Figure 1.
A. Proposed mechanism of MEM-induced h9e peptide self-assembling hydrogelation (SEM image showing the nanofiber scaffold of the hydrogel matrix). B. Storage modulus G′ of 1, 2, and 3 mM peptide hydrogel during the hydrogelation at 37°C. C. SEM image of 1 mM peptide hydrogel. D. SEM image of 3 mM peptide hydrogel.
Figure 2.
Dynamic rheological study of h9e hydrogel.
A. Storage modulus G′ of shear-thinning and recovery test of 1, 2, and 3 mM peptide hydrogel. B. Four times amplitude sweep test with shear strain from 1% to 500% and 1- 5-, and 10-minute breaks. C. Multiple times delivery of peptide hydrogel via pipette; hydrogel was shear thinning but reassembled quickly without permanently destroying hydrogel architecture. D. Temperature profile test of 1, 2, and 3 mM peptide hydrogel between 4°C and 50°C.
Figure 3.
Culturing cells in h9e hydrogel.
A. LSCM image of cells distribution within hydrogel architecture. B. Morphology of cluster cell in 3D hydrogel matrix over 5 days. C. Morphology of cells growth on 2D plastic monolayer over 5 days.
Figure 4.
SEM images of cell cluster in 3D hydrogel matrix.
A.Nanofiber network coating on cell surface. B. A protruding cell cluster from the nanofiber scaffold. C. Interface of the cell surface and the surrounding hydrogel matrix.
Figure 5.
Cell isolation from hydrogel matrix.
A. Cells encapsulating peptide hydrogel diluted in MEM and cell pelleted with 5-minute centrifugation at 4°C. B. Storage G′ and loss G″ moduli hydrogel diluted 15 times at 1 Hz frequency and 1% shear strain at 4°C for 1 hour. C. Viable cells before cell encapsulation and after cell isolation. D. Cell viability before cell encapsulation and after cell isolation. *p<0.05, n = 3, error bars represent standard deviation.
Figure 6.
Cell morphologies at days 1, 3, 5, and 7 in 2D plate and 3D hydrogels.
A. Microscope images of cell growth on 2D monolayer over 7 days. B. Microscope images of cell growth in 1 mM h9e peptide of 3D hydrogel over 7 days. C. Microscope images of cell growth in 2 mM h9e peptide of 3D hydrogel over 7 days. D. Microscope images of cell growth in 3 mM h9e peptide of 3D hydrogel over 7 days.
Figure 7.
Cell viability at days 1, 3, 5, 7 in 2D monolayer and 3D hydrogel. A.
Viable cells of 1, 3, 5, and 7 days cultured in 2D monolayer and 3D hydrogel. B. Cell viability at days 1, 3, 5, 7 in 2D monolayer and 3D hydrogel. *p<0.05, n = 3, error bars represent standard deviation.
Figure 8.
Effect of ciplatin on cell viability via three methods of treatment and IC50 for cisplatin in h9e hydrogel culture.
A. Cells were cultured in hydrogel for 5 days and were treated with 40 µM cisplatin for 0, 1, 3, 5, and 10 days via three methods:first, cells were grown in hydrogel and dosed with cisplatin by placing medium with cisplatin on the top surface of the hydrogel; second,cells were grown in hydrogel on a transwell insert, and medium with cisplatin was placed on top and bottom of the transwell insert; third, ciplatin was mixed with hydrogel and cells prior to hydrogelation. Cell viability was measured using Trypan blue excision method. Data were obtained in three independent experiments and are represented as the mean ± S.D. * P-value is <0.05 compared to control of cells without treatment at appropriate time point. B. MCF-7 cells were cultured in hydrogel for 5 days. After 5 days of colony formation, cells were harvested and treated with various concentrations of cisplatin for additional 5 days in hydrogel culture using “pre-mixed“ method as previously described. Cells were harvested and performed viability assay using Trypan blue excision method. Data were normalized to the control of cells without treatment. Data were obtained in three independent experiments and are represented as the mean ± S.D. The results shows that IC50 of cisplatin in MCF-7 cells under hydrogel culture is 31.25 µM.
Figure 9.
Immunofluorescence assay of cells in 3D hydrogel.
A. MCF-7 cells were cultured in 2D monolayer for 2 days (2D) or in 3D hydrogels for 7 days (3D). Immunofluorescence assay was performed. Red indicates actin and blue indicates DAPI-stained nuclei. B. MCF-7 cells were cultured in hydrogel for 5 days. After 5 days of colony formation, cells were cultured in 3D hydrogel for additional 2 days (Control) or treated with 30 µM cisplatin for 48 hours in hydrogel via “pre-mixed” method (Treatment). Cells were isolated from hydrogels and immunostained with antibodies against actin (a), Ki67 (b), survivin (c), and cleaved caspase-3 (d). Red indicates protein of interest and blue indicates DAPI-stained nuclei. To obtain the 3D images, Z-stack images were taken (0.5–1 µm slices) and reconstituted in ZEN 2010 software. The results represent one of three independent experiments.
Figure 10.
Western blot analysis of cells in 2D monolayer and 3D hydrogel.
MCF-7 cells were cultured in 2D monolayer and 3D hydrogel for 7 days (U) or for 5 days and then treated with 30 µM cisplatin for 48 hours (T). Cells were harvested and whole cell extracts were obtained. Western blot analysis of survivin, procaspase-3, and cleaved caspase-3 was performed. Actin was used as a loading control. The results represent one of three independent experiments.