The authors thank the support of the Zell Family Foundation. We recognize support from the Northwestern Memorial Foundation Dixon Translational Research Grants Initiative, the American Society of Transplant Surgeon&#8217;s Faculty Development Grant, and a Research Grant for the Young Investigator from the National Kidney Foundation of Illinois. We acknowledge support from the Robert R. McCormick Foundation. This work was also supported by NIDDK K08 DK10175 to J.A.W. Imaging and histology cores used for this research is supported by the Mouse Histology and Phenotyping Laboratory, Electron Probe Instrumentation Center (EPIC), and Simpson Querrey Institute Equipment Core at Northwestern University, and a Cancer Center Support Grant (NCI CA060553). The authors would like to acknowledge the Northwestern University Microsurgery Core for rodent kidney procurements. Evaluation of renal tubular epithelia morphology following recellularization conducted in the Fluorescence Microscopy Shared Resource supported by P30 CA118100.

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The authors have nothing to disclose.

Die beschriebene Dezellularisierung Protokoll konsistent erzeugt eine ganz azellulären Nieren ECM, die als 3D-Vorlage für die Kultur von menschlichen Nierenrinden Tubulusepithelzellen (beide proximalen und distalen Tubulus-abgeleitete) dient, zusätzlich zu den vaskulären endothelialen Zellen 7,17. Die Kanüle versehen Nierengefäßsystem dient als wesentliches Merkmal für eine gleichmäßige Abgabe von Reagenzien und Zellen in der gesamten Gerüst in einem Bioreaktor Set-up, so dass die Durchblutung Deze...

An erratum was issued for <a href="http://www.jove.com/video/53271/epithelial-cell-repopulation-preparation-rodent-extracellular-matrix">Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development</a>.
The human RCTEC/RCTE cell stocks used here and in Caralt et al., 2015, Uzarski et al., 2015 were originally provided by Dr. Loghman-Adham (then at St. Louis University) to co-author Dr. Wandinger-Ness (University of New Mexico) through an MTA in 2001. The SV40 immortalized human RCTEC/RCTE cells were characterized as being of distal tubule cell line as detailed in Loghman-Adham et al., 2003. On the basis of recent short tandem repeat (STR) DNA sequencing (Performed by IDEXX BioResearch) of the earliest passages of the RCTEC/RCTE cell stocks it became evident the cells were of mixed lineage. Further analyses of PCR products using QIAxcel capillary electrophoresis demonstrated the presence of a canine product. The product was sequenced and established to be of canine origin. A set of canine specific STR markers were compared to the sample and showed that the sample had a genetic profile with 92% identity to the MDCK cell line.&#160; Later cell stocks that were used in the present publication were STR profiled and showed drift to 100% MDCK lineage. Despite being of canine origin, rather than human as was previously thought, MDCK is similarly a distal tubule epithelial cell line. For this reason, previous interpretation and conclusions drawn using these cells here and in Caralt et al., 2015, Uzarski et al., 2015 remain sound, but for purposes of rigor, reproducibility and experimental validation by others we report on this misidentification. This information will also be reported for listing on the International Cell Line Authentication Committee (ICLAC) database (http://iclac.org).

Erratum: Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

Da die Zahl der Patienten mit end-stage Nierenversagen weiter zu erhöhen, gibt es eine starke und wachsende Knappheit in der Zahl der Spendernieren für die Transplantation zur Verfügung. Die Unfähigkeit, die Nachfrage einer stetig steigenden Zahl von Bewerbern zu treffen für Nierentransplantation Warteliste hat sich die Forschung in Niere Orgel Technik mit dem Ziel der Entwicklung von kundenspezifischen, implantierbare Nierentransplantate auf Anfrage 1,2 aufgefordert werden. Aufbau funktionierender Nierengewebe aus patienteneigenen Zellen würde die Notwendigkeit des lebenslangen Immunsuppression zu beseitigen, verringern die Menge der Zeit verbringen die Patienten an der Dialyse wartet auf eine Nierentransplantation und erstrecken sich lebensrettende Transplantation, mehr Patienten mit chronischer Nierenerkrankung. Der erste Schritt zur Verwendung Bioengineering Patienten stammenden Zellen, die ein Nierengewebe ist es, ein Gerüst, das als Stützsubstrat für Nierenparenchym dient entwickeln (zB Rohr epitheLiAl), Stroma-Fibroblasten und Gefäßzellwachstum. Biomaterial Gerüste aus natürlichen extrazellulären Organ Matrices (ECM) abgeleitet haben mehrere Eigenschaften, die sie für den Einsatz im Tissue Engineering, einschließlich ihrer natürlichen biologischen Zusammensetzung wünschenswert machen; entsprechende Makro- und Mikrostruktur, um physiologische Funktion zu versehen; und zellulären Biokompatibilität Förderung der Zelladhäsion, Migration und Umbau 3 konstruktiven Gewebe. Eine vielversprechende Methode, um Gerüste für Nierengeweberegeneration zu erzeugen, ist durch Dezellularisierung von allogenen oder xenogenen Nieren, die einen Großteil der komplexen natürlichen Proteinzusammensetzung der Niere ECM 4 zu erhalten, behalten die inhärente Komplexität der Architektur der Orgel, und die Überwindung der Schwierigkeiten mit Bottom zugeordnet up-Engineering von dicken cellularized Gewebe durch die Bereitstellung einer Gefäßversorgung in Entwicklungs Zellen nach Gerüst Rezellularisierung 5. Perfusion Dezellularisierung ist ein Verfahren inwelche Detergentien, Enzyme oder andere Zellstörende Lösungen werden gleichmäßig durch das Gefäßnetzwerk des Organs 6 geliefert. Diese Strategie hat sich als effizienter Prozess eingerichtet, um azellulären organ ECM-Gerüste als dreidimensionale (3D), biologische Schablonen für ganzen Organ Engineering 6-8, wie durch die Entwicklung von zellfreien Nieren Vorlagen von weggeworfenen menschlichen Nieren 9 belegt abzuleiten und xenogenen Nieren von Großtier (zB Schwein 10, Ziege 11) und Nagetierquellen 12 erhalten. Insbesondere die Verwendung von kleinen Tiermodellen wie Nagetiere erfordert weniger Zellen und Kulturmedien, die besonders nützlich für die Organ Rezellularisierung Studien, in denen Zellzahlen sind in der Regel begrenzt ist, wie es der Fall mit Stammzellen abgeleiteten Geweben. Das Ziel des beschriebenen Dezellularisierung Protokolls ist es, eine azelluläre Nieren ECM, die als 3D-Gerüst-System zur Regeneration von Nieren Structur verwendet werden können, zu erzeugenES, einschließlich Nephron Tubuli, die im vorliegenden Beispiel mit der menschlichen Nierenrinden Tubulusepithelzellen (RCTE) Zellen wieder besiedelt werden. Wir zuvor beschrieben unserer strengen Bewertung eines optimalen Waschmittelbasis Rattenniere Dezellularisierung Protokoll 7, die mehr schneller (ca. 1 Tag) als andere Methoden zuvor berichtet (Ross et al .- 5 Tage 12, Song et al .- 4,5 Tage 13), und setzt die Orgel zu einer deutlich geringeren Konzentration (0,1%) des Denaturierungsmittels Natriumdodecylsulfat (SDS) bei Dezellularisierung als frühere Berichte 12-15. Eine begrenzte Anzahl von Studien haben die Verwendung von Nagetier-Nieren Dezellularisierung und die anschließende Verwendung als 3D-Gerüst für Zell Repopulation beschrieben (Bewertung anderswo 1) 12-16. In diesem Protokoll stellen wir eine detaillierte Beschreibung unserer zuvor ermittelten optimalen Dezellularisierung Strategie zur Herstellung von azellulären NierengerüsteSprague Dawley-Rattennieren 7. Mit Hilfe speziell entworfenen Perfusion Bioreaktoren in der Lage Dual Aussaat und Pflege Perfusionskultur 17. recellularize wir die azellulären Nieren Gerüste mit menschlichen RCTE Zellen, die konsequent die röhrenförmige Komponente wieder zu bevölkern in diesen dezellularisierten Matrizen, vermehren und überleben in Perfusionskultur über eine Woche. Wir die Verwendung der Resazurin Perfusion Assay zeigen ferner - einem kostengünstigen, nicht-zytotoxischen und nicht-invasive metabolische Beurteilung zuvor für Cytotoxizität verwendet Beispiele 17 -, um eine Anzeige der Lebensfähigkeit der Zellen und die Proliferation innerhalb der rezellularisiertes Nieren über die Zeit 7 bereitzustellen.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TRITON X-100, Proteomics Grade, AMRESCO</td>
			<td>VWR</td>
			<td>M143-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate solution, BioUltra, for molecular biology, 20% in H2O</td>
			<td>Sigma-Aldrich</td>
			<td>05030</td>
			<td></td>
		</tr>
		<tr>
			<td>Peracetic acid solution, purum, ~39% in acetic acid (RT)</td>
			<td>Sigma-Aldrich</td>
			<td>77240</td>
			<td>Peracetic is flammable and corrosive. Prepare within a fume hood using appropriate personal protective equipment (e.g. gloves, goggles).</td>
		</tr>
		<tr>
			<td>200 proof ethanol</td>
			<td>VWR</td>
			<td>V1001TP</td>
			<td></td>
		</tr>
		<tr>
			<td>Sigmacote, siliconizing reagent for glass and other surfaces</td>
			<td>Sigma-Aldrich</td>
			<td>SL2</td>
			<td>For treatment of bioreactor reservoirs. Referred to in text as siliconizing reagent.</td>
		</tr>
		<tr>
			<td>DMEM/F12 media</td>
			<td>Life Technologies</td>
			<td>11320-033</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning cellgro Fetal Bovine Serum Premium, Mediatech</td>
			<td>Corning</td>
			<td>35-010-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution, 100X 10,000 I.U. Penicillin 10,000 &#181;g/mL Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express (1X), Phenol Red</td>
			<td>Life Technologies</td>
			<td>12605-028</td>
			<td>Referred to in text as cell dissociating enzyme.</td>
		</tr>
		<tr>
			<td>Trypan Blue Stain (0.4%)</td>
			<td>Life Technologies</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Resazurin sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>R7017</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex L/S Digital Drive, 600 RPM, 115/230 VAC</td>
			<td>Cole-Parmer</td>
			<td>EW-07522-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex L/S large cartridges for 07519-05 and -06 pump heads.</td>
			<td>Cole-Parmer</td>
			<td>EW-07519-70</td>
			<td>Referred to in text as large pump cartridge.</td>
		</tr>
		<tr>
			<td>Masterflex L/S 8-channel, 4-roller cartridge pump head.</td>
			<td>Cole-Parmer</td>
			<td>EW-07519-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight 6&#34; specimen forceps, serrated</td>
			<td>VWR</td>
			<td>82027-438</td>
			<td></td>
		</tr>
		<tr>
			<td>*Kidney perfusion bioreactor</td>
			<td>WilMad Labglass</td>
			<td>*Custom designs</td>
			<td>Bioreactors are produced as described by WilMad Labglass. The designs have been described in depth in a previous publication.</td>
		</tr>
		<tr>
			<td>Perfusion Circuit Components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24 G x 0.75 in. BD Insyte Autoguard shielded IV catheter (0.7 mm x 19 mm) made of BD Vialon biomaterial. Has notched needle. (50/sp, 200/ca)</td>
			<td>BD Biosciences</td>
			<td>381412</td>
			<td>Referred to in text as 24 gauge catheter.</td>
		</tr>
		<tr>
			<td>Masterflex PharMed BPT Tubing, L/S #14, 25&#39;</td>
			<td>Cole-Parmer</td>
			<td>HV-06508-14</td>
			<td>Referred to in text as peristaltic pump tubing.</td>
		</tr>
		<tr>
			<td>Peroxide-Cured Silicone Tubing, 1/16&#34;id X 1/8&#34;OD, 25 Ft/pack</td>
			<td>Cole-Parmer</td>
			<td>HV-06411-62</td>
			<td>Referred to in text as 1/16&#34; ID silicone rubber tubing.</td>
		</tr>
		<tr>
			<td>Masterflex platinum-cured silicone tubing, L/S 14, 25 ft.</td>
			<td>Cole-Parmer</td>
			<td>HV-96410-14</td>
			<td>Referred to in text as thick-walled 1/16&#34; ID silicone rubber tubing.</td>
		</tr>
		<tr>
			<td>VWR Silicone Tubing, 1/4&#34; ID x 0.5&#34; OD</td>
			<td>VWR</td>
			<td>89068-484</td>
			<td></td>
		</tr>
		<tr>
			<td>Acro 50 Vent Filters, Pall Life Sciences</td>
			<td>VWR</td>
			<td>28143-558</td>
			<td>Referred to in text as 0.2 micron vent filter.</td>
		</tr>
		<tr>
			<td>Cole-Parmer Luer Adapters, Male Luer Lock to 1/8&#34; ID, Nylon, 25/Pk</td>
			<td>Cole-Parmer</td>
			<td>T-45505-11</td>
			<td>Referred to in text as male Luer lock to 1/8&#34; barbed adapter.</td>
		</tr>
		<tr>
			<td>Cole-Parmer Luer Accessory, Male Luer Plug, Nylon, 25/Pk</td>
			<td>Cole-Parmer</td>
			<td>EW-45505-58</td>
			<td></td>
		</tr>
		<tr>
			<td>Female luer x female luer adapter, Nylon, 25/pk</td>
			<td>Cole-Parmer</td>
			<td>EW-45502-22</td>
			<td>Referred to in text as female Luer x female Luer adapter.</td>
		</tr>
		<tr>
			<td>Cole-Parmer Luer Accessory, Female Luer Cap, Nylon, 25/Pk</td>
			<td>Cole-Parmer</td>
			<td>EW-45502-28</td>
			<td></td>
		</tr>
		<tr>
			<td>Smiths Medical Large Bore Hi-Flo Stopcocks # MX4311L - 3-Way Hi-Flo Stopcock with Extended Male Luer Lock, Non-DEHP Formulation, Latex Free (LF), Lipid Resistant, Non-PVD, 50/cs</td>
			<td>Careforde Healthcare</td>
			<td>10254821</td>
			<td>Smiths Medical (vendor) catalogue number is #MX4311L.</td>
		</tr>
		<tr>
			<td>Other Components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL BD Luer-Lok disposable syringe with BD Luer-Lok&#8482; tip.</td>
			<td>BD Biosciences</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm x 10 mm Easy Grip Culture Dish</td>
			<td>BD Biosciences</td>
			<td>353001</td>
			<td>Used to draw cell suspension into syringe for cell seeding.</td>
		</tr>
	</tbody>
</table>

epithelial cell repopulation and preparation of rodent extracellular matrix scaffolds for renal tissue development

Dieses Protokoll beschreibt die Erzeugung von azellulären, noch biofunktionellen, Nierenextrazellulären Matrix (ECM) Gerüste, die als kleine Modellsubstrate für Orgel angelegten Gewebeentwicklung sind. Sprague Dawley-Rattennieren werden durch Einführen eines Katheters in die Nierenarterie über 26 h kanüliert und mit einer Reihe von niedrigkonzentrierten Waschmitteln (Triton X-100 und Natriumdodecylsulfat (SDS)) perfundiert, um intakte, ganze Niere Gerüste mit intakten abzuleiten perfundierbaren Gefäßsystem, Glomeruli und Nierentubuli. Folgende Dezellularisierung wird das Nierengerüst in einem speziell entworfenen Perfusionsbioreaktor Gefäß platziert und der katheterisiert Nierenarterie an eine Perfusionskreislauf, bestehend aus miteinander verbunden sind: einer peristaltischen Pumpe; Schlauch; und wahlweise Sonden für pH, gelöster Sauerstoff und Druck. Nach Sterilisieren des Gerüsts mit Peressigsäure und Ethanol, sowie den Ausgleich der pH-Wert (7,4), der Niere Gerüst zum Impfen mittels Perfusion von Kulturmedium in einem larg zubereitetE-Kapazität bei 37 ° C gehalten und 5% CO 2. Vierzig Millionen Nierenrinden Tubulusepithelzellen (RCTE) Zellen durch die Nierenarterie durch das Gerüst unter Hochstrom (25 ml / min) und Druck (~ 230 mm Hg) für 15 min vor der Verringerung der Fließgeschwindigkeit auf einen physiologischen spritzt und rasch perfundierten (4 ml / min). RCTE Zellen besiedeln vor allem die Rohr ECM Nische in der Nierenrinde, vermehren sich und bilden Rohr Epithelstrukturen über sieben Tagen Perfusionskultur. A 44 uM Resazurinlösung in Kulturmedium wird durch die Niere für 1 h bei Medium tauscht perfundiert, um eine fluorometrische, redox-basierte metabolische Beurteilung der Zell-Lebensfähigkeit und Proliferation während Tubulogenese bereitzustellen. Die Niere Perfusionsbioreaktor ermöglicht nicht-invasive Entnahme von Medium für biochemische Bewertung, und mehrere Einlassöffnungen ermöglichen alternative Rück Aussaat durch die Nierenvene oder Harnleiter. Diese Protokolle können verwendet werden, um Nieren Gerüste mit einem recellularize werdenVielzahl von Zelltypen, einschließlich vaskulären Endothelzellen, Tubulusepithelzellen und stromalen Fibroblasten zur schnellen Auswertung innerhalb dieses Systems.

Nieren nacheinander mit Wasser durchströmt und verdünnte Detergenslösungen (1% Triton X-100, 0,1% SDS) gemäß einer vorher festgelegten, optimalen Dezellularisierung Protokoll (siehe 1A, B) 7, wird über einen 26 Stunden-Zeitraum zunehmend transparent, wie in 2A gezeigt. Das resultierende acellular Nierengerüst ist frei von Zellen und erhält eine zusammenhängende Nieren ECM durch eine intakte Nierenkapsel, die unbeschädigt ist im Anschluss an die Perfusion-Protokoll u...

Watch this Scientific Journal Video about Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development at JoVE.com

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

Epithelialen Zellwiederbevölkerung und Herstellung von Nagetier extrazellulären Matrix Scaffolds für Nierengewebe Entwicklung

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

epithelial-cell-repopulation-preparation-rodent-extracellular-matrix

Research

JoVE Journal

Bioengineering

15.9K Aufrufe.  Northwestern University. This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

Serie: Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Berührungslose, Label-freie Überwachung von Zellen und extrazellulären Matrix mittels Raman-Spektroskopie

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Forschung

Biotechnik

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.

Selbstberichts Gerüste für 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 ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

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

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

Technik Dreidimensionale Epithelgewebe Eingebettet in extrazellulären Matrix

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

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

Herstellung von extrazellulären Matrix Proteinfasern für Brillouin-Spektroskopie

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Tunable Hydrogele aus Pulmonary Extrazellulärmatrix für 3D-Zellkultur

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Die Herstellung von extrazellulären Matrix-derived Foams und Mikroträgern als gewebespezifische Zellkultur und Delivery-Plattformen

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Herstellung einer Niere Cortex extrazelluläre Matrix abgeleitet Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

Herstellung von Decellularized Knorpel abgeleitet Matrix Gerüste

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the interface with a liquid core. The latter is removed post-printing, leaving behind a hollow tube. Integrating an additive manufacturing technique (like the one described here with tailor-made [bio]inks, which structurally and biochemically mimic the native extracellular matrix [ECM]) is an important step towards advanced tissue engineering. However, precise fabrication of well-defined structures requires tailored fabrication strategies optimized for the material in use. Therefore, it is sensible to begin with a set-up that is customizable, simple-to-use, and compatible with a broad spectrum of materials and applications. This work presents an easy-to-manufacture core/shell nozzle with luer-compatibility to explore core/shell printing of woodpile structures, tested with a well-defined, alginate-based scaffold material formulation.

Presented here is a simple-to-use, core/shell, three-dimensional bioprinting set-up for one-step fabrication of hollow scaffolds, suitable for tissue engineering of vascular and other tubular structures.

Core/Shell DruckGerüste für Tissue Engineering von Röhrenstrukturen

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

Pankreasgewebe-abgeleitete extrazelluläre Matrix Bioink für den Druck 3D-Zell-Laden Pankreasgewebe Konstrukte

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...