Name: microhoneycomb monoliths prepared by the unidirectional freeze-drying of cellulose nanofiber based sols: method and extensions
Uploaded: 2018-05-24T13:30:00
Description: Watch this Scientific Journal Video about Microhoneycomb Monoliths Prepared by the Unidirectional Freeze-drying of Cellulose Nanofiber Based Sols: Method and Extensions at JoVE.com

This work was supported by the National Basic Research Program of China (2014CB932400), National Natural Science Foundation of China (Nos. 51525204 and U1607206) and Shenzhen Basic Research Project (No. JCYJ20150529164918735). Also, we would like to thank Daicel-Allnex Ltd. and JSR Co. for kindly supplying polyurethanes and styrene butadiene rubber, respectively.

1. Preparation of 1 wt% 2,2,6,6-Tetramethylpiperidin-1-oxyl (TEMPO)-mediated Oxidized Cellulose Nanofiber (TOCN) Sol
NOTE: The sol is defined as a colloidal suspension of very small solid particles in a continuous liquid medium.
<ol>
	<li>Suspend 66.7 g of Nadelholz Bleached Kraft Pulp (NBKP, containing 12 g of cellulose) in 700 mL of deionized (DI) water with a mechanical agitator at 300 rpm for 20 min.</li>
	<li>Add 20 mL of aqueous TEMPO solution (containing 0.15 g of TEMPO) and 20 mL of ...

<ol>
	<li>Nishihara, H., Mukai, S. R., Yamashita, D., Tamon, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ordered+macroporous+silica+by+ice+templating.">Ordered macroporous silica by ice templating.</a> Chem. Mater. 17, 683-689 (2005).</li><li>Mukai, S. R., Nishihara, H., Yoshida, T., Taniguchi, K., Tamon, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+of+resorcinol-formaldehyde+gels+obtained+through+ice-templating.">Morphology of resorcinol-formaldehyde gels obtained through ice-templating.</a> Carbon. 43 (7), 1563-1565 (2005).</li><li>Mukai, S. R., Nishihara, H., Tamon, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porous+microfibers+and+microhoneycombs+synthesized+by+ice+templating.">Porous microfibers and microhoneycombs synthesized by ice templating.</a> Catal. Surv. Asia. 10 (3-4), 161-171 (2006).</li><li>Nishihara, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+monolithic+SiO2-Al2O3+cryogels+with+inter-connected+macropores+through+ice+templating.">Preparation of monolithic SiO2-Al2O3 cryogels with inter-connected macropores through ice templating.</a> J. Mater. Chem. 16 (31), 3231-3236 (2006).</li><li>Mukai, S. R., Mitani, K., Murata, S., Nishihara, H., Tamon, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembling+of+nanoparticles+using+ice+crystals.">Assembling of nanoparticles using ice crystals.</a> Mater. Chem. Phys. 123 (2), 347-350 (2010).</li><li>Cui, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembled+microhoneycomb+network+of+single-walled+carbon+nanotubes+for+solar+cells.">Self-assembled microhoneycomb network of single-walled carbon nanotubes for solar cells.</a> J. Phy. Chem. Lett. 4 (15), 2571-2576 (2013).</li><li>Xu, T., Wang, C. -. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+two-step+sintering+on+micro-honeycomb+BaTiO3+ceramics+prepared+by+freeze-casting+process.">Effect of two-step sintering on micro-honeycomb BaTiO3 ceramics prepared by freeze-casting process.</a> J. Eur. Ceram. Soc. 36 (10), 2647-2652 (2016).</li><li>Yoshida, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CO2+Separation+in+a+flow+system+by+silica+microhoneycombs+loaded+with+an+ionic+liquid+prepared+by+the+ice-templating+method.">CO2 Separation in a flow system by silica microhoneycombs loaded with an ionic liquid prepared by the ice-templating method.</a> Ind. Eng. Chem. Res. 56 (10), 2834-2839 (2017).</li><li>Mukai, S. R., Nishihara, H., Tamon, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+monolithic+silica+gel+microhoneycombs+(SMHs)+using+pseudosteady+state+growth+of+microstructural+ice+crystals.">Formation of monolithic silica gel microhoneycombs (SMHs) using pseudosteady state growth of microstructural ice crystals.</a> Chem. Commun. (7), 874-875 (2004).</li><li>Gutie&#180;rrez, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macroporous+3D+Architectures+of+Self-Assembled+MWCNT+Surface+Decorated+with+Pt+Nanoparticles+as+Anodes+for+a+Direct+Methanol+Fuel+Cell.">Macroporous 3D Architectures of Self-Assembled MWCNT Surface Decorated with Pt Nanoparticles as Anodes for a Direct Methanol Fuel Cell.</a> J. Phys. Chem. C. 111, 5557-5560 (2007).</li><li>Mukai, S. R., Nishihara, H., Tamon, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+maps+of+ice-templated+silica+gels+derived+from+silica+hydrogels+and+hydrosols.">Morphology maps of ice-templated silica gels derived from silica hydrogels and hydrosols.</a> Micropor. Mesopor. Mat. 116 (1-3), 166-170 (2008).</li><li>Okaji, R., Taki, K., Nagamine, S., Ohshima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+porous+honeycomb+monolith+from+UV-curable+monomer/dioxane+solution+via+unidirectional+freezing+and+UV+irradiation.">Preparation of porous honeycomb monolith from UV-curable monomer/dioxane solution via unidirectional freezing and UV irradiation.</a> J. Appl. Polym. Sci. 125 (4), 2874-2881 (2012).</li><li>Pan, Z. -. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellulose+nanofiber+as+a+distinct+structure-directing+agent+for+xylem-like+microhoneycomb+monoliths+by+unidirectional+freeze-drying.">Cellulose nanofiber as a distinct structure-directing agent for xylem-like microhoneycomb monoliths by unidirectional freeze-drying.</a> ACS Nano. 10 (12), 10689-10697 (2016).</li><li>Saito, T., Nishiyama, Y., Putaux, J. -. L., Vigon, M., Isogai, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homogeneous+Suspensions+of+Individualized+Microfibrils+from+TEMPO-Catalyzed+Oxidation+of+Native+Cellulose.">Homogeneous Suspensions of Individualized Microfibrils from TEMPO-Catalyzed Oxidation of Native Cellulose.</a> Biomacromolecules. 7 (6), 1687-1691 (2006).</li><li>Saito, T., Kimura, S., Nishiyama, Y., Isogai, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellulose+Nanofibers+Prepared+by+TEMPO-Mediated+Oxidation+of+Native+Cellulose.">Cellulose Nanofibers Prepared by TEMPO-Mediated Oxidation of Native Cellulose.</a> Biomacromolecules. 8, 2485-2491 (2007).</li><li>Bekyarova, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiscale+carbon+nanotube&#8722;+carbon+fiber+reinforcement+for+advanced+epoxy+composites.">Multiscale carbon nanotube&#8722; carbon fiber reinforcement for advanced epoxy composites.</a> Langmuir. 23, 3970-3974 (2007).</li><li>Nishihara, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Study on the simultaneous control of the nanostructure and morphology of the porous materials prepared via the ice-templating method [D]. , (2005).</li><li>Zhang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+porous+graphene+sponges+assembled+with+the+combination+of+surfactant+and+freeze-drying.">Three-dimensional porous graphene sponges assembled with the combination of surfactant and freeze-drying.</a> Nano Research. 7 (10), 1477-1487 (2014).</li><li>Tao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+ultrahigh+volumetric+capacitance:+graphene+derived+highly+dense+but+porous+carbons+for+supercapacitors.">Towards ultrahigh volumetric capacitance: graphene derived highly dense but porous carbons for supercapacitors.</a> Sci. Rep. 3, 2975 (2013).</li></ol>

The most critical step for achieving the MHMs is the unidirectional freezing step, during which water solidifies to form columnar ice crystals and push the dispersoid aside to form the framework. The unidirectional freezing process basically involves thermal transfer between the precursor sol and the coolant. In our setup, a dipping machine was used to insert a PP tube containing a precursor sol into the coolant (liquid nitrogen) with a constant velocity. Since liquid nitrogen keeps evaporating all the time, a fluctuant ...

As a brand-new material, microhoneycomb monolith (denoted MHM) has recently attracted tremendous attention from multidisciplinary fields1,2,3,4,5,6,7,8. The MHM was first prepared by S. Mukai et al. through a modified unidirectional freeze-drying (UDF) approach as a monolith with an array of straight microchannels with honeycomb-like cross sections9. MHM possesses the general advantages of honeycomb structures, i.e., efficient tessellation, high strength-to-weight ratio, and low pressure drop. Moreover, compared with the honeycomb monolith with a larger channel size, the MHM has a much larger specific surface area. The UDF method involves the unidirectional growth of ice crystals and simultaneous phase separation upon freezing. After the removal of the ice crystals, a solid component molded by the ice crystal is obtained. The morphology formed upon the phase separation depends on the intrinsic nature of the precursor (sol or gel), and in most of cases, lamella10, fiber11, and fishbone12 structures are likely to form rather than the MHMs. As a result, the formation of MHMs has been reported only in limited precursors, and this has significantly hampered the diversity of their chemical property. We have recently found that cellulose nanofibers have a strong structure-directing function toward forming the MHM structure through the UDF process13. Simply by mixing the cellulose nanofibers with other water-dispersible components, it is possible to prepare a variety of MHMs with different chemical properties. Moreover, their exterior shapes and channel sizes are flexibly and easily controlled13. Thus, MHMs are expected to be used as filters, catalyst supports, flow-type electrodes, sensors and scaffolds for biomaterials.
In this paper, we first explain the basic preparation technique of MHMs from the aqueous dispersion of cellulose nanofibers through the UDF process in detail. Moreover, we demonstrate the preparation of several different types of composite MHMs.

<table border="0" cellpadding="0" cellspacing="0" width="572">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nadelholz Bleached Kraft Pulp</td>
			<td style="width:100px;">Seioko PMC company</td>
			<td style="width:89px;">CSF=600</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TEMPO</td>
			<td style="width:100px;">Macklin Inc.</td>
			<td style="width:89px;">T819129</td>
			<td style="width:167px;">98%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaBr</td>
			<td style="width:100px;">Macklin Inc.</td>
			<td style="width:89px;">S818075</td>
			<td style="width:167px;">AR, 99%</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NaClO</td>
			<td style="width:100px;">Aladin Inc.</td>
			<td style="width:89px;">S101636</td>
			<td style="width:167px;">6-14 wt% active chlorine basis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SBR colloid</td>
			<td style="width:100px;">JSR corp.</td>
			<td style="width:89px;">TRD102A</td>
			<td style="width:167px;">48.5 wt%</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">TiO2</td>
			<td style="width:100px;">Sinopharm Chemical Reagent Co., Ltd.</td>
			<td style="width:89px;">A63725402</td>
			<td style="width:167px;">crystalline anatase phase</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">carbon fiber</td>
			<td style="width:100px;">Shenzhen Xian&#8217;gu Ltd.</td>
			<td style="width:89px;">XGCP-300</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nitric acid</td>
			<td style="width:100px;">Huada Reagent Ltd.</td>
			<td style="width:89px;">7697-37-2</td>
			<td style="width:167px;">65-68 wt%</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mixer</td>
			<td style="width:100px;">Scientific Industries, Inc</td>
			<td style="width:89px;">G-560</td>
			<td style="width:167px;">the mixer&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Mechanical blender</td>
			<td style="width:100px;">Waring Lab Ltd.</td>
			<td style="width:89px;">MX1000XTX</td>
			<td style="width:167px;">For disintegrating cellulose bundles into nanofibers.</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:216px;">Homogenizer</td>
			<td style="width:100px;">Scientz Ltd.</td>
			<td style="width:89px;">HXF-DY</td>
			<td style="width:167px;">For dispersing TiO2 nanoparticles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pH meter&#160;</td>
			<td style="width:100px;">Horiba Ltd.</td>
			<td style="width:89px;">F-74BW</td>
			<td></td>
		</tr>
	</tbody>
</table>

microhoneycomb monoliths prepared by the unidirectional freeze-drying of cellulose nanofiber based sols: method and extensions

Monolithic honeycomb structures have been attractive to multidisciplinary fields due to their high strength-to-weight ratio. Particularly, microhoneycomb monoliths (MHMs) with micrometer-scale channels are expected as efficient platforms for reactions and separations because of their large surface areas. Up to now, MHMs have been prepared by a unidirectional freeze-drying (UDF) method only from very limited precursors. Herein, we report a protocol from which a series of MHMs consisting of different components can be obtained. Recently, we found that cellulose nanofibers function as a distinct structure-directing agent towards the formation of MHMs through the UDF process. By mixing the cellulose nanofibers with water soluble substances which do not yield MHMs, a variety of composite MHMs can be prepared. This significantly enriches the chemical constitution of MHMs towards versatile applications.

The morphologies for different positions of the MHM-TOCN along the direction of unidirectional freezing are investigated and shown in Figure 2. With the position being further away from the bottom part of the MHM-TOCN, a gradual morphology change was revealed (Figure 2, Discussion). By introducing a second component into the TOCN sol to form a homogeneous mixture sol, it is possible to prepare various kinds of co...

Watch this Scientific Journal Video about Microhoneycomb Monoliths Prepared by the Unidirectional Freeze-drying of Cellulose Nanofiber Based Sols: Method and Extensions at JoVE.com

Microhoneycomb Monoliths Prepared by the Unidirectional Freeze-drying of Cellulose Nanofiber Based Sols: Method and Extensions

Here, we present a general protocol to prepare a variety of microhoneycomb monoliths (MHMs) in which fluid can pass through with an extremely low pressure drop. MHMs obtained are expected to be used as filters, catalyst supports, flow-type electrodes, sensors and scaffolds for biomaterials.

Monolithic honeycomb structures have been attractive to multidisciplinary fields due to their high strength-to-weight ratio. Particularly, microhoneycomb ...

This method can help answer key questions about achieving functional MHM materials for applications such as filtration, catalyst supporting, flow type batteries, sensors and bio-scaffolds. The main advantage of this technique is that it utilizes the structure directing function of cellulose nanofibers and can realize constitutional control of the targeted MHMs. This method can help answer key questions about achieving functional MHM materials for applications such as filtration, catalyst supporting, flow type batteries, sensors and bio-scaffolds.Demonstrating the procedure with myself and Cong Wang, a grad student from our laboratory. To begin preparing the tempo-mediated oxidized cellulose nanofiber sol, first combine 66.7 grams of softwood bleached kraft pulp with 700 microliters of deionized water. Mechanically agitate the mixture for 20 minutes at 300 rpm.Over the course of one minute, slowly add to the stirring kraft pulp suspension 20 milliliters each of a 7.5 gram per liter aqueous tempo solution and 75 gram per liter aqueous sodium bromide solution. Begin continuously monitoring the pH of the mixture with a pH meter. Adjust the mixture pH to about 10.5 with a three molar sodium hydroxide solution.Then, over the course of several minutes, slowly pipette into the suspension 63.8 grams of aqueous sodium hypochlorite with 6 to 14%active chlorine. After addition is complete, continue adjusting the pH back to 10.5 whenever it drops to about 10. Once the pH decrease slows, time how long it takes for the pH to drop from 10.5 to 10 to determine how often to check on the mixture during the reaction.Allow the tempo mediated oxidation to proceed normally for a bout 2.5 hours from the start of the sodium hypochlorite addition. Afterwards, filtrate the reaction mixture through a filter. And collect the oxidized cellulose nanofiber.Then, agitate the oxidized cellulose fibers in 1.2 liters of deionized water. Rinse the fibers three times in this way. Dry a small portion of the washed fiber paste overnight in an oven at 60 degrees Celsius to determine the solid content of the paste.Calculate the quantity of water needed to make a 1%by weight sol from the paste. Then transfer the washed fiber paste to laboratory grade blender. Add an appropriate amount of DI water to adjust the concentration of the mixture to 2%by weight.Vigorously blend the paste to disintegrate the oxidized cellulose fibers into nanofibers. Begin with low power blending before vigorously blending the paste at high power. Add one fifth of the needed volume of deionized water to the paste, and blend thoroughly.Repeat this process four more times to obtain the 1%by weight tempo mediated oxidized cellulose nanofiber sol. Store the sol at four degrees Celsius. We hear extracting is constant of the resulting TOCN sol.If it is hot enough, the MHM is likely to be obtained through the newly added process. Normally, the viscosity must be greater than 20, 000 millipascal second. Prior to preparing the tempo mediated oxidized cellulose nanofiber surface oxidized carbon fiber mixed sol, reflux 1.7 grams of 300 mesh carbon fiber in 150 milliliters of concentrated nitric acid to obtain the surface oxidized carbon fibers.Then, place in a 20 milliliter glass vial, 01 grams of the prepared surface oxidized carbon fibers, and 10 grams of the 1%by weight tempo mediated oxidized cellulose nanofiber sol. Manually shake the mixture until the surface oxidized carbon fibers are evenly distributed throughout the sol. Sonicate the mixture for five minutes to obtain an evenly dispersed TOCN SOCF mixed sol.Store the sol at four degrees Celsius. To begin preparing a microhoneycomb monolith, fill the bottom five centimeters of the 13 by 150 millimeter or similarly sized polypropylene tube with glass beads as filler material. Slowly load at least 2.7 milliliters of the chosen sol into the tube, ensuring that the sol level is about 22 millimeters or more above the glass beads.Avoid disturbing the sol unnecessarily to minimize the formation of bubbles in the tube. Use a narrow tipped pipette to carefully remove from the sol any bubbles that were introduced during the loading process. Store the sol sample at four degrees Celsius overnight.Then connect the sample tube to a dipping instrument for a unidirectional freezing. Place a Dewar of liquid nitrogen under the tube. Freeze the sol at an emerging speed of either 50 centimeters per hour for the TOCN sol or 20 centimeters per hour for the mixed sols.Keep the sol frozen once unidirectional freezing is complete. We use our cool time to constantly cool the frozen TOCN sol side hook to prevent it from sleep which will lead to the deterioration of the micromorphology of the resulting monolith. Use a saw to cut open the polypropylene tube.Crack the frozen sol into several pieces using pliers. Freeze dry the frozen sol pieces at minus ten degrees Celsius, minus five degrees Celsius, and zero degrees Celsius for one day each sequentially to obtain the microhoneycomb monolith. Unidirectionally freezing 1%by weight TOCN sol at 50 centimeters per hour without glass beads resulted in a gradual change in orientation and channel size along the freezing direction.The microhoneycomb morphology was retained longitudinally throughout the monolith. The bottom centimeter of the monolith was oriented toward the center of the bulk. A well-aligned honeycomb morphology was obtained two centimeters from the bottom.The microhoneycomb channel size increased from 10 micrometers between the second and third centimeters of the monolith and then remained stable. Overall, the unidirectional freezing effect showed no influence on the morphology past three centimeters. The channel size could be tuned by altering the dipping speed and the temperature of the coolant.Tempo oxidized cellulose nanofibers strongly tended to form the microhoneycomb structure via the unidirectional freezing process even in the presence of a second component. A mixed sol with styrene butadiene rubber yielded a composited microhoneycomb monolith with smooth walls. Tempo oxidized cellulose nanofiber microhoneycomb monoliths also supported titanium oxide nanoparticles in a well-ordered microhoneycomb monolith with nanoparticles adhering to the microhoneycomb walls.A mixed sol with SOCF yielded a novel substructure in which the carbon fibers bridged the neighboring microhoneycomb walls. The unidirectional freeze-drying technique is a novel approach through which many regular microstructures can be obtained. We first had the idea for this method will be so well in random microhoneycomb morphology in a wood pattern.It seems the major component of the wood is cellulose. We began to try imbuing up a similar construction with cellulose. Once mastered, this technique can be used to construct many other functional composite MHMs for targeted applications.

microhoneycomb-monoliths-prepared-unidirectional-freeze-drying

Research

JoVE Journal

Bioengineering

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as Binder

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose and short sisal fibers are dispersed into a water suspension containing bacterial cellulose. The fiber and nanocellulose suspension is then filtered (using vacuum or gravity) and the wet filter cake pressed to squeeze out any excess water, followed by a drying step. This will result in the hornification of the bacterial cellulose network, holding the loose natural fibers together.
Our method is specially suited for the manufacturing of rigid and robust preforms of hydrophilic fibers. The porous and hydrophilic nature of such fibers results in significant water uptake, drawing in the bacterial cellulose dispersed in the suspension. The bacterial cellulose will then be filtered against the surface of these fibers, forming a bacterial cellulose coating. When the loose fiber-bacterial cellulose suspension is filtered and dried, the adjacent bacterial cellulose forms a network and hornified to hold the otherwise loose fibers together.
The introduction of bacterial cellulose into the preform resulted in a significant increase of the mechanical properties of the fiber preforms. This can be attributed to the high stiffness and strength of the bacterial cellulose network. With this preform, renewable high performance hierarchical composites can also be manufactured by using conventional composite production methods, such as resin film infusion (RFI) or resin transfer molding (RTM). Here, we also describe the manufacturing of renewable hierarchical composites using double bag vacuum assisted resin infusion.

We present a novel method of manufacturing rigid and robust short natural fiber preforms using a papermaking process. Bacterial cellulose acts simultaneously as the binder for the loose fibers and provides rigidity to the fiber preforms. These preforms can be infused with a resin to produce truly green hierarchical composites.

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose ...

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (&#949;-caprolactone) (PCL) nanofibers (i.e., PCL/keratin composite fiber). Water soluble keratin was first extracted from human hair and mixed with PCL in different ratios. The blended solution of PCL/keratin was transformed into nanofibrous membranes using a laboratory designed electrospinning set up. Fiber morphology and mechanical properties of the obtained nanofiber were observed and measured using scanning electron microscopy and tensile tester. Furthermore, degradability and chemical properties of the nanofiber were studied by FTIR. SEM images showed uniform surface morphology for PCL/keratin fibers of different compositions. These PCL/keratin fibers also showed excellent mechanical properties such as Young&#39;s modulus and failure point. Fibroblast cells were able to attach and proliferate thus proving good cell viability. Based on the characteristics discussed above, we can strongly argue that the blended nanofibers of natural and synthetic polymers can represent an excellent development of composite materials that can be used for different biomedical applications.

Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

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

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

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

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

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

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with controlled pore structure and mechanical properties. Cellulose nanocrystals (CNCs, 69.62 wt%) based hydrogel ink with matrix (sodium alginate and gelatin) was developed and 3D printed into scaffolds with uniform and gradient pore structure (110-1,100 &#181;m). The scaffolds showed compression modulus in the range of 0.20-0.45 MPa when tested in simulated in vivo conditions (in distilled water at 37 &#176;C). The pore sizes and the compression modulus of the 3D scaffolds matched with the requirements needed for cartilage regeneration applications. This work demonstrates that the consistency of the ink can be controlled by the concentration of the precursors and porosity can be controlled by the 3D printing process and both of these factors in return defines the mechanical properties of the 3D printed porous hydrogel scaffold. This process method can therefore be used to fabricate structurally and compositionally customized scaffolds according to the specific needs of patients.&#160;

The three critical steps of this protocol are i) developing the right composition and consistency of the cellulose hydrogel ink, ii) 3D printing of scaffolds into various pore structures with good shape fidelity and dimensions and iii) demonstration of the mechanical properties in simulated body conditions for cartilage regeneration.

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with ...