This work was financially supported by Swiss National Science Foundation (NRP 62 &#8211; Smart Materials). Also acknowledged is the support of B. Hanselmann, K. Kehl, U. Sch&#252;tz and B. Leuthold.

1. Plasma-initiated Polymerization
<ol>
	<li>Preparing of monomer solution.
	<ol>
		<li>Dissolve HEMA (100 ml; 0.718 mol) in 200 ml water and wash 3x with hexane (100 ml) in a separating funnel. Saturate the aqueous phase with sodium chloride and extract the HEMA with diethyl ether (50 ml). Dry the organic phase over MgSO4 and remove the solvent in vacuo (100 mbar, 40 &#176;C). Distill the HEMA under reduced pressure (15 mbar; 99 &#176;C).</li>
		<li>Prepare a 0.62 M methanolic solution of the inhibitor-free HEMA produced in Section 1.1.1. Pour 30 ml of the solution into a one-necked flask and eliminate oxygen by bubbling Ar through the solution for 1 hr.</li>
	</ol>
	</li>
	<li>Surface-induced polymerization.
	<ol>
		<li>Position two polycarbonate membranes next to each other into the plasma chamber (Figure 2). Place the shiny side of the membrane pointing towards the gas phase.</li>
		<li>Connect the plasma chamber to a high vacuum (20 mbar) for 5 min. Close the valve to the vacuum and open the other valve, which is connected to argon and oxygen gas and purge the chamber with this mixture for 2 hr with 15 sccm argon and 2.5 sccm oxygen.</li>
		<li>Initiate the plasma and reduce the power to the desired power (for polycarbonate membrane: 12 W) and treat the membranes for 4 min with the plasma. Connect the monomer solution with the chamber by opening the corresponding valve. Switch off the plasma and evacuate the chamber.</li>
		<li>Connect the monomer solution with the chamber by opening the corresponding valve and pour the solution into the chamber. Ensure that the membranes are covered with the monomer solution. Open the valve connected to the argon and store the reaction mixture for 12 hr at 20 &#176;C (conditioned room).</li>
		<li>Remove the monomer solution. Wash the membranes with methanol in an ultrasonic bath for 5 min. Repeat the washing procedure with water.</li>
		<li>Dry the membrane in vacuo over molecular sieves for 2 hr.</li>
	</ol>
	</li>
</ol>
2. Postmodification of Coated Membranes
<ol>
	<li>Prepare a solution of spirobenzopyran 1 (Figure 1) (100 mg; 0.27 mmol), N,N-dicyclohexylcarbodiimide (DCC) (55 mg; 0.27 mmol) and dimethyl aminopyridine (DMAP) (33 mg, 0.27 mmol) in tert-butylmetylether (TBME) (12 ml).</li>
	<li>Place a protecting stirrer bar and a protecting grid into a round-bottom flask. Dry the flask and flood the flask with argon.</li>
	<li>Pour the solution into the flask, followed by the coated membrane.</li>
	<li>Stir gently at room temperature for 12 hr.</li>
	<li>Remove the solution and wash the membrane with tert-butylmetylether in an ultrasonic bath for 5 min. Repeat the washing procedure with ethanol and water.</li>
	<li>Dry the membrane in vacuo over molecular sieves for 2 hr.</li>
</ol>
3. Surface Tension Measurements
<ol>
	<li>Fix the membrane with a standard tape on a metal O-ring. Position a drop of nanopure water (3.3 &#956;l) on that part of the membrane surface, which is not in contact with the O-ring. Measure the contact angle (CA) at 5 different spots of the membrane.</li>
	<li>For testing the long term stability of the samples, measure the contact angles at three different spots of the membranes after 0, 1, 2, 3, 7, 14, and 21 days.</li>
</ol>
4. Permeability Tests of the Photochromic Membranes
<ol>
	<li>Fill the receptor chamber of the Franz diffusion cell with water (12 ml).</li>
	<li>Fix the membrane in a Franz diffusion cell. Ensure that the membrane is in contact with the water of the receptor chamber. Fill the donor chamber (the chamber on top of the membrane) with an aqueous caffeine solution (20 mM; 3.0 ml). Irradiate the membrane from the top of the donor chamber with white-light (Figure 3). Collect samples (200 &#956;l) from the receptor cell; for track-edged polycarbonate membranes with a pore diameter of 200 nm, collect samples every 10 min.</li>
	<li>Repeat the experiment as described in step 4.2. but irradiate the membrane with UV-light (366 nm, 80 W/m2) during the entire permeability test.</li>
	<li>Determination of the caffeine concentrations of the collected samples.
	<ol>
		<li>Plot a calibration curve with 15 different caffeine concentrations (between 0.05 mg/ml and 1.5 mM/L) using a UV/Vis spectrometer. Calibrate at 293 nm.</li>
		<li>Determine the concentration of each of the collected samples using the calibration curve.</li>
		<li>Plot the determined concentration vs. the time of the collected samples. Make a linear fit through the points and determine &#916;c from the slope.</li>
	</ol>
	</li>
</ol>

<ol>
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The authors have nothing to disclose.

The plasma process produces a purple gas, which is caused by ionized argon. An orange color would indicate the presence of undesired nitrogen from a leak. The plasma process does not only form radicals on the surface but also etches the membrane7,12. Too much etching can change the pore diameter significantly, which would influence the permeability of the membrane. The controlled reaction conditions of the presented setup allow enhancing the reproducibility of the plasma-initiated grafting process. Nevertheles...

Plasma modification of materials has become an important process in many industrial fields. Cleaning of surfaces and functionalization of surfaces without changing the bulk property of the material has made the plasma treatment an essential process in surface science1-8.
Plasma treatment of polymers results in homolytic bond cleavage. This leads to an edging of the polymeric material and to the formation of radical rich surfaces. By using plasma containing oxygen molecules, the surface becomes oxygen rich and thus more hydrophilic9-11. However, the hydrophilicity of the surfaces is not stable over time12. In order to enhance the long-term stability, the plasma treated surface can be chemically modified after or during the plasma process13-15. This treatment is normally performed by adding a reactive monomer species into the gas phase during the plasma process; these monomers then polymerize from the created radicals of the polymer surface. If the chemical treatment is performed with a nonvolatile monomer, the polymer grafting has to take place after the plasma modification. In order to perform a controlled grafting after the radicals are formed on the surface, a plasma setup is described, which allows the plasma-initiated surface-induced polymerization from the surface in solution under controlled conditions12,16.
The presentation focuses on the modification of track-edged polymer membranes12,17. By modifying the surface tension of these membranes, the permeability rate can be varied12. This clean and fast process allows the creation of very thin layers (&#60;5 nm), which cover the entire membrane surface without changing the bulk property of the polymer membrane. Due to the edging during the plasma process, the pore diameters of the track-edged membranes augment slightly12. The edging rate is depending on the polymer and has a linear behavior.
When using monomers with reactive functional groups, the grafted polymers can be further functionalized. This is demonstrated by the postmodification of a PHEMA-grafted membrane with a carboxylic acid functionalized spiropyran. This results in a photochromic surface, since spiropyran is known to transform into a merocyanine species when irradiated with UV-light. The spiropyran form can be reestablished by irradiating the merocyanine form with visible light (Figure 1)18,19. Since the merocyanine form is more polar than the spiropyran state, the surface tension of the coating can be triggered with light20. The change in surface tension influences the permeability resistance of the membrane towards aqueous solutions. The set-up how to perform the permeability tests of these light-responsive membranes will be shown and the significant change in permeability resistance (decrease in permeability resistance by 97%) is demonstrated. Such a membrane can be integrated in a drug delivery setup or in smart sensing systems.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51680/51680fig1highres.jpg" src="/files/ftp_upload/51680/51680fig1.jpg" /> 
Figure 1. Photoisomerization of spirobenzopyran compound 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

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is presented. The polymerization from the membrane surface is induced by plasma treatment of the membrane, followed by reacting the membrane surface with a methanolic solution of 2-hydroxyethyl methacrylate (HEMA). Special attention is given to the process parameters for the plasma treatment prior to the polymerization on the surface. For example, the influence of the plasma-treatment on different types of membranes (e.g.&#160;polyester, polycarbonate, polyvinylidene fluoride) is studied. Furthermore, the time-dependent stability of the surface-grafted membranes is shown by contact angle measurements. When grafting poly(2-hydroxyethyl methacrylate) (PHEMA) in this way, the surface can be further modified by esterification of the alcohol moiety of the polymer with a carboxylic acid function of the desired substance. These reactions can therefore be used for the functionalization of the membrane surface. For example, the surface tension of the membrane can be changed or a desired functionality as the presented light-responsiveness can be inserted. This is demonstrated by reacting PHEMA with a carboxylic acid functionalized spirobenzopyran unit which leads to a light-responsive membrane. The choice of solvent plays a major role in the postmodification step and is discussed in more detail in this paper. The permeability measurements of such functionalized membranes are performed using a Franz cell with an external light source. By changing the wavelength of the light from the visible to the UV-range, a change of permeability of aqueous caffeine solutions is observed.

The etch rate can be followed by weighing the membrane after different periods of time. As can be seen from Figure 4, the etch rate follows for polyester, polyvinylidene fluoride, and polycarbonate membranes a linear etch rate, which can be determined from the slope of the linear correlation of the etch time versus mass loss. As shown in Figure 4, the polycarbonate membranes show the lowest etch rate of all the three polymer membranes. One consequence of the etching is the chang...

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

A plasma-induced polymerization procedure is described for the surface-initiated polymerization on polymer membranes. Further postmodification of the grafted polymer with photochromic substances is presented with a protocol of conducting permeability measurements of light-responsive membranes.

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 Views.  Empa, Swiss Federal Laboratories for Materials Science and Technology.  Процедуру полимеризации плазменно-индуцированной описан поверхностно-инициатором полимеризации на полимерных мембран. Далее postmodification привитого полимера с фотохромных веществ представлена ​​с протоколом проведения измерений проницаемости легких пробл?...

Video: 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.

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

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.

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.

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.

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.

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.

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

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Surface Engineering of Pancreatic Islets with a Heparinized StarPEG Nanocoating

Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and survival post-transplantation. This protocol aims to achieve surface engineering of pancreatic islets using an ultrathin heparin-incorporated starPEG (Hep-PEG) nanocoating. To generate the Hep-PEG nanocoating for pancreatic islet surface engineering, heparin succinimidyl succinate (Heparin-NHS) was first synthesized by modification of its carboxylate groups using N-(3-dimethylamino propyl)-N'-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The Hep-PEG mixture was then formed by crosslinking of the amino end-functionalized eight-armed starPEG (starPEG-(NH2)8) and Heparin-NHS. For islet surface coating, mouse islets were isolated via collagenase digestion and gradient purification using Histopaque. Isolated islets were then treated with ice cold Hep-PEG solution for 10 min to allow covalent binding between NHS and the amine groups of islet cell membrane. Nanocoating with the Hep-PEG incurs minimal alteration to islet size and volume and heparinization of the islets with Hep-PEG may also reduce instant blood-mediated inflammatory reaction during islet transplantation. This "easy-to-adopt" approach is mild enough for surface engineering of living cells without compromising cell viability. Considering that heparin has shown binding affinity to multiple cytokines, the Hep-PEG nanocoating also provides an open platform that enables incorporation of unlimited functional biological mediators and multi-layered surfaces for living cell surface bioengineering.

This protocol aims to achieve surface engineering of pancreatic islets using a heparin-incorporated starPEG nanocoating via pseudo-bioorthogonal chemistry between the N-hydroxysuccinimide groups of the nanocoating and the amine groups of islet cell membrane.

Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and ...

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.

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

Wet-spinning-based Molding Process of Gelatin for Tissue Regeneration

This article presents an inexpensive method to fabricate gelatin, as a natural polymer, into monofilament fibers or other appropriate forms. Through the wet spinning method, gelatin fibers are produced by smooth extrusion in a suitable coagulation medium. To increase the functional surface of these gelatin fibers and their ability to mimic the features of tissues, gelatin can be molded into a tube form by referring to this concept. Examined by in vitro and in vivo tests, the gelatin tubes demonstrate a great potential for application in tissue engineering. Acting as a suitable filling gap material, gelatin tubes can be used to substitute the tissue in the damaged area (e.g., in the nervous or cardiovascular system), as well as to promote regeneration by providing a direct replacement of stem cells and neural circuitry. This protocol provides a detailed procedure for creating a biomaterial based on a natural polymer, and its implementation is expected to greatly benefit the development of correlative natural polymers, which help to realize tissue regeneration strategies.

We developed and describe a protocol based on the wet spinning concept, for the construction of gelatin-based biomaterials used for the application of tissue engineering.

This article presents an inexpensive method to fabricate gelatin, as a natural polymer, into monofilament fibers or other appropriate forms. Through the ...