2.1 Network Polymerization on TCPS
Costar (Corning; Corning, NY) 12-well tissue culture polystyrene (TCPS) plates were used for all cell culture experiments. 12-well plates were activated by a Plasma Preen II 973 Oxygen Plasma (Plasmatic Systems) set at 1 Torr and 150 W for 1 min. Subsequently, hydrogel network coatings were polymerized directly onto the bottom of each well of a 12-well plate via photoinitiated radical addition polymerization. 200 μL of a solution containing monomer, crosslinker, and photoinitiator was pipetted into each well: 0.15 g/mL N-(3-Aminopropyl)methacrylamide hydrochloride (APMAAm; Polysciences, Warrington, PA), 0.0015 g/mL N,N-methylenebis(acrylamide) (BIS, Polysciences), and 0.005 g/mL Irgacure 2959 (Ciba) in 97:3 (v/v) water:isopropanol (IPA; Sigma Aldrich). The samples underwent photoinitiated polymerization for 1 min using a UV light source (UV light irradiation of 0.36 mW/cm2 at 365 nm). Excess solution was aspirated from wells and then wells were rinsed 3 times with water to remove unreacted materials. Plates were sterilized by soaking in 70:30 (v/v) ethanol:water mix for 20 min followed by 3 rinses in DPBS. All water used in this study was ultra pure ASTM Type I reagent grade water (18 MΩ · cm, pyrogenfree, endotoxin <0.03 EU/ml).
2.2 Network Polymerization on QCM-D crystals and Si wafers
QCM-D sensor crystals (QSX101, Qsense) and Si-wafer pieces (1 cm × 1 cm) were cleaned by soaking in water, acetone, and toluene. Polystyrene (PS) films were spin-coated onto QCM-D sensor crystals and Si-wafers at 2000 rpm for 60 secs from a 1% (w/v) PS solution in toluene as described previously.[23
] PS films were annealed for 48 hours at 110°C and subsequently activated by oxygen plasma (Plasmatic Systems) set at 1 Torr and 150 W for 1 min. Hydrogel coatings were photo-polymerized directly onto the PS layer by flipping the samples upside down in a 6-well PS plate with 500 μL of monomer solution per well. The solution was identical to above aside from the solvent, which was 100% water. The samples underwent photo-initiated polymerization for 1 min using a UV light source (identical parameters as listed in Section 2.1) and were rinsed 3x in water. In addition, the presence of the APMAAm surface was validated by XPS measurements on Si-wafer samples (SI Figure 1
) showing an increase in the N peak as shown in the TCPS-APMAAm samples.
2.3 X-ray Photoelectron Spectroscopy (XPS)
APMAAm-modified 12-well TCPS plates were analyzed with XPS where all spectra were taken on a Surface Science Instruments S-probe spectrometer with a monochromatized Al Kα X-ray, and a low energy electron flood gun for charge neutralization of non-conducting samples. The samples were floated on double sided tape and run as insulators. Three spots were analyzed on each sample. Samples were analyzed with a pass energy of 150 eV for survey spectra and 50 eV for high resolution scans, and a take-off angle of 55°. Service Physics ESCA2000A Analysis Software was used for peak-fitting. The binding energy scale of the high-resolution spectra was calibrated by setting the C 1s to 285.0 eV.
2.4 Contact Angle Goniometry
Water contact angles of as received TCPS and APMAAm substrates (quasi-static advancing (
)) were measured according to methods previously described[24
] using a customized micrometer microscope fitted with a goniometer eyepiece (Gaertner, Chicago, IL). All contact angles were measured at ambient temperature to the nearest degree.
2.5 hES cell cultures
] and H9-hOct4-pGZ (hOct4 promoter driving GFP and Zeo) from Wicell were employed in this work. hESCs were cultured on APMAAm gels and Matrigel™
controls in chemically defined mTeSR™
1 media (Stem Cell Technologies, Vancouver, BC). Cells were fed daily and passaged 1:3–1:6 every 3–5 days by exposure to Collagenase IV (Gibco Invitrogen; Carlsbad, Ca) at 200 U/mL in Knockout DMEM (KO-DMEM; Gibco Invitrogen) for 5 min at room temperature (RT). Cells were then washed on the dish with Dulbecco’s Phosphate Buffered Saline (DPBS; Gibco Invitrogen), followed by mTeSR™
1 media containing 5μM Rock Inhibitor (Ri; Calbiochem EMD Chemicals). Cells were gently scraped and pipetted into smaller colonies, and passaged in mTeSR™
1 media supplemented with Ri. For controls, Matrigel™
was diluted 1:30 in KO-DMEM at 4°C, allowed to adsorb for more than 10 min at RT, and then aspirated immediately before use.
2.6 hES cell differentiation
H1s and H9-hOct4-pGZ were differentiated by normal passaging and suspension in 20% FBS in KO DMEM (Gibco Invitrogen). At Day 8, EBs were plated on gelatin-coated TCPS wells and immunostaining experiments were carried out at day 40.
After 10 passages on APMAAm, hESCs were passaged back onto Matrigel™ and brought to Children’s Hospital Oakland Cytogenics laboratory for karyotyping by GTG-banding.
2.8 Immunostaining and Quantitative Analysis
Samples in 12-well plates were fixed using 4% (v/v) paraformaldehyde in DPBS at 37°C for 10 min. Samples were then rinsed 3x in PBS and kept at 4°C. Cells were permeabilized with 0.1% Triton-X (Sigma) for 10 min; for intracellular markers, cells were further permeabilized with 0.5% SDS for 5 min. Cells were incubated with a 1:100 dilution of primary antibody [Mouse Anti-Oct-4 IgG (Santa Cruz Biotechnology); Mouse Anti-SSEA-4 IgG (Millipore); Mouse Anti-Tra-1-60 IgM (Millipore); Rabbit Anti-Desmin IgG (Thermo Scientific); Rabbit Anti-α-Smooth Muscle Actin IgG (Millipore); Mouse Anti-Human B Tubuliin III IgG (Millipore)] overnight at 4°C. The next day, cells were incubated with an appropriate Alexa Fluor secondary antibody 1:300 for 1 hr at RT [Goat Anti-Mouse AlexaFluor 488 IgG (Molecular Probes), Goat Anti-Mouse AlexaFluor 488 IgM (Molecular Probes), Goat Anti-Rabbit AlexaFluor 546 IgG (Molecular Probes)]. Finally, cell nuclei were stained with 4′,6-diamino-2-diamidino-2-phenylindole, dilactate (DAPI; Molecular Probes) for 5 min at RT. All staining steps were followed by 3 washes in PBS. Cells were visualized immediately as follows: Epifluorescent imaging (Axiovert), whole plate imaging (ImageXpressMicroscope), and Confocal imaging (Zeiss). For quantitative image analysis, the Metamorph software was used and the ‘Cell scoring’ algorithm was applied. Briefly, the cell nuclei were identified by DAPI staining and the corresponding area positive for a specific wavelength (i.e. AlexaFluor 488, AlexaFluor 546) was measured. The percent of cells that were associated with a positive stain was quantified for 49 different sites (1000 μm2/site) on each well. Data is presented as the mean percentage of positive cells on all sites ± SEM.
2.9 Quantitative RT-PCR
RNA and cDNA were attained from cells using TaqMan Fast Cells-to-CT Kit (Applied Biosystems) according to the manufacturer’s instructions for n=3 biological replicates. cDNA and RT-PCR reactions were performed with a StepOnePlus instrument (Applied Biosystems) using TaqMan Fast Universal PCR Master Mix, and TaqMan Gene Expression Assays (GAPDH HS9999905_m1; Oct4 Hs00742896_s1; Nanog Hs02387400_g1; Sox2 Hs00602736_s1) according to the manufacturer’s instructions.
2.10 Quartz Crystal Microbalance with Dissipation (QCM-D) experiments
In a QCM-D, an AC voltage is pulsed across an AT-cut piezoelectric quartz crystal, causing it to oscillate in shear mode at its resonant frequency. The resonant frequency of the crystal is recorded in real time and depends on the total oscillating mass and the intrinsic properties of the quartz crystal. The Sauerbrey relationship[26
] states that a change in the mass (ΔM) of a film or adlayer is directly proportional to a change in the normalized resonant frequency of the crystal (ΔF):
where C is the mass sensitivity constant of −17.7 ng cm−2
and n is the overtone number. The ΔF is due to the change in total coupled mass, including hydrodynamically coupled water and water associated with adsorbed molecules. The dampening of the shear wave is also recorded simultaneously with the resonant frequency of the crystal as the dissipation factor (D
), which is the ratio of the dissipated energy to the stored energy. For this work, APMAAm-modified sensor crystals were loaded into the QCM-D (E4, Biolin Scientific, Sweden) and solutions were flowed over the surface of the crystal at 400 μL/min using a Peristaltic pump (Ismatec IPC-N4; Glattbrugg, Switzerland). Frequency (F) and dissipation were recorded in real time at 4 different overtones: n=1, 3, 5, and 7. Initially, DPBS flowed over the APMAAm surface to establish a baseline for the F and D values of the crystal. Once F and D were stabilized, different solutions were introduced sequentially until the measurement reached equilibrium. All calculations were done with n=7 overtone since it contained the least amount of noise.
2.11 Cell Attachment Studies
hESCs were passaged normally and plated in the specified media. After 24 h, hESCs were washed in PBS and frozen at −20°C immediately. Cyquant (Molecular Probes) was used to quantify cell attachment according to the manufacturer’s instructions. Briefly, cells were simultaneously lysed and incubated with a proprietary green fluorescent dye, which fluoresces when bound to nucleic acids for 5 min. After the incubation time, solution was pipetted onto a 96-well black plate (Costar) and the fluorescence was measured employing a Spectramax GeminiXS spectrofluorometer (Molecular Devices, CA; ex/em/cutoff, 480/520/515 nm). The population doubling time (PDT) was calculated according to the following equation:[27
Where T1 was day 1, T2 was day 4, N1 was the number of cells at T1, and N2 was the number of cells at T2.
2.12 Protein Spreading Experiments
QCM-D sensor crystals were modified as described above with spin-coated PS, followed by synthesis of APMAAm films. First, F and D measurements of the modified crystals were allowed to stabilize in PBS in the QCM-D. Next, one of four BSA solutions in PBS: 0.01 mg/mL BSA, 0.025 mg/mL BSA, 0.05 mg/mL BSA, and 0.1 mg/mL BSA was introduced into each chamber. Solutions were flowed over the surface of the crystal at 200 μL/min until the F and D signals were equilibrated. Finally samples were rinsed with PBS. The Sauerbrey relationship was used with F7/7 measurements to calculate the total mass of the film adsorbed. The τ–75 was defined as the time for each experiment to reach 75% of the total saturated mass adsorbed. From this total mass, the footprint size was calculated.
All data were expressed as the average of at least three replicate experiments ± the standard error of the mean. Statistical comparisons were performed by ANOVA (P < 0.05) followed by Holms t -tests (P <0.05) for significance.