1. Aggregate preparation for measurement of tissue surface tension. 
For adherent cells, spheroids can be formed by using either the hanging drop method or by generating a coherent sheet of cells that can then be cut into 1 mm fragments.
Aggregate formation by the hanging drop method:
<ol>
<li>Near-confluent adherent cell cultures should be grown to 90% confluence, whereupon monolayers should be rinsed twice with PBS. After draining well, add 2 mls (for 100 mm plates) of 0.05% trypsin-1 mM EDTA, and incubate at 37&deg;C until cells detach. Stop trypsinization by adding 2 mls of complete medium and gently use a 5 ml pipette to triturate the mixture until cells are in suspension. Transfer cells to a 15 ml conical tube.</li>
<li>Add 40 &mu;l of a 10 mg/ml DNAse stock solution and incubate for 5 minutes at RT. Vortex briefly and centrifuge at 200 x g for 5 minutes.</li>
<li>Discard supernatant and wash pellet with 1 ml complete tissue culture medium. Repeat, then resuspend cells in 2 mls of complete tissue culture medium.</li>
<li>Count the cells using a hemacytometer, or automated cell counter and adjust concentration to 2.5 x 106 cells/ml.</li>
<li>Remove the lid from a 60 mm tissue culture dish and place 5 mls of PBS in the bottom of the dish. This will act as a hydration chamber.</li>
<li>Invert the lid and use a 20 &mu;l pipette to deposit 10 &mu;l drops onto the bottom of the lid. Make sure that drops are placed sufficiently apart so as to not touch. It is possible to place at least 20 drops per dish.</li>
<li>Invert the lid onto the PBS-filled bottom chamber and incubate at 37&deg;C/5% CO2/95% humidity, monitor the drops daily and incubate until either cell sheets or aggregates have formed.</li>
<li>Once sheets form, they can be transferred to round-bottom glass shaker flasks containing 3 mls of complete medium and incubated in a shaking water bath at 37&deg;C and 5% CO2 until spheroids form.</li>
</ol>
Aggregate formation by the cell sheet method:
<ol start="9">
<li>Single cell suspensions are prepared as described above but concentration is adjusted to 1x106 cells/ml.</li>
<li>Transfer 3 ml of the cell suspension to 10 ml round-bottom flasks (Belco, Vineland, NJ).</li>
<li>Incubate flasks in a gyratory water bath/shaker (New Brunswick Scientific, Edison, NJ) at 37&deg;C, 5% CO2 for 4h at 120 rpm until cells recover from trypsinization.</li>
<li>Transfer cells to a glass round-bottom centrifuge tube and centrifuge at 200 X g into a thin pellet. Incubate for another 24 h until a coherent sheet forms.</li>
<li>Gently swirl the culture tube to dislodge the sheet and pour the contents of the culture tube into a small, sterile glass tissue culture dish.</li>
<li>Use micro scalpels to cut the sheets into fragments of various sizes.</li>
<li>Incubate fragments at 37&deg;C on the gyratory waterbath shaker at 120 rpm under 5% CO2 for 2 to 3 days or until they become spherical.</li>
</ol>
2. Measurement of aggregate surface tension
<ol>
<li>The tissue surface tensiometer. The apparatus is shown in Figs. 1 and 2. The compression cell (Fig. 3) is composed of two chambers. The outer chamber (OC) is connected to a 37&deg;C circulating water pump, and serves to regulate the temperature of the inner chamber (IC). The chambers are constructed of milled Delrin and contain quartz windows for visualization of the aggregate. The lower assembly (LA) screws into the base of the inner chamber and is used to 1) position the aggregate in the inner chamber; 2) seal the bottom of the inner chamber; 3) elevate the aggregate to initiate compression; and (4) control the distance between the parallel plates and hence the compression of the aggregate. The central core (CC) of the assembly is adjustable. The tip of the central core (the pedestal) is composed of smooth Teflon and acts as the lower compression plate (LCP). The upper compression plate (UCP) is a Teflon cylinder 15mm long that hangs from the balance arm (B) by a flame-straightened nickel-chromium wire (NCW)*. During the course of an experiment, the cell aggregate (A) is positioned on the lower plate and raised until it contacts the upper plate. The upper plate is connected to the balance arm (B). Compression of the aggregate causes displacement of the balance arm. The balance is a Cahn/Ventron model 2000 recording electrobalance, which operates on the null balance principle. The fulcrum of the balance arm has an armature within a permanent magnetic field. When the balance is operating, it continuously modulates the current passing through the electromagnetic assembly, which in turn maintains the balance arm in the horizontal position. When an object is suspended from the balance arm, the voltage, which the balance applies to keep the arm in the horizontal position, is proportional to the object's weigh. This voltage measures the external compressive force applied to the aggregate. The aggregate's shape is monitored by visual observation through a 25 x Nikon SMZ10A stereoscope coupled to a computer equipped with a frame grabber. In order to minimize adhesion of cell aggregates to the compression plates, both the lower and upper compression plates were coated with poly (2-hydroxyethylmethacrylate) {poly(HEMA)}, a polymeric material to which cells do not adhere 16. 
* The wire is flame-straightened by hanging a 15-inch length of wire from a retort stand and clamping a small binder clip to the end. A Bunsen burner is then run up and down the length of the wire until the wire glows red. The straightened wire can then be cut into the appropriate lengths. A small hook is formed by bending the wire approximately &frac14; of an inch from the end using two razor blades. The other end is then inserted into the barrel of the lower compression plate.</li>
<li>Aggregate compression.
<ol>
<li>The inner chamber is filled with pre-warmed CO2-independent tissue culture medium (Gibco/BRL, NY).</li>
<li>Aggregates ranging in size from about 200-300 &mu; are positioned on the lower compression plate (Fig. 4A). Aggregates are loaded by aspirating an aggregate in tissue culture medium half-way up the tip of a Pasteur pipette fitted with a silicone bulb, transferring the pipette to the inner chamber, and positioning the tip of the pipette above the LCP. The aggregate is then gently expelled onto the LCP by gently squeezing the bulb. Alternatively, the aggregate is allowed to fall by gravity onto the LCP.</li>
<li>The upper compression plate (UCP) is positioned above the aggregate and allowed to settle, establishing a pre-compression apparent UCP weight baseline.</li> 
<li>The LCP is then raised until the aggregate is compressed against the UCP (Fig. 4B). Adjusting the height of the inner core of the lower apparatus will control different degrees of compression. Compression is monitored by observation through a dissecting microscope equipped with a CCD video camera.</li>
<li>Aggregate images are captured, digitized, and analyzed using ImageJ. Apparent UCP weight change is continuously recorded on a strip chart recorder, achievement of shape equilibrium being denoted by the leveling-off of the Cahn balance's voltage output. Each aggregate is subjected to 2 different degrees of compression.</li>
</ol>
</li>
<li>Calculation of aggregate surface tension. 
At shape equilibrium, the cohesivity of an aggregate of cells compressed between parallel plates to which it does not adhere can be obtained from the Young-Laplace equation (Fig. 5), where &sigma; is cohesivity, F is the force acting to compress the aggregate, &pi;r32 is the area of the surface of the aggregate upon which force F is exerted, and R2 and R3 are, respectively, the radius of the equator of the compressed aggregate and the radius of an arc defining its surface profile normal to the compressing plates and extending between them (Fig. 6). Measuring the compressive force and geometry at force and shape equilibrium and applying these measurements to the Young-Laplace equation generates numerical values of apparent tissue surface tension. Upon reaching equilibrium and calculation of &sigma;1, aggregates are decompressed and allowed to approach a second equilibrium and &sigma;2 is calculated as described above.</li>
<li>Confirmation of aggregate liquidity. 
The calculated surface tension of a liquid aggregate, when subjected to two different compressions will remain constant. In such aggregates the ratio of &sigma;2/&sigma;1 will be equal to 1 and will be less than the ratio of the force applied at each successive compression (F2/F1). In contrast, the calculated surface tension of an elastic aggregate will obey Hooke's law and increase proportionately to the applied force. For elastic aggregates the ratio of &sigma;2/&sigma;1 will not be equal to 1 but will instead approach the ratio of F2/F1. The surface tension of liquid aggregates will also be independent of aggregate size 2,17. Only measurements in which surface tension is independent of the applied force and size are used to calculate average &sigma; for each cell line. 
</li>
</ol>
3. Representative results:
Below is a table of typical TST results for aggregates of rat prostate fibroblasts (RPF) and rat prostate smooth muscle cells (RPSMC). As can be seen in Fig. 7 aggregates of RPF cells have a surface tension of 22.8 &plusmn; 1.1 dynes/cm. Moreover, the mean surface tension values measured after compression 1 and after compression 2 were statistically identical when compared by a paired t-test. We also compared the ratios of &sigma;2/&sigma;1 and F2/F1 to ensure that these aggregates did not obey Hooke's law, as they would if they behaved as elastic solids. As demonstrated in Table 1, the ratio of &sigma;2/&sigma;1 does indeed approach 1.0. Moreover, the ratio of F2/F1 was significantly greater than &sigma;2/&sigma;1 (paired t-test, P &lt; 0.05), further confirming that these aggregates do not obey Hooke's law and in fact behave as liquids. In contrast RPSMCs obeyed Hooke's law. As is evident in Table 1, the ratio of &sigma;2/&sigma;1 is significantly greater than 1 and was not statistically different than that of F2/F1. To further demonstrate liquid-like behavior, we explored the relationship between surface tension (&sigma;) and aggregate volume. As can be seen in Fig. 8, volume is independent of sigma for RPF cells (red regression line, r2 = 0.002), whereas there appears to be some dependence of sigma on volume for RPSMCs (blue regression line, r2 = 0.146). These data further confirm that RPF aggregates behave in a liquid-like manner, whereas those of RPSMCs appear to behave more as elastic solids. Only those mesurements obtained from aggregates behaving as liquids would be used to calculate surface tension.
<img src="/files/ftp_upload/2739/2739fig1.jpg" alt="Figure 1"/> 
Figure 1. Overview of the tissue surface tensiometer.
<img src="/files/ftp_upload/2739/2739fig2.jpg" alt="Figure 2"/> 
Figure 2. A more detailed view of the tensiometer chamber (right panel).
<img src="/files/ftp_upload/2739/2739fig3.jpg" alt="Figure 3"/> 
Figure 3. Schematic view of the tensiometer chamber.
<img src="/files/ftp_upload/2739/2739fig4.jpg" alt="Figure 4"/> 
Figure 4. Images of uncompressed (A) and compressed (B) aggregates.
<img src="/files/ftp_upload/2739/2739fig5.jpg" alt="Figure 5"/> 
Figure 5. The Laplace Equation.
<img src="/files/ftp_upload/2739/2739fig6.jpg" alt="Figure 6"/> 
Figure 6. Diagram of a liquid droplet compressed between two parallel plates to which it adheres poorly, at shape equilibrium. R1 and R2 are the two primary radii of curvature, at the droplet's equator and in a plane through its axis of symmetry, respectively. R3 is the radius of the droplet's circular area of contact with either compression plate. H is the distance between upper and lower compression plates. X is one side of a right-angled triangle with hypotenuse R2 extending to a point of contact between the droplet's surface and either compression plate.

<table border="1">
 <tbody> 
 <tr>
 <td>&nbsp;</td>
 <td>&sigma;1(dynes/cm &plusmn; SEM)</td>
 <td>&sigma;2(dynes/cm &plusmn; SEM)</td>
 <td>P&sigma;1 vs &sigma;2</td>
 <td>&sigma;1,2(dynes/cm &plusmn; SEM)</td>
 <td>&sigma;2/&sigma;1</td>
 <td>F2/F1</td>
 <td>P&sigma;2/&sigma;1 and F2F1</td> 
 </tr> 
 <tr>
 <td>RPF</td>
 <td>22.6 &plusmn; 1.7</td>
 <td>22.9 &plusmn; 1.4</td>
 <td>&gt; 0.05*</td>
 <td>22.8 &plusmn; 1.1</td>
 <td>1.04 &plusmn; 0.04</td>
 <td>1.47 &plusmn; 0.06</td>
 <td>&lt; 0.05</td>
 
 </tr>
 <tr>
 <td>RPSMC</td>
 <td>15.0 &plusmn; 2.8</td>
 <td>23.0 &plusmn; 3.2</td>
 <td>0.039</td>
 <td>NA</td>
 <td>1.9 &plusmn; 0.3</td>
 <td>1.6 &plusmn; 0.1</td>
 <td>0.16*</td>
 
 </tr> 
 </tbody>
</table>
Figure 7. TST measurements and confirmation of aggregate liquidity for aggregates of rat prostate fibroblasts and smooth muscle cells.
<img src="/files/ftp_upload/2739/2739fig8.jpg" alt="Figure 8"/>
 Figure 8. Relationship between sigma and volume for aggregates of RPF (red line) and RPSMCs (blue line).

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No conflicts of interest declared.

Measuring aggregate cohesion by TST is relatively straightforward. There are, however, key steps that must be mastered in order to generate useable TST data; 1) aggregates must be "healthy". This can be controlled by ensuring that aggregate formation begins with cells that are at optimal confluence prior to detachment. Aggregate size and time in culture must also be controlled to minimize the development of a necrotic core within the aggregate; 2) Another parameter that can influence TST measurements is the degree of adh...

<ul>
	<li>water bath/shaker (New Brunswick Scientific, Edison, NJ)</li> 
	<li>10 ml round-bottom flasks (Belco, Vineland, NJ)</li>
</ul>

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.

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Measurement of Aggregate Cohesion by Tissue Surface Tensiometry

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 ...

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12.5K Views.  UMDNJ-Robert Wood Johnson Medical School. 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.

Video: Measurement of Aggregate Cohesion by Tissue Surface Tensiometry

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

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 ...

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.

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 ...

Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. Kelvin probe force microscopy (KPFM) is a module of atomic force microscopy (AFM) that measures the contact potential difference between surfaces at the nano-scale. The combination of KPFM with AFM allows for the simultaneous generation of surface potential and topographical maps of biological samples such as bacterial cells. Here, we employ KPFM to examine the effects of surface potential on microbial adhesion to medically relevant surfaces such as stainless steel and gold. Surface potential maps revealed differences in surface potential for microbial membranes on different material substrates. A step-height graph was generated to show the difference in surface potential at a boundary area between the substrate surface and microorganisms. Changes in cellular membrane surface potential have been linked with changes in cellular metabolism and motility. Therefore, KPFM represents a powerful tool that can be utilized to examine the changes of microbial membrane surface potential upon adhesion to various substrate surfaces. In this study, we demonstrate the procedure to characterize the surface potential of individual methicillin-resistant Staphylococcus aureus USA100 cells on stainless steel and gold using KPFM.

Here, we present a protocol explaining the use of Kelvin probe force microscopy as a tool for generating high resolution nano-scale surface potential maps. This tool was applied to assess the role of surface potential on the binding capacity of microorganisms to substrate surfaces.

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. ...

Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM)

Exosomes are microvesicular structures that play a mediating role in intercellular communication. It is of interest to study the internal cargo of exosomes to determine if they carry disease discriminatory biomarkers. For performing exosomal analysis, it is necessary to develop a method for extracting and analyzing exosomes from target biofluids without damaging the internal content.
Electric field-induced release and measurement (EFIRM) is a method for specifically extracting exosomes from biofluids, unloading their cargo, and testing their internal RNA/protein content. Using an anti-human CD63 specific antibody magnetic microparticle, exosomes are first precipitated from biofluids. Following extraction, low-voltage electric cyclic square waves (CSW) are applied to disrupt the vesicular membrane and cause cargo unloading. The content of the exosome is hybridized to DNA primers or antibodies immobilized on an electrode surface for quantification of molecular content.
The EFIRM method is advantageous for extraction of exosomes and unloading cargo for analysis without lysis buffer. This method is capable of performing specific detection of both RNA and protein biomarker targets in the exosome. EFIRM extracts exosomes specifically based on their surface markers as opposed to size-based techniques.
Transmission electron microscopy (TEM) and assay demonstrate the functionality of the method for exosome capture and analysis. The EFIRM method was applied to exosomal analysis of 9 mice injected with human lung cancer H640 cells (a cell line transfected to express the exosome marker human CD63-GFP) in order to test their exosome profile against 11 mice receiving saline controls. Elevated levels of exosomal biomarkers (reference gene GAPDH and protein surface marker human CD63-GFP) were found for the H640 injected mice in both serum and saliva samples. Furthermore, saliva and serum samples were demonstrated to have linearity (R = 0.79). These results are suggestive for the viability of salivary exosome biomarkers for detection of distal diseases.

Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement EFIRM

Exosomes are microvesicular structures found within biofluids that potentially carry important disease discriminatory biomarkers. Here, a novel method is used to specifically extract exosomes and rapidly test the exosomal cargo for both RNA/protein targets following the disruption of exosomes using non-uniform electric cyclic square waves.

Exosomes are microvesicular structures that play a mediating role in intercellular communication. It is of interest to study the internal cargo of ...

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. In this article we outline a valid and reliable technique to prepare and test permeabilized skeletal muscle fibers in vitro.

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle ...

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 ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.

Biomaterials doped with Bone Morphogenetic Protein 2 (BMP2) have been used as a new therapeutic strategy to heal non-union bone fractures. To overcome side effects resulting from an uncontrollable release of the factor, we propose a new strategy to site-directly immobilize the factor, thus creating materials with improved osteogenic capabilities.

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present ...

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.