The authors would like to thank Kelly A. Burke for technical assistance with ACC substrate preparation. Based on the article published in Biomaterials, Davis KA, et al., Dynamic cell behavior on shape memory polymer substrates, Biomaterials, doi:10.1016/j.biomaterials.2010.12.006, Copyright Elsevier (2011). This material is based upon work supported by NSF under Grant No. DMR-0907578.

1. Isothermal UV-Curing of NOA63
<ol>
<li>A custom curing chamber was developed using a glass slide (75 mm x 25 mm x 1 mm), a 1 mm thick Teflon spacer, and an aluminum plate (75 mm x 25 mm x 3 mm) as shown in Figure 1. The chamber is held together using small binder clips.</li>
<li>Inject the NOA63 into the chamber through a hole in the Teflon spacer using an 18 gauge needle. The NOA63 can be gently heated to ease injection.</li>
<li>Place the chamber on a hot plate set at 125 &deg;C and allow to heat to a uniform temperature for 5 min.</li>
<li>Pre-cure the NOA63 in a UV lamp chamber (&lambda;max = 365 nm; see Table) for 20 min with the lamp 6.5 cm from the surface of the glass.</li>
<li>Remove the NOA63 from the chamber while warm using a razor blade.</li>
<li>Post-cure the NOA63 under the UV light for 3 h 40 m on the hot plate at 125 &deg;C.</li>
<li>Store the NOA63 desiccated at -20 &deg;C.</li>
</ol>
2. Shape Memory Characterization of NOA63
<ol>
<li>Prepare a dumbbell specimen by hot-pressing a cured NOA63 film with a customized punch (see Table), whose dimensions are shown in Figure 2.</li>
<li>Load the specimen into a dynamic mechanical analyzer (DMA; see Table) with tensile fixture. Set the instrument to "force controlled" mode, then program the testing procedure as follows:
<ol>
<li>Equilibrate at 95.00 &deg;C</li>
<li>Isothermal for 10.00 min</li>
<li>Ramp force 0.300 N/min to 2.500 N</li>
<li>Isothermal for 5.00 min</li>
<li>Ramp 2.00 &deg;C/min to 20.00 &deg;C</li>
<li>Isothermal for 10.00 min</li>
<li>Ramp force 0.300 N/min to 0.015 N</li>
<li>Isothermal for 5.00 min</li>
<li>Ramp 2.00 &deg;C/min to 95.00 &deg;C</li>
<li>Repeat steps 2-9 two more times</li>
</ol></li>
</ol>
3. Preparation of Active Cell Culture Substrates
<ol>
<li>Individual samples can be prepared from the isothermally cured SMP film. Cut the SMP film with a razor to desired sample size. Place the SMP on a hot plate set at a temperature higher than the Tg to reduce the modulus and ease cutting.</li>
<li>The temporary shape can be fixed in a number of different ways. Here we use a bench top hydraulic press with heated/cooled platens to emboss a temporary topography. Set the temperature of the platens at a temperature above the Tg.</li>
<li>An embosser was made by curing epoxy on a vinyl record. This will produce a temporary shape of parallel grooves. Here, the embosser had triangular peaks 35 to 40 &mu;m high and 60 &mu;m wide, spaced 80 &mu;m apart. The embosser can be made from other materials and with different topographies, but must be stiffer than NOA63 at the embossing temperature. Place the SMP samples face down on the embosser and place the samples and embosser in the press.</li>
<li>Apply a ~100 kPa preload to make contact between the heating platens and the samples and hold for ~5 minutes to allow the samples to reach a uniform temperature.</li>
<li>Apply 1-6 MPa to the samples and hold for 1 minute. A stress of 4.7 MPa leads to incomplete replication of the embosser topography. The temporary topography produced has rounded peaks 25 to 35 &mu;m high and 150 &mu;m wide. The SMP will fracture if a larger stress is applied. Apply smaller stresses to introduce topographies with smaller amplitudes.</li>
<li>Reduce the temperature below the Tg. Here we use the water cooling capability of the press platens.</li>
<li>When the temperature is below the Tg, remove the applied force.</li>
<li>The samples can be stored desiccated at -20 &deg;C. When stored under these conditions, we have observed less than a 1 &mu;m decrease in amplitude recovery after two months for samples embossed with the vinyl record embosser.</li>
</ol>
4. Active Cell Culture Experiment
<ol>
<li>The UV light of a biological safety cabinet (BSC) is used to sterilize the samples. Arrange the samples face down in sterile dishes without lids and turn on the BSC UV light for 6 h.</li>
<li>Flip samples to new sterile dishes face up. Turn on the BSC UV light for 6 h.</li>
<li>The samples now need to be equilibrated to a relatively stable state before plating with cells. Place the samples in a 96 well plate and add 150 &mu;l of complete growth medium.</li>
<li>Put the plate in a 30 &deg;C incubator with 5% CO2 until reaching the desired partial recovery. Here we use 30 h to produce samples with large enough amplitude to align cells.</li>
<li>The samples can now be plated with cells. Place the samples in a new 96 well plate.</li>
<li>Add 150 &mu;l of cell solution to the samples. Here we use C3H/10T1/2 mouse embryonic fibroblasts at 20,000 cells/ml to achieve isolated cells (generally not in contact with other cells).</li>
<li>To allow the cells to attach and spread on the temporary topography, place in a 30 &deg;C incubator for 9.5 h.</li>
<li>To assay cell morphology before transition, remove samples and perform staining and fluorescence imaging of the samples. This material exhibits autofluorescence through most of the UV and visible range. Fluorophores in the far red end of the spectrum such as Alexa Fluor 647 are recommended to reduce background.</li>
<li>To trigger samples to recover, move the plate to a 37 &deg;C incubator and continue culture for 19 h to allow the material to recover and allow the cells to adapt their morphology to the new topography.</li>
<li>Remove samples and apply appropriate stain (phalloidin for filamentous actin imaging) and imaging procedures.</li>
</ol>
5. Representative Results: 
Cured NOA63 is a transparent, glassy solid that has excellent shape memory properties as shown in Figure 3. In this case the material was cured as in Protocol 1 above and shows a uniform Tg of 51.1 &deg;C (determined from the onset of E' drop). It is observed from the one way shape memory cycles (heating, deforming, cooling, recovering, Figure 3) that, a large percentage of strain was fixed after unloading at 20 &deg;C, corresponding to a fixing ratio15 (Rf) of 89.3% (averaged over three cycles; the same below for Rr). The fixed strain recovered at a recovery ratio (Rr) of 84.4% in a relatively small temperature range during heating. Furthermore, the shape memory performance showed no deterioration up to three cycles, in that all the curves follow almost exactly with each other.
NOA63 was used in this protocol because it is readily available from the manufacturer and supplied as an easily cured solvent-free prepolymer with photoinitiator. However, its composition is not disclosed by the supplier. It was found to allow high cell attachment and viability. Finally, the transition temperature could be tuned to allow a meaningful magnitude of recovery between two cell compatible temperatures. A number of other polymer systems could also be used with this protocol if the transition temperature is compatible with cell culture and if they promote cell adhesion and viability. 
The amplitude of the temporary topography (grooves) decreases over time at 30 &deg;C. By 30 h at 30 &deg;C, the amplitude has been reduced by ~50% (Figure 4, time 0)16. It reduces another 10% over the next 9.5 h. When recovery is triggered by increasing the temperature to 37 &deg;C, the amplitude reduces to 0.5% of the initial amplitude within 9.5 h. For the embosser used and an embossing stress of 4.9 MPa, this corresponds to a functional change of 13 &mu;m grooves to a nearly flat surface.
An example of a cell behavior controlled through the use of active cell culture substrates is a change in cytoskeleton organization. On temporary grooved substrates before recovery is triggered, the actin microfilaments align along the direction of the grooves (Figure 5a)16. After recovery by temperature increase, the microfilaments have reorganized and are randomly oriented. Control samples that have static grooves or a static flat surface do not reorganize after the increase in temperature (Figure 5b,c).
<img src="/files/ftp_upload/2903/2903fig1.jpg" alt="Figure 1"/> Figure 1: Schematic for NOA63 curing chamber. Cross-section view (left) and top-down view without glass cover (right).
<img src="/files/ftp_upload/2903/2903fig2.jpg" alt="Figure 2"/> Figure 2: The dumbbell geometry used for bulk shape memory characterization. W: width of narrow section, L: length of narrow section, G: gage length, WO: width overall, LO: length overall, D: distance between grips, R: radius of fillet, and RO: outer radius.
<img src="/files/ftp_upload/2903/2903fig3.jpg" alt="Figure 3"/> Figure 3: The bulk one-way shape memory of a single NOA63 cure, repeated 3 times (the asterisk indicates experimental onset). At the point denoted with an asterisk, the polymer has been heated and is then deformed by applying a stress to define its temporary shape. This strain is held constant, and the temperature is decreased to fix the temporary shape below the Tg of the polymer. The temperature is then increased, and the material recovers to its permanent shape as the temporary strain is reduced.
<img src="/files/ftp_upload/2903/2903fig4.jpg" alt="Figure 4"/> Figure 4: SMP recovery can be triggered under cell-culture-compatible temperatures. The amplitude of 25.6 &plusmn; 0.8 &mu;m present following embossing recovered to 12.6 &plusmn; 1.5 &mu;m after 30 h equilibration at 30 &deg;C (time 0, black circles). After the samples were moved to a 37 &deg;C incubator (9.5 h), the amplitude recovered to 1.1 &plusmn; 0.2 &mu;m within 3.5 h. The amplitude recovered to ~0.3 &plusmn; 0.1 &mu;m within 9.5 h and no detectable decrease over the final 9.5 h was observed (red triangles). Error bars represent one standard deviation (n = 4-6). Traces are contact profilometry scans of representative samples.
<img src="/files/ftp_upload/2903/2903fig5.jpg" alt="Figure 5"/> Figure 5: Cell actin cytoskeleton rearranges following topographic transition. a, Confocal images of cells stained with phalloidin on embossed substrates show microfilaments aligned with groove direction (white arrow) before transition and temperature increase. After transition, microfilaments have rearranged are randomly oriented. b, Cells on flat control substrates show randomly oriented microfilaments before and after temperature increase. c, Cells on grooved control substrates show microfilaments aligned with groove direction before and after temperature increase. Scale bar is 100 &mu;m. Traces are as in Figure 4.

<ol>
	<li>Liu, C., Qin, H., Mather, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+progress+in+shape-memory+polymers.">Review of progress in shape-memory polymers.</a> J. Mater. Chem. 17, 1543-1543 (2007).</li><li>Mather, P. T., Luo, X. F., Rousseau, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shape+Memory+Polymer+Research.">Shape Memory Polymer Research.</a> Annu. Rev. Mater. Res. 39, 445-445 (2009).</li><li>Lendlein, A., Kelch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shape+Memory+Polymers.">Shape Memory Polymers.</a> Angew. Chem. Int. 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No conflicts of interest declared.

The Tg of NOA63 can be easily controlled via the curing temperature. We used this to generate SMP substrates that can be triggered in a cell compatible range. NOA63 is plasticized by water which lowers the dry Tg, so we increased the dry Tg by curing at 125 &deg;C to move the wet Tg range between 30 and 37 &deg;C.
The active cell culture substrates demonstrated are able to control cell behavior. The results of microfilament reorganization highlight the potentia...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent or instrument</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>NOA63</td>
			<td>Norland Products Inc.</td>
			<td>NOA63</td>
			<td>Lot number 111</td>
		</tr>
		<tr>
			<td>Dogbone Punch</td>
			<td>TestResource, Inc. Shakopee, MN</td>
			<td>&nbsp;</td>
			<td>Scaled-down Type IV dogbone (ASTM D638-03)</td>
		</tr>
		<tr>
			<td>Benchtop Hydraulic Press</td>
			<td>Carver</td>
			<td>3851</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>C3H10T1/2 Mouse Embryonic Fibroblasts</td>
			<td>ATCC</td>
			<td>CCL-226</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biological Safety Cabinet</td>
			<td>Thermo Fisher</td>
			<td>1357</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>UV Lamp</td>
			<td>Spectroline</td>
			<td>SB-100PC</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dynamic Mechanical Analyzer (DMA)</td>
			<td>TA Instruments, Inc.</td>
			<td>Q800</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Inverted Fluorescence Microscope</td>
			<td>Leica</td>
			<td>Leica DMI 4000B</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Confocal Laser Scanning Microscope</td>
			<td>Zeiss</td>
			<td>LSM 710</td>
			<td>20x/0.8 NA air or a 40x/1.30 NA oil objective</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Shape Memory Polymers for Active Cell Culture at JoVE.com

Shape Memory Polymers for Active Cell Culture

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 ...

shape-memory-polymers-for-active-cell-culture

Research

JoVE Journal

Bioengineering

13.4K ビュー.  Syracuse Biomaterials Institute. 培養中に地形を変更する機能を細胞培養基板を開発するための方法が説明されています。このメソッドは永続的な形状を記憶する能力を持つ形状記憶ポリマーとして知られているスマート材料を利用しています。この概念は、材料と幅広い用途に適応可能です。

ビデオ: Shape Memory Polymers for Active Cell Culture

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

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 ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to the success of this approach is a scaffold that is able to conformally fit within an irregular defect while also having the requisite biodegradability, pore interconnectivity and bioactivity. By nature of their shape recovery and fixity properties, shape memory polymer (SMP) scaffolds could achieve defect &#8220;self-fitting.&#8221; In this way, following exposure to warm saline (~60 &#186;C), the SMP scaffold would become malleable, permitting it to be hand-pressed into an irregular defect. Subsequent cooling (~37 &#186;C) would return the scaffold to its relatively rigid state within the defect. To meet these requirements, this protocol describes the preparation of SMP scaffolds prepared via the photochemical cure of biodegradable polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method. A fused salt template is utilized to achieve pore interconnectivity. To realize bioactivity, a polydopamine coating is applied to the surface of the scaffold pore walls. Characterization of self-fitting and shape memory behaviors, pore interconnectivity and in vitro bioactivity are also described.

Fabrication of a Bioactive, PCL-based &quot;Self-fitting&quot; Shape Memory Polymer Scaffold

Scaffolds capable of fitting within cranio-maxillofacial (CMF) bone defects while exhibiting osteoconductivity and bioactivity are of interest. This protocol describes the preparation of a shape memory scaffold based on polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method employing a fused salt template and application of a bioactive polydopamine coating.

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to ...

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity.
In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported protocol is of general validity and can be straightforwardly extended to other biological preparations.

A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

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 ...