Name: wet chemistry and peptide immobilization on polytetrafluoroethylene for improved cell-adhesion
Uploaded: 2016-08-15T14:00:00.000+00:00
Duration: 6 min 15 s
Description: Watch this Scientific Journal Video about Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion at JoVE.com

The authors would like to acknowledge the help of Walter Scholdei (Max-Planck-Institute for Polymer Research, Mainz, Germany.

Sodyum naftalinid Aktive Çözüm ve Yüzey Aktivasyon 1. hazırlanması Not: iyi havalandırılan bir davlumbaz tepkilere yürütmek. yüksek derecede yanıcı çözücüler ve metalik sodyum gibi aşındırıcı metallerin işlemek için genel kurallara uyun. Naftalin Çok az miktarlarda dahi çok hoş olmayan bir koku (mothball), var! aksi belirtilmedikçe reaksiyonlar, oda sıcaklığında gerçekleştirilir. Sodyum azid son derece zehirlidir! THF moleküler elek (hacim olarak) üzerinde yaklaşık% 20 muhafaza edildi (% 99.9, Malzeme listesine bakınız). sodyum üzerinde gözle görülür bir su içeriği ile THF damıtmak. su eser miktarlarda mevcut olması durumunda, sodyum naftalenid oluşumu oluşmaz. <ol><li> (3 A moleküler elek üzerinde kurutuldu, THF,), 20 ml tetrahidrofuran içinde naftalen 1.4 g (10.9 mmol) içindeki bir çözeltisine, bir PTFE ile donatılmış bir vidalı kapaklı 100 ml&#39;lik bir cam şişede, 0.25 g (10.9 mmol) sodyum metal ilave kaplanmış manyetik karıştırma çubuğu. Not: Dissolu(- 40 ° C ila 35) yon ölçüde küçük parçalar ve mütevazı ısıtma halinde sodyum kesilerek arttırılabilir. Nihai çözüm karanlık, biraz yeşilimsi rengi vardır ve kesinlikle kuru koşullarda saklanabilir. </li><li> 0.5 mm kalınlığında folyo malzemeden 12 mm çapında PTFE diskleri yumruk. Mark bir tarafı (örneğin, bir mektup damga yumruk kullanarak) izopropanol ile ve temiz. </li><li> 2 dk kullanarak forseps - 1 için aktive etme çözeltisi (adım 1.1) ayrı ayrı PTFE örnekleri inkübe. Not: koyu kahverengi, beyaz renk değişimi başarılı bir şekilde tedavi gösterir. </li><li> Daha sonra, izo-propanol ile iki kez THF ile ve daha sonra durulanır. Not: naftalenid çözüm tükendiğinde bir sulu açık kahverengi rengi değişir. (Muhtemelen metal sodyum ihtiva eden) Kullanılmayan naftalenid çözeltisi yavaş yavaş tasfiye etmeden önce, izo-propanol ilave edilerek dekompoze edilmesi gerekir. </li><li> Hidrojen peroksit (% 30) 3 saat süre ile trikloroasetik asit (ağ / hac)% 20 ihtiva eden tedavi örnekleri okside eder. Yıkamasu ve kuru ile. Yüzey şimdi hafif kahverengimsi bir görünüme sahiptir. Not: Aktivasyon ve oksidasyon sonucu yüzeyinin büyük ölçüde artmıştır ıslatılabilme içinde. Bu bulgu, daha önce 14 ayrıntılı olarak incelenmiştir. </li><li> 2 saat boyunca kuru THF içinde% 50 (h / h) heksametilen diizosiyanat (HMDI) ile oksitlenir diskleri tedavi THF ile durulanır ve kurumaya bırakın. </li><li> 3 saat ve kuru - 2 suda izosiyanat rulman örnekleri hidrolize. Not: Nihai amino işlevselleştirilmiş PTFE yüzeyi çok daha stabil izosiyanat taşıyan numuneler daha ayrıntılı bağlanma için birçok standart çapraz bağlayıcı ile uyumludur. </li></ol> 2. Peptit İmmobilizasyonu <ol><li> karbonat tamponu, pH 9, 50 mM% 20 (h / h) dietilenglikol diglisidil eter (diepoksit) içindeki bir çözeltisi hazırlandı. </li><li> ayrı ayrı 24 plaka içine belirgin tarafı yukarı ile amine diskleri yerleştirin ve epoksit çözeltisi 1.5 ml ekleyin. Örneklerin tam kapsama sağlamak. 2 saat boyunca inkübe ve iki yıkamakatı, suyla ve bir kez karbonat tamponu ile yıkandı. </li><li> Taze 24 plaka, tek tek oyuklara altına, NaN3% 0.01 ihtiva eden 50 mM bir 0.5 mg / ml peptid (ör REDV) karbonat tamponu, pH 9, 50 ul ekle ve dikkatli bir şekilde, epoksi-fonksiyonlu diskler ters yer aşağıya doğru peptid solüsyonu damlasına üzerine (yani aşağı yan işaretlenmiştir). Kuyunun dibine PTFE disk arasındaki boşluk tamamen nedeniyle kılcal eylem ıslanan olduğundan emin olun. </li><li> Islak odasında inkübe (örneğin, sıkı bir kapama kapağı ile bir sızdırmaz plastik kutu) nemli bir atmosferde gece boyunca en az 3 saat ya da için (alt ıslak kağıt mendil yerleştirerek). Not: REDV motifi gibi iyi karakterize kabul edilebilir olsa bir karıştırılmış veya ters bir amino asit sekansı, ek bir negatif kontrol olarak dahil edilebilir. </li><li> su ile üç kez yıkanır ve en az 30 dakika boyunca,% 50 izo-propanol / su içinde sterilize edilir. Tohumlama, rin hücre Öncekisteril fosfat tamponlu tuzlu su (PBS) içinde örnekleri e. </li></ol> 3. Hücre Yayımlama <ol><li> Standart hücre kültürü prosedürleri 17,18 kullanarak HUVECler büyütün. </li><li> 24 yuvalı plakalar içinde tadil edilmiş tarafı yukarı peptit-konjuge numuneler. Bir kontrol olarak işlenmemiş PTFE diskleri kullanın. </li><li> Tohum 5 x 10 4 oyuk başına 2 ml&#39;lik ortam içinde HUVECler ve bir kuluçka makinesi içinde 37 ° C de 4 saat ve% 5 CO2 inkübe edilir. </li><li> diskleri çıkarın, unattached hücreleri çıkarmak ve yeni bir 24 plaka aktarmak dikkatlice yıkayın. İstenen süre boyunca taze ortam 2 ml ilave edilir ve kuluçkaya (örneğin, 24 saat, w, 1, vb.) her iki günde orta değiştirin. </li><li> dimetil sülfoksit (DMSO) içinde 1 mg / ml Calcein-AM bir stok çözelti hazırlayın. </li><li> Büyümeden sonra hücreler, orta kaldırmak, PBS ile yıkayın ve PBS ihtiva eden 1 ul / ml Calcein-AM leke ekleme (örneğin, ml PBS her bir stok çözeltisi 1 ul). Hafifçe ajitasyon ve incubatKaranlıkta 45 dakika boyunca 37 ° C &#39;de, e. </li><li> Standart FITC filtreleri ile donatılmış bir floresan mikroskop mikrograflar almak (Ör 488 nm, Em 515 nm) hemen PBS ile yıkayın ve. 100-kat büyütme kullanın. değiştirilmemiş gelen triplicates gibi muamele örnekleri gerçekleştirin. </li><li> ImageJ yazılımı kullanarak hücreler tarafından kolonize alanı belirlemek. Alternatif elle hücreleri saymak veya ImageJ kullanın. Her iki yöntem de ilgili yüzeylerde eki ve hücrelerin büyümesi ile ilgili benzer bilgiler verecektir. </li></ol>

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The authors have nothing to disclose.

PTFE yüzey modifikasyonu protokolünün ayrıntılı açıklaması Şekil 6&#39;da gösterildiği gibi, polimer omurgasından flor ortadan kaldırılması ile başlayarak ardışık adımlardan oluşur. Bunun bir sonucu olarak, bir katman konjuge karbon-karbon çift bağlarının bol miktarda içerir, meydana gelen naftalenid tedavisi üzerine geliştirilen koyu kahverengimsi renk ile uyum. Asidik hidrojen peroksit ile standart oksidasyon soluk kahverengi aydınlatıcı eşliğinde hidroksile yüzey, b...

Yani gelişmiş biyouyumluluk, hücre yapışma eksikliği, fibrotik kapsülleme ve trombojenitesine uyarılmasını göstermezler süredir onaylanan tıpta kullanılan çeşitli polimerler, birkaç söz. biyo ve biyolojik sistemi arasındaki etkileşimler özellikle implantın yüzeyinde yer alır. Bunun bir sonucu olarak, araştırma etkilenmez malzemenin yığın özelliklerini bırakarak istenilen uygulama için uygun özellikleri oluşturmak için yüzey değişimi üzerine odaklanmıştır. Fizyolojik olarak inert bir polimer olarak politetrafloroetilen (PTFE) gibi fıtığı cerrahi mesh 1, ve en önemlisi, tıbbi bağlantı noktaları 2 ve, vasküler greftler 3 gibi birçok tıbbi alanlarda kullanılır. Özellikle kan temas durumlarda PTFE hidrofobik doğası çoğu zaman thromboti sonuçlanan bir sonucu platelet yapışması, plazma bileşenlerinin gibi spesifik olmayan adsorpsiyon neden olurc olayları ve greft 4 tıkanması. Bundan başka PTFE, en polimerleri gibi, arzu edilen bir özellik, vasküler greft 5, iç (lümen) yüzey üzerinde endotelyal hücreleri (EC) faydalı bir tabakanın oluşumuna yol olacaktır hücre yapışması ve kapsamı desteklemez. Bir biomimetic endotel özellikle onun antitrombojenik özelliklerini 6, doğal eşdeğeri işlevlerin çoğunu yerine getirmesi beklenir. Genel biomimetic değişiklik stratejisi etkilenmemiş malzemeler dökme özelliklerini bırakarak sadece hücre yapışma malzemeyi endowing kavramına dayanmaktadır. Buna ek olarak, platelet yapışma önleyici bir yapışkan (anti-kirlenme) 7 özellikleri dahil edilmesiyle azaltılabilir. Çeşitli peptiplere - çoğunlukla hücre-dışı matrisin proteinlerden türetilen - güçlü integrin 8 sınıfına ait, hücresel reseptörlere bağlanarak hücre yapışmasını geliştirmek olduğu tarif edilmiştir. olmakBu konuda bir örnek st bilinen çoğu hücre tipi ile etkileşim peptid Arg-Gly-Asp (RGD) &#39;dir. Diğer amino asit dizileri özel olarak spesifik hücreler üzerinde eksprese edilen integrinler tarafından tanınır. Örneğin, Arg-Glu-Asp-Val (REDV) ve Tyr-Ile-Gly-Ser-Arg (YIGSR) belirli bir şekilde 9 EC bağlamak için tespit edilmiştir. Peptidler kovalent immobilizasyon metaller ve polimerler 10,11 içeren doğal olmayan yapışkan malzemelerin bir bolluk üzerinde gerçekleştirilmiştir. Gözenekli PTFE, daha doğrusu PTFE (ePTFE) genişletilmiş - polietilen tereftalat (PET) ile birlikte - vasküler üretimi için en önemli malzeme 12 greftleri olduğunu. Plazma modifikasyonu 13 veya fotokimyasal yöntemlerle 14 tarafından uygun tedaviler için kurulan fiziksel teknikler, gözenekli ve / veya boru şeklinde yapılar sırasıyla gözenekler ya da lümene içinde kolayca tedavi edilebilir olmadığı gerçeği ile engellenmektedir. Islak kimyasıPTFE üzerinde çünkü çoğu kimyasal saldırılar 15 direnen fluorin içeren polimer derece etkisiz doğası zor bir iştir. Bu yazıda bir kovalent modifikasyon stratejisi için nispeten basit bir yöntem açıklanmaktadır. PTFE bondable işlemek için bir prosedür uyarlanan fonksiyonel gruplar biyolojik olarak aktif moleküllerin daha fazla bağlanması için çapa noktaları olarak hizmet malzemeler yüzeyinde oluşturulmuştur.

<table><tbody><tr><td>PTFE foil 0.5 mm</td><td>Cadillac Plastic&amp;nbsp;</td><td>n/a</td><td></td></tr><tr><td>REDV peptide</td><td>Genecust</td><td>n/a</td><td>custom synthesis &gt;95% purity</td></tr><tr><td>iso-propanol</td><td>Sigma Aldrich</td><td>34965</td><td></td></tr><tr><td>tetrahydrofurane (THF)</td><td>Sigma Aldrich</td><td>401757</td><td></td></tr><tr><td>dimethylsulfoxide</td><td>Sigma Aldrich</td><td>D8418</td><td></td></tr><tr><td>molecular sieve 3 &amp;Aring;</td><td>Sigma Aldrich</td><td>208574</td><td></td></tr><tr><td>sodium metal</td><td>Sigma Aldrich</td><td>483745</td><td></td></tr><tr><td>phosphate buffered saline (PBS)</td><td>Sigma Aldrich</td><td>D8537</td><td></td></tr><tr><td>naphthalene</td><td>Sigma Aldrich</td><td>147141</td><td></td></tr><tr><td>hydrogen peroxide 30%</td><td>Sigma Aldrich</td><td>95321</td><td></td></tr><tr><td>trichloroacetic acid</td><td>Sigma Aldrich</td><td>T6399&amp;nbsp;</td><td></td></tr><tr><td>diethylene glycol diglycidyl ether</td><td>Sigma Aldrich</td><td>17741</td><td></td></tr><tr><td>hexamethylene diisocyanate (HMDI)</td><td>Sigma Aldrich</td><td>52650</td><td></td></tr><tr><td>Calcein-AM</td><td>Sigma Aldrich</td><td>56496</td><td></td></tr><tr><td>sodium bicarbonate</td><td>Sigma Aldrich</td><td>S6014&amp;nbsp;</td><td></td></tr><tr><td>sodium azide</td><td>Sigma Aldrich</td><td>71290</td><td></td></tr><tr><td>24 well plates</td><td>Greiner-Bio-One</td><td>662 160</td><td></td></tr><tr><td>ATR-FTIR spectrophotometer Nicolet Magna-IR 850</td><td>&amp;nbsp;Nicolet</td><td>n/a</td><td></td></tr><tr><td>fluorescence microscope Olympus X-70</td><td>Olympus</td><td>n/a</td><td></td></tr><tr><td>humbilical vein endothelial cells (HUVECs)</td><td>Lonza</td><td>n/a</td><td></td></tr><tr><td>ePTFE vascular graft</td><td>Gore</td><td>n/a</td><td></td></tr></tbody></table>

wet chemistry and peptide immobilization on polytetrafluoroethylene for improved cell-adhesion

malzemeler hücre yapışma özelliklerine sahip yüzey endowing biyomalzeme araştırma ve doku mühendisliğinde yaygın bir stratejidir. Bu durum, bu maddeler de önlenebilir yeni sentezlenmiş polimerlerden tanıtımı ile bağlantılı özelliği ve yasal sorunlar nedeniyle tıpta uzun süreli kullanımı vardır önceden onaylanmış bir polimer için özellikle ilgi çekicidir. Politetrafloroetilen (PTFE), vasküler greft üretimi için en sık kullanılan malzeme biri ama polimerin özelliklerini teşvik hücre yapışmasını yoksundur. Endotelizasyon örneğin, endotel hücrelerinin konfluent tabakası ile greft iç yüzeyi tam kapsamı esas olarak yapay bir arayüz trombojenisitesini azaltmak suretiyle, en iyi performans için anahtar kabul edilir. Bu çalışma, peptit ile modifiye edilmiş PTFE üzerinde endotelyal hücrelerin büyümesini incelenmiş ve modifiye edilmemiş alt tabaka üzerine elde edilen bu sonuçlar karşılaştırır. ile Kavramaendotelyal hücre yapışma peptid Arg-Glu-Asp-Val (REDV) daha sonra konjügasyon adımları, ardından reaktif sodyum naftalenid ile fluorin-içeren polimerin aktivasyonu yoluyla gerçekleştirilir. Hücre kültürü, bir, iki haftalık bir süre boyunca gösterilmiştir peptid hareketsizleştirilmiş malzeme İnsan Umbilikal Ven Endotelyal Hücreleri (HUVECs) ve mükemmel hücresel büyüme kullanılarak gerçekleştirilir.

Önemli kimyasal reaksiyon adımlarının sonuçlar IR spektroskopisi (Şekil 1) ile izlendi. sodyum naftalenid ile ilk aktivasyon çift bağları oluşturur - ve küçük bir ölçüde - OH-işlevleri. Sinyal gösteren C = C bağının hemen hemen tamamen, hidroksil grubu taşıyan bir yüzey verecek şekilde oksidasyon ortadan kalkar. Daha fazla standart konjugasyon adımların analizi burada gösterilmemiştir. Aktivasyonu ve oksidasyona bağlı renk değişiklikleri ...

Watch this Scientific Journal Video about Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion at JoVE.com

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

Geliştirilmiş Hücre yapışma Politetrafloroetilen Islak Kimya ve Peptit İmmobilizasyonu

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

wet-chemistry-peptide-immobilization-on-polytetrafluoroethylene-for

Research

JoVE Journal

Bioengineering

7.7K Görüntüleme.  Sidra Cardiovascular Research. Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

Video: Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young&#8217;s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.&#160;primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.

Microwave-assisted Functionalization of Polyethylene glycol and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques.

Mikrodalga destekli Poli işlevselleştirilmesi (etilen glikol) ve zincir polimerizasyonu ve Hidrojel formasyonunda kullanılan On-reçine Peptides

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large ...

Kimya

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

Protein bazlı Hydrogels mimarilerde kolayca idare hücre kültür uygulamaları için

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Biyokimya

Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. Compared to other analytical separation techniques, such as chromatography, CE is cheap, robust and effectively requires no sample preparation (for a number of complex natural matrices or polymeric samples). CE is fast and can be used to follow the evolution of mixtures in real time (e.g., chemical reaction kinetics), as the signals observed for the separated compounds are directly proportional to their quantity in solution.
Here, the efficiency of CE is demonstrated for monitoring the covalent grafting of peptides onto chitosan films for subsequent biomedical applications. Chitosan&#39;s antimicrobial and biocompatible properties make it an attractive material for biomedical applications such as cell growth substrates. Covalently grafting the peptide RGDS (arginine - glycine - aspartic acid - serine) onto the surface of chitosan films aims at improving cell attachment. Historically, chromatography and amino acid analysis have been used to provide a direct measurement of the amount of grafted peptide. However, the fast separation and absence of sample preparation provided by CE enables equally accurate yet real-time monitoring of the peptide grafting process. CE is able to separate and quantify the different components of the reaction mixture: the (non-grafted) peptide and the chemical coupling agents. In this way the use of CE results in improved films for downstream applications.
The chitosan films were characterized through solid-state NMR (nuclear magnetic resonance) spectroscopy. This technique is more time-consuming and cannot be applied in real time, but yields a direct measurement of the peptide and thus validates the CE technique.

Free solution capillary electrophoresis is a fast, cheap and robust analytical method that enables the quantitative monitoring of chemical reactions in real time. Its utility for rapid, convenient and precise analysis is demonstrated here through analysis of covalent peptide grafting onto chitosan films for improved cell adhesion.

Kapiler Elektroforez Real Time Kitosan Films üzerine Peptit Aşılama Monitör

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

Proteinler kovalent immobilizasyon için tek molekül kuvvet spektroskopisi

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

Doku Mühendisliği Thermoresponsive Nanostructured Yüzeylerin Hazırlanması

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Biyomühendislik

Covalent Attachment of Single Molecules for AFM-based Force Spectroscopy

Atomic force microscopy (AFM)-based single molecule force spectroscopy is an ideal tool for investigating the interactions between a single polymer and surfaces. For a true single molecule experiment, covalent attachment of the probe molecule is essential because only then can hundreds of force-extension traces with one and the same single molecule be obtained. Many traces are in turn necessary to prove that a single molecule alone is probed. Additionally, passivation is crucial for preventing unwanted interactions between the single probe molecule and the AFM cantilever tip as well as between the AFM cantilever tip and the underlying surface. The functionalization protocol presented here is reliable and can easily be applied to a variety of polymers. Characteristic single molecule events (i.e., stretches and plateaus) are detected in the force-extension traces. From these events, physical parameters such as stretching force, desorption force and desorption length can be obtained. This is particularly important for the precise investigation of stimuli-responsive systems at the single molecule level. As exemplary systems poly(ethylene glycol) (PEG), poly(N-isopropylacrylamide) (PNiPAM) and polystyrene (PS) are stretched and desorbed from SiOx (for PEG and PNiPAM) and from hydrophobic self-assembled monolayer surfaces (for PS) in aqueous environment.

Covalent attachment of probe molecules to atomic force microscopy (AFM) cantilever tips is an essential technique for the investigation of their physical properties. This allows us to determine the stretching force, desorption force and length of polymers via AFM-based single molecule force spectroscopy with high reproducibility.

AFM tabanlı Kuvvet Spektroskopisi için Tek Moleküllerin Kovalent Eki

Atomic force microscopy (AFM)-based single molecule force spectroscopy is an ideal tool for investigating the interactions between a single polymer and ...

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

Biyoaktif proteinler veya peptidler hidrojel Photochemistry biyolojik uygulamalarda kullanarak üzerinde desenlendirme

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...

Mitigation of Blood Borne Cell Attachment to Metal Implants through CD47-Derived Peptide Immobilization

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, improving the biocompatibility of metal stents remains a significant challenge. The goal of this protocol is to describe a robust technique of metal surface modification by biologically active peptides to increase biocompatibility of blood contacting medical implants, including endovascular stents. CD47 is an immunological species-specific marker of self and has anti-inflammatory properties. Studies have shown that a 22 amino acid peptide corresponding to the Ig domain of CD47 in the extracellular region (pepCD47), has anti-inflammatory properties like the full-length protein. In vivo studies in rats, and ex vivo studies in rabbit and human blood experimental systems from our lab have demonstrated that pepCD47 immobilization on metals improves their biocompatibility by preventing inflammatory cell attachment and activation. This paper describes the step-by step protocol for the functionalization of metal surfaces and peptide attachment. The metal surfaces are modified using polyallylamine bisphosphate with latent thiol groups (PABT) followed by deprotection of thiols and amplification of thiol-reactive sites via reaction with polyethyleneimine installed with pyridyldithio groups (PEI-PDT). Finally, pepCD47, incorporating terminal cysteine residues connected to the core peptide sequence through a dual 8-amino-3,6-dioxa-octanoyl spacer, are attached to the metal surface via disulfide bonds. This methodology of peptide attachment to metal surface is efficient and relatively inexpensive and thus can be applied to improve biocompatibility of several metallic biomaterials.

Presented here is a protocol for appending peptide CD47 (pepCD47) to metal stents using polybisphosphonate chemistry. Functionalization of metal stents using pepCD47 prevents the attachment and activation of inflammatory cells thus improving their biocompatibility.

CD47-Türetilmiş Peptid İmmobilizasyonu Ile Metal İmplantlara Kan Yoluyla Bulaşan Hücre Bağlılığının Azaltılması

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, ...

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat soft substrates that deform under cell traction (2D-TFM). TFM relies on the use of linear elastic materials, such as polydimethylsiloxane (PDMS) or polyacrylamide (PA). For 2D-TFM on PA, the difficulty in achieving high throughput results mainly from the large variability of cell shapes and tractions, calling for standardization. We present a protocol to rapidly and efficiently fabricate micropatterned PA hydrogels for 2D-TFM studies. The micropatterns are first created by maskless photolithography using near-UV light where extracellular matrix proteins bind only to the micropatterned regions, while the rest of the surface remains non-adhesive for cells. The micropatterning of extracellular matrix proteins is due to the presence of active aldehyde groups, resulting in adhesive regions of different shapes to accommodate either single cells or groups of cells. For TFM measurements, we use PA hydrogels of different elasticity by varying the amounts of acrylamide and bis-acrylamide and tracking the displacement of embedded fluorescent beads to reconstruct cell traction fields with regularized Fourier Transform Traction Cytometry (FTTC).
To further achieve precise recording of cell forces, we describe the use of a controlled dose of patterned light to release cell tractions in defined regions for single cells or groups of cells. We call this method local UV illumination traction force microscopy (LUVI-TFM). With enzymatic treatment, all cells are detached from the sample simultaneously, whereas with LUVI-TFM traction forces of cells in different regions of the sample can be recorded in sequence. We demonstrate the applicability of this protocol (i) to study cell traction forces as a function of controlled adhesion to the substrate, and (ii) to achieve a greater number of experimental observations from the same sample.

Near-UV lithography and traction force microscopy are combined to measure cellular forces on micropatterned hydrogels. The targeted light-induced release of single cells enables a high number of measurements on the same sample.

Çekiş Kuvveti Mikroskopisinde Uygulamalar için Hidrojel Desenleme Teknikleri Kullanılarak Hücre Yapışıklıklarının Kontrolü

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat ...

Biyofonksiyonel Kendiliğinden Birleşen Peptitlerin Hücre Adezyonu ve Morfolojisi için Değerlendirilmesi

Fabrication and Characterization of Colorectal Cancer Organoids from SW1222 Cell Line in Ultrashort Self&#45;Assembling Peptide Matrix

Ultrashort self-assembling peptides (SAPs) can spontaneously form nanofibers that resemble the extracellular matrix. These fibers allow the formation of hydrogels that are biocompatible, biodegradable, and non-immunogenic. We have previously proven that SAPs, when biofunctionalized with protein-derived motifs, can mimic the extracellular matrix characteristics that support colorectal organoid formation. These biofunctional peptide hydrogels retain the original parent peptide's mechanical properties, tunability, and printability while incorporating cues that allow cell-matrix interactions to increase cell adhesion. This paper presents the protocols needed to evaluate and characterize the effects of various biofunctional peptide hydrogels on cell adhesion and lumen formation using an adenocarcinoma cancer cell line able to form colorectal cancer organoids cost-effectively. These protocols will help evaluate biofunctional peptide hydrogel effects on cell adhesion and luminal formation using immunostaining and fluorescence image analysis. The cell line used in this study has been previously utilized for generating organoids in animal-derived matrices.

Yazar Spotlight: Kolorektal Kanser Organoidleri için Ultra Kısa Peptit Matrislerinin Optimizasyonu

This protocol aims to evaluate biofunctional self-assembling peptides for cell adhesion, organoid morphology, and gene expression by immunostaining. We will use a colorectal cancer cell line to provide a cost-effective way of obtaining organoids for intensive testing.

Ultra Kısa Kendi Kendine Birleşen Peptit Matrisinde SW1222 Hücre Hattından Kolorektal Kanser Organoidlerinin İmalatı ve Karakterizasyonu

Ultrashort self-assembling peptides (SAPs) can spontaneously form nanofibers that resemble the extracellular matrix. These fibers allow the formation of ...