Directed growth of fibroblasts into three dimensional micropatterned geometries via self-assembling scaffolds.

Chemicals and materials Cell culture

L929 mouse fibroblast cells were purchased from ATCC (cat # CCL-1). The following chemicals were purchased from Sigma-Aldrich (Sigma): Minimum Essential Medium Eagle, horse serum, sodium pyruvate, nonessential amino acids and fibronectin from bovine plasma. Phosphate-buffered saline (PBS) was purchased from Gibco.

Fluorescent microscopy Calcein

AM was purchased from both Sigma and Invitrogen. Ethidium homodimer-1 was purchased from Invitrogen. Fabrication Si wafers were purchased from Montco Silicon Technologies. Gold (Au) and copper (Cu) slugs were purchased from Alfa Aesar, chromium (Cr) rods from Fil-Tech Inc, Au plating solution from Technic Inc, and the following chemicals from MicroChem Corp.: SC 1800 series photoresist (PR), 351 developer, SU-8 2015 photoresist (SU-8), and SU-8 developer. Iron (III) chloride was purchased from Sigma. Acetone, 2-propanol, and ethanol solvents were purchased from J.T. Baker and Fisher Chemicals.

Scanning electron microscopy

Sodium cacodylate, formaldehyde, glutaraldehyde, and uranyl acetate were purchased from Electron Microscopy Sciences. The following chemicals were purchased from J.T. Baker: sucrose, sodium hydroxide, sodium acetate anhydrous, hydrochloric acid, and osmium tetroxide. Calcium chloride was purchased from Fischer Chemicals and sodium barbiturate from Sigma.

Fabrication of the scaffolds

The fabrication process is an adaptation of that used in prior work [ 23 , 24 ] specifically designed to include the bio-inert materials Au and SU-8 for cell culture. The details of the fabrication process are as follows: Si wafer substrates were first rinsed with acetone, 2-propanol, and distilled water and were then dried under nitrogen gas. A 15 nm Cr adhesion layer followed by a 150 nm Cu sacrificial layer were thermally evaporated on the wafer ( Fig. 2a ) at a pressure of approximately 10 −5 Torr. Next, a lift-off metallization step was used to precisely pattern the stress driving layer that was composed of Cr and Au. Briefly, a 2.7 μm layer of PR was spin-coated at 3000 rpm and baked for 1 minute at 115°C on a hot plate. The micrometer features were patterned by selectively exposing the Si wafer to ultraviolet light through a photomask that was designed using AutoCAD and laser printed on plastic film at 40,640 dots per inch by Fineline Imaging, Inc. The wafer was developed in Microposit 351 Developer resulting in regions of bare Cu and regions of PR. For structures with a single direction of curvature, a stressed bilayer of 140 nm Cr and 30 nm Au was thermally evaporated as before, and upon removing the PR in acetone, only the patterned bilayer remained ( Fig. 2b ). The lithography step was repeated in order to electrodeposit 270 nm of Au. A similar process was used to deposit 11.5/245/59 nm of Cr/Au/Cr which is necessary for the bi-directionally folded structures. Once the Cr/Au bilayer or Cr/Au/Cr trilayer was complete, we patterned the areas that would become rigid panels in the folded scaffolds. This was achieved by either electrodepositing a 3 μm thick layer of Au or photopatterning a 10 μm thick layer of SU-8 ( Fig. 2c ). Finally, PR was photopatterned atop the flexible hinge regions ( Fig. 2d ) and the 2D templates were released by dissolution of the Cu sacrificial layer in a 40 wt % aqueous solution of iron (III) chloride with 5 wt % hydrochloric acid ( Fig. 2e ). On dissolution at 40 o C, the templates lifted-off and spontaneously assembled into 3D scaffold geometries in less than one minute ( Fig. 2f ). They were rinsed multiple times in deionized water and stored in 1x PBS, pH 7.4, for cell culture.

Preparation of substrates for cell culture

Scaffolds were transferred from PBS at room temperature to chilled (4°C) PBS containing 25 ug/mL bovine fibronectin. They were incubated for at least 1.5 hours in a bio-safety hood at room temperature before being rinsed with warm 1x PBS, pH 7.4, followed by one rinse with warm cell culture medium as specified in the following section.

Cell culture

L929 mouse fibroblast cells were cultured in 75 cm 2 culture flasks containing 85% Minimum Essential Medium Eagles with l-glutamine, sodium bicarbonate, 10% horse serum, and less than 1% of both sodium pyruvate and nonessential amino acids. Cells were maintained at 37 °C in a humidity controlled incubator with 5% CO 2 . Cells were suspended using trypsin just prior to culturing on the scaffolds. After rinsing the fibronectin coated scaffolds with media, they were moved to a freshly suspended fibroblast solution (~10 4 cells/mL) and incubated for 2 hours to allow cells to adhere. The scaffolds were then rinsed with fresh media to remove non-adhered cells. Finally, they were pipetted into multi-well plates and placed in the incubator as above, with media exchanged daily.

Cell viability assay

The materials used in the construction of the scaffolds (namely Au, Cr, Cu, SU-8, and PR) were deposited on Si wafers and screened for viability using a two-color fluorescence assay. Cells were first grown to approximately 80% confluency in a tissue culture treated BD Falcon clear 96-well plate. Wells were then exposed to approximately 25 mm square pieces of each test material as well as bare Si. The positive and negative controls were media and latex, respectively. Cells were stained for viability by incubating with calcein AM (2.5 μM) and ethidium homodimer-1 (4 μM) in warm PBS after 24 and 48 hours of incubation with test substrates. Fluorescence measurements were made using a Spectra Max Gemini XPS fluorescent spectrophotometer and Softmax Pro analyzer (Molecular Devices). Wells were read at nine discrete sections and samples were done in duplicates. An analysis of variance was performed on the cell viability data obtained along with a post-hoc Scheffe test to investigate the biocompatibility of the individual components of the scaffolds.

Fluorescence microscopy of cell culture on self-assembling sheets

Cells were stained for viability by incubating with calcein AM (2.5 μM in warm PBS) for 45 minutes prior to imaging. Fluorescence microscopy was primarily conducted using a Nikon AZ100 multi-zoom microscope fitted with a Nikon DS-Fi1 camera. Focused 3D images were acquired by taking multiple images in the z plane using a motorized stage. The focused images were created using the extended depth of field (EDF) algorithm within Nikon’s NIS-Elements software. The multiday cell study ( Fig. 3 ) was performed by taking a z-stack manually on a Nikon Labophot fitted with a QImaging camera and the composite image was again created via EDF using NIS-Elements. Scanning electron microscopy (SEM) Samples were prepared for SEM following a fixation and post-fixation protocol by Perkins et al [ 25 ]. Briefly, cells were fixed for one hour in a solution containing 0.1M sodium cacodylate, 3.0% formaldehyde, 1.5% glutaraldehyde, 2.5% sucrose, and 5 mM calcium chloride at a pH of 4. Samples were then washed three times in 0.1M sodium cacodylate with 2.5% sucrose at a pH of 7.4 for 15 minutes each, post-fixed with Palade’s osmium tetroxide [ 25 ] for one hour on ice, then rinsed and incubated at room temperature with Kellenberger’s uranyl acetate for two hours in the dark before being dehydrated in chilled ethanol. Afterwards, the samples were dried using a Tousimis SAMDRI-795 critical point dryer, mounted with carbon tape, and sputter-coated with 3 nm of platinum before being imaged using a JEOL JSM-6700F cold cathode field emission SEM.

Chemicals and materials Cell culture

L929 mouse fibroblast cells were purchased from ATCC (cat # CCL-1). The following chemicals were purchased from Sigma-Aldrich (Sigma): Minimum Essential Medium Eagle, horse serum, sodium pyruvate, nonessential amino acids and fibronectin from bovine plasma. Phosphate-buffered saline (PBS) was purchased from Gibco.

Fluorescent microscopy Calcein

AM was purchased from both Sigma and Invitrogen. Ethidium homodimer-1 was purchased from Invitrogen. Fabrication Si wafers were purchased from Montco Silicon Technologies. Gold (Au) and copper (Cu) slugs were purchased from Alfa Aesar, chromium (Cr) rods from Fil-Tech Inc, Au plating solution from Technic Inc, and the following chemicals from MicroChem Corp.: SC 1800 series photoresist (PR), 351 developer, SU-8 2015 photoresist (SU-8), and SU-8 developer. Iron (III) chloride was purchased from Sigma. Acetone, 2-propanol, and ethanol solvents were purchased from J.T. Baker and Fisher Chemicals.

Scanning electron microscopy

Sodium cacodylate, formaldehyde, glutaraldehyde, and uranyl acetate were purchased from Electron Microscopy Sciences. The following chemicals were purchased from J.T. Baker: sucrose, sodium hydroxide, sodium acetate anhydrous, hydrochloric acid, and osmium tetroxide. Calcium chloride was purchased from Fischer Chemicals and sodium barbiturate from Sigma.