Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Summary

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.

Abstract

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.

Introduction

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 stimuli^1-4.

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 interface^5-8. 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 methods^9-12. 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 segments^13-17. In addition, the number of nanostructures on a surface is regulated by controlling the area per molecule (A_m) 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 surface^19-27. PIPAAm and its copolymers exhibit a lower critical solution temperature (LCST) in aqueous media at temperatures near 32 °C. The culture surface also exhibited a temperature-responsive alternation between hydrophobicity and hydrophilicity. At 37 °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 °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 demonstration^{26, 27}. A Langmuir film of block copolymers composed of polystyrene (P(St)) and PIPAAm (St-IPAAm) was fabricated. The Langmuir film with a specific A_m 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.

Protocol

1. Synthesis of Polystyrene-block-poly(N-isopropylacrylamide) by Two-step Reversible Addition-fragmentation Chain Transfer (RAFT) Radical Polymerization

1. Dissolve styrene (153.6 mmol), 4-cyano-4-(ethylsulfanylthiocarbonyl) sulfanylpentanoic acid (ECT; 0.2 mmol), and 4,4'-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.
2. Obtain the polystyrene (PSt) (Mw: 13,500) as a macro RAFT agent by polymerization at 70 °C for 15 hr in an oil bath.
3. Precipitate PSt macro RAFT agent with 800 ml of ether and dry in vacuo.
4. 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.
5. Remove the oxygen in the solution by the freeze-pump-thaw degassing cycles as mentioned in step 1.1.
6. Perform a polymerization at 70 °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.

2. Preparation of Silanized Hydrophobic Modified Glass Substrates

1. Wash glass substrates (24 mm x 50 mm) with an excess of acetone and ethanol and sonicate for 5 min to remove surface contaminants.
2. Dry the substrates in an oven at 65 °C for 30 min. Then use oxygen plasma (400 W, 3 min) to activate the surfaces of the substrates at RT.
3. Immerse the substrates in toluene containing 1% hexyltrimethoxysilane overnight at RT to silanize the substrate.
4. Wash the silanized substrates in toluene and immerse in acetone for 30 min to remove unreacted agents.
5. Anneal substrates for 2 hr at 110 °C to thoroughly immobilize the surface.
6. Cut the silanized substrates by a glass cutter to 25 mm x 24 mm to fit the cell culture dishes (dish size: φ35 mm).

3. Preparation of Langmuir Films and Film-transferred Surface

1. Place the Langmuir film instrument in a cabinet to prevent the accumulation of dust.
2. Wash the Langmuir trough (size: 580 mm x 145 mm) and barriers with distilled water and ethanol to remove contaminants.
3. 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.
4. 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.
5. Zero the surface-pressure-measurement instrument according to manufacturer'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.
6. Aspirate small contaminants until the surface pressure is nearly 0 mN/m.
7. Reposition the barriers on both sides, and add distilled water to compensate for the decrease of distilled water from step 3.6.
8. 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.
9. Gently drop 27 µl of St-IPAAm dissolved in chloroform onto the trough using a microsyringe or micropipette.
10. 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.
11. Measure the surface pressure (π)-A_m isotherms with the platinum Wilhelmy plate attached to the surface-pressure-measurement instrument during compression according to manufacturer's protocol.
12. 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.
13. 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.
14. Lift the substrate horizontally with the transfer apparatus and dry for 1 day in a desiccator.

4. Culturing Cells and Optimizing Cell Adhesion and Detachment on the Langmuir Film Transferred Surface

1. To prepare cell suspensions, culture bovine carotid artery endothelial cells (BAECs) to one third confluence at 37 °C in 5% CO₂ and 95% air on tissue culture polystyrene (TCPS) with Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 100 U/ml penicillin.
2. After confluence is reached, treat BAECs with 3 ml of 0.25% trypsin-EDTA for 3 min at 37 °C in 5% CO₂ and 95% air.
3. 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.
4. Centrifuge at 120 x g for 5 min, and aspirate the supernatant. Re-suspend the cells with 10 ml of the DMEM.
5. Place the St-IPAAm surfaces under ultraviolet light on a clean bench for sterilizing for 5 min.
6. 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 °C with 5% CO₂ and 95% air.
   Note: Sterilize the St-IPAAm surfaces by ultraviolet light equipped to a clean bench.
7. Record time-lapse images of adherent BAECs for approximately 24.5 hr at 37 °C by a phase-contrast microscope with 10X magnification. After BAEC adhesion, record detachment of the BAECs from the St-IPAAm surface at 20 °C for approximately 3.5 hr.

5. Cell Sheet Fabrication on the Langmuir Film-transferred Surfaces

1. Culture BAECs used in the same manner described in Section 4.
2. Seed a total of 1.0 x 10⁵ cells/cm² on St-IPAAm surfaces and incubate for 3 days at 37 °C in 5% CO₂. Confluent BAECs spontaneously detached at 20 °C.

Results

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...

Discussion

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...

Materials

List of materials used in this article

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