組織工学のための熱応答性ナノ構造表面の調製

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.