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  • br Roche The cDNA samples were analyzed for

    2020-08-30


    (Roche). The cDNA samples were analyzed for expression of E- variance with Tukey-Kramer post hoc testing, and significance was
    hypoxia-inducible factor 1a (HIF-1a), and vimentin, relative to
    glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an inter- 3. Results and discussion
    nal standard for sample normalization. In addition, the cDNA
    samples of day 3 were analyzed for expression of snail family zinc 3.1. Viscoelasticity of AC gels
    Fig. S1(a)-(c) (Supplementary data) shows the typical frequency
    2.7. Cellular migration
    u dependence of linear viscoelasticity of the gel substrate. We can
    clearly see that both moduli are almost independent of u, with the
    MDA-MB-231 and MCF-7 AUY 922 were seeded on the substrates exception of decrease in G0(u) at the u region < 10 rad/s. The value
    and cultured under normoxic or hypoxic conditions. Both cells
    suggesting the system is a viscoelastic solid-like gel. The storage
    were seeded at the density of 5
    cultured for 24 h to attach the cells to the substrates. An environ- mid, and 32 Pa for AC-soft.
    ment of 37 C temperature and normoxic or hypoxic condition was
    Fig. S1(d) (Supplementary data) shows the temperature
    applied during the time lapse measurement. The cells were stained
    dependence of both dynamic moduli of the TCP at the temperature
    range of 20e40 C. As expected, both moduli are not sensitive to the
    experiment. The blue fluorescent images of the tracers were ob-
    framework of the experimental temperature because of the solid
    tained every 15 min for 16 h by an EVOS FL Auto fluorescent mi-
    state of PS, where the storage modulus is around 1.4 GPa and the
    croscope (Life Technologies). To
    track the cell movement, the
    glass transition temperature is ~100 C.
    position of 20 individual centroids [x(t), y(t)] of the cell nucleus was
    The viscoelastic features of four substrates are characterized by
    obtained by a Mosaic particle tracker for ImageJ developed by
    the damping coefficient (tand ¼ G00(u)/G0(u)) and storage modulus
    Helmuth et al. [21]. The start points of each cell were defined at the
    same position.
    (Fig. 1). Generally, in the viscoelastic materials, hard and stiff ma-
    terials such as metals and ceramics show high stiffness and low
    2.7.1. Evaluation of cellular displacement and direction
    loss. On the other hand, soft materials have high damping and low
    stiffness. The solid line indicates a viscoelastic figure of merit r> rection Dq were calculated as equations (1) and (2).
    aluminum, poly(methyl methacrylate), TCP (PS), and natural rub-
    Dt
    y t Dt y t 2 (1) ber. This was found in traditional solid materials [25]. With tand
    D
    ð
    qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
    seven orders of magnitude lower than that of solid materials. This is
    cos 1 ðxðt þ DtÞ xðtÞÞ
    (2) achieved by soft matrix with cross-linker BIS molecule. The
    biomedical properties of a tissue in terms of stiffness (elastic
    ð
    Dr
    modulus) are shown in Fig. 1. Compliant tissue such as lung exhibits
    where Dt is the time interval of the time lapse measurement. The low stiffness AUY 922 (400 Pa), whereas tissues exposed to high mechanical
    loading, such as bone or skeletal muscle, exhibit high stiffness with
    average of the calculated Dr (x,y) and Dq (x,y) was plotted as a polar
    histogram.
    3.2. Effect of viscoelasticity of AC gels on cellular morphology
    2.7.2. Characterizing cellular migration using the persistent random
    Fig. 2 shows the cellular morphology of MDA-MB-231 and MCF-
    walk model
    7 breast cancer cells cultured on different substrates of AC gels and
    The obtained x, y values
    were first transferred into mean
    the TCP coated with type I collagen (TCP-coat) under hypoxic
    squared displacement (MSD) as equation (3).
    condition at day 3. For comparison, the cellular morphology
    MSD t hD x t þ t Þ x t ÞÞ 2
    t y t ÞÞ 2 Ei (3) cultured on the TCP without collagen coating at day 3 is shown in
    Fig. S2 (Supplementary data). The corresponding morphological
    ð
    n
    parameters (cytoplasm roundness, AN/AC, and nuclear elongation