Name: Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering (Video) | JoVE Journal
Uploaded: 2016-03-01T14:00:00.000+00:00
Duration: 12 min 22 s
Description: Learn to fabricate thermoresponsive nanostructured surfaces using the Langmuir-Schaefer method for controlled cell adhesion and detachment in tissue engineering applications.

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5 minutes

This study was financially supported by the Creation of Innovation Centers for Advanced Interdisciplinary Research Program's Project for Developing Innovation Systems "Cell Sheet Tissue Engineering Center (CSTEC)" of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

1. Synthesis of Polystyrene-block-poly(N-isopropylacrylamide) by Two-step Reversible Addition-fragmentation Chain Transfer (RAFT) Radical Polymerization
<ol>
	<li>Dissolve styrene (153.6 mmol), 4-cyano-4-(ethylsulfanylthiocarbonyl) sulfanylpentanoic acid (ECT; 0.2 mmol), and 4,4&#39;-Azobis(4-cyanovaleric acid) (ACVA; 0.04 mmol) in 40 ml of 1,4-dioxane. Freeze the solution in liquid nitrogen under vacuum for 15-20 min to remove the reactive species and gradually thaw at RT. Make sure that the solution is completely thawed and repeat this freeze-pump-thaw degassing cycle three times.</li>
	<li>Obtain the polystyrene (PSt) (Mw: 13,500) as a macro RAFT agent by polymerization at 70 &#176;C for 15 hr in an oil bath.</li>
	<li>Precipitate PSt macro RAFT agent with 800 ml of ether and dry in vacuo.</li>
	<li>Dissolve IPAAm monomer (4.32 mmol), PSt macro RAFT agent (0.022 mmol), and ACVA (0.004 mmol) in 4 ml of 1,4-dioxane.</li>
	<li>Remove the oxygen in the solution by the freeze-pump-thaw degassing cycles as mentioned in step 1.1.</li>
	<li>Perform a polymerization at 70 &#176;C for 15 hr in an oil bath after degassing. Obtain synthesized St-IPAAm molecule (Mw: 32,800) in the same manner as the PSt macro RAFT agent.</li>
</ol>
2. Preparation of Silanized Hydrophobic Modified Glass Substrates
<ol>
	<li>Wash glass substrates (24 mm x 50 mm) with an excess of acetone and ethanol and sonicate for 5 min to remove surface contaminants.</li>
	<li>Dry the substrates in an oven at 65 &#176;C for 30 min. Then use oxygen plasma (400 W, 3 min) to activate the surfaces of the substrates at RT.</li>
	<li>Immerse the substrates in toluene containing 1% hexyltrimethoxysilane overnight at RT to silanize the substrate.</li>
	<li>Wash the silanized substrates in toluene and immerse in acetone for 30 min to remove unreacted agents.</li>
	<li>Anneal substrates for 2 hr at 110 &#176;C to thoroughly immobilize the surface.</li>
	<li>Cut the silanized substrates by a glass cutter to 25 mm x 24 mm to fit the cell culture dishes (dish size: &#966;35 mm).</li>
</ol>
3. Preparation of Langmuir Films and Film-transferred Surface
<ol>
	<li>Place the Langmuir film instrument in a cabinet to prevent the accumulation of dust.</li>
	<li>Wash the Langmuir trough (size: 580 mm x 145 mm) and barriers with distilled water and ethanol to remove contaminants.</li>
	<li>Dry the trough and barriers by wiping with a lintless towel. Then fill the trough with approximately 110 ml of distilled water, and set the barriers on both sides of the trough. Note that distilled water should be added without spilling in the following steps from 3.5 to 3.13.</li>
	<li>Heat a platinum Wilhelmy plate (perimeter: 39.24 mm) for monitoring the surface tension with a gas burner until the plate turns red and then wash with distilled water to remove contaminants. Suspend the Wilhelmy plate on a wire attached to the surface-pressure-measurement instrument.</li>
	<li>Zero the surface-pressure-measurement instrument according to manufacturer&#39;s protocol. Compress the air-water interface on the trough by the barriers on the both sides of the trough until the interface reaches approximately 50 cm2 without any drops of polymer.</li>
	<li>Aspirate small contaminants until the surface pressure is nearly 0 mN/m.</li>
	<li>Reposition the barriers on both sides, and add distilled water to compensate for the decrease of distilled water from step 3.6.</li>
	<li>Dissolve 5 mg of the synthesized St-IPAAm molecule in 5 ml of a development solution of chloroform. 
	Note: Dichloromethane or toluene can also be used as the solvent.</li>
	<li>Gently drop 27 &#181;l of St-IPAAm dissolved in chloroform onto the trough using a microsyringe or micropipette.</li>
	<li>After waiting for 5 min to allow complete evaporation of chloroform, move both barriers horizontally to compress the St-IPAAm molecule at the interface. Maintain compression rate of the barriers at 0.5 mm/sec until the target area of 50 cm2 is reached. 
	Note: A rapid compression rate causes defects in the Langmuir film.</li>
	<li>Measure the surface pressure (&#960;)-Am isotherms with the platinum Wilhelmy plate attached to the surface-pressure-measurement instrument during compression according to manufacturer&#39;s protocol.</li>
	<li>After reaching the target area size, maintain the surface for 5 min to allow the St-IPAAm molecules to relax; the molecules do not reach equilibrium immediately after compression.</li>
	<li>Transfer the Langmuir film to a hydrophobically modified glass substrate using a transfer apparatus for 5 min to robustly adsorb the film. Fix the hydrophobic glass substrate in parallel on the device. Connect the device to an alignment stage and move perpendicularly.</li>
	<li>Lift the substrate horizontally with the transfer apparatus and dry for 1 day in a desiccator.</li>
</ol>
4. Culturing Cells and Optimizing Cell Adhesion and Detachment on the Langmuir Film Transferred Surface
<ol>
	<li>To prepare cell suspensions, culture bovine carotid artery endothelial cells (BAECs) to one third confluence at 37 &#176;C in 5% CO2 and 95% air on tissue culture polystyrene (TCPS) with Dulbecco&#39;s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 100 U/ml penicillin.</li>
	<li>After confluence is reached, treat BAECs with 3 ml of 0.25% trypsin-EDTA for 3 min at 37 &#176;C in 5% CO2 and 95% air.</li>
	<li>Deactivate the trypsin-EDTA by adding 10 ml of the DMEM containing 10% FBS, and collect the cell suspension to a 50 ml conical tube.</li>
	<li>Centrifuge at 120 x g for 5 min, and aspirate the supernatant. Re-suspend the cells with 10 ml of the DMEM.</li>
	<li>Place the St-IPAAm surfaces under ultraviolet light on a clean bench for sterilizing for 5 min.</li>
	<li>Seed the recovered cells on the St-IPAAm surfaces at a concentration of 1.0 x 104 cells/cm2 counted by a disposable hemocytometer and observe the cells on the surfaces by a microscope equipped with an incubator at 37 &#176;C with 5% CO2 and 95% air. 
	Note: Sterilize the St-IPAAm surfaces by ultraviolet light equipped to a clean bench.</li>
	<li>Record time-lapse images of adherent BAECs for approximately 24.5 hr at 37 &#176;C by a phase-contrast microscope with 10X magnification. After BAEC adhesion, record detachment of the BAECs from the St-IPAAm surface at 20 &#176;C for approximately 3.5 hr.</li>
</ol>
5. Cell Sheet Fabrication on the Langmuir Film-transferred Surfaces
<ol>
	<li>Culture BAECs used in the same manner described in Section 4.</li>
	<li>Seed a total of 1.0 x 105 cells/cm2 on St-IPAAm surfaces and incubate for 3 days at 37 &#176;C in 5% CO2. Confluent BAECs spontaneously detached at 20 &#176;C.</li>
</ol>

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</ol>

All authors contributed equally to writing the manuscript and have approved the final version. The authors declare that they have no competing financial interests.

A temperature-responsive surface was fabricated by the Langmuir-Schaefer method, and the surface properties for cell adhesion/detachment and cell sheet recovery were optimized. When using this method for the fabrication of surfaces, several steps are critical. The molecular composition of the St-IPAAm molecules has a great effect on the surface structure and the stability of the surface, and by extension, on cell adhesion and detachment. In particular, the St-IPAAm molecules should have a narrow molecular weight distribu...

Nanostructured surfaces have recently attracted substantial attention due to their various potential applications, including patterning, cell culture, cleaning, and surface switching. For example, superhydrophobic surfaces inspired by the nanostructure of the lotus leaf and other responsive surfaces are capable of reacting to external stimuli1-4.
The Langmuir film is one of the most widely studied polymer coatings. A Langmuir film is formed by dropping amphiphilic molecules onto an air-water interface5-8. The film can then be transferred onto a solid surface by physical or chemical adsorption, and the molecular conformation on a solid surface can be controlled using vertical and horizontal transfer methods9-12. The density of the Langmuir film can be precisely regulated by compressing the air-water interface. Recently, this method has also proven effective for fabricating nanoscaled sea-island structures by utilizing amphiphilic block copolymers. The nanostructures are assumed to consist of a core of hydrophobic segments and a shell of hydrophilic segments13-17. In addition, the number of nanostructures on a surface is regulated by controlling the area per molecule (Am) of the block copolymer at the interface.
We have focused on an original, unique scaffold-free tissue engineering approach, cell sheet engineering, using a temperature-responsive culture surface. The developed technology has been applied to regenerative therapies for various organs18. A temperature-responsive culture surface was fabricated by grafting poly(N-isopropylacrylamide) (PIPAAm), a temperature-responsive molecule, onto a surface19-27. PIPAAm and its copolymers exhibit a lower critical solution temperature (LCST) in aqueous media at temperatures near 32 &#176;C. The culture surface also exhibited a temperature-responsive alternation between hydrophobicity and hydrophilicity. At 37 &#176;C, the PIPAAm-grafted surface became hydrophobic, and cells readily attached and proliferated on the surface as well as on conventional tissue culture polystyrene. When the temperature was lowered to 20 &#176;C, the surface became hydrophilic, and cells spontaneously detached from the surface. Therefore, cultured confluent cells on the surface could be harvested as an intact sheet by changing the temperature. These cell adhesion and detachment properties were also displayed by a surface fabricated by Langmuir film coating for laboratory demonstration26, 27. A Langmuir film of block copolymers composed of polystyrene (P(St)) and PIPAAm (St-IPAAm) was fabricated. The Langmuir film with a specific Am could be horizontally transferred to a hydrophobically modified glass substrate. In addition, cell adhesion on and detachment from the prepared surface in response to temperature were evaluated.
Here, we describe protocols for the fabrication of a nanostructured Langmuir film composed of thermo-responsive amphiphilic block copolymers on a glass substrate. Our method may provide an effective fabrication technique for organic nanofilms in various fields of surface science and may facilitate more effective control of cell adhesion on and spontaneous detachment from a surface.

<table><tbody><tr><td>&lt;em&gt;N&lt;/em&gt;-isopropylacrylamide</td><td>Kohjin</td><td></td><td>No catalog number</td></tr><tr><td>Azobis(4-cyanovaleric acid)</td><td>Wako Pure Chemicals</td><td>016-19332</td><td></td></tr><tr><td>Styrene</td><td>Sigma-Aldrich</td><td>S4972</td><td></td></tr><tr><td>1,3,5-trioxane</td><td>Sigma-Aldrich</td><td>T81108</td><td></td></tr><tr><td>1,4-Dioxane</td><td>Wako Pure Chemicals</td><td>045-24491</td><td></td></tr><tr><td>DMEM</td><td>Sigma&amp;nbsp;</td><td>D6429</td><td></td></tr><tr><td>PBS</td><td>Nakarai</td><td>11482-15</td><td></td></tr><tr><td>Streptomycin</td><td>GIBCO BRL</td><td>15140-163</td><td></td></tr><tr><td>Penicillin</td><td>GIBCO BRL</td><td>15140-122</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Sigma</td><td>T4174</td><td></td></tr><tr><td>FBS</td><td>Japan Bioserum</td><td>JBS-11501</td><td></td></tr><tr><td>BAECs</td><td>Health Science Reserch Resources Bank</td><td>JCRB0099</td><td></td></tr><tr><td>Cover Glasses</td><td>Matsunami Glass Industry</td><td>C024501</td><td></td></tr><tr><td>AFM NanoScope V</td><td>Veeco</td><td></td><td></td></tr><tr><td>&lt;sup&gt;1&lt;/sup&gt;H NMR INOVA 400</td><td>Varian, Palo Alto</td><td></td><td></td></tr><tr><td>ATR/FT-IR NICOLET 6700</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>GPC HLC-8320GPC</td><td>Tosoh</td><td></td><td></td></tr><tr><td>TSKgel Super AW2500, AW3000, AW4000</td><td>Tosoh</td><td></td><td></td></tr><tr><td>Langmuir-Blodgett Deposition Troughs&amp;nbsp;</td><td>KSV Instruments</td><td>KN 2002</td><td>KSV NIWA Midium trough</td></tr><tr><td>Nikon ECLIPSE TE2000-U</td><td>Nikon</td><td></td><td></td></tr></tbody></table>

preparation of thermoresponsive nanostructured surfaces for tissue engineering

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Block copolymers composed of polystyrene and poly(N-isopropylacrylamide) (St-IPAAms) with specific molecular weights were synthesized by RAFT radical polymerization. ECT was prepared as a chain-transfer agent as described in Moad et al.28. Two St-IPAAm molecules of different PIPAAm chain lengths were synthesized, and the obtained block polymers were characterized by 1H nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC). The mol...

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

The overall goal of this Langmuir-Schaefer method with block co-polymers is to fabricate cell sheets on various conditions of the temperature-responsive surface, by controlling strength of cell adhesion and attachments. This method can help answer key questions in the biomaterials field, such as biocompatible materials, cell culture surface, and bioseparation. The main advantage of this technique is that the interaction between temperature-responsive surface and cells can be controlled, and the modifying conditions for cell sheet recovery can be optimized.Though this method can provide insight into interaction between cell and surface, it can also be applied to other system such as bioseparation and biofilm. To begin, dissolve styrene, ECT, and ACVA in 40 milliliters of 1, 4-Dioxane. Freeze the solution in liquid nitrogen under vacuum for 15 to 20 minutes to remove the reactive species.After gradually thawing the solution at room temperature, repeat this freeze, pump, thaw, degassing cycle three times. Obtain the polystyrene as a macro-RAFT agent by polymerization at 70 degrees Celsius for 15 hours in an oil bath. Precipitate the polystyrene macro-RAFT agent with 800 milliliters of ether, and dry in vacuo.Next, dissolve IPAAm monomer, polystyrene macro-RAFT agent, and ACVA in four milliliters of 1, 4-Dioxane. Remove the oxygen in the solution using freeze, pump, thaw degassing cycles as before. Perform a polymerization at 70 degrees Celsius for 15 hours in an oil bath after degassing.Finally, obtain the synthesized St-IPAAm molecule in the same manner as the polystyrene macro-RAFT agent. Wash glass substrates with an excess of acetone and ethanol, and sonicate for five minutes to remove surface contaminants. Dry the substrates in an oven at 65 degrees Celsius for 30 minutes.Then, use oxygen plasma to activate the surfaces of the substrates at room temperature. Immerse the substrates in toluene containing 1%hexatrimethoxysilane overnight at room temperature to sylanise the substrate. Then, wash the sylanised substrates in toluene and immerse in acetone for 30 minutes to remove the unreacted agents.Anneal substrates for two hours at 110 degrees Celsius to thoroughly immobilize the surface. Finally, cut the sylanised substrates by a glass cutter to 25 millimeters by 24 millimeters to fit the cell culture dishes. Place the Langmuir film instrument in a cabinet to prevent the accumulation of dust.Wash the Langmuir trough in barriers with distilled water and ethanol to remove the contaminants. Dry the trough and barriers by wiping with a lintless towel. Then fill the trough with approximately 110 milliliters of distilled water, and set the barriers on both sides of the trough.Next, heat a platinum Wilhelmy plate for monitoring the surface tension, with a gas burner until the plate turns red. Then wash the plate with distilled water to remove the contaminants. Suspend the Wilhelmy plate on a wire attached to the surface pressure measurement instrument.Zero the surface pressure measurement instrument according to the manufacturer's protocol. Compress the air, water interface on the trough by the barriers on both sides of the trough until the interface reaches approximately 50 square centimeters without any drops of polymer. Aspirate any small contaminants until the surface pressure is nearly zero millinewtons per meter.Reposition the barriers on both sides, and add distilled water to compensate for the decrease of distilled water. Next, dissolve five milligrams of the synethesized St-IPAAm molecule in five milliliters of a development solution of chloroform. Then, gently drop 27 microliters of St-IPAAm dissolved in chloroform, onto the trough using a microsyringe or micropipette.After waiting for five minutes to allow complete evaporation of chloroform, move both barriers horizontally to compress the St-IPAAm molecule at the surface. Maintain compression rate of the barriers at 0.5 millimeters per second until the target area of 50 square centimeters is reached. Measure the surface pressure Pi-A isotherms with the platinum Wilhelmy plate attached to the surface pressure measurement instrument during compression, according to the manufacturer's protocol.After reaching the target area size, maintain the surface for five minutes to allow the St-IPAAm molecules to relax. The molecules do not reach equilibrium immediately after compression. Then, transfer the Langmuir film to a hydrophobically-modified glass substrate using a transfer apparatus for five minutes to robustly absorb the film.Fix the hydrophobic glass substrate in parallel on the device. Connect the device to an alignment stage and move perpendicularly. Lift the substrate horizontally with the transfer apparatus and dry for one day in a dessicator.To prepare cell suspensions, culture bovine carotid artery endothelial cells, or BAECs, to 1/3 confluence at 37 degrees Celsius in 5%CO2 and 95%air, on tissue culture polystyrene with DMEM, containing FBS and Penicillin. After confluence is reached, treat BAECs with three milliliters of 0.25%trypsin EDTA for three minutes at 37 degrees Celsius in 5%CO2 and 95%air. Deactivate the trypsin EDTA by adding 10 milliliters of the DMEM containing 10%FBS.And collect the cell suspension in a 50-milliliter conical tube. After centrifuging at 120 times g for five minutes, aspirate the supernatent and resuspend the cells in 10 milliliters of the DMEM. Seed the recovered cells on the St-IPAAm surfaces at a concentration of 10, 000 cells per square centimeter, counted by a disposable hemocytometer.Observe the cells on the surfaces by a microscope equipped with an incubator at 37 degrees Celsius with 5%CO2 and 95%air. Next, sterilize the St-IPAAm surfaces by an ultraviolet light equipped to a clean bench. Record timelapse images of adherent BAECs for approximately 24.5 hours at 37 degrees Celsius by a phase-contrast microscope with 10x magnification.After BAEC adhesion, record detatchment of the BAECs from the St-IPAAm surface at 20 degrees Celsius for approximately 3.5 hours. Following optimization of cell adhesion and detachment on the Langmuir film transferred surface, fabricate the cell sheets by first culturing BAECs as before. Seed a total of 100, 000 cells per square centimeter on St-IPAAm surfaces.And incubate for three days at 37 degrees Celsius in 5%CO2. Confluent BAECs spontaneously detached at 20 degrees Celsius. Preparation of a temperature-responsive Langmuir film transferred surface is shown here.The synthesized St-IPAAm chloroform solution was gently dropped onto an air, water interface. Two barriers were used to compress the St-IPAAm molecules on the interface until a target area of 50 square centimeters was reached. The surface pressure was measured during compression to detect any defects in the Langmuir film.After compression, a hydrophobically-modified cover glass substrate was horizontally placed on the interface with an alignment stage. The substrate was lifted horizontally and dried for one day. After this step, St-IPAAm surfaces were ready for use in cell culture experiments.Representative results of atomic force microsopy topographic images of St-IPAAm surfaces are shown here. Nanostructures were observed on both of the St-IPAAm surfaces. The size and shape of the nanostructures were strongly dependant on AM, and the composition of St-IPAAm.Here, a macroscopic image of a recovered cell sheet on a Langmuir film transferred surface with St-IPAAm 480, and an AM of 40 square nanometers per molecule, is shown. Cells reached confluence after three days in culture on St-IPAAm 170 and St-IPAAm 480 surfaces at 37 degrees Celsius. And the cell sheet was rapidly recovered after reducing the temperature from 37 to 20 degrees Celsius.After watching this video, you should have a good understanding of how to fabricate, how to design molecular composition of polymer, fabricate and transfer Langmuir film, control cell adhesion and detachment, and recover cell sheet. After its development, this technique paves the way for researchers in the field of the biomaterials and tissue engineering to explore interactions between cells and surfaces and cell culture strategies for use in degenerative medicine. The implication of this technique extend towards therapy of cell transplantation, because this technique has potential to fabricate cell sheets of various cell types, provided from patients.

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Research

JoVE Journal

Bioengineering

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Learn to fabricate thermoresponsive nanostructured surfaces using the Langmuir-Schaefer method for controlled cell adhesion and detachment in tissue engineering applications.

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

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