Martin Danner örnekleri ve hücre kültür yordamlar kurulması hazırlarken onun yardımını kabul edilmektedir. Yazarlar Biesterfeld Spezialchemie GmbH (Hamburg, Almanya), özellikle Robert Radsziwill sürekli destek ve sohbet için teşekkür etmek istiyorum. Bu sonuçlar için önde gelen araştırma Avrupa Araştırma Konseyi Avrupa Birliği'nin yedinci çerçeve programı (FP/2007-2013) ERC Hibe Sözleşmesi n. 340929 altında fon aldı.

<ol>
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Yazarlar ifşa gerek yok.

Kompozit yapıların tasarım Young katsayısı veya örnekleri kalınlığı gibi malzeme özelliklerine basit ayarlama sağlar. Young katsayısı PDMS, iki bileşenler arasındaki karıştırma oranı değiştirme veya karışımları farklı silikon elastomer30,31kullanarak üretim geniş bir alanda etkin bir şekilde değiştirilebilir. Açıklanan yöntemleri geçerli araştırmada kullanılan PDMS'ın sınırlı değildir, ama özellikle yapışkanlı performans güçlü kullanılan belirli türüne göre değişir. Bu iletişim kuralı içinde önemli bir adım bileşik filimler (Şekil 1) üretim sürecidir. Bu film kalınlığı önemli ölçüde farklı yüzeylerde (Şekil 5 ve Şekil 6) deri de dahil olmak üzere, tarih filmleri yapışma davranışını etkiler gösterilmiştir. Film kalınlığı yanı sıra zaman ve sıcaklık kür işlemi sırasında malzeme özellikleri32etkiler. Bu nedenle, parametre olarak polimer katman kalınlığı dikkatli bir şekilde uyarlanmıştır ve doğrulanmadı gerekir.
İnce filmlerin yapışkanlı özelliklerinin çözümlemenin Ra kadar farklı yüzey pürüzlülük ile iki cam yüzeylerde kullanarak normal kuvvet yapışma ölçüleri ile 0.338 µm (Şekil 3) =. Genel olarak, pürüzlülük yapışma yüzeylerinin, elastik malzeme33,34özellikle önemli ölçüde etkiler. Cam pürüzlülüğü farklı sertliklerin boyutlarda, bu nedenle daha yüksek pürüzlülük değerleri21sergilenmesi yüzeylerde imalatı izin zımpara taşlama tarafından kolayca değiştirilebilir. Buna ek olarak, diğer malzemeler, örneğin epoksi reçine yüzeylerde15,35üretim için kullanılabilir. Bu sunulan Protokolü'nün bir önemli değişiklik strateji olabilir. Örneğin, Eğer farklı yüzey serbest enerji sergilenmesi yüzeylerde gerekli veya belirli Topografyaları gereklidir. Burada, çekme-off stres ve iş ayrılık PDMS ve SSA üretilen ince filmlerin özel olarak oluşturulmuş bir kurulum (makroskopik yapışma ölçüm cihazı (MAD, Şekil 4)) ile analiz edildi. 36 optik uyum substrat ve uç ölçüm sonuçlarının analizi için kritik bir adımdır. Bu nedenle, tilt açı düzeltilmesi mümkün olduğunca kesin Gonyometre ile yapılması gerekiyor. Bu el ile yatay bir kişi elde kadar film yüzeyine temas substrat getirerek yeterli hassasiyetle elde edilebilir.
Geçerli protokol tutun zaman bir saniye (Şekil 5 ve Şekil 7) sabit tutuldu. Özellikle yapışkanlı elastik bir filmi bir kaba substrat yüzey ile performansını incelenmesi için uzantı tutun zaman ek bilgi sağlar. Örneğin, bildirilen8tutun zaman artan stres çekme-off bir artış olmuştur. Geçerli protokol, diğer yöntemlerle gerçekleştirilen ölçümler yanı sıra örneğin kabuğu testleri, yapışma performansı37daha kapsamlı bir soruşturma izin yerine olabilir.
Bileşik Filmler (Şekil 7) farklı film yumuşak deri yapıştırıcı kalınlıkları belirlendi sergilenmesi yapışkanlı özellikleri. Bu film kalınlığı çekme-off stres artış müşteri adayına, bir düşüş gösteren hapsi, yani, substrat çapı ve film kalınlığı, artar38,39 arasındaki oran olarak yayımlanmış veri doğrultusunda bizim sonuçları. . Bu sonuçlar ve Şekil 7' de tasvir veri dayalı, biz yaklaşık 100 µm (yaklaşık 60 µm PDMS film için yaklaşık 40 µm kalınlığında ile uygulanan SSA tabakası kalınlığı) toplam kalınlığı ile bileşik filmleri olumlu yapışma p sergi sonucuna zellikleri pürüzlü yüzeylerde.
Daha sonra biyolojik karakterizasyonu ile ilgili deneyler bozulmamış bileşik filmler gerçekleştirilmiş ve plazma bileşik Filmler (Şekil 8) tedavi. Silikon elastomer tedavisinde plazma yüzeyler ve hücresel eki ve hücresel40,41yayılmasını teşvik hidrofilik özelliklerini artırmak için sık sık uygulanan, çok yönlü bir tekniktir. Silikonlar kendi düşük toksisite ve yüksek biostability için bilinen ama artık monomerleri veya fizyolojik süreçler, ayrıca sitotoksisite42,43için önde gelen etkileyen katalizörler içerebilir. Yapılan deneyler biz gözlemledim daha az içinde karaciğer kullanan bir göstergesi ve bir Trypan mavi dışlama tahlil %5 sitotoksisite. Sunulan protokolü, tüm hücresel nüfus ilişkisi kesildi yüzeye yayılması Analizi (Şekil 9B) analiz formu hücresel toplamları dahil olmak üzere. Bir değişiklik Protokolü'nün daha farklı sonuçlar üretebilir. Her örnek için müstakil hücresel toplamları içeren süpernatant olabilir bir ayrı tepki tüp transfer ve enzimatik polimer yüzeyden kaldırıldı hücreleri ile birlikte değil. Bu yüzeye bağlı hücre tam değerlendirme izin ve sonunda polimerler etkisi hücre adezyon işlemiyle ilgili daha ayrıntılı bir kararlılık ortaya koyuyor. Burada sunulan immunocytochemical yöntemlere ek olarak, hücrelerin protein ifade detaylı bir kantitatif değerlendirilmesi sağlayan immunoblot yöntemlerini ile soruşturma için hasat.
Özet olarak, üretim koşulları uygulamalar Gelişmiş hücre kültür araştırma için ince elastomerik bileşik filmlerin üretimi için kurduk. Ayrıca, bu ince filmler yüksek adaptasyon pürüzlülük, cilt yapıştırıcıların sofistike tasarımı etkinleştirme cilde sahip.

Bu protokol için üretim ince elastomer filmlerin detaylı yöntemleri sunulmaktadır. Yaygın olarak kullanılan Doktor blade tekniği ince bileşik film üretimi için kullanılmaktadır. İmalat tekniği polyethylenterephtalate (PET) folyo, büyük ölçekte bu filmlerin sonraki üretim etkinleştirme üzerinde yapıldı. Vurgu bu protokol tekrarlanabilirlik, üretim kompozit filmlerin farklı katmanları ve bileşik yama final biyolojik ve yapışma özelliklerinin belirlenmesi hassas bir değerlendirme olduğunu. Silikon elastomer poly-(dimethylsiloxane) (PDMS) yoğun üretim cilt yapıştırıcılar, havacilik uygulamalar ve ek araştırma alanları1,2,3 de dahil olmak üzere Biyomedikal teknolojisinde kullanılan ,4. Son zamanlarda, yumuşak cilt yapıştırıcılar (SSAs) sözde PDMS, başka bir alt sınıfı olmuştur tanıttı, bağ ve de-bağ nazik cilt için belirli.
Silikon SSAs functionalized vinil elastomerler, benzer polimerler silis5takviye devamsızlık göre farklı vardır. Diğer PDMS, SSA'ın Barcelona'da Young katsayısı geniş bir alanda modülasyonlu cross-linker konsantrasyon veya kuruma süresi6,7,8tarafından adapte edilebilir. Bu değişikliği Young katsayısı silikonlu elastomerler, malzemenin yapışkanlı özellikleri önemli ölçüde etkiler ve ayrıca üstünde yüzey9,10 kültürlü prokaryotik ve ökaryotik hücrelerde derin sonuçları vardır , 11. biyolojik hücresel düzeyde, bu gösterildi, ökaryotik hücreleri sinyal iletim düzeyde bir modülasyon matris esneklik veya kalınlığı yüzey9,10,12 yanıt ,13,14. Bu yüzden, hücre kültür uygulamaları ayarlanabilir mekanik özelliklere sahip polimerlerin geniş bir ilgi var. Önemlisi, özünde düşük yüzey enerji Silikon esaslı elastomerler ökaryotik hücrelerin hücre kültürü için optimal koşullar sağlamaz. Oksijen Plazma tedavidir çekme-off gücünü bir donanım için önde gelen PDMS düşük yüzey enerji geçici olarak artırmak için yaygın olarak kullanılan bir teknik eki, yayılmasını teşvik paralel iken moleküllerin yüzey adsorpsiyon azalma ve Ökaryotik hücreler15,16,17,18yayılması.
Malzeme özelliklerine ek olarak, yüzey topografyası hücresel yapışma ve yapışkanlı etkileşim iki malzeme19,20,21,22arasında büyük ölçüde etkiler. Yüzey pürüzlülüğü iki yüzey arasında iletişim oluşumu üzerinde çeşitli etkileri vardır: azaltma iletişim alanının yüksek elastik enerji etkisi çatlamak yaymayı yapıştırıcı gücü23, değiştirebilirsiniz yanı sıra asperities çevreleyen depolanan 24. İnsan derisi için kendi yapışkan filimler yapışma gelişmekte olan uygulama alanı, Örneğin, yara sosları, ECG elektrotlar fiksasyonu veya diğer taşınabilir elektronik cihazlar25,26,27olduğunu, 28. Öz-yapıştırıcılar ile ilgili olarak yüzey topografyası yapışkanlı performansını ölçmek için normal yapışma ölçümleri8,21' pürüzlülük değişen derecelerde ile cam yüzeylerde kullanılabilir. Burada, iki cam yüzeylerde yapıştırıcı polimer filmler özelliklerini araştırmak için seçilmiştir. İlk, bileşik filmler PDMS destek katman karıştırma oranında PDMS tarafından farklı karıştırma oranı ile kaplı 10-1 ağırlık parçaların ile karakterize. İkinci adımda yapışkanlı bir SSA katman her iki bileşenin miktarda eşit ağırlık ve destekleyici bir PDMS film üstüne film kalınlığı değişen hazırlanmıştır.

<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

Bu protokol için biz ince elastomer bileşik filmleri Gelişmiş hücre kültür uygulamaları için ve cilt yapıştırıcılar gelişimi için imal etmek yöntemleri mevcut. İki farklı poly-(dimethyl siloxanes) (PDMS ve yumuşak deri yapıştırıcı (SSA)), kullanılmıştır için biyolojik etkileri ve yapışkanlı özellikleri derinlik soruşturmada. Bileşik filmleri esnek destek katmanı ve yapışkan bir üst kaplama oluşur. Her iki katmanı Doktor blade uygulama tekniği ile imal edilmiştir. Mevcut araştırmada, katman kalınlığı bir fonksiyonu veya Young katsayısı üst tabaka bir varyasyon olarak kompozit filmleri yapışkanlı davranışını soruşturma. Young katsayısı PDMS in Bankası crosslinker karıştırma oranı değişen tarafından değiştirildi. Buna ek olarak, SSA film kalınlığı yaklaşık 320 µm. taramalı elektron mikroskobu için (SEM) yaklaşık 16 µm çeşitli ve optik mikroskobu kalınlığı ölçümleri için kullanılan. Elastomer filmlerin yapışkanlı Özellikler güçlü film kalınlığı, Young katsayısı polimerler ve yüzey özelliklerine bağlıdır. Bu nedenle, yumuşak ve sert yüzeyler sergileyen cam yüzeyler üzerinde bu filmlerin normal yapışma araştırıldı. Çekme-off stres ve iş ayrılık silikon elastomer karıştırma oranına bağlıdır.
Ayrıca, üzerinde bir destekleyici destek katmanı yerleştirilen yumuşak deri yapıştırıcı kalınlığı cilt uygulamaları için yamalar üretmek için çeşitli. Sitotoksisite, yayılmasını önleme ve hücresel yapışma L929 fare fibroblastlar (karıştırma oranı 10:1) PDMS ve (karıştırma oranı 50: 50) SSA filmleri üzerinde yapılmıştır. Biz burada, ilk kez yan yana karşılaştırma iki polimerler üretilen ince bileşik filmlerin göstermiştir ve biyolojik ve yapışkan özelliklerinin incelenmesi mevcut.

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

Polimer ince film Kompozit yapıların farklı Young'un dönmeler ya da kalınlıkları sahip üretim süreci için bir protokol sunulmuştur. Filmler için Gelişmiş hücre kültür çalışmaları veya deri yapıştırıcı olarak üretilmektedir.

İnce Film kompozit silikon elastomer hücre kültürü ve cilt uygulamaları için: üretim ve karakterizasyonu

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

10.6K Görüntüleme.  INM - Leibniz Institute for New Materials. Polimer ince film Kompozit yapıların farklı Young'un dönmeler ya da kalınlıkları sahip üretim süreci için bir protokol sunulmuştur. Filmler için Gelişmiş hücre kültür çalışmaları veya deri yapıştırıcı olarak üretilmektedir.

Video: 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.

Mekanik Dinamik Hücre Kültürü için Microfabricated platformlar

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

Biyomühendislik

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.

Hücresel Self-Meclis Biyomekanik Analizi ve Doku Mühendisliği, Hücre-Türetilmiş Doku Yüzüklerin Üretiyor yönelik

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.

Için İpek Film Kültür Sistemi In vitro Analizi ve Tasarımı Biyomateryal

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.

Hücre hücre Etkileşim kurgulama için Silikon mikroçipler

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

Biyoloji

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.

Microfabricated Kanallar Hücre Göç Çalışması

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.

PDMS Zarında Micropatterned Hücreler germe

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.

Kantitatif, Deri Yara Yönetimi için bir konformal Cilt benzeri elektronik Sistemi İmalat ve Karakterizasyonu

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.

3D Mekansal Hücre Kültürleri için hücre yapı iskeleleri olarak biyo-uyumlu sıvı kristal Elastomer Köpüklerin sentezi

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.

Kültür Ultrathin Porated elastik Hydrogels bir Biomimetic membran için çift olarak hücre

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.

Mikromakine Mikroakışkan Platformda Basitleştirilmiş Üç Boyutlu Çip Üzerinde Cilt Modeli Üretimi

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