(A) Viability assessment of human islets encapsulated in a 3D NFC hydrogel platform. Top: Schematic of the 3D NFC hydrogel platform. Bottom: Representative images of an islet after 21 days cultured in NFC hydrogel platform, followed by LIVE/DEAD viability staining and imaged for morphology (bright-field), viable cells (green fluorescence), and apoptotic cells (red fluorescence). Inset: Representative image of an islet cultured for 7 days in standard (CIT) medium, followed by LIVE/DEAD staining. Scale bar: 100 μm. (B) Human islets cultured for 14 or 21 days in NFC hydrogel with CIT media were functionally similar to their freshly isolated (day 1) counterparts. The experiments were conducted using islets from the same donors as in Figure 2. Results were obtained from 3 independent experiments. Each experiment was performed in duplicate or triplicate with islets isolated from the same donor. The number of donors used in each condition (n = 2 or n = 3) is indicated. Values are shown as mean ± SEM. Insulin values were normalized to DNA content of each sample. (C) A comparison of human islets cultured for 42 days in Transwell or with 3D NFC hydrogels. Human islets cultured in Transwell for 42 days exhibited significantly higher levels of basal insulin secretion in response to low glucose (1 mM and 3 mM) compared with fresh islets (day 1) or hydrogel-cultured islets. Experiments were conducted 4 times using samples from 4 different cadaveric donors. Each experiment was performed in duplicate or triplicate with islets isolated from the same donor. Values were shown as mean ± SEM. Insulin values were normalized to DNA content of each sample. Multiple-comparisons t test was performed, followed by Holm-Šidák correction. (D) Representative images of human islets following long-term NFC hydrogel culture. Cultured human islets were retrieved on day 14, 21, 42, or 83 by treatment with cellulase and imaged for morphology (bright-field). Scale bars: 100 μm.