機械的にダイナミックな細胞培養のための微細加工プラットフォーム

The overall goal of the following experiment is to micro fabricate devices used to mechanically stimulate cells two and three dimensional culture environments. This is achieved by using a multi-layer sandwich molding and stacking technique to produce an array of vertically actuated micro posts. As a second step, the vertically actuated micro post array is modified to stretch a culture film, which applies well controlled strains to cells cultured on the film.

Alternatively, the array can be modified to compress micro patterned biomaterial constructs in order to apply mechanical stimulation to cells. Cultured in an array of three dimensional hydrogels results are obtained that show the simultaneous application of a range of two dimensional echo biaxial and three dimensional compression strains across the array based on image analysis of fluorescent fiduciary markers. Hi, I'm Chris Mariahs from the labs of Craig Simmons and U Sun in the Department of Mechanical and Industrial Engineering and the Institute of Biomaterials and Biomedical Engineering at the University of Toronto.

Today we'll show you a procedure to fabricate micro devices used to mechanically stimulate cells. We use this procedure in our lab to study how cells respond to a variety of mechanical forces in in vitro culture. So let's get started.

The process of sandwich mold fabrication includes placing an overhead transparency onto uncured poly dimethyl Sloane or PDMS on an SU eight master. The sandwich is placed in a foam glass and metal stack and cured in an oven under compression. Finally, the stack is disassembled and the transparency peeled away retaining the patterned PDMS layer.

To begin this process thoroughly mix PDMS, monomer and crosslinker. In the standard 10 to one ratio, deposit three milliliters of uncured PDMS mixture onto a previously fabricated SU eight master and DGAs the polymer in a vacuum chamber. Next place the foam pad and SU eight master on a plexiglass base plate.

Carefully lower the smooth side of an inkjet transparency onto the uncured PDMS, making sure to avoid trapping any air bubbles. Then position a Siloized glass slide on top of the transparency beneath the second foam pad and a plexiglass top plate. Clamp the sandwich together using a C clamp.

Cure the PDMS sandwich in an oven at 80 degrees Celsius for at least four hours. Following curing, remove the sandwich from the oven and allow it to cool. Once cooled, remove the clamp and disassemble the sandwich.

Carefully peel the transparency away from the SU eight master retaining the patent PDMS film. Trim the edges of the pattern film to remove excess PDMS store the pattern layers in a dust-free environment until they're ready for use. The next portion of this protocol involves the construction of a micro fabricated array of vertically actuated posts.

A multi-layer fabrication process is required to make the array of micro posts. The lower most patterned PDMS layer is prepared by sandwich mold fabrication. For these experiments, circular patterns ranging from 600 to 1, 200 microns in diameter were used.

In order to transfer the PDMS layer to a clean glass slide, use a Corona discharge unit to treat the PDMS and glass surfaces with oxygen plasma. Then place the two surfaces in contact with each other. Place the stack on a hot plate at 80 degrees Celsius for 10 minutes to complete the bonding process.

Peel the transparency away and discard. This process ensures that the patterned PDMS film is never released from a rigid substrate, which prevents shrinkage of the individual layers. The second sandwich molded layer is formed as a negative replica mold of the desired diaphragm and post structure.

In this demonstration, circular posts 500 microns in diameter were used to form this layer. First, temporarily affix the negative replica mold to a glass slide using double-sided tape and siloized in a vacuum desiccate deposit uncured PDMS onto the sandwich, mold layer, Degas and spin coat for 45 seconds At 1000 rotations per minute yielding an actuation diaphragm 60 microns thick, partially cure the spin coated PDMS layer at 80 degrees Celsius for approximately 20 minutes or until the PDMS is sticky but does not deform permanently. When touched, remove the glass slide and double-sided tape from the partially cured PDMS layer and transparency Film affix it to a pair of glass handling slides and plasma.

Treat the partially cured PDMS, then invert the PDMS and place it into a custom made aligner. The aligner consists of a vacuum chuck mounted onto a micro manipulator. Next plasma treat the first PDMS layer, then place it on a rotary stage beneath the vacuum chuck.

Observe alignment between the two layers with an avatar 12 times zoom machine vision system. Using the manipulator, align the layers and carefully bring them into contact. Apply light pressure with a finger to stick the layers together.

Then remove the sandwich from the aligner. Remove the glass handling slides and make sure that the layers are in full contact with each other. Curing an oven at 80 degrees Celsius for an additional 30 minutes.

After 30 minutes, the transparency and siloized replica PDMS mold can be peeled from the device and discarded. The device is then fully cured at 80 degrees Celsius for at least four hours to conduct experiments for cells cultured on a deforming two dimensional substrate. The micro actuator array is modified by bonding a third PDMS layer to the device.

The third PDMS layer consists of a two layer structure. The lower circle matches the size of the actuation cavity beneath the actuated micro posts. The diameter of the upper circle is 100 microns greater than the diameter of the loading posts.

A 200 micron wide micro channel connects these features to a connection area and will be used to lubricate the loading posts and culture film. To modify the device for 2D culture first fabricate and bond the connectors to it as described in the written protocol. Then spin coat a 10 to 15 micron layer of PDMS onto the smooth side of a transparency cure.

The thin PDMS film at 80 degrees Celsius for at least four hours. The next step is to apply a vacuum to the actuators and bond the PDMS layer to the device. To accomplish this.

First, apply a low level vacuum to the actuators lowering the array of posts plasma treat and bond. The spin coated PDMS film to the actuated micro device as described in the written protocol, the posts and culture film will remain hydrophilic for a short time. Then connect the lubricant syringe to the device and fill the lubrication channels with a 90%glycerol in deionized water solution.

In addition to lubrication, this formulation maintains the hydrophilicity of the PDMS indefinitely reducing friction coefficients. Place the device on a hot plate at 40 degrees Celsius and continue to inject lubricant into the channel, causing a small pressure increase. After a few minutes, the air bubbles were diffuse through the PDMS and all surfaces will be lubricated.

For the final step, remove the negative pressure releasing the posts using a scalpel. Cut around the thin PDMS film. Carefully peel the transparency backing away from the device plasma bond.

A hand cut PDMS gasket around the device culture area. Sterilize the device by soaking it in 70%ethanol for five minutes, followed by treatment under a germicidal UV light in a biological safety cabinet for 45 minutes. Plasma, treat the surface and incubate with 100 micrograms per milliliter collagen overnight at four degrees Celsius.

Following overnight incubation, wash the device with sterile phosphate buffered saline or PBS and seed cells at 10 to 15, 000 cells per square centimeter. Following standard cell culture protocols, the micro actuator array can be modified to conduct experiments for cells cultured in three dimensional photo patterned hydrogel constructs. First, a multi-layer PDMS based platform is fabricated.

After covering the base platform with a meth, a related glass cover slip hydrogel precursor solution is injected. Then a photo polymer reservable biomaterial is micro patterned into the array and the remaining precursor solution is flushed away. The array of vertically actuated loading posts can then be used to apply compressive mechanical forces to cells cultured in three dimensional biomaterials with pieces of scotch tape masking the areas around the posts coat the device with paraline C in a PDS 2010 lab COTER two system.

This will improve polymerization kinetics for the biomaterial system. Once coated, remove the device from the coating system and peel the scotch tape away, leaving a conformal layer of paraline C over the posts. Then meth ACRL glass cover slip by immersion in a 2%volume per volume solution of three trimeth oxy profile Methacrylate in 95%ethanol for two minutes.

Rinse the cover. Slip in 100%ethanol, followed by baking at 100 degrees Celsius, leaving methacrylate groups on the surface. Cast a 200 micron thick PDMS spacer.

Film in a Petri dish and cut it into small sections. Plasma bond four of these small sections around the edge of the meac related cover slip. Then plasma bond the spaces to the PDMS areas of the device covering the Lene C coated.

Finally sterilized the device by washing in 70%ethanol and expose it to germicide or UV light in a biological safety cabinet for 45 minutes. To photo lithographically pattern the cell laden PEG hydrogels into the devices. Prepare the precursor and photo initiator solutions as described in the written protocol and thoroughly mix them to obtain a solution with a concentration of 0.4%weight per volume of photo initiator.

Filter the mixture through a 0.45 micron syringe filter to sterilize and remove particulates, trypsin ice cell cultures, and prepare a cell suspension in culture media at twice the desired endpoint cell concentration. Then mix the cell suspension and the filtered hydrogel precursor photo initiator solution in a one-to-one ratio. Inject the solution into the micro device using a long 25 gauge needle.

Carefully avoid trapping bubbles in the device. Align a printed transparency mask to alignment marks on the device surface. Set up a UV illumination system to provide a 365 nanometer wavelength UV dose of 17.5 milliwatt per square centimeter at the device surface.

Illuminate the hydrogel through the mask for 195 seconds following illumination. Wash away un polymerized hydrogel precursor solution with PBS and replace the PBS with culture media actuate the loading posts to compress the hydrogel constructs. Here, a completed sample device for 2D mechano stimulatory culture is shown.

Red dye is used to mark the actuating pressure delivery channels and blue D is used to mark the lubrication channels strain characterization. Experiments can be used to determine applied strain fields by tracking fluorescent bead movement on the substrates. Red spots represent the unformed location of the beads while green spots represent the deformed locations.

A sample device for three dimensional compressive experiments can be seen here where green dye is used to mark actuation pressure channels. The top down view of the micro post array reveals the hydrogel construct patterned on top of the post side reconstructed images of fluorescent beads in the hydrogel construct. Under rest and 55 kilo pascal actuation pressures demonstrate the strain characterization process in a three dimensional culture system.

So we've just shown you how to fabricate devices used to mechanically stimulate cells in either two or three dimensional culture systems. When doing this procedure, it's important to remember to keep your devices as clean and dust free as possible and to be patient, especially during the alignment steps. So that's it.

Thanks for watching and good luck with your experiments.