Name: micropatterning and assembly of 3d microvessels
Uploaded: 2016-09-09T15:00:00.000+00:00
Duration: 13 min 5 s
Description: Watch this Scientific Journal Video about Micropatterning and Assembly of 3D Microvessels at JoVE.com

Die Autoren möchten die Lynn und Mike Garvey Imaging Labor am Institut für Stammzell und Regenerative Medizin, sowie die Washington Nanofabrication Einrichtung an der Universität von Washington zu bestätigen. Sie erkennen auch die finanzielle Unterstützung des National Institute of Health gewährt DP2DK102258 (zu YZ) und die Ausbildung gewährt T32EB001650 (bis SSK und MAR) und T32HL007312 (MAR).

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Engineered microvessels sind ein in vitro - Modell , wo physiologische Eigenschaften wie luminalen Geometrie, hydrodynamischen Kräfte und mehrzelligen Wechselwirkungen vorhanden und abstimmbaren sind. Diese Art von Plattform ist in leistungsstark , dass es bietet die Möglichkeit , endothelial Verhalten in einer Vielzahl von Kontexten zu modellieren und zu studieren , wo die in vitro Kulturbedingungen können zu der der Mikroumgebung in Frage abgestimmt werden. Zum Beispiel werden die Antriebsmechanis...

Die Mikrovaskulatur in jedem Organ hilft , die Gewebemikroumgebung definieren, Gewebe - Homöostase und Entzündung regulieren, Permeabilität, Thrombose und Fibrinolyse 1,2 aufrechtzuerhalten. Mikrovaskulären Endothel, insbesondere ist die Schnittstelle zwischen der Blutströmung und dem umgebenden Gewebe und somit eine kritische Rolle spielt Gefäß- und Organfunktion in Reaktion auf Reize wie hydrodynamischen Kräfte in Modulieren und zirkulierende Cytokine und Hormone 3 bis 5. Das Verständnis der detaillierten Wechselwirkungen zwischen dem Endothel, Blut, und das umgebende Gewebe Mikroumgebung ist für die Untersuchung der Gefäßbiologie und Fortschreiten der Krankheit wichtig. Allerdings sind die Fortschritte , diese Interaktionen in das Studium wurde durch in - vitro - Tools beschränkt behindert , die in vivo mikrovaskulären Struktur rekapitulieren nicht und Funktion 6,7. Als Ergebnis hat sich das Feld und therapeutischen Fortschritt stark auf kostspielige und zeit verlassenraubend Tiermodelle , die oft nicht zum Erfolg beim Menschen 8 zu übersetzen - 10. Während in vivo - Modellen bei der Untersuchung von Krankheitsmechanismen und vaskuläre Funktionen sind von unschätzbarem Wert, sind sie komplex und fehlt oft eine genaue Kontrolle der einzelnen zellulären, biochemischen und biophysikalischen Signale. Gefäßsystem im ganzen Körper verfügt über eine breite hierarchische Struktur in Verbindung mit expansive kapillaren Betten, optimierte Perfusion und Nährstofftransport gleichzeitig 11 bereitstellt. Zunächst Gefäßformen als primitive Plexus , die 12,13 während der frühen Entwicklung zu einem hierarchisch verzweigten Netzwerk reorganisiert. Obwohl viele der in diesen Prozessen beteiligt sind Signale gut 14 verstanden werden - 16, bleibt es schwer , wie solche Gefäßmuster 15 bestimmt wird. Im Gegenzug diesen Prozess in vitro zu rekapitulieren organisierten Gefäßnetze zu konstruieren hat Bienen schwierig. Viele existierende in vitro Plattformen Vaskulatur, wie zweidimensionale Endothelzellkulturen zu modellieren, fehlen wichtige Eigenschaften wie mehrzelligen Nähe dreidimensionale luminalen Geometrie, Durchfluss- und extrazellulären Matrix. Rohrbildungstests in 3D Hydrogele (Collagen oder Fibrin) 17-19 oder Invasionsassays 20,21 verwendet wurden 23 Endothelfunktion in 3D und ihre Wechselwirkungen mit anderen vaskulären 17,22 oder Gewebezelltypen zu untersuchen. Allerdings montiert Lumen in diesen Tests fehlt Verbunds, der hämodynamischen Strömungs und entsprechende Perfusion. Darüber hinaus verhindert die Neigung zur Gefäß Regression in diesen Rohrbildungstests 24 Langzeitkultur und Reifung , die den Grad der funktionellen Studien begrenzt, die durchgeführt werden können. Somit gibt es einen wachsenden Bedarf in vitro Plattformen der mikrovaskulären Netzwerke zu konstruieren , die in geeigneter Weise modellieren endothelial Eigenschaften und sind in der Lage Langzeitkultur. Eine Vielzahl von vaskulären Engineering-Techniken haben in den letzten Jahren für medizinische Anwendungen entstanden bei Patienten mit Gefäßerkrankung zu ersetzen oder zu umgehen betroffenen Schiffe. Mit großem Durchmesser Gefäße aus synthetischen Materialien, wie Polyethylenterephthalat (PET), und Polytetrafluorethylen (ePTFE) , haben erhebliche therapeutische Erfolge mit Langzeitdurchgängigkeit (durchschnittlich 95% Durchgängigkeit über 5 Jahre) 25 hatte. Obwohl mit kleinem Durchmesser synthetische Transplantate (&lt;6 mm) in der Regel Komplikationen konfrontiert wie Intimahyperplasie und Thrombopoese 26 - 28, Gewebe mit kleinem Durchmesser Transplantate mit biologischen Material entwickelt haben erhebliche Fortschritte gemacht 29,30. Trotz Fortschritten dieser Art entwickelt, Schiffe, die auf der Mikroskala haben eine Herausforderung geblieben. Um adäquat microvasculature zu modellieren, ist es notwendig, komplexe Netzwerkstrukturen mit suf zu erzeugenreichend mechanische Festigkeit Durchgängigkeit und mit einer Matrix-Zusammensetzung zu erhalten, die für die parenchymalen Zellen und Zellumbau für beide Nahrungsmittelpermeation ermöglicht. Dieses Protokoll stellt einen neuartigen künstlichen perfundierbaren Gefäßnetz , das eine native in vivo nachahmt mit einem abstimmbaren und steuerbaren Mikroumgebung schaffen 31-34. Das beschriebene Verfahren erzeugt Engineered microvessels mit einem Durchmesser in der Größenordnung von 100 &amp; mgr; m. Ausgeführt microvessels werden hergestellt durch Perfusion Endothelzellen durch einen mikrofluidischen Kanal, der in weichen Typ eingebettet ist I-Hydrogel-Kollagen. Dieses System hat die Fähigkeit, gemusterten Netzwerke mit offenen Struktur luminalen erzeugen, replizieren multi-zellulären Interaktionen modulieren extrazellulären Matrixzusammensetzung und gelten physiologisch relevanten hämodynamischen Kräften.

<table><tbody><tr><td>&lt;strong&gt;Wafer Fabrication&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>AutoGlow Plasma System</td><td>AutoGlow</td><td></td><td></td></tr><tr><td>Headway Spin Coater</td><td>Headway Research, Inc&amp;nbsp;</td><td>PWM32 Spin Coater&amp;nbsp;</td><td></td></tr><tr><td>ABM Contact Aligner</td><td>AB-M</td><td></td><td></td></tr><tr><td>Alpha Step Profilometer</td><td>Tencor</td><td>Alpha Step 200</td><td></td></tr><tr><td>SU-8 Developer</td><td>Microchem</td><td>Y020100</td><td></td></tr><tr><td>SU-8 Resist</td><td>Microchem</td><td>SU-8 2000</td><td></td></tr><tr><td>8&quot; silicon wafer</td><td>Wafer World Inc.</td><td></td><td></td></tr><tr><td>Tabletop Micro Pattern Generator</td><td>Heidelberg Instruments</td><td>&amp;mu;PG 101</td><td>For generation of photomask</td></tr><tr><td>Hot plate</td><td>VWR</td><td>97042-646</td><td></td></tr><tr><td>Ispropyl alcohol</td><td>Avantor Performance Materials</td><td>9088</td><td></td></tr><tr><td>Petri dishes (120 x 120 mm, square)</td><td>Sigma-Aldrich</td><td>Z617679</td><td></td></tr><tr><td>Trichloro(3,3,3-trifluoropropyl)silane</td><td>Sigma-Aldrich</td><td>MKBG3805V</td><td></td></tr><tr><td>Polydimethylsiloxane (PDMS) elastomer base and curing agent</td><td>Dow Corning</td><td>Sylgard 184</td><td>Mixed at 10:1 (w/w)</td></tr><tr><td>Vacuum desiccator</td><td>Sigma-Aldrich</td><td>Z119024-1EA</td><td></td></tr><tr><td>Oven</td><td>VWR</td><td>9120976</td><td></td></tr><tr><td>&lt;strong&gt;Device Fabrication and Culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>poly(methyl methacrylate) (PMMA)</td><td>Plexiglas</td><td></td><td></td></tr><tr><td>Corona Treater</td><td>Electro-Technic Products, Inc.</td><td>BD-20</td><td>Handheld device for plasma treatment of PMMA devices and PDMS molds</td></tr><tr><td>Soldering Iron</td><td>Weller&amp;nbsp;</td><td>WTCPS</td><td></td></tr><tr><td>Stainless Steel Truss Head Slotted Machine Screw</td><td>McMaster-Carr&amp;nbsp;</td><td>91785A096</td><td></td></tr><tr><td>Stainless steel dowel pins</td><td>McMaster-Carr&amp;nbsp;</td><td>93600A060</td><td></td></tr><tr><td>Tweezers&amp;nbsp;</td><td>Miltex</td><td>24-572</td><td>Any similar tweezers may be used</td></tr><tr><td>Spatula (Micro Spoon)</td><td>Electron Microscopy Services</td><td>62410-01</td><td></td></tr><tr><td>Screw driver</td><td></td><td></td><td>Any flat head screwdriver may be used, autoclaved</td></tr><tr><td>Glass coverslips (22 x 22 mm)</td><td>Fisher Scientific</td><td>12-542B</td><td></td></tr><tr><td>Bleach</td><td>Clorox</td><td>4460030966</td><td></td></tr><tr><td>Petri dishes (150 x 25 mm)</td><td>Corning</td><td>430599</td><td></td></tr><tr><td>Petri dishes (100 x 20 mm)</td><td>Corning</td><td>2909</td><td></td></tr><tr><td>Cotton, cut into 1 cm x 3 cm pieces</td><td></td><td></td><td>Autoclaved</td></tr><tr><td>Polyethyleneimine (PEI)</td><td>Sigma-Aldrich</td><td>P3143</td><td>Dilute to 1% in cell culture grade water</td></tr><tr><td>Glutaraldehyde</td><td>Sigma-Aldrich</td><td>G6257</td><td>Dilute to 0.1% in cell culture grade water</td></tr><tr><td>Sterile H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>Autoclaved DI H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Type I collagen, dissolved in 0.1% acetic acid</td><td></td><td></td><td>Isolated from rat tails as described in Rajan &lt;i&gt;et. al.&lt;/i&gt; 2006 (ref #37)</td></tr><tr><td>1 ml syringe</td><td>BD</td><td>309659</td><td></td></tr><tr><td>10 ml syringe</td><td>BD</td><td>309604</td><td></td></tr><tr><td>15 ml conical tubes</td><td>Corning</td><td>352097</td><td></td></tr><tr><td>30 ml conical tubes</td><td>Corning</td><td>352098</td><td></td></tr><tr><td>M199 10x Media&amp;nbsp;</td><td>Life Technologies</td><td>11825-015</td><td></td></tr><tr><td>1 N NaOH (sterile)</td><td>Sigma-Aldrich</td><td>415413</td><td>Dilute to 1 N in cell culture grade water</td></tr><tr><td>HUVECs</td><td>Lonza</td><td></td><td></td></tr><tr><td>Endothelial growth media</td><td>Lonza</td><td>CC-3124</td><td></td></tr><tr><td>Trypsin</td><td>Corning</td><td>25-052-CI</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Thermofisher Scientific</td><td>10082147</td><td></td></tr><tr><td>Dextran from Leuconostoc spp. (70&amp;nbsp;kDa)</td><td>Sigma-Aldrich</td><td>31390</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>Hemocytometer</td><td>Hausser Scientific Co.</td><td>3200</td><td></td></tr><tr><td>Gel loading tips</td><td>VWR</td><td>37001-152</td><td></td></tr><tr><td>18 G Blunt Fill Needle</td><td>BD&amp;nbsp;</td><td>305180</td><td></td></tr><tr><td>20 G Stainless Steel Dispensing Needle</td><td>McMaster-Carr</td><td>75165A123</td><td></td></tr><tr><td>Tygon 1/32&amp;rdquo; ID, 3/32&quot; OD Silicon Tubing</td><td>Cole-Parmer</td><td>EW-95702-00</td><td></td></tr><tr><td>1/16&quot; Tube-to-tube Coupling</td><td>McMaster-Carr</td><td>5116K165</td><td></td></tr><tr><td>90&amp;deg; Elbow Connectors, Tube-to-Tube</td><td>McMaster-Carr</td><td>5121K901</td><td></td></tr><tr><td>Luer Lock Coupling (Female, 1/16&quot; ID)</td><td>McMaster-Carr</td><td>51525K211</td><td></td></tr><tr><td>Plastic Forceps, with Jaw Grips</td><td>Electron Microscopy Services</td><td>72971</td><td></td></tr><tr><td>Dual Syringe Pump</td><td>Harvard Apparatus</td><td>70-4505</td><td></td></tr><tr><td>5 ml Polystyrene Round-bottom tube</td><td>Fisher Scientific</td><td>14-959-2A</td><td></td></tr><tr><td>&lt;strong&gt;Device Analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Formaldehyde</td><td>Sigma-Aldrich</td><td>F8775</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A8806-5G</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T-9284</td><td></td></tr><tr><td>Rabbit anti-hCD31</td><td>Abcam</td><td>ab32457</td><td>1:25 working dilution</td></tr><tr><td>FITC conjugated anti-von Willebrand Factor antibody</td><td>Abcam</td><td>ab8822</td><td>1:100 working dilution</td></tr><tr><td>Goat anti-rabbit 568 secondary antibody</td><td>Thermofisher Scientific</td><td>A-11011</td><td>1:100 working dilution</td></tr><tr><td>Hoescht</td><td>Thermofisher Scientific</td><td>H1399</td><td>Resuspended in DMSO</td></tr><tr><td>Sodium cacodylate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C0250</td><td>To make 0.2 M cacodylate buffer</td></tr><tr><td>Ethanol</td><td>VWR International</td><td>BDH1164-4LP</td><td></td></tr><tr><td>40 kDa FITC-conjugated Dextran</td><td>Sigma-Aldrich</td><td>FD40S&amp;nbsp;</td><td></td></tr><tr><td>&lt;strong&gt;Additional Culture Reagents&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CHIR-99021</td><td>Selleck Chem</td><td>S2924</td><td>Small molecule GSK-3 inhibitor</td></tr><tr><td>Human recombinant VEGF</td><td>Peprotech</td><td>100-20</td><td></td></tr><tr><td>Human recombinant bFGF</td><td>Peprotech</td><td>AF-100-18B</td><td></td></tr></tbody></table>

micropatterning and assembly of 3d microvessels

In - vitro - Plattformen Endothelzellen und Gefäßbiologie zu studieren , um 2D - endothelialen Zellkultur, Strömungskammern mit Polymer oder Glas basierenden Substraten und Hydrogel-basierte Rohrbildungstests weitgehend begrenzt sind. Diese Tests, während informativ, nicht rekapitulieren nicht Lumen Geometrie, richtige extrazellulären Matrix und mehrzelligen Nähe, die bei der Modulation der Gefäßfunktion eine Schlüsselrolle spielen. Dieses Manuskript beschreibt ein Spritzgussverfahren Engineered Gefäße mit einem Durchmesser in der Größenordnung von 100 &amp; mgr; m zu erzeugen. Microvessels werden durch Impfen Endothelzellen in einem mikrofluidischen Kanal innerhalb einer nativen Typ-I-Kollagen-Hydrogel eingebettet hergestellt. Durch die Integration von Bildung Parenchymzellen in der Kollagenmatrix vor dem Kanal können spezifische Gewebe-Mikroumgebungen modelliert und untersucht werden. Zusätzliche Modulationen der hydrodynamischen Eigenschaften und Medienzusammensetzung ermöglichen Steuerung von komplexen Gefäßfunktion innerhalb des gewünschten Mikro.Diese Plattform ermöglicht die Studie von perivaskulären Zellrekrutierung, Blut-Endothel-Interaktionen, Flussantwort und Gewebe-mikrovaskulären Wechselwirkungen. Ausgeführt microvessels bieten die Möglichkeit, den Einfluss von einzelnen Komponenten eines vaskulären Nische zu isolieren und zu steuern, genau seine chemischen, mechanischen und biologischen Eigenschaften der Gefäßbiologie sowohl in Gesundheit und Krankheit zu studieren.

Die konstruierte Schiff Plattform schafft funktionelle microvasculature in einem natürlichem Kollagen Typ I Matrix eingebettet und ermöglicht eine strenge Kontrolle der zellulären, biophysikalische und biochemische Umgebung in vitro. Engineered microvessels, humane Nabelschnurvenen-Endothelzellen (HUVECs) herzustellen sind durch die kollagen eingebettet mikrofluidischen Netzwerk durchbluteten, wo sie legen ein Patent Lumen und konfluenten Endothel zu bilden. Wie in 1A...

Watch this Scientific Journal Video about Micropatterning and Assembly of 3D Microvessels at JoVE.com

Micropatterning and Assembly of 3D Microvessels

Dieses Manuskript stellt ein Spritzgussverfahren microvessels zu entwickeln, die physiologischen Eigenschaften des Endothels rekapitulieren. Die Mikrofluidik-basierten Prozess erstellt Patent 3D Gefäßnetze mit tailor Bedingungen, wie Durchfluss, zelluläre Zusammensetzung, Geometrie und biochemische Steigungen. Das Herstellungsverfahren und Beispiele für mögliche Anwendungen beschrieben.

Mikrostrukturierung und Montage von 3D-Micro-Ader

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

micropatterning-and-assembly-of-3d-microvessels

Research

JoVE Journal

Bioengineering

11.7K Aufrufe.  University of Washington.  Dieses Manuskript stellt ein Spritzgussverfahren microvessels zu entwickeln, die physiologischen Eigenschaften des Endothels rekapitulieren. Die Mikrofluidik-basierten Prozess erstellt Patent 3D Gefäßnetze mit tailor Bedingungen, wie Durchfluss, zelluläre Zusammensetzung, Geometrie und biochemische Steigungen. Das Herstellungsverfahren und Beispiele für mögliche Anwendungen beschrieben. 

Serie: Micropatterning and Assembly of 3D Microvessels

Process of Making Three-dimensional Microstructures using Vaporization of a Sacrificial Component

Vascular structures in natural systems are able to provide high mass transport through high surface areas and optimized structure. Few synthetic material fabrication techniques are able to mimic the complexity of these structures while maintaining scalability. The Vaporization of a Sacrificial Component (VaSC) process is able to do so. This process uses sacrificial fibers as a template to form hollow, cylindrical microchannels embedded within a matrix. Tin (II) oxalate (SnOx) is embedded within poly(lactic) acid (PLA) fibers which facilitates the use of this process. The SnOx catalyzes the depolymerization of the PLA fibers at lower temperatures. The lactic acid monomers are gaseous at these temperatures and can be removed from the embedded matrix at temperatures that do not damage the matrix. Here we show a method for aligning these fibers using micromachined plates and a tensioning device to create complex patterns of three-dimensionally arrayed microchannels. The process allows the exploration of virtually any arrangement of fiber topologies and structures.

The Vaporization of a Sacrificial Component (VaSC) process is used to fabricate microvascular structures. This procedure uses sacrificial poly(lactic) acid fibers to form hollow microchannels with precise 3D geometric positioning provided by laser micromachined guide plates.

Verfahren zur Herstellung von dreidimensionalen Mikrostrukturen mit Verdampfung eines Opferbauteil

Vascular structures in natural systems are able to provide high mass transport through high surface areas and optimized structure. Few synthetic material ...

Forschung

Ingenieurwesen

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

Einer mikrofluidischen Vorrichtung mit Groove Patterns für ein Studium Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

Biologie

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

Microfabrication der Chip-Größe Gerüste für die dreidimensionale Kultivierung der Zellen

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Capillary-based Centrifugal Microfluidic Device for Size-controllable Formation of Monodisperse Microdroplets

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal microfluidic device. W/O microdroplets have recently been used in powerful methods that enable miniaturized chemical experiments. Therefore, developing a versatile method to yield monodisperse W/O microdroplets is needed. We have developed a method for generating monodisperse W/O microdroplets based on a capillary-based centrifugal axisymmetric co-flowing microfluidic device. We succeeded in controlling the size of microdroplets by adjusting the capillary orifice. Our method requires equipment that is easier-to-use than with other microfluidic techniques, requires only a small volume (0.1-1 &#181;l) of sample solution for encapsulation, and enables the production of hundreds of thousands number of W/O microdroplets per second. We expect this method will assist biological studies that require precious biological samples by conserving the volume of the samples for rapid quantitative analysis biochemical and biological studies.

Here, we demonstrate a simple production method for size-controllable, monodisperse, water-in-oil (W/O) microdroplets using a capillary-based centrifugal microfluidic device. This method requires only a small sample volume and enables high-yield production. We expect this method will be useful for rapid biochemical and cellular analyses.

Kapillar-basierten Kreisel Mikrofluidik-Vorrichtung zur Größe steuerbare Bildung von monodispersen Mikrotröpfchen

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal ...

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

Bildung Biomembrane Microarrays mit einem Rakel-basierten Montageverfahren

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

Biotechnik

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

Lipiddoppelschicht Vesicle Erzeugung anhand Mikrofluidik Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, including studies of microbial physiology and interactions. At the simplest level, accurate spatial patterning of microorganisms would enable reliable, long-term imaging of large numbers of individual cells and transform the ability to quantitatively study distance-dependent microbe-microbe interactions. More uniquely, coupling accurate spatial patterning and full control over environmental conditions, as offered by microfluidic technology, would provide a powerful and versatile platform for single-cell studies in microbial ecology. 
This paper presents a microfluidic platform to produce versatile and user-defined patterns of microorganisms within a microfluidic channel, allowing complete optical access for long-term, high-throughput monitoring. This new microfluidic technology is based on capillarity-assisted particle assembly and exploits the capillary forces arising from the controlled motion of an evaporating suspension inside a microfluidic channel to deposit individual microsized objects in an array of traps microfabricated onto a polydimethylsiloxane (PDMS) substrate. Sequential depositions generate the desired spatial layout of single or multiple types of micro-sized objects, dictated solely by the geometry of the traps and the filling sequence. 
The platform has been calibrated using colloidal particles of different dimensions and materials: it has proven to be a powerful tool to generate diverse colloidal patterns and perform surface functionalization of trapped particles. Furthermore, the platform was tested on microbial cells, using Escherichia coli cells as a model bacterium. Thousands of individual cells were patterned on the surface, and their growth was monitored over time. In this platform, the coupling of single-cell deposition and microfluidic technology allows both geometric patterning of microorganisms and precise control of environmental conditions. It thus opens a window into the physiology of single microbes and the ecology of microbe-microbe interactions, as shown by preliminary experiments.

We present a technology that uses capillarity-assisted assembly in a microfluidic platform to pattern micro-sized objects suspended in a liquid, such as bacteria and colloids, into prescribed arrays on a polydimethylsiloxane substrate.

Strukturierung von Mikroorganismen und Mikropartikeln durch sequentielle kapillaritätsunterstützte Montage

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

Bildgestützte, laserbasierte Herstellung von Mikrofluidik-Networks-Vascular abgeleitet

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

Eine vielseitige Methode von Patterning Proteinen und Zellen

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular compartments while circumventing complications arising from natural cell-to-cell variations. To standardize cell shape, cells are either confined in 3D molds or controlled for adhesive geometry through adhesive islands. However, traditional micropatterning techniques based on photolithography and deep UV etching heavily depend on clean rooms or specialized equipment. Here we present an infrared laser assisted micropatterning technique (microphotopatterning) modified from Doyle et al. that can be conveniently set up with commercially available imaging systems. In this protocol, we use a Nikon A1R MP+ imaging system to generate micropatterns with micron precision through an infrared (IR) laser that ablates preset regions on poly-vinyl alcohol coated coverslips. We employ a custom script to enable automated pattern fabrication with high efficiency and accuracy in systems not equipped with a hardware autofocus. We show that this IR laser assisted micropatterning (microphotopatterning) protocol results in defined patterns to which cells attach exclusively and take on the desired shape. Furthermore, data from a large number of cells can be averaged due to the standardization of cell shape. Patterns generated with this protocol, combined with high resolution imaging and quantitative analysis, can be used for relatively high throughput screens to identify molecular players mediating the link between form and function.

The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories.

Kontrolle der Zellgeometrie durch Laser Assisted Micropatterning

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular ...