Este trabalho foi apoiado financeiramente pela Swiss National Science Foundation (NRP 62 - Materiais inteligentes). Também é reconhecido o apoio de B. Hanselmann, K. Kehl, U. Schütz e B. Leuthold.

1. A polimerização iniciou-Plasma <ol><li> Preparação da solução de monómero. <ol><li> Dissolve-HEMA (100 ml; 0,718 mol) em 200 ml de água e lava-se 3x com hexano (100 ml) numa ampola de decantação. Saturar a fase aquosa com cloreto de sódio e extrair o HEMA com éter dietílico (50 ml). Seca-se a fase orgânica sobre MgSO4 e remover o solvente sob vácuo (100 mbar, 40 ° C). Destila-se o HEMA sob pressão reduzida (15 mbar, 99 ° C). </li><li> Prepare uma solução de 0,62 M metanólico do HEMA sem inibidor produzido na Seção 1.1.1. Verter 30 ml da solução para um balão de uma tubuladura e eliminar oxigénio fazendo borbulhar Ar através da solução durante 1 hora. </li></ol></li><li> Polimerização induzida por Surface. <ol><li> Posição duas membranas de policarbonato ao lado do outro para a câmara de plasma (Figura 2). Coloque o lado brilhante da membrana apontando para a fase gasosa. </li><li> Ligue a câmara de plasma para ahlta vácuo (20 mbar) durante 5 min. Fechar a válvula de vácuo e abre a outra válvula, o qual está ligado ao árgon e gás de oxigénio e de purga da câmara com esta mistura durante 2 horas com 15 sccm de árgon e 2,5 sccm de oxigénio. </li><li> Iniciar o plasma e reduzir a potência para a potência desejada (por membrana de policarbonato: 12 W) e tratar as membranas de 4 min, com o plasma. Ligue a solução de monómero com a câmara através da abertura da válvula correspondente. Desligue o plasma e evacuar a câmara. </li><li> Ligue a solução de monómero com a câmara através da abertura da válvula correspondente e verter a solução para dentro da câmara. Certifique-se de que as membranas são cobertas com a solução de monómero. Abrir a válvula ligada ao árgon e armazenar a mistura de reacção durante 12 horas a 20 ° C (ambiente climatizado). </li><li> Remover a solução de monómero. Lavar as membranas com metanol num banho de ultra-sons durante 5 min. Repita o procedimento de lavagem com água. </li><li> Seca-se a membrana emvácuo sobre peneiras moleculares durante 2 horas. </li></ol></li></ol> 2. Postmodification de membranas revestidas <ol><li> Prepara-se uma solução de spirobenzopyran 1 (Figura 1) (100 mg; 0,27 mmol), N, N-diciclohexilcarbodiimida (DCC) (55 mg; 0,27 mmol) e dimetil-aminopiridina (DMAP) (33 mg, 0,27 mmol) em terc-butylmetylether (TBME) (12 ml). </li><li> Colocar uma barra de agitação e uma grelha de protecção para proteger um balão de fundo redondo. Seca-se o balão e inundar o balão com árgon. </li><li> Verter a solução para o balão, seguindo-se a membrana revestida. </li><li> Agita-se suavemente à temperatura ambiente durante 12 horas. </li><li> Remover a solução e lava-se a membrana com terc-butylmetylether num banho de ultra-sons durante 5 min. Repetir o processo de lavagem com etanol e água. </li><li> Seca-se a membrana sob vácuo sobre peneiras moleculares durante 2 horas. </li></ol> 3. Medidas tensão superficial <ol><li&gt; corrigir a membrana com um padrão de fita sobre um metal de O-ring. Posicionar uma gota de água nanopura (3,3 ul) sobre a parte da superfície da membrana, que não está em contacto com o O-ring. Meça o ângulo de contato (AC) em 5 pontos diferentes da membrana. </li><li> Para testar a estabilidade a longo prazo das amostras, medir os ângulos de contacto em três pontos diferentes das membranas após 0, 1, 2, 3, 7, 14 e 21 dias. </li></ol> 4. Testes permeabilidade das membranas fotossensíveis <ol><li> Encher a câmara do receptor da célula de difusão de Franz com água (12 ml). </li><li> Fixar a membrana numa célula de difusão de Franz. Assegure-se que a membrana está em contacto com a água da câmara receptora. Encher a câmara doadora (câmara em cima da membrana) com uma solução aquosa de cafeína (20 mM, 3,0 ml). Irradiar a membrana a partir do topo da câmara de dador com luz branca (Figura 3). Coletar amostras (200 mL) a partir da célula receptora; for gumes pista membranas de policarbonato com um diâmetro de poro de 200 nm, coletar amostras a cada 10 min. </li><li> Repetir a experiência conforme descrito no passo 4.2. mas irradiar a membrana com luz UV (366 nm, 80 W / m 2) durante todo o ensaio de permeabilidade. </li><li> Determinação das concentrações de cafeína das amostras recolhidas. <ol><li> Traça-se a curva de calibração com concentrações de cafeína 15 diferentes (entre 0,05 mg / ml e 1,5 mM / L), utilizando um de UV / Vis espectrómetro. Calibrar a 293 nm. </li><li> Determinar a concentração de cada uma das amostras recolhidas utilizando a curva de calibração. </li><li> Traçar a concentração determinada vs. o tempo das amostras coletadas. Fazer um ajuste linear pelos pontos e determinar Ac a partir do declive. </li></ol></li></ol>

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O processo produz um plasma de gás de roxo, o qual é causado por árgon ionizado. Uma cor de laranja que indicaria a presença de azoto indesejada a partir de uma fuga. O processo de plasma não só formar radicais sobre a superfície mas também grava a membrana de 7,12. Demasiada gravação pode alterar o diâmetro do poro significativamente, o que iria influenciar a permeabilidade da membrana. As condições de reacção controlada da configuração apresentada permitir melhorar a reprodutibilidade do pr...

Modificação de plasma de materiais tornou-se um processo importante em muitos campos industriais. Limpeza de superfícies e de funcionalização de superfícies, sem alterar a propriedade de grandes quantidades de material fez o tratamento por plasma de um processo essencial em superfície ciência 1-8. Tratamento de plasma de polímeros resulta em homolítica clivagem da ligação. Isto leva a um debrum do material polimérico e para a formação de superfícies ricas radicais. Usando plasma contendo moléculas de oxigênio, a superfície torna-se oxigênio rico e, portanto, mais hidrofílica 9-11. No entanto, a hidrofilicidade das superfícies não é estável ao longo do tempo 12. A fim de melhorar a estabilidade a longo prazo, a superfície tratada a plasma pode ser quimicamente modificada depois ou durante o processo de plasma de 13-15. Este tratamento é normalmente realizado através da adição de uma espécie monomérica reactiva para a fase gasosa, durante o processo de plasma, estes monómeros, em seguida, polimerizarde entre os radicais criados de superfície do polímero. Se o produto químico de tratamento é realizado com um monómero não-volátil, o enxerto de polímero tem de ter lugar após a modificação do plasma. A fim de realizar um enxerto controlada após os radicais são formados na superfície, uma instalação de plasma é descrito, o qual permite que a polimerização induzida por superfície iniciada por plasma a partir da superfície em solução, sob condições controladas, 12,16. A apresentação centra-se na modificação de membranas de polímeros gumes-track 12,17. Ao modificar a tensão de superfície destas membranas, a taxa de permeabilidade pode ser variada 12. Este processo de limpeza e rápido permite a criação de camadas muito finas (&lt;5 nm), que cobrem toda a superfície da membrana sem alterar a propriedade de grandes quantidades de a membrana de polímero. Devido ao debrum durante o processo de plasma, os diâmetros de poro das membranas gumes pista aumentar ligeiramente 12. A taxa de afiação é depending do polímero e tem um comportamento linear. Quando se utiliza monómeros com grupos funcionais reactivos, os polímeros enxertados podem ser ainda mais funcionalizados. Isto é demonstrado pela postmodification de uma membrana enxertada com PHEMA com um ácido carboxílico spiropyran funcionalizado. Isto resulta numa superfície fotocrómico, desde spiropyran é conhecido para transformar uma espécie de merocianina quando irradiado com luz UV. A forma spiropyran pode ser restabelecida por irradiação da forma merocianina com luz visível (Figura 1) 18,19. Uma vez que a forma de merocianina é mais polar do que o estado spiropyran, a tensão de superfície do revestimento pode ser disparado com luz 20. A alteração na tensão superficial influencia a resistência à permeabilidade da membrana para as soluções aquosas. O set-up como realizar os testes de permeabilidade destes membranas de luz sensível neste período ea mudança significativa na resistência à permeabilidade (diminuir in resistência permeabilidade de 97%) é demonstrada. Tal membrana pode ser integrado numa instalação de entrega de droga ou em sistemas de sensores inteligentes. <img alt="Figura 1" fo:content-width="5in" fo:src="/files/ftp_upload/51680/51680fig1highres.jpg" src="/files/ftp_upload/51680/51680fig1.jpg" /> Figura 1. Fotoisomerização do composto spirobenzopyran 1.

<table border="0" cellpadding="0" cellspacing="0" style="width:943px;" width="943">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Name of Reagent/ Equipment</td>
			<td style="width:284px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">2-Hydroxyethyl methacrylate, 97%</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td style="width:119px;">128635</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Hexane 99%</td>
			<td style="width:284px;">Biosolve</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Magnesium sulfate (MgSO4, anhydrous)</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Methanol, 99%&#160;</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td style="width:119px;">14262</td>
			<td>dried over molecular sieves</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">N,N-Dicylcohexylcarbodiimide, 99%</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Dimethyl aminopyridine, 99%</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td style="width:119px;">107700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Tert-butylmethylether, 98%</td>
			<td style="width:284px;">Fluka</td>
			<td style="width:119px;">306975</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Polycarbonate membrane</td>
			<td style="width:284px;">Whatman</td>
			<td style="width:119px;"></td>
			<td>Nanopore Track Etched (TE) (1.0 &#956;m, 0.2 &#956;m, 0.1 &#956;m, 50 nm, 30 nm and 15 nm pore diameter; 47 mm or 25 mm membrane diameter)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Caffeine (reagent plus)</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td style="width:119px;">C0750&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Franz diffusion cell (12 ml)</td>
			<td style="width:284px;">SES-Analysesysteme</td>
			<td style="width:119px;">6C010015</td>
			<td>15mm unjacheted Franz Cell, 12 ml Receptor volume, Flat ground, clear glass, stirbat and clamp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">UV-Lamp</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td>UV irradiation (366 nm, 15 W/m2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">White light lamp</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td>White light irradiation (500 W bulb)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">UV/Vis spectrophotometer</td>
			<td style="width:284px;">Varian 50Bio/50MPR</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:373px;">Polyester membranes</td>
			<td style="width:284px;">Sterlitech</td>
			<td style="width:119px;">PET0225100</td>
			<td>Polyester Membrane Filters, 0.2 &#956;m pore diameter, 25 mmm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Polyvinylidene fluoride membranes</td>
			<td style="width:284px;">Millipore</td>
			<td style="width:119px;"></td>
			<td>PVDF Membranes Durapore (0.22 &#956;m pore diameter; 47 mm membrane diameter)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Argon (99.9995%)</td>
			<td style="width:284px;">Alphagaz</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Dressler Cesar RF Power Generator</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td>Plasma chamber setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">MKS Multi Gas Controller 647C</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td>Plasma chamber setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">MKS Mass-Flow controllers</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td>Plasma chamber setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Vacuubrand RE 2.5 rotary vane vacuum pump</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td>Plasma chamber setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Contact angle measurement device Kr&#252;ss G10</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Balances Mettler Toledo AB204-S and Mettler ME30</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of light-responsive membranes by a combined surface grafting and postmodification process

A fim de modificar a tensão de superfície de membranas de polímeros gumes traçado disponíveis comerciais, um processo de polimerização iniciou-superfície é apresentado. A polimerização da superfície da membrana é induzida por tratamento com plasma da membrana, seguida pela reacção a superfície da membrana com uma solução metanólica de 2-hidroxietil metacrilato (HEMA). É dada especial atenção para os parâmetros do processo para o tratamento de plasma, antes da polimerização sobre a superfície. Por exemplo, a influência do tratamento de plasma em diferentes tipos de membranas (por exemplo, de poliéster, de policarbonato, de fluoreto de polivinilideno) é estudada. Além disso, a estabilidade dependente do tempo das membranas de superfície enxertada é mostrado por medidas de ângulo de contacto. Quando enxerto poli (metacrilato de 2-hidroxietil) (PHEMA), desta forma, a superfície pode ser ainda modificado por esterificação da porção álcool do polímero com uma função de ácido carboxílico da substância desejada.Estas reacções podem ser utilizadas para a funcionalização da superfície da membrana. Por exemplo, a tensão de superfície da membrana pode ser alterada ou uma funcionalidade desejada como o apresentado luz-resposta pode ser inserido. Isto é demonstrado pela reacção de PHEMA com um ácido carboxílico funcionalizado unidade spirobenzopyran o que leva a uma membrana de luz que respondem. A escolha do solvente desempenha um papel importante na etapa postmodification e é discutido em mais pormenor no presente documento. As medições de permeabilidade de tais membranas funcionalizadas são realizados usando uma célula de Franz com uma fonte de luz externa. Ao alterar o comprimento de onda da luz do visível para a gama de UV, uma alteração da permeabilidade das soluções aquosas de cafeína é observado.

A taxa de corrosão pode ser seguido por pesagem da membrana após diferentes períodos de tempo. Como pode ser visto a partir da Figura 4, a taxa de corrosão segue para o poliéster, o fluoreto de polivinilideno, policarbonato e membranas de uma taxa de corrosão linear, que pode ser determinada a partir do declive da correlação linear do tempo etch contra a perda de massa. Como mostrado na Figura 4, as membranas de policarbonato mostram a menor taxa de corrosão de todas a...

Watch this Scientific Journal Video about Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process at JoVE.com

Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

Um processo de polimerização induzida por plasma é descrito para a polimerização iniciada na superfície em membranas poliméricas. Além disso postmodification do polímero enxertado com substâncias fotossensíveis é apresentado com um protocolo de realização de medições de permeabilidade das membranas de luz que respondem.

Preparação de Membranas de Luz respondem por um enxerto de superfície combinada e Processo Postmodification

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is ...

preparation-light-responsive-membranes-combined-surface-grafting

Research

JoVE Journal

Bioengineering

11.7K visualizações.  Empa, Swiss Federal Laboratories for Materials Science and Technology.  Um processo de polimerização induzida por plasma é descrito para a polimerização iniciada na superfície em membranas poliméricas. Além disso postmodification do polímero enxertado com substâncias fotossensíveis é apresentado com um protocolo de realização de medições de permeabilidade das membranas de luz que respondem. 

Vídeo: Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

Measurement of Aggregate Cohesion by Tissue Surface Tensiometry

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium. We have developed tissue surface tensiometry (TST) specifically to measure the surface free energy of interaction between cells. The biophysical concepts underlying TST have been previously described in detail1,2. The method is based on the observation that mutually cohesive cells, if maintained in shaking culture, will spontaneously assemble into clusters. Over time, these clusters will round up to form spheres. This rounding-up behavior mimics the behavior characteristic of liquid systems. Intercellular binding energy is measured by compressing spherical aggregates between parallel plates in a custom-designed tissue surface tensiometer. The same mathematical equation used to measure the surface tension of a liquid droplet is used to measure surface tension of 3D tissue-like spherical aggregates. The cellular equivalent of liquid surface tension is intercellular binding energy, or more generally, tissue cohesivity. Previous studies from our laboratory have shown that tissue surface tension (1) predicts how two groups of embryonic cells will interact with one another1-5, (2) can strongly influence the ability of tissues to interact with biomaterials6, (3) can be altered not only through direct manipulation of cadherin-based intercellular cohesion7, but also by manipulation of key ECM molecules such as FN8-11 and 4) correlates with invasive potential of lung cancer12, fibrosarcoma13, brain tumor14 and prostate tumor cell lines15. In this article we will describe the apparatus, detail the steps required to generate spheroids, to load the spheroids into the tensiometer chamber, to initiate aggregate compression, and to analyze and validate the tissue surface tension measurements generated.

We describe a method of measuring binding energy, expressible as tissue surface tension, between cells within 3D tissue-like aggregates. Differences in tissue surface tension have been demonstrated to correlate with invasiveness of lung, muscle, and brain tumors, and are fundamental determinants of establishing spatial relationships between different cell types.

Medição de Coesão agregados por tensiometria superfície do tecido

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium.  We ...

Bioengenharia

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Andaimes auto-relato para cultura de células 3-Dimensional

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

Geração e Alongamento das embarcações da engenharia de tecidos em um modelo de rato

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Adaptação do Processo Electrospinning para fornecer três ambientes únicos para um Tri-camadas<em&gt; In Vitro</em&gt; Modelo da parede das vias aéreas

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

Caracterização qualitativa da fração aquosa de Hydrothermal Liquefação de algas por cromatografia gasosa 2D com Time-of-flight Espectrometria de Massa

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using an automated robotic dispensing system. The particular bioink was selected as it allows cells to sediment towards and sense the features. The dispensing system bioprints viable cells in hydrogel bioinks using a backpressure assisted print head. However, by replacing the print head with a sharpened stylus or scalpel, the dispensing system can also be employed to create topographical cues through surface etching. The stylus movement can be programmed in steps of 10 &#181;m in the X, Y and Z directions. The patterned grooves were able to orientate mesenchymal stem cells, influencing them to adopt an elongated morphology in alignment with the grooves&#39; direction. The patterning could be designed using plotting software in straight lines, concentric circles, and sinusoidal waves. In a subsequent procedure, fibroblasts and mesenchymal stem cells were suspended in a 2% gelatin bioink, for bioprinting in a backpressure driven extrusion printhead. The cell bearing bioink was then printed using the same programmed coordinates used for the etching. The bioprinted cells were able to sense and react to the etched features as demonstrated by their elongated orientation along the direction of the etched grooves.

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

Técnica Automated Robotic dispensação de Orientação da superfície e bioprinting de Células

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

Deteção da endotoxina em Nano-formulações usando Limulus Amoebocyte Lysate (LAL) ensaios

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the cognitive powers of the brain with comparable efficiency and density. To date, silicon-based three-terminal transistors and two-terminal memristors have been widely used in neuromorphic circuits, in large part due to their ability to co-locate information processing and memory. Yet these devices cannot achieve the interconnectivity and complexity of the brain because they are power-hungry, fail to mimic key synaptic functionalities, and suffer from high noise and high switching voltages. To overcome these limitations, we have developed and characterized a biomolecular memristor that mimics the composition, structure, and switching characteristics of biological synapses. Here, we describe the process of assembling and characterizing biomolecular memristors consisting of a 5 nm-thick lipid bilayer formed between lipid-functionalized water droplets in oil and doped with voltage-activated alamethicin peptides. While similar assembly protocols have been used to investigate biophysical properties of droplet-supported lipid membranes and membrane-bound ion channels, this article focuses on key modifications of the droplet interface bilayer method essential for achieving consistent memristor performance. Specifically, we describe the liposome preparation process and the incorporation of alamethicin peptides in lipid bilayer membranes, and the appropriate concentrations of each constituent as well as their impact on the overall response of the memristors. We also detail the characterization process of biomolecular memristors, including measurement and analysis of memristive current-voltage relationships obtained via cyclic voltammetry, as well as short-term plasticity and learning in response to step-wise voltage pulse trains.

Soft, low-power, biomolecular memristors leverage similar composition, structure, and switching mechanisms of bio-synapses. Presented here is a protocol to assemble and characterize biomolecular memristors obtained from insulating lipid bilayers formed between water droplets in oil. The incorporation of voltage-activated alamethicin peptides results in memristive ionic conductance across the membrane.

Montagem e caracterização de Biomolecular Memristors consiste em membranas de lipídios dopado com canal iônico

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the ...

Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is deleterious for the cells as it reduces their ability to form a mature and functional layer. Here, we present a method for establishing and subculturing human and sheep corneal endothelial cell cultures that minimizes the loss of cell-to-cell contact. Explants of corneal endothelium/Descemet&#39;s membrane are taken from donor corneas and placed into tissue culture under conditions that allow the cells to collectively migrate onto the culture surface. Once a culture has been established, the explants are transferred to fresh plates to initiate new cultures. Dispase II is used to gently lift clumps of cells off tissue culture plates for subculturing. Corneal endothelial cell cultures that have been established using this protocol are suitable for transferring to biomaterial membranes to produce tissue-engineered cell layers for transplantation in animal trials. A custom-made device for supporting biomaterial membranes during tissue culture is described and an example of a tissue-engineered graft composed of a layer of corneal endothelial cells and a layer of corneal stromal cells on either side of a collagen type I membrane is presented.

This protocol describes the critical steps required to establish and grow corneal endothelial cell cultures from explants of human or sheep tissue. A method for subculturing corneal endothelial cells on membranous biomaterials is also presented.

Crescimento de Camadas de Células Endoteliais De Córnea Humana e Ovelha em Membranas Biomateriais

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is ...

Homogeneous Glycoconjugate Produced by Combined Unnatural Amino Acid Incorporation and Click-Chemistry for Vaccine Purposes

Genetic code expansion is a powerful tool to introduce unnatural amino acids (UAAs) into proteins to modify their characteristics, to study or create new protein functions or to have access to protein conjugates. Stop codon suppression, in particular amber codon suppression, has emerged as the most popular method to genetically introduce UAAs at defined positions. This methodology is herein applied to the preparation of a carrier protein containing an UAA harboring a bioorthogonal functional group. This reactive handle can next be used to specifically and efficiently graft a synthetic oligosaccharide hapten to provide a homogeneous glycoconjugate vaccine. The protocol is limited to the synthesis of glycoconjugates in a 1:1 carbohydrate hapten/carrier protein ratio but amenable to numerous pairs of biorthogonal functional groups. Glycococonjugate vaccine homogeneity is an important criterion to ensure complete physico-chemical characterization, thereby, satisfying more and more demanding drug regulatory agency recommendations, a criterion which is unmet by classical conjugation strategies. Moreover, this protocol makes it possible to finely tune the structure of the actual conjugate vaccine, giving rise to tools to address structure-immunogenicity relationships.

Genetic code expansion is applied for the introduction of an unnatural amino acid bearing a biorthogonal functional group on a carrier protein at a defined site. The biorthogonal function is further used for the site-selective coupling of a carbohydrate antigen to provide a homogeneous glycoconjugate vaccine.

Glicoconjugate homogêneo produzido por incorporação combinada de aminoácidos não naturais e click-química para fins de vacina

Genetic code expansion is a powerful tool to introduce unnatural amino acids (UAAs) into proteins to modify their characteristics, to study or create new ...