br For comparison type I collagenecoated plates were prepare
For comparison, type I collagenecoated plates were prepared by adding Cellmatrix I-C solution onto 24-well tissue culture plates (TCPs) and heated at 37 C for 2 h. Then, the added solution was removed.
Rheological measurements were conducted using a RDAII in-strument with a torque transducer capable of measurements in the range of 0.2e200 g cm. Dynamic oscillatory shear measurements were performed by applying a time-dependent strain of g(t) ¼ go sin(ut), where go is the strain amplitude, u is the frequency, and t is the time. The resultant shear stress is s(t) ¼ go [G0 sin(ut) þ G00cos(ut)], with G0 and G00 being the storage and loss modulus, respectively. Measurements were conducted by using a set of 25-mm diameter parallel plates with a sample thickness of ~1.12 mm at 25 C. The strain amplitude was fixed to 20% for AC gels without surface modification to obtain reasonable signal intensities even at elevated temperature or low frequency (u) to avoid the non-linear response. For sample investigated, the limits of linear viscoelasticity were determined by performing strain sweeps at a series of fixed u's .
Dynamic mechanical properties of polystyrene (PS) TCP (23.7 12.3 0.78 mm3 ¼ width length thickness) were measured using RDAII in the tension-torsion mode . The tem-perature dependence of dynamic storage modulus (G0) and loss modulus (G00) was measured at a constant frequency (u) of 10.0 rad/ s, at a strain amplitude of 0.05%, and in the temperature range of 20e40 C with a heating rate of 2 C/min. The data in these two different measurements are compatible with each other.
Human breast adenocarcinoma cell lines, MDA-MB-231 Amer-ican Type Culture Collection (ATCC) and MCF-7 (ATCC), were cultured in high-glucose DMEM (Nacalai Tesque) supplemented with 10% (v/v) FBS, 100 unit/mL penicillin (Nacalai Tesque, Kyoto), and 100 mg/mL streptomycin (Nacalai Tesque, Kyoto), grown at 37 C under 5% CO2 Brequinar and 95% relative humidity (nor-moxia) or hypoxic condition (94% N2, 5% CO2, and 1% O2) at 37 C. The cells were grown to 70e80% confluence under normal culture condition before being seeded onto the fiber substrates.
2.5. Immunofluorescence staining
MDA-MB-231 and MCF-7 cells were seeded at the density of 1.0 104 cells cm¡2 on AC gels modified with type I collagen substrates (AC-soft, AC-mid, and AC-stiff) and type I collagene-coated 24-well TCP under normoxic or hypoxic conditions for a period of 3 days. The cells were fixed with 4% paraformaldehyde for 15 min at room temperature. The cells were then washed with PBS (Nacalai Tesque) and permeabilized with 0.1% Triton X (Nacalai Tesque) for 6 min. The fixed cells were washed twice with PBS and blocked with 2% bovine serum albumin (Wako) in PBS for 60 min. The cell cytoskeleton and nuclei were stained using Alexa Fluor 488 phalloidin (Life Technologies) and Hoechst 33342 (Life Technolo-gies) for 20 min. All stained samples were imaged using a fluo-rescent microscope (EVOS FL Auto, Life Technologies).
The cellular morphologies (nuclear elongation factor, cytoplasm roundness, and nuclear area [AN]etoecytoplasm area [AC] ratio [AN/ AC]) were manually quantified by following the contour of each cell (n ¼ 32). The nuclear elongation factor, cytoplasm roundness, and AN/AC were calculated as the major axis/minor axis of the nucleus, 4 (area)/(p(major axis)2) of the cytoplasm, and area of nuclear (AN)/ area of the cytoplasm (AC), respectively. By definition, the cyto-plasm roundness is equal to 1 for a completely round cell.
2.6. Real-time polymerase chain reaction
MDA-MB-231 and MCF-7 cells were seeded at the density of 2.0 104 cells cm¡2 on the chamber slide and cultured under normoxic or hypoxic condition for a period of 3 days. The total RNA was extracted from the cells cultured for three days using the Fast Gene™ RNA Premium Kit (Nippon genetics). Then, the RNA was subjected to reverse transcription using a Transcriptor Universal
cDNA Master kit (Roche) following the manufacturer's instructions. where S is the cellular migration speed, and P is the persistent time.
The reaction solutions included 5 mL of KAPA SYBR® FAST qPCR Cellular diffusivity D is calculated as D ¼ S2P/2.
Master Mix (KAPA BIOSYSTEMS), 200 nM forward and reverse
volume. The resulting cDNA yield was then subjected to real-time
polymerase chain reaction using the LightCycler® 96 system
All data presented are expressed as mean and standard devia-
(Roche). The results were analyzed using LightCycler® 96 software tion. Statistical analysis was performed using one-way analysis of