Fig. 2

Cytocompatibility testing of IIZK peptide hydrogel. (A) Cell viability assessment of AML cell lines after 14 days of 3D culture with IIZK peptide hydrogel. Cells were stained with calcein-AM (green, live cells) and ethidium homodimer-1 (red, dead cells). Upper: Fluorescent images of cell viability (scale bar, 200 µM). Bottom: Flow cytometry analysis of cell viability; x-axis: calcein-AM, y-axis: ethidium homodimer. Representative images of 3 independent experiments (n = 3). (B) Assessment of AML cell proliferation with 3D IIZK peptide hydrogel compared to Matrigel and 2D cultures. (C) SEM images of the HL-60 AML cell line after 1 and 4 days of 3D culture within IIZK peptide scaffold. (D) Clonogenicity potential of HL-60 and MV4-11 in 3D culture with IIZK peptide scaffold or in 2D culture. Left panel: Light microscopy images of the colony formation using methylcellulose media for 2D- and 3D-cultured cells (scale bar, 100 μm). Right panel: Percentage of colonies formed in Methylcellulose (**p < 0.01). (E) In vivo fluorescence imaging of mice after receiving DIR-stained KG1a cells from 2D or 3D models (squared image). NSG mice were randomly assigned to 4 groups and intravenously injected with: (i) HBSS (n = 4), (ii) DiR-labeled KG1a cells harvested from 2D culture (2D group; n = 4), (iii) DiR-labeled KG1a cells harvested from 3D culture (3D group; n = 4), or (iv) DiR-labeled peptides (PEP-DiR; n = 4). Mice were placed at various positions for IVIS imaging to track the distribution of KG1a cells. Mice were imaged 2 h (2 H) and 48 h (48 H) after the injection. After 48 h, the mice were euthanized, and the major organs were collected (bones = spine/femur/tibia, heart, kidney, liver, spleen) for ex vivo imaging using the IVIS Spectrum. Representative images of the organs are shown. The average luminescence signal (n = 4 mice per group) is graphed for each organ. Significant differences are indicated (*p < 0.05 and **p < 0.001). Except for the liver, significant differences were only observed in comparisons with the control groups [(i) and (iv)]