Journal: bioRxiv

Article Title: 3D Droplet-Based Bioprinting of Customized In Vitro Head and Neck Cancer Tumor Microenvironment Models

doi: 10.64898/2026.03.27.714925

Figure Lengend Snippet: FaDu and 2A3 cells were suspended directly into a matrix metalloproteinase (MMP) labile bio-thiol activator which was then printed with its respective 4-arm maleimide PEG bioink to establish hydrogels upon component contact at room temperature. Both cell lines were printed into 8 different matrix conditions (PEG versus PEG fnc in stiffnesses of 0.7, 1.1, 3.0, and 4.8 kPa) for a total of 16 matrix conditions investigated in this study. High-throughput printing was achieved through the use of 96-well plates, with the printing process allowing for spatial control of different cell and gel types to create a customized system based on user-preference.

Article Snippet: FaDu (HTB-43) and 2A3 (CRL-3212) human-derived cell lines were purchased from ATCC, and cultured using ATCC-suggested media for each line.

Techniques: High Throughput Screening Assay, Control

Journal: bioRxiv

Article Title: 3D Droplet-Based Bioprinting of Customized In Vitro Head and Neck Cancer Tumor Microenvironment Models

doi: 10.64898/2026.03.27.714925

Figure Lengend Snippet: Viability stains demonstrate high viability of cell clusters over time. Hydrogel samples were collected at 1, 4, and 7 days of culture, stained to assess viability, and imaged via confocal microscopy as z-stacks. (a) Images of FaDu cells, in a PEG hydrogel of 1.1 kPa stiffness, at 1, 4, and 7 days of culture, show high cell viability (green) and few dead cells (red). The development of multicellular clusters over time tracks with observations from light microscopy in . (b) Final images of each cell type and gel condition, recorded at day 7 of culture, reinforce the varied cell response to each condition. Scale: x and y dimensions reflect 826 × 826 μm, and z dimension reflects 200 μm depth. Blue: DAPI (nuclei), Green: calcein-AM (live cells), Red: ethidium homodimer (dead cell nuclei); (c) Quantified viability from reconstructed z-stacks for FaDu (left) and 2A3 (right) cells, printed in each hydrogel stiffness, either in PEG or peptide-functionalized PEG hydrogels. Each bar reflects triplicate specimens, error bars reflect 95% CI. Comparisons were conducted for cell viability between PEG and PEG fnc matrices at each stiffness, * = p < 0.05. For Day 7 samples at the end of the experiment, letters identify statistically different results across four stiffnesses within the PEG (a, b) matrix or the PEG fnc (a’, b’) matrix, α=0.05.

Article Snippet: FaDu (HTB-43) and 2A3 (CRL-3212) human-derived cell lines were purchased from ATCC, and cultured using ATCC-suggested media for each line.

Techniques: Staining, Confocal Microscopy, Light Microscopy

Journal: bioRxiv

Article Title: 3D Droplet-Based Bioprinting of Customized In Vitro Head and Neck Cancer Tumor Microenvironment Models

doi: 10.64898/2026.03.27.714925

Figure Lengend Snippet: Hydrogels enable cell survival and rapid proliferation to form multicellular clusters. Light microscopy images of each cell type in each hydrogel condition were captured over 7 days to assess cell proliferation and morphology changes. (a) Images of FaDu cells, encapsulated as single cells in a PEG hydrogel of 1.1 kPa stiffness, at 0, 2, 4, and 6 days of culture show progressive cell proliferation and formation of multicellular clusters. (b) Final images of each cell type and gel condition, recorded at day 7 of culture, demonstrate the varied cell response to each condition. All samples were seeded initially as single-cell suspensions under identical cell concentrations. Corresponding day 0 images for panel (b) are provided in Supporting Information Figure SI-1. Scale bar = 100 μm. (c) Mean cluster counts for FaDu and 2A3 cells in each matrix, normalized to the day 1 count. Each point represents triplicate repeats, and error bars reflect the 95% confidence interval. Despite some variation, cluster counts are statistically indistinguishable for all conditions.

Article Snippet: FaDu (HTB-43) and 2A3 (CRL-3212) human-derived cell lines were purchased from ATCC, and cultured using ATCC-suggested media for each line.

Techniques: Light Microscopy, Single Cell

Journal: bioRxiv

Article Title: 3D Droplet-Based Bioprinting of Customized In Vitro Head and Neck Cancer Tumor Microenvironment Models

doi: 10.64898/2026.03.27.714925

Figure Lengend Snippet: Cluster morphology measures and modeling at day 7 timepoint identify impacts of cell type and matrix parameters. 3D reconstructions of calcein-AM green-stained cell clusters for each cell type, matrix stiffness, and composition permutation were analyzed in Imaris to obtain per-cluster measurements of (a) surface area and (b) volume, and generate (c) complexity as a measure of invasiveness. (a-c) Graphs show the median of each value, ± 95% CI, n= 2-3 replicate gels (red, blue, green, offset for visibility) per condition, and n∼50-200 clusters per gel replicate. (d) Population data were modeled using lmer to generate plots of predicted cluster area, volume, and complexity as a function of stiffness. Cluster area and volume measures at day 7 varied with both stiffness and cell type and were cross-correlated. Area and volume magnitudes were correlated with matrix composition at day 7, but their change with stiffness was independent of matrix. Predicted complexity values aligned for FaDu cells in both matrices and for 2A3 cells in PEG fnc , but with unique behavior for 2A3 cells in PEG.

Article Snippet: FaDu (HTB-43) and 2A3 (CRL-3212) human-derived cell lines were purchased from ATCC, and cultured using ATCC-suggested media for each line.

Techniques: Staining