Journal: Materials Futures

Article Title: A clinically defined and xeno-free hydrogel system for regenerative medicine

doi: 10.1088/2752-5724/ae4e4d

Figure Lengend Snippet: Substrate characterisation in 2D and 3D cell culture—biological responses and physical properties with different compositions. (a) hPSC confluence in 2D growth factor-reduced Matrigel (Geltrex), laminin 521, fibrin-laminin hydrogel, and fibrin gel after 4 d; scale bar = 100 μ m. (b) hPSCs cultured in 3D hydrogels. Top panel: fibrin (5 mg ml −1 ) gel. Bottom panel: Alphagel containing structures resembling pluripotent spheroids; scale bar = 200 μ m. Scanning electron microscopy of (c) fibrin gel and (d) Alphagel; scale bar = 1 μ m, magnification 20 K X, iProbe = 13 pA, 2.00 kV, Working Distancee = 4.4 mm for both images. (e) Young’s moduli in hydrogels with varying fibrin and laminin concentrations. One-way ANOVA; ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. (f) Cell viability of hPSCs cultured in Alphagel, fibrin-only hydrogels, and 2D standard substrates. Mean ± sandard error of the mean displayed. t -test; * = p < 0.05. (g) ELISA of laminin 521 in culture media used with acellular Alphagel and (h) the calculated amount of fibrin-bound laminin.

Article Snippet: Apolipoprotein B (APOB) secretion in the supernatant was quantified using the human APOB ELISA quantification kit (Mabtech, no. 3715-1H-6) according to the product literature.

Techniques: Cell Culture, Electron Microscopy, Enzyme-linked Immunosorbent Assay

Journal: Materials Futures

Article Title: A clinically defined and xeno-free hydrogel system for regenerative medicine

doi: 10.1088/2752-5724/ae4e4d

Figure Lengend Snippet: Characterisation of iHeps derived in Alphagel, fibrin-only hydrogels, and Matrigel. (a) Key hepatocyte markers in Alphagel-derived iHeps. ALB = albumin, HNF = hepatocyte nuclear factor, CYP2A6 = Cytochrome P450 2A6, CD147 = cluster of differentiation protein 147, and E-CAD = E-cadherin. Scale bar = 25 μ m. (b) Key hepatocyte markers by gene expression (qPCR): CCAAT/enhancer-binding protein alpha (CEBPA), T-box transcription factor 3= TBX3, alpha-fetoprotein = AFP. (c) Albumin secretion (ELISA) and (d) CYP3A4 activity (P450-Glo TM ) in PHHs versus iHeps cultured in various gels (day 22). HCM = Hepatocyte Culture Media (Lonza). (e) LDL uptake (red) in Alphagel-derived iHeps versus hPSCs. Scale bar = 100 μ m. (f) CDFDA secretion (green) in Alphagel-derived iHeps versus hPSCs. Top panel scale bar = 20 μ m; bottom panel scale bar = 50 μ m. One-way ANOVA was used; * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001.

Article Snippet: Apolipoprotein B (APOB) secretion in the supernatant was quantified using the human APOB ELISA quantification kit (Mabtech, no. 3715-1H-6) according to the product literature.

Techniques: Derivative Assay, Gene Expression, Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Cell Culture

Journal: Materials Futures

Article Title: A clinically defined and xeno-free hydrogel system for regenerative medicine

doi: 10.1088/2752-5724/ae4e4d

Figure Lengend Snippet: Characterisation of iHeps cultured in Hepatogel and its effect on cell retention after intra-hepatic cell transplantation. (a) Differentially expressed genes in iHeps: Hepatologel, Alphagel, Matrigel, and adult PHHs. (b) A heat map summarising the differential gene expression of a hepatic 24-gene panel across replicates of Matrigel, Alphagel, and Hepatogel (normalised to hPSC). (c) Albumin ELISA of culture media and (d) luciferin-based measure of CYP3A4 activity: 2 d after completion of iHep differentiation and 2 d after plating PHHs. One-way ANOVA; * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. (e) Mouse livers 3 d after intra-hepatic injection with H-iHeps in Hepatogel and 0.9% saline. Red box = area magnified. * = site of injection, dotted white lines demarcate engrafted cell mass. Scale bar = 1 mm. (f) Human albumin (stained red) in engrafted iHeps 3 d after intra-hepatic injection. Scale bar = 100 μ m. (g) H-iHeps identified by albumin staining on liver histology 3 d after intra-hepatic injection. (h) ELISA of mouse serum for human albumin after injection with H-iHeps in Hepatogel and 0.9% saline over time. Day 0 = serum levels before injection. T -test; *** = p < 0.001 and **** = p < 0.0001.

Article Snippet: Apolipoprotein B (APOB) secretion in the supernatant was quantified using the human APOB ELISA quantification kit (Mabtech, no. 3715-1H-6) according to the product literature.

Techniques: Cell Culture, Transplantation Assay, Gene Expression, Enzyme-linked Immunosorbent Assay, Activity Assay, Injection, Saline, Staining

Journal: PLOS Pathogens

Article Title: Nicotinamide metabolism is essential for Hepatitis C Virus replication and the production of infectious Lipo-Viro-Particles

doi: 10.1371/journal.ppat.1014165

Figure Lengend Snippet: Huh7 cells were treated or not with 100 µM 6-AN for 72h of culture. (A-B) ApoB and ApoE secreted in cells supernatant quantified by ELISA. Are presented means ± SEM (n = 3, student’s t-test, **p < 0.0021, ***p < 0.0002). (C) Quantification of intracellular triglycerides in total cell extracts. Are presented means ± SEM (n = 3, student’s t-test, *p < 0.01). (D) Representative picture of 3D lipid droplets reconstruction using IMARIS software in Huh7 cells after Oil-red-O (yellow) and Hoechst (blue) staining. (E) The mean total volume of lipid droplets per cell was determined by analyzing their intracellular content of 50 cells for each condition with the IMARIS software. Are presented means ± SEM, one-way ANOVA for multiple comparison, n.s. non significative, ****p < 0.0001.

Article Snippet: ApoB and ApoE concentrations in medium and gradients fractions were determined by ELISA as previously described [ ], using the following antibodies: anti-human ApoB mAbs (LDL 20/17), anti-human ApoB mAb (LDL 11) biotin, anti-human ApoE mAb (E276), anti-human ApoE mAb (E887) biotin (MABTECH, Sweden).

Techniques: Enzyme-linked Immunosorbent Assay, Software, Staining, Comparison

Journal: PLOS Pathogens

Article Title: Nicotinamide metabolism is essential for Hepatitis C Virus replication and the production of infectious Lipo-Viro-Particles

doi: 10.1371/journal.ppat.1014165

Figure Lengend Snippet: Huh7 cells were treated or not with 100 µM 6-AN for 72h of culture. (A-B) ApoB and ApoE secreted in cells supernatant quantified by ELISA. Are presented means ± SEM (n = 3, student’s t-test, **p < 0.0021, ***p < 0.0002). (C) Quantification of intracellular triglycerides in total cell extracts. Are presented means ± SEM (n = 3, student’s t-test, *p < 0.01). (D) Representative picture of 3D lipid droplets reconstruction using IMARIS software in Huh7 cells after Oil-red-O (yellow) and Hoechst (blue) staining. (E) The mean total volume of lipid droplets per cell was determined by analyzing their intracellular content of 50 cells for each condition with the IMARIS software. Are presented means ± SEM, one-way ANOVA for multiple comparison, n.s. non significative, ****p < 0.0001.

Article Snippet: ApoB and ApoE concentrations in medium and gradients fractions were determined by ELISA as previously described [ ], using the following antibodies: anti-human ApoB mAbs (LDL 20/17), anti-human ApoB mAb (LDL 11) biotin, anti-human ApoE mAb (E276), anti-human ApoE mAb (E887) biotin (MABTECH, Sweden).

Techniques: Enzyme-linked Immunosorbent Assay, Software, Staining, Comparison