einer 96-Well-Zellkulturplatte Zuweisung eines Gerüst pro Vertiefung. (10 min) </li><li> Tauchen die Gerüste in Zellkulturmedium equilibriert diese vor der Saatgutzelleing. Inkubation für 1 h bei 37 ° C. (10 min) </li><li> Saugt den Überschuß des Mediums von den Gerüsten. </li><li> Apply 100 ul Zellsuspension / Schafott. (10 min) </li><li> Inkubieren der Zellen bei 37 ° CO / N, um die Zellanheftung zu dem Gerüst zu ermöglichen. </li><li> Am folgenden Morgen saugen die fraktionslosen Zellen und gelten 200 ul / Vertiefung frisches Kulturmedium. (10 min) </li></ol></li><li> Gerüst Einbettung mit Kollagenmatrix. (2 h) <ol><li> Zeigen 10x PBS, Wasser, 1 N NaOH und Rattenschwanz-I-Kollagen auf Eis. Bereiten Sie eine Arbeitslösung von Kollagen nach den Anweisungen des Herstellers. Pflegen Sie auf Eis, bis die Zellen ausgesät Konstrukte sind bereit (bis 1 h). </li><li> Entfernen Sie die Zelle ausgesät Seidenkonstrukte aus dem Inkubator und saugen Sie den Überschuß von Medium. </li><li> Mit einer sterilen Pinzette übertragen Sie die Gerüste zu den leeren Brunnen auf dem Well-Platte und tauchen jedes Gerüst in 100 ul 3 mg / ml Kollagenlösung. Setzen Sie den Gewebekulturplatte zurückim Brutschrank für 30 Minuten, um die Polymerisation des Kollagens zu erlauben. </li><li> Apply 100 ul vorgewärmten Zellkulturmedium / well. Kultur die Konstrukte für eine Woche Wechsel des Mediums jeden Tag durch den Austausch nur die Hälfte des Volumens des Mediums. </li></ol></li></ol> 4. Mikroskopie Analyse <ol><li> Live / Dead-Färbung (1 Stunde) <ol><li> Bereiten Stammlösung von Propidiumiodid (PI) 1 mg / ml (1,5 mm) in destilliertem Wasser. </li><li> Bereiten Stammlösung von Fluoresceindiacetat (FDA) 2 mg / ml in Aceton. </li><li> Bereiten Sie Arbeitslösung, die 10 &amp; mgr; M PI und 0,15 uM FDA in PBS verdünnt. Vorwärmen die Lösung auf 37 ° C in einem Wasserbad. </li><li> Waschen Sie die Zellen mit vorgewärmten PBS, um die Serum-Esterasen zu entfernen. Saugen Sie das PBS. </li><li> Tragen Sie die vorgewärmte Arbeitslösung auf die Zellen für 2 Minuten und waschen Sie es mit PBS. </li><li> Verwenden Epifluoreszenzmikroskop für Bild in die Zellen (PI ex λ = 490 nm, λ em = 570 nm; FDA ex λ = 490 nm, λ em = 514 nm). Der Fleck bleibt über 40 Minuten stabil. Längerer Färbung kann in unspezifische Anfärbung von PI-Aufnahme durch die Zellen und Co-Lokalisation von PI / FDA in Zellen, was zur Folge haben. </li></ol></li><li> Immunfärbung <ol><li> Bei dem gewünschten Zeitpunkt der Kultur zu fixieren Sie die Zellen mit 4% Paraformaldehyd (PFA) für 30 min bei RT. Aufgrund der Toxizität des PFA Rauch Der Fixierschritt sollte im Abzug durchgeführt werden. </li><li> Waschen Sie Gerüste mit 3x PBS Haltepunkt:. Die PFA-Fest Konstrukte können in PBS bei 4 ° C für mehrere Tage, bevor mit weiteren Schritten fortfahren gespeichert werden. </li><li> Die Gerüste in 0,2% Triton, 0,25% Rinderserumalbumin (BSA) in PBS mit Primärantikörper O / N Inkubieren bei 4 ° C. </li><li> Am folgenden Tag entsorgen Sie die Farblösung und waschen Sie die Gerüste 3 x 30 min mit PBS, um den ungebundenen primären Antikörper zu entfernen. </li><li> Der Proben mit einem sekundären Antikörper in 0,2% verdünnt Triton, 0,25% BSA in PBS für 2 inkubierenh bei RT. </li><li> Entfernen Sie die Farblösung und waschen Sie die Gerüste 3 x 30 min mit PBS, um den ungebundenen sekundären Antikörper zu entfernen. </li><li> Bild die Gerüste mit einem konfokalen Mikroskop mit 20x-Objektiv, indem 100 &amp; mgr; z-Abschnitte der Probe mit 1 &amp; mgr; m Schritt. Um die dünne Neuriten die Bildauflösung sollte mindestens 1024 x 1024 Pixel groß sein zu visualisieren. </li></ol></li><li> RNA, DNA und Proteinisolierung Hinweis: RNA, DNA und Proteinisolierung wird mit einem handelsüblichen AllPrep DNA / RNA / Protein Mini Kit nach den Anweisungen des Herstellers durchgeführt. <ol><li> Mit einer sterilen Pinzette übertragen Sie die Gerüste aus der Kulturplatte in ein 2 ml sterilen Röhrchen. Legen Sie ein Gerüst pro Röhrchen. </li><li> Gib 200 ul RLT-Puffer zu jedem Röhrchen. </li><li> Stören das Konstrukt durch Fragmentierung mit sterilen Mikroschere. </li><li> Um das Gewebe zu homogenisieren gestört, übertragen den Inhalt des Rohrs, um die Spin-Säule. Spin für 2 min bei voller Geschwindigkeit in einer Mikrozentrifuge.Das Lysat wird homogenisiert, während es durch die Spinsäule passiert. </li><li> Sammeln Sie die Strömung durch und verwenden Sie es sofort an RNA, DNA und Protein nach dem Hersteller-Protokoll zu isolieren. </li><li> Quantifizierung der Ausbeute an Nukleinsäuren andere kompatible Methode (zB Nanodrop). </li><li> Quantifizierung der Proteinausbeute mit BCA-Protein-Assay gemäß den Anweisungen des Herstellers. Messen Sie die Ergebnisse der Test unter Verwendung von Mikroplatten-Reader bei einer Wellenlänge von 562 nm. Anmerkung: Die erwartete Ausbeute an Nukleinsäuren und Protein pro Konstrukt in Bezug auf die Zelldichte ist in Figur 4 dargestellt ist. </li></ol></li></ol>

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Here we described the guidelines to assemble a compartmentalized 3D brain-like tissue model. The model is characterized by dense polarized culture of neurons resulting in the development of two morphologically different compartments: one containing densely packed cell bodies, second containing pure axonal networks. Overall, the scaffold architecture and growth pattern mimic brain cortical tissue including the six laminar layers and white matter tracts21.
The donut-shaped silk protein-based tissue model allows for modifications and tuning of mechanical properties, versatile structural forms, hydrogel fillers and cellular components. Thus, this tissue model establishes a base platform for a wide range of studies. Silk is a favorable candidate for biomaterial platforms due to its biocompatibility, aqueous-based processing, and robust mechanical and degradation properties22. The silk matrix also serves as a stable anchor to reduce collagen gel contraction over time in culture. The properties such as silk concentration and porosity of the scaffold used in this model have been previously adjusted to achieve optimal cell growth and mechanical properties resulting in brain-like tissue elasticity18,23,24. We suggest to keep these parameters constant to ensure the successful outcome and reproducibility of the experiments.
The 3D neuronal network formation was achieved by combining two types of biomaterials with different mechanical properties: a stiff scaffold to provide neuronal anchoring and a softer gel matrix to permit axon penetration and connectivity in 3D. Selective material preferences of the silk scaffold and soft hydrogel provide the underlying principle for compartmentalizing the neurons from the axonal connections. Due to the inert nature of the silk scaffold, functionalization with poly-D-lysine is required for neuronal attachment. However, other cell adhesion promoting factors can be applied such as RGD25 or fibronectin26. To achieve the 3D network formation the silk scaffold needs to be filled with hydrogel in a timely manner as soon as neurons are attached to the scaffold. Likewise, the collagen matrix filler can be replaced by other hydrogels, thus allowing the study of the influence of 3D extracellular environments on axonal network formation to serve as a platform to evaluate novel hydrogels in terms of neuronal compatibility. Additionally, apart from rat cortical neurons other neuronal sources such as hippocampal neurons or induced pluripotent cell (iPSc)-derived neurons can be utilized. Moreover, the tissue model can be used to study heterocellular interactions by including glial cells along with neurons and to build more complex brain-like tissue.
As shown previously, our model can be utilized to evaluate neuronal functionality in 3D microenvironment with a variety of assays such as cell viability, gene expression, LC-MS/MS and electrophysiology, thus demonstrating physiological relevance of the model18. Other methods, which are frequently utilized to evaluate 3D tissue-engineered constructs, such as immunostaining and microscopy8,9,11 can also be used to assess cell distribution and extent of axonal network formation. However, it has to be noted, that due to the high density of the collagen matrix, the penetration of antibodies and depth of the imaging is limited to few hundred micrometers. Moreover, the signal to noise ratio may be affected by nonspecific background fluorescence. This can be overcome by using lipophilic cell tracers and genetically expressed fluorescent proteins which diminish the need for immunostaining11. Alternatively, the imaging can be performed with 2-photon microscopy instead of usual one-photon technique, which may reduce the signal loss, photobleaching, and can be extended to several hundred micrometers of depth.
Summarizing, the silk and collagen-based brain-like tissue offers a robust platform to study neuronal networks in 3D and is a starting point for the development of more advanced models of neurological disorders in the future. Independent from the mode of evaluation, the relative simplicity of this tissue model supports its utility, success and reproducibility.

Das Zentralnervensystem (ZNS) kann durch eine Vielzahl von Erkrankungen mit vaskulärer, strukturellen, funktionellen, infektiösen oder degenerativen berührt. Schätzungsweise 6,8 Millionen Menschen sterben jedes Jahr als Folge von neurologischen Erkrankungen, die eine wachsende sozioökonomische Belastung weltweit darstellt 1. Nur einige der Erkrankungen haben jedoch verfügbaren Behandlungen. Daher gibt es einen kritischen Bedarf an innovativen therapeutischen Strategien für Patienten mit neurologischen Störungen. Leider scheitern viele ZNS-Therapeutika gezielt in klinischen Studien, zum Teil auf der Nutzung der unzureichenden präklinischen Forschung Modelle, die nicht die Beurteilung der akuten und chronischen Auswirkungen mit physiologisch-relevanten Funktionsanzeigen zulassen. Trotz erheblicher Forschungsanstrengungen in den letzten Jahrzehnten, gibt es eine große Menge nicht bekannt über die Struktur und Funktion des ZNS. Um mehr Wissen zu erlangen, sind Tiermodelle häufig unsed zu modellieren pathologischen Zuständen, wie traumatische Hirnverletzungen (TBI) oder Demenz, vor allem in präklinischen Studien. Unterscheiden sich jedoch deutlich von Tieren Menschen sowohl in der Anatomie des ZNS sowie in der Funktion, die Genexpression und Stoffwechsel 2-4. Auf der anderen Seite, sind 2D-in-vitro-Kulturen die gemeinsame Methode zur Zellbiologie untersuchen und werden routinemäßig für die Wirkstoffforschung eingesetzt. Jedoch 2D Zellkulturen fehlt die Komplexität und die physiologische Bedeutung im Vergleich zu menschlichen Gehirns 5-7. Zwar gibt es keinen Ersatz für die niedrigen Kosten und die Einfachheit der 2D-Zellkulturstudien oder die Komplexität von Tiermodellen zur Verfügung gestellt, könnte 3D-Tissue Engineering verbesserte Forschungsmodelle zu generieren, um die Lücke, die in vitro und in vivo Ansätze zwischen der 2D besteht zu schließen. 3D-Tissue Engineering bietet mehrere physiologisch relevanten durch 3D-Zell-Zell-Interaktionen und extrazelluläre Signale von den Biomaterial Gerüsten versehen ist experimentellen Bedingungen. Despite die deutlichen Anzeichen dafür, hinter dem Wert von 3D-Kulturen, gibt es derzeit nur wenige 3D-ZNS-Gewebe-Modelle wie Stammzellen abgeleiteten organoiden Kulturen 8-10, neurospheroids 11 und verteilt Hydrogel Kulturen 12,13. Erweiterte technische Methoden, einschließlich Mehrschichtlithographie 14 und 3D-Druck 15 wurden für die Untersuchung der Lunge, Leber und Nierengewebe verwendet worden. Jedoch gibt es fehlende 3D CNS Modell für compartmentalized neuronale Wachstum zu ermöglichen, wie beispielsweise Nachahmen des kortikalen Architektur und Biologie. Getrennt Wachstum von Neuriten von neuronalen Zellkörpern zuvor in 2D-Kulturen unter Verwendung von Mikrofabrikations 16,17, die die Untersuchung der Axon-Trakt Tracing Calciumeinstrom, Netzarchitektur und Aktivitäten gezeigt. Diese Idee hat uns inspiriert, um einen 3D-polarisierte Nervengewebe in dem Zellkörper und axonalen Bahnen sind in verschiedenen Fächern imitiert die Schichtenarchitektur des Gehirns 18 befindet entwickeln </sup&gt;. Unser Ansatz basiert auf der Verwendung von einzigartigen Seide Gerüst Design, hohe Dichte von Zellen beherbergt in einem begrenzten Volumen und ermöglicht Auswuchs von Axonen dichten Netzes in einem Kollagengel basiert. Hier zeigen wir die volle Montageverfahren des Gehirns artiges Gewebe einschließlich der Gerüstherstellung und neuronalen Kultur.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Na2CO3</td>
			<td>Sigma-Aldrich</td>
			<td>223530</td>
			<td>-</td>
		</tr>
		<tr>
			<td>LiBr</td>
			<td>Sigma-Aldrich</td>
			<td>213225</td>
			<td>-</td>
		</tr>
		<tr>
			<td>MWCO 3500 dialysis cassettes&#160;</td>
			<td>Thermo Fisher</td>
			<td>66110</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Heating plate</td>
			<td>Fisher Scientific</td>
			<td>Isotemp</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Centrifuge 5804 R</td>
			<td>Eppendorf</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sieve</td>
			<td>Fisher Scientific</td>
			<td>-</td>
			<td>No. 270, No. 35, No. 30</td>
		</tr>
		<tr>
			<td>PTFE mold</td>
			<td>-</td>
			<td>-</td>
			<td>made in house (Figure 1) 10 cm diameter, 2 cm height</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>71382</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Biopsy Punch 5mm</td>
			<td>World Precision Instrument</td>
			<td>501909</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Biopsy Punch 2mm</td>
			<td>World Precision Instrument</td>
			<td>501908</td>
			<td>-</td>
		</tr>
		<tr>
			<td>poly-D-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P6407-5MG</td>
			<td>final concentration 10 ug/ml, dissolved in water</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D1283-500ML</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>T4049-500ML</td>
			<td>-</td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Roche</td>
			<td>10104159001</td>
			<td>final concentration 0.3%</td>
		</tr>
		<tr>
			<td>Soybean protein</td>
			<td>Sigma-Aldrich</td>
			<td>T6522-100MG</td>
			<td>final concentration 1 mg/ml</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>warm up to 37&#176;C before use</td>
		</tr>
		<tr>
			<td>B27 supplement 50x</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Penicilin Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Collagen I, rat tail, 100 mg</td>
			<td>Corning</td>
			<td>354236</td>
			<td>final concentration 3 mg/ml</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma-Aldrich</td>
			<td>S2770</td>
			<td>corrosive</td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Sigma-Aldrich</td>
			<td>P4170-10MG</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>Fluorescein Diacetate</td>
			<td>Sigma-Aldrich</td>
			<td>F7378-5G</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Olympus MVX10</td>
			<td>Olympus</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>toxic, final concentration 4%</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906-10G</td>
			<td>-</td>
		</tr>
		<tr>
			<td>anti-&#946;IIITubulin antibody&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T2200</td>
			<td>rabbit 1:500</td>
		</tr>
		<tr>
			<td>anti-rabbit Alexa-546</td>
			<td>Molecular Probes</td>
			<td>A11010</td>
			<td>goat 1:250</td>
		</tr>
		<tr>
			<td>goat serum</td>
			<td>Sigma-Aldrich</td>
			<td>G9023-5ML</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Leica SP2 confocal microscope</td>
			<td>Leica</td>
			<td>-</td>
			<td>objective 20x</td>
		</tr>
		<tr>
			<td>QIAshredder</td>
			<td>Qiagen</td>
			<td>79654</td>
			<td>-</td>
		</tr>
		<tr>
			<td>AllPrep DNA/RNA/Protein Mini Kit</td>
			<td>Qiagen</td>
			<td>80004</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ethyl alcohol, Pure</td>
			<td>Sigma-Aldrich</td>
			<td>E7023&#160;</td>
			<td>-</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>63689</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000c/2000 UV-Vis Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>BCA protein assay</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Plate reader Spectramax M2</td>
			<td>Molecular Devices</td>
			<td>-</td>
			<td>absorbance 562 nm</td>
		</tr>
		<tr>
			<td>Micro Scissors, Economy, Vannas-type</td>
			<td>TedPella</td>
			<td>1346</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

engineered 3d silk-collagen-based model of polarized neural tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Solide Seiden Schwämmen vorgeschnitten in die Donut-Form stellen eine einzigartige und einfache Idee, eine unterteilte Architektur ähnelt Nervengewebe (2A) zu erreichen. Das hochporöse Struktur des Gerüsts mit einer Porengröße von etwa 500 um entstehen außerordentlich großer Oberfläche ermöglicht die Aussaat und das Wachstum der hohen Zelldichte in einem kleinen Volumen (2x10 7 / ml) (2B). Zusätzlich wird die hohe Porosität des Trägermaterials ermöglicht die uneingeschränkte Diffusion von Nährstoffen und Abfallstoffen, was zu überlegenen Lebensfähigkeit dichten Zellkulturen über längere Zeiträume. Nach der Aussaat der Neuronen schnell und gleichmäßig zu befestigen, um die Oberfläche der Gerüstporen. Nach Zellanheftung abgeschlossen ist und die Zellen werden auf der Seide Substrat äquilibriert wird der Schwamm-Gerüst mit dem weichen Kollagenmatrix, um eine 3D-Umgebung für axonale Netzwerkbildung (Figur 3 bereitzustellen gefüllten </strong&gt;). In Abwesenheit der Kollagenmatrix der Kompartimentierung von Axonen und Zellkörper ist nicht möglich, Neuronen wachsen Erweiterungen nur an der Oberfläche der Poren, was zu gemischten Netzen mit 2D Flächen (3B) gefunden. Im Gegensatz dazu liefert das Kollagengel eine Maschenwerk, die umfangreiche axonalen Wachstums in 3D unterstützt, während neuronalen Zellkörpern bleiben mit dem Seiden Gerüst (3C) befestigt sind. Dieses Wachstumsverhalten führt zu Kompartimentierung der Zellkörper und reinem axonalen Netze (Figur 3D), die die grauen und weißen Substanz des Kortex im Gehirn ähnelt. Abgesehen von Mikroskopie kann die Konstrukte mit einer Vielzahl von anderen Methoden 18 ausgewertet werden, abhängig von der Frage, hinter dem Experiment. Zum Beispiel kann die Isolierung von DNA, RNA und Protein aus den Konstrukten leicht unter Verwendung kommerzieller Kits (Abbildung 4). Die Ausbeute an Nukleinsäuren und Protein per Konstrukt, hängt von der Anzahl der Zellen, der ursprünglich auf die Seide Gerüste geimpft. Je mehr Zellen ausgesät desto höher ist die Ausbeute an DNA, RNA und Protein. <img alt="Abbildung 1" src="/files/ftp_upload/52970/52970fig1.jpg" /> Abbildung 1. PTFE-Form für Seide Schwamm Gerüstherstellung verwendet Die Abmessungen:.. 10 cm Durchmesser, 2 cm Höhe <a href="https://www.jove.com/files/ftp_upload/52970/52970fig1large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 2" src="/files/ftp_upload/52970/52970fig2.jpg" /> Abbildung 2. 3D-Gehirn-ähnliche Gewebemodell. (A) Poröse Seiden Schwamm Schafott. (B) lebend / tot-Färbung von Neuronen am Tag 1 nach der Aussaat auf Seide Gerüst vor dem Kollagen (grün-lebende Zellen, red-dead-Zellen) die Einbettung. Panels auf der rechten Seite zeigen die magnification der Bereich im roten Rahmen eingefangen. <a href="https://www.jove.com/files/ftp_upload/52970/52970fig2large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 3" src="/files/ftp_upload/52970/52970fig3.jpg" /> Abbildung 3. Neuronale compartmentalized Auswuchs in der 3D-Gehirn-ähnliche Gewebemodell (A) Scaffold Fächer:. Red-Zellkörper Fach, Blau-axonalen Fach. (B) Die neuronale Wachstumsmuster am Tag 1 nach der Aussaat vor Kollagen Einbettung. (C). Neuronale Auswachsen am Tag 7 nach der Aussaat in den Zellkörper Fach. (D) Neuronale Auswuchs am 7. Tag nach der Aussaat im axonalen Kompartiment. Grün -. ΒIIITubulin <a href="https://www.jove.com/files/ftp_upload/52970/52970fig3large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version davon zu sehen</a>Abbildung. <img alt="Figur 4" src="/files/ftp_upload/52970/52970fig4.jpg" /> Abbildung 4. Erwartete Rendite aus (A) RNA und DNA, und (B) Protein pro Konstrukt in Bezug auf die Menge an Zellen ausgesät pro Gerüst (± SD, n = 5). <a href="https://www.jove.com/files/ftp_upload/52970/52970fig4large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen .</a>

Watch this Scientific Journal Video about Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue at JoVE.com

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

Engineered 3D Silk-Kollagen-basierten Modell von polarisiertem Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

engineered-3d-silk-collagen-based-model-of-polarized-neural-tissue

Research

JoVE Journal

Bioengineering

12.4K Aufrufe.  Tufts University. Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

Serie: Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Electric Field-gesteuerte gerichtete Wanderung von neuralen Vorläuferzellen in 2D-und 3D-Umgebungen

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Forschung

Biotechnik

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

Protokoll für Relative Hydrodynamische Beurteilung der Tri-Polymer Merkblatt Ventile

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Tissue Engineering eines Menschen 3D<em&gt; In-vitro-</em&gt; Tumor-Testsystem

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

Generierung und Pfropfen von Tissue Engineering Gefäße in einem Maus-Modell

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

Herstellung, Charakterisierung und Einsatzmöglichkeiten von 3D-Tissue-Engineering Menschen Ösophagus-Schleimhaut-Modell

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining&#39;s, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its in silico expansion provides an additional value for preclinical drug testing in more realistic conditions than in 2D cell culture.

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

Ein kombiniertes 3D-Gewebezüchtungen<em&gt; In Vitro</em&gt; /<em&gt; In Silico</em&gt; Lungentumormodell für Hintergründe in bestimmten Mutations Drug Wirksamkeit vorhersagen

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

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 von Knorpel und Haut-Gewebe-Analoga nutzen eine neuartige Passive Einheit Mischtechnik für Bioink Precellularization

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

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

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

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

Aussaat und Implantation von einem biosynthetischen Tissue-Engineering trachealen Transplantat in einem Mausmodell

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

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...

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.

3D-Bioprinting von murinen kortikalen Astrozyten für die Entwicklung von neuralähnlichem Gewebe

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