Journal: Disease Models & Mechanisms

Article Title: Compound design of a patient-derived 3D cell culture system modelling early peritoneal endometriosis

doi: 10.1242/dmm.052436

Figure Lengend Snippet: Characterisation of primary human peritoneal mesothelial cells (HPMCs) and human peritoneal fibroblasts (HPFs) compared to the LP-9 mesothelial cell line and normal human dermal fibroblasts (NHDFs). (A) Representative phase-contrast micrographs of LP-9, HPMCs, NHDFs and HPFs. Mesothelial and fibroblast cells exhibit cobblestone and spindle-like morphologies, respectively. (B) We observed localised expression of the cytoskeletal markers cytokeratin (CK) and vimentin (VIM) in LP-9 cells and HPMCs, while VIM, but not CK, was expressed in NHDFs and HPFs. HPMCs ( n =6) showed a high percentage of CK + /VIM + cells [98.20±1.05% (mean±s.d.)], while HPFs ( n =3) exhibited 81.36±5.63% CK − /VIM + cells. Scale bars: 200 µm (A); 50 µm (B).

Article Snippet: NHDFs were cultured in Fibroblast Growth Medium (Promocell, C-23110) containing 1 ng/ml recombinant human basic fibroblast growth factor and 5 μg/ml recombinant human insulin, supplemented with 2 mM L-glutamine and 100 μg/ml Primocin.

Techniques: Expressing

Journal: Disease Models & Mechanisms

Article Title: Compound design of a patient-derived 3D cell culture system modelling early peritoneal endometriosis

doi: 10.1242/dmm.052436

Figure Lengend Snippet: Establishing composite 3D hydrogel constructs using peritoneal mesothelial cells and fibroblasts. (A) Schematic illustration of model construction and culture timeline. (B) Representative axial view [also seen in C (M3)] and Haematoxylin and Eosin (H&E)-stained section of a hydrogel construct showing the formation of a mesothelial monolayer (ML) and submesothelial layer (SML) on a transwell membrane (TM). (C) Representative images of hydrogel matrices using M1 (collagen I), M2 (70:30 collagen I:Matrigel ratio), M3 (50:50 collagen I:Matrigel ratio) and M4 (collagen I+human fibronectin). Construct generated with matrix combination M3 demonstrated minimal contraction in LP-9/NHDF and HPMC/HPF trials. (D) Lactate dehydrogenase (LDH) cytotoxicity assay in M3 composite hydrogel constructs containing HPMC/HPF ( n =3 donors) over a 10-day culture period. (E) Dual immunofluorescence staining of cleaved caspase-3 (CC-3) and VIM to detect apoptotic HPMCs/HPFs in M3 constructs on day 3 and day 10 of culture ( n =3). Scale bars: 300 µm (B); 100 µm (C); 50 µm (E).

Article Snippet: NHDFs were cultured in Fibroblast Growth Medium (Promocell, C-23110) containing 1 ng/ml recombinant human basic fibroblast growth factor and 5 μg/ml recombinant human insulin, supplemented with 2 mM L-glutamine and 100 μg/ml Primocin.

Techniques: Construct, Staining, Membrane, Generated, LDH Cytotoxicity Assay, Immunofluorescence

Journal: Disease Models & Mechanisms

Article Title: Compound design of a patient-derived 3D cell culture system modelling early peritoneal endometriosis

doi: 10.1242/dmm.052436

Figure Lengend Snippet: Histological and functional analysis of the human parietal peritoneum and peritoneal layer models. (A) Histological staining of transverse sections through parietal peritoneum and composite 3D hydrogel constructs composed of LP-9/NHDFs and HPMCs/HPFs. Immunofluorescence using antibodies against the mesothelial markers podoplanin (PDPN) and mesothelin (MSLN), and submesothelial markers fibroblast specific protein 1 (FSP1) and tumor endothelial marker 1 (TEM1). (B) Colocalisation of MSLN and collagen IV (COLIV) suggesting spontaneous basal lamina formation. (C) Human tissue plasminogen activator (tPA) enzyme-linked immunosorbent assay (ELISA) to determine the functionality of the mesothelial cells in models assembled with HPMCs from three different donors over a 10-day culture period. Scale bars: 50 µm; 15 µm (insets in B).

Article Snippet: NHDFs were cultured in Fibroblast Growth Medium (Promocell, C-23110) containing 1 ng/ml recombinant human basic fibroblast growth factor and 5 μg/ml recombinant human insulin, supplemented with 2 mM L-glutamine and 100 μg/ml Primocin.

Techniques: Functional Assay, Staining, Construct, Immunofluorescence, Marker, Enzyme-linked Immunosorbent Assay

Journal: PLOS One

Article Title: Fatty acid-binding protein 5 (FABP5) modulates limbal epithelial cell homeostasis by regulating the expression of key genes under both normal and inflammatory conditions , in vitro

doi: 10.1371/journal.pone.0347228

Figure Lengend Snippet: PAX6, TP53, TP63, FOSL2, FOXC1, PPARγ and NFκB mRNA expression (A) and PAX6 and FOSL2 protein levels (B) in LECs transfected with FABP5 siRNA or control (ctrl) siRNA. Western blot image from a single biological replicate (C) . Data are shown as mean ± SEM. Statistical analysis has been performed using two-way ANOVA followed by Tukey test (n = 7). p-values below 0.05 were considered statistically significant. PAX6 mRNA level was significantly lower in FABP5 knockdown LECs than in ctrl siRNA LECs, in KSFM medium or following LPS and IL-1β treatment (p ≤ 0.022). Furthermore, PAX6 protein level was significantly lower following FABP5 knockdown, than in ctrl siRNA LECs without treatment and after IL-1β treatment (p ≤ 0.031). TP63 mRNA level was significantly higher in ctrl siRNA LECs following LPS or IL-1β treatment, than without treatment (p ≤ 0.015). FOSL2 mRNA level was significantly higher in ctrl siRNA LECs following IL-1β treatment, than without treatment (p = 0.033). Under LPS and IL-1β treatment, FOSL2 mRNA levels were significantly lower in FABP5 knockdown cells than in control (transfected) cells (p ≤ 0.025).

Article Snippet: After centrifugation at 200g for 5 minutes, cells were cultured in Keratinocyte growth medium (KSFM) (Cat. Nr. C-20111, Promocell, Heidelberg, Germany), at 37 oC, 5% CO 2 .

Techniques: Expressing, Transfection, Control, Western Blot, Knockdown