A variant hESC line (BG01v) was cultured on MEF feeders that have been inactivated with mitomycin-C. Cells were cultured in hESC medium, which consisted of Dulbecco's modified Eagle's medium (DMEM)/F12 medium (Gibco, Cat. No. 11320–033) supplemented with 20% knockout serum replacement (KSR; Gibco, Cat No. 10828–028), 1 mM L-glutamine (Gibco, Cat. No. 25030–081), 0.1 mM minimal essential medium nonessential amino acids (Gibco, Cat. No. 11140–050), 50 U/ml penicillin and 50 μg/ml streptomycin (Gibco, Cat. No. 15070–063), 4 ng/ml basic fibroblast growth factor (Sigma, Cat. No. F-0291), and 0.1mM β-mercaptoethanol (Sigma, Cat. No. M7522). Cells were passaged every 3–5 days by treatment for 2–3 minutes with 1 mg/ml collagenase type IV (Gibco, Cat. No. 17104–019) in ES cell medium, followed by a 40 second treatment with 0.05% trypsin. Cells were plated at 50,000 cells/35 mm plate and grown at 37°C and 5% CO2.
Substrate synthesis and characterization
The PDMS substrate was prepared from the commercially available Sylgard 184 silicone elastomer kit (Dow Corning, Midland, MI) by mixing the base and the curing agent in varying ratios. Specifically, PDMS with base: crosslinker w/w ratio 10:1, 20:1, and 40:1 were prepared. The pre-polymer mixtures were mixed thoroughly for at least 5 minutes, degassed, and poured into 35 mm polystyrene tissue culture Petri dishes. PDMS was then cured for at least 60 hours at 22-33°C. Samples were stored at room temperature in a vacuum desiccator.
Tensile testing was done to characterize the bulk mechanical properties of the substrate. Specifically, 1 mm dog-bone shaped strips were subjected to a tensile load at a strain rate of 10 mm/min and the test was conducted to failure. The elastic modulus was determined manually by calculating the slope of the stress strain curve within linear limits.
Topographical and phase images were taken on an MFP-3D AFM (Asylum Research, Santa Barbara, CA). Images were obtained in non-contact (AC/tapping) mode and height, amplitude and phase images were taken using a silicon cantilever (AC-240 TS, Olympus Instruments) at a scan speed 1 Hz at 512 pixels/line. The scan size was 20 μm × 20 μm.
Surface wetting properties of the various substrates were evaluated by measuring the static water contact angles via the sessile drop method using a Ramé-Hart Goniometer/ Tensiometer (Model 500) equipped with a special optical system and a CCD camera and the image was analyzed using DROPImage Advanced for contact angle determination.
Cell culture on PDMS substrates
Before conducting cell culture experiments, PDMS substrates were sterilized by treating them with ethanol under ultraviolet light for 1 hour, followed by a second round of UV exposure for another 30 minutes. To promote cell attachment to the various substrates studied, plates coated with various formulations of PDMS and the polystyrene plates were treated with 2.5 mL of 10 μg/mL of human plasma fibronectin (Chemicon, Cat. No. FC010) overnight at 37°C. Substrates were then washed twice with PBS and cells were seeded at 50,000 cells per 35 mm plate. To promote differentiation, cells were grown in differentiation medium (hESC medium without bFGF). Cells grown on inactivated MEF feeders in hESC medium were used as controls. Medium was changed on the second day and on every following day. On the 4th, 7th, and 10th day cells were collected from the plates by treating them with collagenase and trypsin as described above. Cells that remained attached following enzymatic passaging were collected using a rubber cell scraper. Cell counts were performed using a hemocytometer. The collected cells were snap frozen in liquid nitrogen and stored at -80°C for subsequent gene expression analysis. All experiments were performed with three replicates per condition.
Alkaline phosphatase activity
Some of the collected cells were also seeded onto plates with inactivated MEF feeders and propagated in hESC medium for 4 days after which an Alkaline Phosphatase Substrate Kit (Vector Labs, Cat. No. VC-SK-5100-KI01) was used to assay for alkaline phosphatase (AP) activity.
Cell surface area calculations
To study how substrates affect morphology independent of cell-cell contact, cells were plated at a density of 10,000 cells per 35 mm plate and grown in hESC growth medium. Cells seeded at the same density on acellularized MEF layers were used as controls. After 12 hours, images of cells from different experimental conditions were captured using a Nikon Eclipse TS100 inverted microscope and a Nikon Coolpix 5000 digital camera. Area was calculated using ImageJ software by manually outlining the cell perimeter with each area measurement performed twice. Cell density was calculated by manually counting the number of cells in the field of vision and extrapolating to the cell density in each of the 35 mm dishes. Each condition was performed with three replicate plates, and images of multiple cells were captured from each plate. All cells that were in contact with other cells were excluded from the analysis.
Gene expression analysis
For gene expression analysis, samples were prepared by isolating total RNA using TRIZOL (Invitrogen, Cat. No. 10296–010, Carlsbad, CA) according to manufacturer's instructions. Briefly, cell pellets were treated with TRIZOL and chloroform, RNA from the aqueous phase was precipitated in isopropyl alcohol, washed with 75% ethanol, and dissolved in water. RNA was quantified using a UV–vis Spectrophotometer (Biomate 3, Thermo Scientific, Waltham, MA). cDNA was reverse-transcribed from 1 μg of total RNA according to manufacturer's protocols using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Cat. No. 4368814 Foster City, CA). The reactions were incubated for 10 minutes at 25°C and for 120 minutes at 37°C.
Expression of pluripotent and differentiation genes was analyzed using quantitative real time RT-PCR using Taqman primers (Applied Biosystems, Foster City, CA); performed in an ABI HT7900 system (Applied Biosystems, Foster City, CA) and the data were acquired using sequence detection system software (SDS v2.2.1, Applied Biosystems, Foster City, CA). The original replicates (n = 3) for each condition were tested in duplicate, and all failed reactions (termed “undetermined” by the software) were excluded from the analysis. ΔCt values were obtained by normalizing the Ct values against the endogenous 18S ribosomal RNA. Data analysis for differential expression between the different samples was conducted in triplicate and Student’s t-test was conducted to ascertain the significance of differential expression.
Expression Index was used to detect the relative differentiation state of cells and was based on average Ct values from triplicate measurements and used the mathematical model described in Noaksson et al. [31
] wherein EI is given by the equation
where E is the PCR efficiency, Ct is the threshold cycle, m and n are the number of genes that are upregulated and downregulated, respectively, upon hESC differentiation. KRS is the relative sensitivity constant (accounts for the differences in fragment lengths of templates) was not determined since it does not affect the relative comparison of samples.