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    2020-07-29

    124 ± 8 Pa and from 12 ± 8 to 50 ± 5 Pa, when the concentration of CDEAC hydrogel increased from 1 to 5 wt%, respectively (Fig. 4E). This storage modulus range is comparable to 30–500 Pa characteristic of Matrigel which is widely used for CS growth [49]. The reversibility of gelation/liquefaction conversion was estimated in the SCN−/ClO− cycles by periodically exchanging the concentration of two ions (Fig. 4F). The suspension with concentration 3 wt% formed a hydrogel with G’ = 102 Pa. When the hydrogel was treated with ClO−, the value of G′ was decreased to 30 Pa, and the value of G′ was smaller than the loss moduli, G″, indicating that the hydrogel structure has been de-stroyed. However, the G′ was increased to 86 Pa when the SCN− was
    bars are 50 μm, CCDEAC = 2 wt%. (H) Plots profile of RGB quantitative co-localization calculation of red mark in Fig. 5G. (I) Variation in the average diameter of CSs cultured in CDEAC hydrogel with different CCMC at a different time. For each bar, 200 CSs were measured. The error bars represent the standard deviation. Student's t-
    test showed that the average CS diameter significantly varied with CCDEAC. On day 0, no statistical difference existed in the size of Loxapine Succinate (p > 0.05). From day 5 to day 15, significant statistical differences were observed in the size of CSs grown in CDEAC hydrogel from 1 to 4 wt% (p < 0.05). For each CCDEAC, CSs showed significant growth for each successive time period (p < 0.005). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of
    this article.)
    Fig. 6. (A) Bright-field image of CSs released from CDEAC hydrogel on 7-day culture. (B) Fluorescence image of CSs released from the CDEAC hydrogel on 7-day culture. In A and B, CCMC = 2 wt%. The CSs were stained with Calcein AM (green) and Ethidium Homodimer I (red). The scale bars are 100 μm. (C–F) Fluorescence images of the representative CSs released from the hydrogels. CSs were stained by Hoechst (Blue) and Alexa Fluor® 488 E-Cadherin Rabbit monoclonal antibody (green). Fluorescence of CMC-DPY-Eu-AMBA-CMC hydrogel was shown in (E). The merged fluorescence image of the CSs was shown in (F). The scale bars are 50 μm in (C–F). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
    Fig. 7. (A–B) Representative bright field images of MCF-7 CSs in fibrin gel after day 0 (A) and day 10 culture (B). The scale bar is 100 μm in (A) and (B). (C) Variation in the average diameter of CSs at different culture times. For each data point, at least 200 CSs were measured. The error bars represent the standard deviation. The error bars represent the standard deviation. Student's t-test showed that the average CS diameter significantly varied with the time period. CSs showed significant growth for each successive time period (p < 0.005). (D–G) Confocal microscopy images of released CSs re-cultured in fibrin gel for 10 days. The CSs were stained with Hoechst (blue), Alexa Fluor® 488 E-Cadherin Rabbit monoclonal antibody (green) and Alexa Fluor-568 Phalloidin (red). The scale bar is 50 μm. (For inter-pretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
    added to the above system. The recovery of G′ and G″ upon repetitive adding SCN− was 85% and 95%, respectively.
    3.3. Multicellular tumor spheroid formation and release from the nanocellulose hydrogel
    Before the modified CMCs were used in biological applications, we assessed their cytotoxicity by MTT assay. As shown in Fig. 5A and B, MCF-7 cells did not show apparent toxicity when cultured with Eu(III) complex-CMC or K-DPY-CMC alone for 15 days. To demonstrate the utility of this ion-responsive fluorescence hydrogel for cell encapsula-tion, MCF-7 breast cancer cells, including 1 × 105 cells/mL, were mixed with the Eu(III) complex-CMC suspension in PBS. Upon adding K-DPY-CMC, the mixed suspension formed a hydrogel, and then the cells were cultured in this hydrogel for 15 days. As shown in Supplementary Fig. S13, when the excess hydrogel is present, the cell picture was blurry, due to the light scattered by the CMC hydrogel. However, they were clearly observed by washing the excess hydrogels with PBS. Notably, the CMC hydrogel did not dissociate from the CSs under these condi-tions, because the cell surface emitted the red fluorescence of CMC hydrogel (Supplementary Fig. S14). We tracked the individual CSs grown at different times in the CMC hydrogel complex by phase con-trast microscope images. As shown in Figs. 5C–G, S15, and S16, the MCF-7 cells cultured in the 3D matrix displayed a rounded, clustered morphology, and the average diameter of CSs increased with increasing time. Based on plots profile of RGB quantitative co-localization calcu-lation (Fig. 5H), the spatial distributions of the cell nuclei, cell mem-brane, and the hydrogel were obviously observed, in which CDEAC hydrogel was surround in the cell spheroids. In addition, the con-centration of CDEAC hydrogel has an effect on the growth profile of CSs (Fig. 5I). We also cultured the CSs in Matrigel and found that the size, shape, and growth profile were similar with that of CSs formed in the CDEAC hydrogel (Supplementary Fig. S17). Moreover, the viability of encapsulated cells in the hydrogels was visualized by using live/dead staining in confocal microscopy; it was observed that there are few dead (red) cells in the CDEAC hydrogel (Supplementary Fig. S18). Moreover, we test CDEAC hydrogel with several cancer cell lines (HeLa, HepG2 and HL60 cells) and found that they could grow into corresponding cancer spheroids (Supplementary Figs. S19 and S20), indicating the advantages of such hydrogel over the previously published systems.