Journal: Engineering in Life Sciences

Article Title: The cultivation conditions affect the aggregation and functionality of β‐cell lines alone and in coculture with mesenchymal stromal/stem cells

doi: 10.1002/elsc.202100168

Figure Lengend Snippet: Images of the spheroids from static culture for morphological analysis and the determination of properties such as spheroid size and circularity ( n = 12; data are means ± STDV). (A) Morphological differences can be seen during the aggregation of the different β‐cell lines within 7 days. The images of INS‐1, MIN6, and MSCs were reduced by 60%, whereas the 1.1B4 images were reduced by 73% ( * ). In both cases the scale bar represents 100 μm. (B) The growth kinetics of the spheroids reveal the differences between cell lines. The INS‐1 cells (squares) showed fast aggregation and a continuous increase in spheroid size, whereas the MIN6 cells (circles) needed 3 days to form stable spheroids before slow growth was observed. The 1.1B4 cells (triangles) aggregated slowly over 5–7 days, represented by a peak on day 2 followed by a decline in size until a stable spheroid was formed and a volume increase was observed. The hMSC‐TERTs (crosses) and ad‐MSCs (stars) showed no growth and the spheroid size declined over time. 1.1B4, a cell line formed by the electrofusion of primary human pancreatic islets and PANC‐1 cells; hMSC‐TERT, human mesenchymal stromal/stem cells immortalized with reverse transcriptase telomerase; INS‐1, rat insulinoma‐1 cell line; MIN6, mouse insulinoma‐6 cell line; MSC, mesenchymal stromal/stem cell; STDV, standard deviations

Article Snippet: The insulin in the supernatants was measured in duplicates, using the corresponding enzyme‐linked immunosorbent assay (ELISA) kit: the human ultra‐sensitive insulin ELISA (EIA‐2337) for 1.1B4 cells, the rat insulin ELISA (EIA‐2049) for INS‐1 cells, and the mouse insulin ELISA (EIA‐3439) for MIN6 cells (all kits from DRG Instruments).

Techniques: Electrofusion

Journal: Engineering in Life Sciences

Article Title: The cultivation conditions affect the aggregation and functionality of β‐cell lines alone and in coculture with mesenchymal stromal/stem cells

doi: 10.1002/elsc.202100168

Figure Lengend Snippet: The viability of spheroids from static cultures determined by staining with calcein AM and ethidium after 7 days. (A) INS‐1 spheroids featured a dead core and a viable mantle, whereas MIN6 spheroids featured a heterogenous distribution of dead cells. The loose structure of the 1.1B4 spheroids promoted sufficient mass transfer resulting in a high viability. (B) Insulin secretion profiles of INS‐1 cells cultured as monolayers and spheroids cultured under static (96‐well plate) and dynamic (shaking flask) conditions ( n = 3; data are means ± STDV; significance intervals are * p < 0.05, ** p < 0.01, and *** p < 0.001). 1.1B4, a cell line formed by the electrofusion of primary human pancreatic islets and PANC‐1 cells; INS‐1, rat insulinoma‐1 cell line; MIN6, mouse insulinoma‐6 cell line; STDV, standard deviations

Article Snippet: The insulin in the supernatants was measured in duplicates, using the corresponding enzyme‐linked immunosorbent assay (ELISA) kit: the human ultra‐sensitive insulin ELISA (EIA‐2337) for 1.1B4 cells, the rat insulin ELISA (EIA‐2049) for INS‐1 cells, and the mouse insulin ELISA (EIA‐3439) for MIN6 cells (all kits from DRG Instruments).

Techniques: Staining, Cell Culture, Electrofusion

Journal: Engineering in Life Sciences

Article Title: The cultivation conditions affect the aggregation and functionality of β‐cell lines alone and in coculture with mesenchymal stromal/stem cells

doi: 10.1002/elsc.202100168

Figure Lengend Snippet: The insulin profiles of β‐cell spheroids in static culture was measured by GSIS ( n = 3; error = STDV)

Article Snippet: The insulin in the supernatants was measured in duplicates, using the corresponding enzyme‐linked immunosorbent assay (ELISA) kit: the human ultra‐sensitive insulin ELISA (EIA‐2337) for 1.1B4 cells, the rat insulin ELISA (EIA‐2049) for INS‐1 cells, and the mouse insulin ELISA (EIA‐3439) for MIN6 cells (all kits from DRG Instruments).

Techniques:

Journal: Engineering in Life Sciences

Article Title: The cultivation conditions affect the aggregation and functionality of β‐cell lines alone and in coculture with mesenchymal stromal/stem cells

doi: 10.1002/elsc.202100168

Figure Lengend Snippet: Aggregation of INS‐1 (upper row), MIN6 (middle row) and 1.1B4 (lower row) cells with hMSC‐TERTs at different ratios after 24 h. MSCs were stained blue (VPD) and β‐cells were stained with the green dye CFSE. Starting with monospheroids in the first (MSCs, blue) and second (β‐cells, green) columns, the cell ratios increase from left to right. Due to different scaling of the images, the MSC spheroids seem to have a different size in each setup, but the seeding density was always 1000 cells per well. In all cases the scale bar represents 100 μm. 1.1B4, a cell line formed by the electrofusion of primary human pancreatic islets and PANC‐1 cells; CFSE, 5‐(and 6)‐carboxyfluorescein diacetate, succinimidyl ester; hMSC‐TERT, human mesenchymal stromal/stem cells immortalized with reverse transcriptase telomerase; INS‐1, rat insulinoma‐1 cell line; MIN6, mouse insulinoma‐6 cell line; MSC, mesenchymal stromal/stem cell

Article Snippet: The insulin in the supernatants was measured in duplicates, using the corresponding enzyme‐linked immunosorbent assay (ELISA) kit: the human ultra‐sensitive insulin ELISA (EIA‐2337) for 1.1B4 cells, the rat insulin ELISA (EIA‐2049) for INS‐1 cells, and the mouse insulin ELISA (EIA‐3439) for MIN6 cells (all kits from DRG Instruments).

Techniques: Staining, Electrofusion

Journal: Engineering in Life Sciences

Article Title: The cultivation conditions affect the aggregation and functionality of β‐cell lines alone and in coculture with mesenchymal stromal/stem cells

doi: 10.1002/elsc.202100168

Figure Lengend Snippet: Bright‐field and viability images (at day 7) of monospheroids (0–1 = MSC only; 1–0 = β‐cell only) and heterospheroids INS‐1 (upper row), MIN6 (middle row), and 1.1B4 (lower row) cocultured with hMSC‐TERTs at different cell ratios. The stated viabilities of the spheroids were assessed by measuring the red (core) and green (whole spheroid) diameter and the resulting volume to describe the real “3D viability,” but the displayed images only represent two dimensions of the spheroids, which could provide a deceptive impression. Scale bar = 100 μm. 1.1B4, a cell line formed by the electrofusion of primary human pancreatic islets and PANC‐1 cells; hMSC‐TERT, human mesenchymal stromal/stem cells immortalized with reverse transcriptase telomerase; INS‐1, rat insulinoma‐1 cell line; MIN6, mouse insulinoma‐6 cell line; MSC, mesenchymal stromal/stem cell

Article Snippet: The insulin in the supernatants was measured in duplicates, using the corresponding enzyme‐linked immunosorbent assay (ELISA) kit: the human ultra‐sensitive insulin ELISA (EIA‐2337) for 1.1B4 cells, the rat insulin ELISA (EIA‐2049) for INS‐1 cells, and the mouse insulin ELISA (EIA‐3439) for MIN6 cells (all kits from DRG Instruments).

Techniques: Electrofusion

Journal: Frontiers in Immunology

Article Title: High humidity environment increases FBG by impairing the intestinal barrier

doi: 10.3389/fimmu.2025.1625609

Figure Lengend Snippet: High humidity induces increased expression of the pancreatic MDP-NOD2 pathway, which inhibits GABA synthesis and promotes GC secretion. (A) Visualization of MDP in pancreatic tissue, with scale bars indicating 100μm. (B) IF staining of NOD2 in pancreatic β-cells. Pancreatic β-cells are marked in green, NOD2 in red, and nuclei with Dapi in blue. Scale bars = 60μm. (C) Western blot analysis of NOD2 expression in various groups (n = 6). obNC VS obH was analyzed by Welch’s t test, t=5.303, P =0.003. obH VS obHA was analyzed by Welch’s t test, t=6.568, *** P < 0.001, ** P < 0.01. (D, E) Western blot analysis of NOD2 (D) and GAD67 (E) in MIN6 cells post-MDP treatment. n = 3, D was analyzed by student’s two-tailed t test, t=11.38, P =0.0003. (E) was analyzed by student’s two-tailed t test, t=10.79, P =0.0004. *** P < 0.001. (F) : GABA levels in MIN6 cells treated with MDP. n = 3, F was analyzed by student’s two-tailed t test, t=11.11, P =0.0004. *** P < 0.001. (G) Western blot analysis of NOD2 in MIN6 cells post-NOD2 knockdown. n = 3, G was analyzed by student’s two-tailed t test, t=6.635, P =0.0027. ** P < 0.01. (H) : Western blot analysis of GAD67 in MIN6 cells post-NOD2 knockdown and MDP treatment. n = 3, (H) was analyzed by student’s two-tailed t test, t=5.904, P =0.0041. ** P < 0.01. (I) Quantification of GABA levels in MIN6 cells and NOD2-knockdown MIN6 cells treated with MDP. n = 3, (I) was analyzed by student’s two-tailed t test, t=11.04, P =0.0004. *** P < 0.001. (J) Western blots of GABA A β3 in α-TC1–6 cells treated with GABA. n = 3, (J) was analyzed by student’s two-tailed t test, t=7.29, P =0.0019. ** P < 0.01. (K) IF staining of GABA A β3 in α-TC1–6 cells GABA treatment, with green indicating GABA A β3 and blue for Dapi. Scale bars=60μm. (L) Measurement of GC levels in α-TC1–6 cells post-GABA treatment. n = 3, L was analyzed by student’s two-tailed t test, t=8.854, P =0.0009. *** P < 0.001. (M-P) GABA (M) and GC (N) levels in co-cultured MIN6 and α-TC1–6 cells. GABA (O) and GC levels (P) in co-cultured α-TC1–6 and NOD2 knockdown MIN6 cells treated with MDP. n = 3. M was analyzed by student’s two-tailed t test, t=3.938, P =0.017. (N) was analyzed by student’s two-tailed t test, t=5.011, P =0.0074. (O) was analyzed by student’s two-tailed t test, t=8.731, P =0.0009. (P) was analyzed by student’s two-tailed t test, t=8.211, P =0.0012. *** P < 0.001, ** P < 0.01, * P < 0.05. Data are shown as means ± SD. GABA, Gamma-Aminobutyric Acid; GAD67, Glutamate Decarboxylase 67; GABA A β3, Gamma-Aminobutyric Acid Receptor Subunit Beta-3; NOD2, Nucleotide-binding Oligomerization Domain-containing Protein 2.

Article Snippet: MIN6 cells, were procured from Servicebio (China).

Techniques: Expressing, Staining, Western Blot, Two Tailed Test, Knockdown, Cell Culture, Binding Assay