Martin Danner ist bekannt für seine Hilfe bei der Vorbereitung von Proben und Definition der Zelle Kultur Verfahren anerkannt. Die Autoren möchten Biesterfeld Spezialchemie GmbH (Hamburg, Deutschland), vor allem Robert Radsziwill für kontinuierliche Unterstützung und Diskussionen zu danken. Die Forschung führt zu diesen Ergebnissen wird finanziell vom European Research Council unter EU Fp7 (FP/2007-2013) ERC Grant Agreement n. 340929.

<ol>
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Die Autoren haben nichts preisgeben.

Das Design der composite-Strukturen ermöglicht die einfache Anpassung der Materialeigenschaften, wie des Elastizitätsmoduls oder die Dicke der Proben. Elastizitätsmodul von PDMS kann effektiv in einem breiten Spektrum durch das Mischungsverhältnis zwischen den beiden Komponenten zu verändern oder die Herstellung der Mischungen mit einem unterschiedlichen Silikon Elastomer30,31geändert werden. Die beschriebenen Methoden sind nicht beschränkt auf die PDMS in dieser Untersuchung verwendet, aber vor allem die selbstklebende Leistung hängt stark von der spezifischen Art verwendet. Ein wichtiger Schritt innerhalb dieses Protokolls ist der Herstellungsprozess von Verbundfolien (Abbildung 1). Es zeigte sich, dass die Dicke der Filme erheblich beeinträchtigt die Haftung Verhalten der Filme auf verschiedenen Substraten, einschließlich Haut (Abbildung 5 und Abbildung 6). Neben der Foliendicke Zeit und Temperatur während des Trocknungsprozesses wirkt sich die Materialeigenschaften32. Parameter wie die Dicke der Polymeren Schichten müssen daher sorgfältig angepasst und überprüft werden.
Analyse der selbstklebenden Eigenschaften dünner Schichten erfolgte mit Normalkraft Adhäsion Messungen mit zwei Glassubstraten mit verschiedenen Rauheit der Oberfläche bis zu Ra = 0.338 µm (Abbildung 3). Im Allgemeinen wirkt Rauheit deutlich die Haftung von Oberflächen, insbesondere von elastischen Materialien33,34. Die Rauheit des Glases kann leicht variiert werden, durch Schleifen mit Schleifpapier verschiedener Schroffheit Größen, daher erlauben die Herstellung von Substraten, die höhere Rauheit Werte21ausstellen. Darüber hinaus andere Materialien, z.B. Epoxidharz für die Herstellung von Substraten15,35einsetzbar. Dies könnte eine wichtige Modifikation Strategie des vorliegenden Protokolls sein. Zum Beispiel wenn Substrate mit verschiedenen Oberflächen freie Energien benötigt oder spezielle sind Topographien erforderlich. Hier wurden abziehen Stress und der Trennung von den Dünnschichten des PDMS und SSA hergestellt mit einem maßgeschneiderten Setup (makroskopische Adhäsion Messgerät (MAD, Abbildung 4)) analysiert. 36 optische Ausrichtung des Substrats und Eindringkörper ist ein entscheidender Schritt für die Analyse der Messergebnisse. Einstellung des Neigungswinkels muss daher mit dem Goniometer, so präzise wie möglich durchgeführt werden. Dies kann mit ausreichender Genauigkeit erreicht werden, indem Sie manuell das Substrat in Kontakt mit der filmoberfläche bis ein horizontale Kontakt entsteht.
Das aktuelle Protokoll wurde die Haltezeit auf eine Sekunde (Abbildung 5 und Abbildung 7) konstant gehalten. Vor allem für die Untersuchung der Klebstoff Leistung einen elastischen Film auf eine grobe Substratoberfläche stellt eine Erweiterung der Haltezeit Zusatzinformationen. Zum Beispiel wurde eine Zunahme der abziehen Stress mit zunehmendem Haltezeit gemeldeten8. Neben der Messungen in das aktuelle Protokoll, andere Methoden könnte zum Beispiel Peel Tests durchgeführt werden ermöglicht eine umfassendere Untersuchung der Haftung Leistung37.
Die Hafteigenschaften von Verbundfolien ausstellen anderen Film, die Dicke des Klebers weiche Haut ermittelt wurden (Abbildung 7). Unsere Ergebnisse sind in Übereinstimmung mit veröffentlichten Daten zeigen, dass eine Abnahme der Film Dicke führen zu einer Erhöhung der Belastung abziehen wie die Entbindung, d.h., das Verhältnis zwischen Durchmesser und Film Substratdicke, erhöht38,39 . Basierend auf diesen Ergebnissen und die Daten, die in Abbildung 7dargestellt, schließen wir, dass Verbundfolien mit einer Gesamtdicke von ca. 100 µm (Dicke der SSA-Schicht von ca. 60 µm aufgetragen zu einem PDMS-Film mit einer Dicke von ca. 40 µm) günstige Adhäsion p ausstellen Roperties auf rauen Oberflächen.
Als nächstes Experimente im Zusammenhang mit der biologischen Charakterisierung auf unberührten Verbundfolien durchgeführt wurden und Plasma behandelt Verbundfolien (Abbildung 8). Plasmabehandlung von Silikon-Elastomeren ist eine häufig angewandte, vielseitige Technik, um die hydrophilen Eigenschaften von Oberflächen und Förderung der zellulären Anlage und Mobilfunk Verbreitung40,41zu erhöhen. Silikone sind bekannt für ihre geringe Toxizität und hohe-Kompatibilität aber enthält möglicherweise Restmonomere oder Katalysatoren, die physiologische Prozesse, was auch zu Zytotoxizität42,43beeinflussen könnten. In die Experimente, die wir beobachtet haben, weniger als 5 % Zytotoxizität mit LDH Version als Indikator und ein Trypan blau-Ausschluss-Assay. Im vorgestellten Protokoll der gesamten zellulären Bevölkerung getrennt zellulären Aggregate einschließlich Form, die die Oberfläche für die Verbreitung Analyse (Abbildung 9 b) analysiert wurde. Eine Änderung des Protokolls könnte mehr differenzierte Ergebnisse produzieren. Für jede Probe konnte der Überstand mit freistehenden zellulären Aggregate auf einem separaten Reaktionsgefäß übertragen und nicht in Kombination mit der Zellen enzymatisch aus der Polymeroberfläche entfernt. Dies ermöglicht die genaue Beurteilung der Zellen an der Oberfläche befestigt und schließlich offenbaren eine genauere Bestimmung des Einflusses der Polymere auf die zelluläre Klebevorgang. Neben den hier vorgestellten Immunocytochemical Methoden möglicherweise Zellen für die Untersuchung mit Immunoblot Methoden, so dass eine detaillierte quantitative Bewertung der Proteinexpression geerntet werden.
Zusammenfassend haben wir Herstellungsbedingungen für die Herstellung von dünnen Elastomeren Verbundfolien für Anwendungen in der fortgeschrittenen Kultur Zellforschung etabliert. Darüber hinaus besitzen diese Dünnfilme hohen Anpassungsfähigkeit auf Haut, Rauheit, anspruchsvolles Design der Haut Klebstoffe ermöglichen.

In diesem Protokoll werden detaillierte Methoden für die Herstellung von dünnen Elastomer Filme vorgestellt. Die allgemein verfügbaren Arzt Klinge Technik wurde für die Herstellung von dünnen Verbundfolien eingesetzt. Die Herstellungstechnik wurde auf Polyethylenterephtalate (PET) Folien, spätere Produktion dieser Filme in großem Maßstab durchgeführt. Schwerpunkt dieses Protokolls ist die Bewertung der Reproduzierbarkeit, präzise Herstellung von den verschiedenen Schichten der Verbundfolien und Bestimmung der biologischen und Adhäsion Eigenschaften des endgültigen zusammengesetzte Patches. Die Silikon-Elastomer-poly-(dimethylsiloxane) (PDMS) wird weitgehend verwendet in der biomedizinischen Technologie, einschließlich der Herstellung von Haut-Klebstoffen, Mikrofluidik Anwendungen und zusätzliche Forschung Felder1,2,3 ,4. Vor kurzem, eine andere Unterklasse von PDMS, so genannte weiche Haut Klebstoffe (SSAs) wurden eingeführt, insbesondere für sanfte Haut kleben und de-bonding.
Silikon-SSAs sind Vinyl funktionalisiert Elastomere, abweichend von Analog Polymere durch den Mangel an Kieselsäure5zu stärken. Ähnlich wie bei anderen PDMS, SSA kann Elastizitätsmodul in einem breiten Spektrum von modulierenden Vernetzer Konzentration oder heilende Zeit6,7,8angepasst werden. Diese Veränderung des Elastizitätsmoduls von Siliconelastomeren die klebtechnischen Eigenschaften des Materials erheblich beeinträchtigt und hat auch tiefgreifende Auswirkungen auf die prokaryotischen und eukaryotischen Zellen kultiviert auf der Oberfläche9,10 , 11. auf der Zellebene biologische wurde gezeigt, daß Eukaryotische Zellen reagieren auf den Signalpegel Transduktion zu einer Modulation der Matrix Elastizität oder Dicke der Oberfläche9,10,12 ,13,14. Daher ein breites Interesse in Zelle Kultur Anwendungen von Polymeren mit einstellbaren mechanischen Eigenschaften vorhanden. Wichtig ist, bietet intrinsisch niedrige Oberflächenenergie von Siliconelastomeren basiert nicht optimale Bedingungen für die Zellkultur von eukaryotischen Zellen. Sauerstoff-Plasma-Behandlung ist eine weit verbreitete Technik, PDMS niedrige Oberflächenenergie vorübergehend, führt zu einer Steigerung der Haftzugfestigkeit erhöhen verringerte Oberfläche Adsorption von Molekülen, während parallel Anlage, Verbreitung zu fördern und Verbreitung von eukaryotischen Zellen15,16,17,18.
Neben den Materialeigenschaften beeinträchtigt der Oberflächentopographie erheblich, zelluläre Adhäsion und die selbstklebende Interaktion zwischen zwei Materialien19,20,21,22. Rauheit der Oberfläche hat verschiedene Auswirkungen auf die Kontakt zwischen zwei Flächen: Reduktion der Kontaktfläche, hohe gespeicherte elastische Energie rund um Unebenheiten sowie Einfluss auf die Rissausbreitung Haftfestigkeit23, verändern kann 24. Haftung von selbstklebenden Folien auf die menschliche Haut ist ein aufstrebendes Gebiet der Anwendung, z. B. Wundauflagen, Fixierung der EKG-Elektroden oder andere tragbare elektronische Geräte25,26,27, 28. Um den Klebstoff Leistungsmessung von selbst-Klebstoffen in Bezug auf Topographie können Glassubstrate mit unterschiedlichem Grad der Rauheit im normalen Adhäsion Messungen8,21verwendet werden. Hier wurden zwei Glassubstraten ausgewählt, um die Hafteigenschaften von Polymerfolien untersuchen. Zeichneten sich erste, zusammengesetzte Filme mit einer Trägerschicht PDMS im Mischungsverhältnis 10: 1 Gewicht Teile mit unterschiedlichen Mischungsverhältnis von PDMS abgedeckt. In einem zweiten Schritt wurde eine Klebeschicht SSA mit gleichem Gewicht Mengen beider Komponenten und mit unterschiedlichen Schichtdicken auf PDMS Vorfilm vorbereitet.

<table><tbody><tr><td>2-Propanol, 97%</td><td>Stockmeier Chemie</td><td>1000452610000</td><td>Isopropanol</td></tr><tr><td>Abrasive diamnod hand pad</td><td>Bohle</td><td>MO 5007522</td><td>Grit: 220</td></tr><tr><td>Accutase</td><td>Capricorn Scientific</td><td>ACC-1B</td><td></td></tr><tr><td>Albumin Fraktion V</td><td>Roth</td><td>0163.2</td><td>BSA</td></tr><tr><td>Alexa Fluor 488 Phalloidin</td><td>ThermoFischer Scientific</td><td>A12379</td><td>highly toxic</td></tr><tr><td>Aquamount</td><td>Polysciences</td><td>18606-20</td><td>water soluble mounting medium</td></tr><tr><td>CytoTox-ONE Homogeneous Membrane Integrity Assay</td><td>Promega</td><td>G7890</td><td></td></tr><tr><td>DPBS, without Ca&lt;sup&gt;2+&lt;/sup&gt;, Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>ThermoFischer Scientific</td><td>14190094</td><td></td></tr><tr><td>Fetal bovine serum gold</td><td>GE Health Care Life Science</td><td>A15-151</td><td>FBS</td></tr><tr><td>Goniometer OCA35</td><td>Dataphysics</td><td></td><td>for the determination of the static water contact angle</td></tr><tr><td>Hoechst Dye 33342</td><td>Sigma-Aldrich</td><td>B1155-100MG</td><td>bisBenzimide H 33342 trihydrochloride, highly toxic</td></tr><tr><td>Microscope Axiovert 25</td><td>Zeiss</td><td></td><td>Microscope used for cell culture documentation</td></tr><tr><td>Microscope Eclipse LV100ND</td><td>Nikon</td><td></td><td>Microscope used for film thickness determination</td></tr><tr><td>Paraformaldehyde, aqueous solution 16%</td><td>Electron Microscopy Sciences</td><td>RT 15710</td><td>electron microscopy grade</td></tr><tr><td>penicillin und streptomycin solution</td><td>Sigma-Aldrich</td><td>P4333-100ML</td><td></td></tr><tr><td>Phenom XL Scanning Electron Microscope (SEM)</td><td>Phenom</td><td></td><td></td></tr><tr><td>Poly-(vinyl alcohol) 4-88, MW 31000</td><td>Sigma-Aldrich</td><td>81381-1KG</td><td>Mowiol 4-88</td></tr><tr><td>Poly-dimethyl siloxanes, Sylgard 184</td><td>Dow Corning</td><td>(400)000108351397</td><td>PDMS</td></tr><tr><td>RPMI 1640 basal medium</td><td>ThermoFischer Scientific</td><td>21875034</td><td></td></tr><tr><td>soft skin adhesive (SSA)</td><td>Dow Corning</td><td>(400)000108251792</td><td>MG 7-9800 Soft Skin Adhesive (SSA)</td></tr><tr><td>speed mixer DAC 600.2 VAC-P</td><td>Hauschild</td><td></td><td></td></tr><tr><td>stylus profilomter</td><td>Zeiss</td><td></td><td>Model: SURFCOM 1500SD3</td></tr><tr><td>Tecan Infinite M200 pro</td><td>Tecan</td><td></td><td>fluorescence plate reader</td></tr><tr><td>Triton X 100</td><td>Calbiochem</td><td>648466</td><td></td></tr><tr><td>Trypan Blue solution</td><td>Sigma-Aldrich</td><td>T8154-100ML</td><td>highly toxic</td></tr><tr><td>Trypsin/EDTA solution</td><td>PAN-Biotech</td><td>P10-023500</td><td>0.05% Trypsin, 0.02% EDTA in PBS</td></tr><tr><td>UV glue</td><td>Bohle</td><td>BO MV76002</td><td>medium viscosity</td></tr></tbody></table>

thin film composite silicon elastomers for cell culture and skin applications: manufacturing and characterization

In diesem Protokoll präsentieren wir Methoden, um dünne Elastomer Verbundfolien für fortgeschrittene Zelle Kultur Anwendungen und für die Entwicklung der Haut Klebstoffe zu fabrizieren. Zwei verschiedene Poly--(dimethyl siloxanes) (PDMS und weiche Haut Klebstoff (SSA)), werden seit in tiefe Untersuchung von biologischen Wirkungen und Hafteigenschaften. Die Verbundfolien bestehen aus einer flexiblen Trägerschicht und eine selbstklebende Deckschicht. Beide Schichten wurden durch Arzt Klinge Applikationstechnik hergestellt. In der vorliegenden Untersuchung ist der Klebstoffe Verhalten der Verbundfolien als Funktion der Schichtdicke oder eine Variation des Elastizitätsmoduls der obersten Ebene untersucht worden. Der Elastizitätsmodul des PDMS wurde geändert durch Variation der Ausgangspunkt, um Vernetzer Mischungsverhältnis. Darüber hinaus die Dicke der SSA Filmen variierte von ca. 16 µm bis ca. 320 µm. Scanning Electron Microscopy (SEM) und optische Mikroskopie für Dickenmessung verwendet worden. Die Hafteigenschaften der Elastomer-Filme hängen stark von der Schichtdicke, der Elastizitätsmodul von Polymeren und Oberflächeneigenschaften. Also, normale Haftung dieser Filme auf Glassubstraten mit glatten und rauen Oberflächen untersucht worden ist. Abziehen-Stress und Arbeit der Trennung sind abhängig von dem Mischungsverhältnis von Siliconelastomeren.
Außerdem variierte die Dicke der weiche Hautkleber auf eine Unterstützende Trägerschicht platziert um Patches für Haut Anwendungen zu produzieren. Zytotoxizität, Verbreitung und zelluläre Adhäsion L929 murine Fibroblasten auf PDMS Filme (Mischungsverhältnis 10:1) und SSA-Filme (Verhältnis 50: 50 mischen) wurden durchgeführt. Wir haben hier zum ersten Mal die Gegenüberstellung von zusammengesetzten Dünnfilmen beide Polymere hergestellt gezeigt und die Untersuchung ihrer biologischen und Klebstoff Eigenschaften präsentieren.

Watch this Scientific Journal Video about Thin Film Composite Silicon Elastomers for Cell Culture and Skin Applications: Manufacturing and Characterization at JoVE.com

Thin Film Composite Silicon Elastomers for Cell Culture and Skin Applications: Manufacturing and Characterization

Ein Protokoll für die Herstellung von Polymeren Dünnschicht Verbundstrukturen besitzen unterschiedliche Youngs Moduli oder dicken wird vorgestellt. Filme sind für fortgeschrittene Zelle Kulturwissenschaft oder als Haut Klebstoffe hergestellt.

Dünnen Film Composite Silikon Elastomere für Zellkultur und Haut Anwendungen: Herstellung und Charakterisierung

In this protocol, we present methods to fabricate thin elastomer composite films for advanced cell culture applications and for the development of skin ...

thin-film-composite-silicon-elastomers-for-cell-culture-skin

Research

JoVE Journal

Developmental Biology

10.6K Aufrufe.  INM - Leibniz Institute for New Materials. Ein Protokoll für die Herstellung von Polymeren Dünnschicht Verbundstrukturen besitzen unterschiedliche Youngs Moduli oder dicken wird vorgestellt. Filme sind für fortgeschrittene Zelle Kulturwissenschaft oder als Haut Klebstoffe hergestellt.

Serie: Thin Film Composite Silicon Elastomers for Cell Culture and Skin Applications: Manufacturing and Characterization

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

Mikrofabrizierten Plattformen für Mechanisch Dynamische Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Forschung

Biotechnik

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

Directed Cellular Self-Assembly to Cell-Derived Tissue Ringe für biomechanische Analyse und Tissue Engineering Fabricate

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment 1,2 . These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications 3-8 . In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications 9-13. The culture system methodology presented here represents a scalable approach for rapid assessments of cell-silk film surface interactions. Of particular interest is the use of surface patterned silk films to study differences in cell proliferation and responses of cells for alignment 12,14 . The seeded cultures were cultured on both micro-patterned and flat silk film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the silk film in vitro culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields.

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are a novel class of biomaterials readily customizable for an array of biomedical applications. The presented silk film culture system is highly adaptable to a variety of in vitro analyses. This system represents a biomaterial design platform offering in vitro optimization before direct translation to in vivo models.

Silk Film Culture-System für In-vitro- Analyse und Design-Biomaterial

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment ...

Silicon Microchips for Manipulating Cell-cell Interaction

The role of the cellular microenvironment is recognized as crucial in determining cell fate and function in virtually all mammalian tissues from development to malignant transformation.&nbsp; In particular, interaction with neighboring stroma has been implicated in a plethora of biological phenomena; however, conventional techniques limit the ability to interrogate the spatial and dynamic elements of such interactions.
In Micromechanical Reconfigurable Culture (RC), we employ a micromachined silicon substrate with moving parts to dynamically control cell-cell interactions through mechanical repositioning. Previously, this method has been applied to investigate intercellular communication in co-cultures of hepatocytes and non-parenchymal cells, demonstrating time-dependent interactions and a limited range for soluble signaling 1.
Here, we describe in detail the preparation and use of the RC system. We begin by demonstrating the handling of the device parts using tweezers, including actuating between the gap and contact configurations (cell populations separated by a narrow 80-&micro;m gap, or in direct intimate contact). Next, we detail the process of preparing the substrates for culture, and the multi-step cell seeding process required for obtaining confluent cell monolayers. Using live microscopy, we then illustrate real-time manipulation of cells between the different possible experimental configurations. Finally, we demonstrate the steps required in order to regenerate the device surface for reuse: toluene and piranha cleaning, polystyrene coating, and oxygen plasma treatment.

This article describes an experimental approach for dynamic regulation of cell-cell interactions between adherent cells on a micrometer scale. Manipulation of intercellular communication between hepatocytes and stromal cell is demonstrated. The developed platform enables investigation of cell-cell interactions in a variety of biological processes, including development and pathogenesis.

Silizium-Mikrochips zur Manipulation Zell-Zell-Interaktion

The role of the cellular microenvironment is recognized as crucial in determining cell fate and function in virtually all mammalian tissues from ...

Biologie

Study of Cell Migration in Microfabricated Channels

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which impose a polarized phenotype to cells by physical constraints. Once inside channels, cells have only two possibilities: move forward or backward. This simplified migration in which directionality is restricted facilitates the automatic tracking of cells and the extraction of quantitative parameters to describe cell movement. These parameters include cell velocity, changes in direction, and pauses during motion. Microchannels are also compatible with the use of fluorescent markers and are therefore suitable to study localization of intracellular organelles and structures during cell migration at high resolution. Finally, the surface of the channels can be functionalized with different substrates, allowing the control of the adhesive properties of the channels or the study of haptotaxis. In summary, the system here described is intended to analyze the migration of large cell numbers in conditions in which both the geometry and the biochemical nature of the environment are controlled, facilitating the normalization and reproducibility of independent experiments.

A quantitative method to study spontaneous migration of cells in a one-dimensional confined microenvironment is described. This method takes advantage of microfabricated channels and can be used to study migration of large number of cells under different conditions in single experiments.

Studium der Zellmigration in Microfabricated Kanäle

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which ...

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

Stretching-Cells auf einer mikrostrukturierten PDMS-Membran

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Fabrication and Characterization of a Conformal Skin-like Electronic System for Quantitative, Cutaneous Wound Management

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of cutaneous wound healing on the skin. Existing methods, however, still rely on visual inspections through various microscopic tools and devices that normally include high-cost, sophisticated systems and require well trained personnel for operation and data analysis. Here, we describe methods and protocols to fabricate a conformal, skin-like electronics system that enables conformal lamination to the skin surface near the wound tissues, which provides recording of high fidelity electrical signals such as skin temperature and thermal conductivity. The methods of device fabrication provide details of step-by-step preparation of the microelectronic system that is completely enclosed with elastomeric silicone materials to offer electrical isolation. The experimental study presents multifunctional, biocompatible, waterproof, reusable, and flexible/stretchable characteristics of the device for clinical applications. Protocols of clinical testing provide an overview and sequential process of cleaning, testing setup, system operation, and data acquisition with the skin-like electronics, gently mounted on hypersensitive, cutaneous wound and contralateral tissues on patients.

This article presents methods to fabricate and characterize a conformal, skin-like electronic system and protocols for the use in clinical applications, particularly on cutaneous wound management.

Herstellung und Charakterisierung eines Conformal-Haut-like Electronic System for Quantitative, kutane Wundmanagement

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

Synthese von biokompatiblen Liquid Crystal Elastomerschäume als Zellgerüste für 3D Spatial Zellkulturen

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

Ultradünne derzei-elastischen Hydrogele als eine biomimetische Basalmembran für Dual Zellkultur

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...

Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of concept, a simplified and undifferentiated human skin containing a dermal (stromal) and an epidermal (epithelial) compartment has been modelled. To accomplish this, a versatile and robust, vinyl-based device divided into two chambers has been developed, overcoming some of the drawbacks present in microfluidic devices based on polydimethylsiloxane (PDMS) for biomedical applications, such as the use of expensive and specialized equipment or the absorption of small, hydrophobic molecules and proteins. Moreover, a new method based on parallel flow was developed, enabling the in situ deposition of both the dermal and epidermal compartments. The skin construct consists of a fibrin matrix containing human primary fibroblasts and a monolayer of immortalized keratinocytes seeded on top, which is subsequently maintained under dynamic culture conditions. This new microfluidic platform opens the possibility to model human skin diseases and extrapolate the method to generate other complex tissues.

Here, we present a protocol to generate a three-dimensional simplified and undifferentiated skin model using a micromachined microfluidic platform. A parallel flow approach allows the in situ deposition of a dermal compartment for the seeding of epithelial cells on top, all controlled by syringe pumps.

Generierung eines vereinfachten dreidimensionalen Skin-on-a-Chip-Modells in einer mikrobearbeiteten mikrofluidischen Plattform

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of ...