We acknowledge funding by DFG (1116/17-1) and the Leipzig School of Natural Sciences &#34;BuildMoNa&#34; (GSC 185). This work has been supported through the Fraunhofer Attract project 601 683. T. H. acknowledges funding from the European Social Fund (ESF-100077106).

1. Preparation of n HTs
NOTE: Here, n denotes the number of different single DNA strands involved in the formation of the helix tubes of a certain size. For n=8, eight different single DNA strands make up a unit ring.
<ol>
	<li>Purchase DNA sequences (HPLC purity grade or higher) from a suitable DNA synthesis service or perform high-quality synthesis and purification (exemplary sequences given in Table 1).</li>
	<li>Resuspend lyophilized oligonucleotides in purified water and follow the further resuspension steps given in the corresponding manual from the company. Adjust the amount of water to obtain a final concentration of 200 &#181;M.</li>
	<li>Determine concentrations of the single DNA strands to verify the values given by the distributor (e.g., via UV-Vis spectroscopy at 260 nm) since the exact stoichiometry is crucial for the assembly process. Calculate the concentration of the oligonucleotides, taking into account the specific molar weight and the extinction coefficient for each single-stranded DNA oligonucleotide. 
	NOTE: The extinction coefficient values inherently vary for the different sequences and are stated in the documentation of commercially purchased DNA. They can also be calculated according to the nearest-neighbor model using a number of freely available online tools. To comply with the linear range of the spectrometer, consider diluting the sample used for concentration determination.</li>
	<li>Using a micropipette, mix the DNA strands-each at the same concentration-in a container suitable for the thermocycler (e.g., a 200 &#181;L PCR tube) to form the desired nHTs (final buffer conditions: 1xTE (10 mM Tris and 1 mM EDTA, pH 8) and 12.5 mM MgCl2). 
	NOTE: Final DNA nHT samples consist of n- 1 strands, U1&#8722; Un&#8722;1, and one Tn strand. The concentration per strand can vary from sub-micromolar to more than 10 &#181;M, depending on the needs of the user (e.g., &#60;1 &#181;m per strand for the subsequent dilution to observe single filaments or higher concentrations for the examination of dense network mechanics).</li>
	<li>Hybridize the nHTs in a thermocycler (denaturation for 10 min at 90 &#176;C, with a subsequent quick drop to 65 &#176;C; slow temperature decrease from 65 &#176;C to 55 &#176;C, with complementary base pairing in 20 temperature steps of -0.5 K for 30 min each; and a quick drop from 55 &#176;C to 20 &#176;C); see Figure 2A. 
	NOTE: The slow decrease steps from 65 &#176;C can be lengthened to increase the average tube length; however, the subsequent quick drop to 20 &#176;C is crucial to avoid the aggregation of polymerized tubes.</li>
	<li>Store the hybridized nHTs for up to 3 weeks at 4 &#176;C without detectable degradation. 
	NOTE: For fluorescence microscopy, labeled nHTs are hybridized by (partly) substituting U1 with the modified U1-Cy3 (Table 1). For longer observation times, the addition of established anti-bleaching agents is advisable.</li>
	<li>Carefully dilute the sample to the desired final concentration (e.g., 4 &#181;M for networks or 20 nM for single-filament observations) using the sample buffer, if necessary. To minimize possible error sources, assemble samples at the desired final concentration.</li>
</ol>
2. Shear Rheology
<ol>
	<li>Choose an appropriate geometry (plate-plate or cone-plate) for the dynamic shear rheometer. For small volumes (i.e., below 200 &#181;L), use the cone-plate geometry.</li>
	<li>Load the sample on the dynamic shear rheometer.</li>
	<li>Passivate the air-water interface of the sample and environment using the following steps to prevent potential evaporation effects.
	<ol>
		<li>Surround the sample with 2.5 mL of the sample buffer bath.</li>
		<li>Add a surfactant just below the micelle concentration.</li>
		<li>Use a Hamilton syringe to surround the sample with a lipid to avoid direct contact of the sample with air.</li>
	</ol>
	</li>
	<li>Seal the sample chamber with a cap equipped with wet sponges and, if possible, with an additional water bath (not in contact with the sample) to further suppress evaporation.</li>
	<li>Start the measurement using the rheometer-specific software at room temperature; alternatively, perform the measurement at different temperatures, such as 37 &#176;C (if physiological conditions are needed). 
	NOTE: Cooling the sample is often accompanied by the condensation of water vapor from the surrounding air, while heating leads to enhanced evaporation. Both effects can uncontrollably change the concentration of the sample, so all measurements in a series should be performed at a constant temperature.</li>
	<li>Allow the sample to equilibrate for 2 h at room temperature. The time evolution may be monitored with a time sweep (one data point per minute; strain &#947; = 5%; frequency f = 1 Hz).</li>
	<li>Measure the mechanical properties with the desired test protocols. Start with a series of frequency sweeps (f-sweeps) because they leave the sample intact and allow for more measurements. 
	NOTE: Additionally, short f-sweeps should be carried out before and after longer measurements (such as long f-sweeps with a much higher resolution) to verify that the sample remained unchanged throughout the full measurement protocol.
	<ol>
		<li>Perform a short f-sweep (e.g., &#947; = 5%; f = 0.01 Hz to 30 Hz; 5 data points per decade).</li>
		<li>Perform a long f-sweep (e.g., &#947; = 5%; f = 0.001 Hz to 30 Hz; 21 data points per decade); the low-frequency regime can be omitted if not necessary to reduce measurement time.</li>
		<li>Perform a short f-sweep.</li>
		<li>Perform a &#947;-sweep (e.g., f = 1 Hz; &#947; = 0.0125% to 100%; 20 data points per decade). 
		NOTE: Use the short f-sweep first, as it is usually inconsistent with previous f-sweeps because networks usually rupture during &#947;-sweep or detach from the plates. Alternatively, use &#947;-ramp (e.g., = 0.025 s-1, t = 60 s); however, the ramps usually require changing the motor mode, so subsequent short f-sweeps would be falsified.</li>
	</ol>
	</li>
	<li>Bin and/or smooth the raw data with a Gaussian kernel.</li>
</ol>
3. Fluorescence-assisted Analysis of DNA Annealing
<ol>
	<li>Prepare 8 aliquots of 12 &#181;L for each sample using 1-4 &#181;M DNA per strand, 12.5 mM MgCl2 in 1x TE buffer, and 1x nucleic acid gel stain from a 10,000x DMSO stock. Make a negative control with no DNA, but include nucleic acid gel stain.</li>
	<li>Transfer the aliquots to a real-time PCR plate and seal with adhesive film (e.g., a 96-well reaction plate). 
	NOTE: The high temperatures used in the thermal annealing protocols will evaporate samples. Thus, make sure that the wells are tightly sealed with adhesive film to prevent water evaporation and concentration increase, particularly during long-term experiments.</li>
	<li>Centrifuge the plate at 100 x g for 1 min at room temperature to remove the gas bubbles; if the gas bubbles expand, the wells cannot be analyzed.</li>
	<li>Load the sealed plate into a real-time quantitative PCR system.</li>
	<li>Run an annealing program. Denature the DNA at 90 &#176;C for 10 min. Drop the temperature quickly to 72 &#176;C. Then, decrease the temperature slowly by 0.5 &#176;C every 30 min. After the signal has decayed, accelerate the temperature ramp. 
	NOTE: 72 &#176;C is sufficiently above the real hybridization temperature; thus, the signal is recorded over a broad temperature range suitable for determining the necessary temperature range.</li>
	<li>Make sure that the lid of the cycler heats to at least 100 &#176;C to prevent the condensation of evaporated sample on the adhesive film.</li>
	<li>During assembly, excite the samples with an argon-ion laser at 488 nm and detect the fluorescence at 550 nm when using a nucleic acid gel stain (or spectrally similar). For other dyes, choose an appropriate excitation/emission combination. Measure the fluorescence signal as often as possible to increase the overall signal quality using the built-in camera. 
	NOTE: Here, measurements were taken every 9 s.</li>
	<li>If preset melting and annealing programs are applied, use the provided software to analyze the data. If not, subtract the previous mean value from the mean value for each temperature step to obtain the relative change of the fluorescent signal.</li>
</ol>
4. AFM Imaging
<ol>
	<li>Cleave mica (e.g., mica &#34;V1&#34;) by attaching commercially available adhesive tape and ripping it off; the uppermost layer of the mica should be removed.</li>
	<li>Using a micropipette, deposit pre-formed nHT in Mg2+ containing folding buffer on the freshly cleaved mica and let it settle for 10 min.</li>
	<li>Spin the samples in short 3-s intervals at 2,000 x g on a small table centrifuge to remove the remaining liquid. Several nHTs should still be attached to the mica surface.</li>
	<li>Use a cantilever tip with a high stiffness (e.g., 54 Nm-1) and determine its resonance frequency with the internal AFM software.</li>
	<li>Record the height profile with the AFM in air in tapping mode at low scan rates (e.g., 1 line/s) to avoid damaging or displacing the nHTs.</li>
</ol>

The authors have nothing to disclose.

To obtain properly formed networks, assembling the DNA nanotubes is a crucial step. Errors during the synthesis process negatively impact the tube quality; therefore, it is recommended that HPLC or a more stringent process be used to purify the oligonucleotides. Since the formation of discrete rather than aggregated DNA nanotubes, as well as their length distribution, depends upon the equimolar stoichiometry of the n constituent oligonucleotides within the set, it is necessary to remeasure the concentrations of ...

Due to the complex behaviors enabled by their unique mechanical properties, semiflexible polymers are fundamental building blocks of living matter. In contrast to flexible polymers, semiflexible polymers adopt an outstretched configuration due to their non-vanishing backbone stiffness while still remaining subject to strong thermal fluctuations1. Thus, purely stochastic models cannot be applied to their behaviors, as with the extremes of fully flexible or rigid polymers. The so-called worm-like chain model2,3,4 was developed to quantify this stiffness via the lp, which is the decay constant of the tangent-tangent correlation along the filament4. If lp is comparable to the contour length (lc) of the filament, the polymer is considered semiflexible1. Analogous to the poles of a tent, their arrangements in networks or bundles stabilizes the entire collective system at low volume fractions, leading to unusual viscoelastic properties5,6,7,8,9. These structures provide high elasticities at large mesh sizes10, maintaining mechanical integrity while still facilitating diffusive and active transport processes. This property is especially suitable for biological systems such as the cytoskeleton or the extracellular matrix, but it is also widely used in food engineering1,11,12.
Going beyond their significance to living matter, it is crucial to comprehensively examine the physical properties of these structures in order to have the tools to develop biomimetic materials or novel hydrogels. In terms of semiflexible polymers, this implies the systematic determination of the collective properties of networks resulting from single-filament properties such as lp and the development of a descriptive theoretical framework. In pioneering studies, the cellular biopolymer actin was established as a model system for semiflexible polymers and is still widely considered the gold standard5,13,14,15,16,17. However, exhaustive studies are limited with this system since they are bound to the inherent properties of this protein. Various theoretical approaches have aimed at building a description of the non-trivial mechanical behaviors at the single-filament level and have led to notably different scaling predictions for the dependence of the linear elastic plateau shear modulus, G0 (i.e., the &#34;elasticity&#34; of the network), with respect to concentration (c) and lp6,7,13,14,15,18,19,20,21,22,23. While the concentration scaling is readily accessible in experiments with actin-based or other model systems and while theoretical predictions have been rigorously verified13,16,24,25, the scaling with respect to lp has remained experimentally inaccessible. This, however, is a major limitation since lp is also an independent variable that is the defining quantity of semiflexible polymers.
This central, natural limitation imposed by the fixed lp of actin or other biologically-derived polymers such as collagen has recently been resolved by employing tile-based DNA tubes, which are tunable in their mechanical properties9,26,27,28. Slight variations in the architectures of the tubes (e.g., different numbers of constituent DNA strands within the unit ring) yield distinct values for lp, which can be evaluated via fluorescence microscopy, either by analyzing one fluctuating tube or by evaluating the curved configurations of several adhered tubes, as described previously9,28. These analyses revealed that lp values of the different tube populations vary over more than one order of magnitude and that different evaluation techniques yield consistent results9,28.
Surprisingly, the overall scaling of the linear elastic plateau shear modulus G0 with respect to the concentration and lp has been reported to be inconsistent with all previous theoretical approaches9, in particular demonstrating a much stronger than predicted dependence upon lp. These findings emphasize the value of a new model system to study the central properties of semiflexible polymers. Employing n-helix DNA tubes dramatically broadens the scope of these investigations. Not only can lp be freely varied without changing the basic material, but the inherent programmable nature of DNA can enable the systematic examination of additional elements, such as crosslinks or kinetic switching processes. Additionally, these tubes are soluble in water and, in contrast to most proteins, stable in adequate pH and ionic conditions for several weeks, without detectable degradation9.
To assemble these tubes, a discrete set of DNA oligonucleotides is used, each of which contains two domains that share complementary base sequences to two neighboring strands (due to the specific sequences, a single strand cannot form structures such as hair pins). The complementary sequences hybridize in a cyclic manner, forming enclosed, half-overlapping rings of n interconnected double-helical segments (Figure 1A and B). These rings form at a discrete diameter (Figure 1C), and their half-overlapping configuration exposes axial sticky ends complementary to the sticky ends of another ring. This selective addition of matching oligonucleotides triggers a stacking of the rings, leading to the effective polymerization of filamentous DNA helix tubes of size n (nHT). Their contour lengths typically measure several microns in length, and their length distribution is comparable to that of actin filaments9,26,27,28. It has been shown for similar DNA nanotubes that they indeed exhibit polymerization kinetics similar to those of actin filaments and microtubules29. Depending on the number n of individual DNA strands making up the basic ring structure, the nHT architecture, as well as its circumference and diameter, can be controllably varied. Using more DNA strands increases the circumference of the rings/tubes, and the corresponding architectural change shifts the mechanical properties to higher lp values (Figure 1C), corresponding to a higher rigidity. On the mesoscopic scale, these larger lp values translate into less bent conformations due to the higher stiffness (Figure 1D and E).

<table border="0" cellpadding="0" cellspacing="0" width="667">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:169px;">AFM cantilever ACTA</td>
			<td style="width:196px;">AppNano</td>
			<td style="width:123px;"></td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">AFM - NanoWizard 3</td>
			<td style="width:196px;">JPK Instruments</td>
			<td style="width:123px;"></td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">CCD camera</td>
			<td style="width:196px;">Andor</td>
			<td style="width:123px;">iXon DV887</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">DMSO</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:123px;">D2650</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:169px;">DNA oligonucleotides</td>
			<td style="width:196px;">Biomers.net</td>
			<td style="width:123px;"></td>
			<td style="width:180px;">For sequences see Table 1</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:169px;">DNA Cy3-labeled oligonucleotides</td>
			<td style="width:196px;">Biomers.net</td>
			<td style="width:123px;"></td>
			<td style="width:180px;">For sequence see Table 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">EDTA</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:123px;">E-9884</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:169px;">Epi-fluorescence micro-scope</td>
			<td style="width:196px;">Leica</td>
			<td style="width:123px;">DM-IRB</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:169px;">MgCl2</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:123px;">M-8266</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:169px;">Mica &#34;V1&#34;, 12 mm round</td>
			<td style="width:196px;">Plano GmbH</td>
			<td style="width:123px;">50-12</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:169px;">MicroAmp&#174; Fast Optical 96-Well Reaction Plate</td>
			<td style="width:196px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:123px;">4346907</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:169px;">MicroAmp&#174; Optical Adhesive Film</td>
			<td style="width:196px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:123px;">4306311</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:169px;">NanoDrop 1000 Spectrophotometer</td>
			<td style="width:196px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:123px;"></td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">100x objective</td>
			<td style="width:196px;">Leica5</td>
			<td style="width:123px;">506168</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:169px;">Purified water</td>
			<td style="width:196px;">Merk Millipore - Milli-Q &#38; Elix</td>
			<td style="width:123px;"></td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Sapphire PCR tubes</td>
			<td style="width:196px;">Greiner Bio-One</td>
			<td style="width:123px;">683271</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:169px;">TProfessional Standard PCR Thermocycler</td>
			<td style="width:196px;">Core Life Sciences Inc.</td>
			<td style="width:123px;">070- Standard</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="42" style="height:42px;width:169px;">7900HT Fast Real-Time PCR System</td>
			<td style="width:196px;">Applied Biosystems</td>
			<td style="width:123px;">4351405</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Rheometer</td>
			<td style="width:196px;">TA Instruments</td>
			<td style="width:123px;">ARES</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:169px;">SYBR&#174; Green I nucleic acid gel stain</td>
			<td style="width:196px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:123px;">S7567</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Tris</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:123px;">T4661</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:169px;">Triton X-100</td>
			<td style="width:196px;">Sigma-Aldrich Co.</td>
			<td style="width:123px;">X-100</td>
			<td style="width:180px;">Suppresses evaporation of sample at air-water interface</td>
		</tr>
	</tbody>
</table>

dna nanotubes as a versatile tool to study semiflexible polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

The assembly of DNA nanotubes via a temperature ramp (Figure 2) is a very reliable method to form these artificial semiflexible polymers. These polymers have comparable characteristics to their naturally occurring counterparts, such as actin filaments, but provide a much broader experimental framework since their mechanical properties can be controllably altered9,27. Like biopolymers, they can be arra...

Watch this Scientific Journal Video about DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers at JoVE.com

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

dna-nanotubes-as-a-versatile-tool-to-study-semiflexible-polymers

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JoVE Journal

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6.8K Views.  Fraunhofer Institute for Cell Therapy and Immunology. Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

Video: DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the engineered pDNA is administered to the vaccines, it is transcribed and translated into immunogen proteins that can elicit responses from the immune system. Many ways of delivering DNA vaccines have been investigated; however each delivery route has its own advantages and pitfalls. Skin tattooing is a novel technique that is safe, cost-effective, and convenient. In addition, the punctures inflicted by the needle could also serve as a potent adjuvant. Here, we a) demonstrate the intradermal delivery of plasmid DNA encoding enhanced green fluorescent protein (pCX-EGFP) in a mouse model using a tattooing device and b) confirm the effective expression of EGFP in the skin cells using confocal microscopy.

Skin tattooing is a potent and safe way to delivery DNA vaccine intradermally. Here, a DNA plasmid encoding EGFP is delivered by tattooing to the skin of a laboratory mouse, and the expression of EGFP in the skin cells is then inspected by confocal microscopy.

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the ...

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...