96 well alvetex scaffold plate  (ReproCELL)

Journal: bioRxiv

Article Title: Inflammatory Synovial Fibroblast Culture in 3D Systems: A Comparative Transcriptomic and Functional Study

doi: 10.1101/2022.12.21.521283

Figure Lengend Snippet: A) Schematic representation of 3D Alvetex ® scaffolds. Cells were expanded in 2D and then seeded on 3D fibronectin-coated scaffolds (seeding side indicated as ‘top’). 0.5×10 6 , 1×10 6 and 2×10 6 SFs were initially seeded in 6-well inserts and then cultured for 14 weeks (left panels). 0.5×10 6 SFs were seeded in 6-well inserts and cultured for 7, 14 and 21 days (right panels). Scaffolds were then fixated in formalin, and longitudinal sections (7 μm) were stained with hematoxilin and eoxin to evaluate cell number and distribution. B) Haemotoxylin and Eosin staining on naïve SFs cultured in non-coated scaffolds alone (PBS) or fibronectin (FN) coated. Images were taken on a EVOS brightfield microscope at x10 magnification (scale bar = 500 μm). Heatmaps show the percentage of cells in the indicated area of the scaffold (calculated with ImageJ software), as mean from technical triplicates from three independent experiments. Statistical significance was evaluated by twoway ANOVA, *p<0.05. C) Hematoxilin/eosin staining and immunofluorescence to detect vimentin (red) and DAPI (blue) was conducted in sections (7 μm) of FN-coated scaffolds containing naïve SFs (1×10 6 cells, 9 days in culture). Whole discs were fixed and stained with phalloidin (green) to visualise cytoskeleton organization and cell shape prior to acquisition of Z-stack images. DAPI (blue) was used as a counterstaining to visualise cell nuclei. Images were acquired using a Zeiss Confocal microscope (phalloidin/DAPI) staining and 3D reconstructions were done with Imaris software.

Article Snippet: Alvetex ® scaffolds (AMS Biotechnology (Europe) Lt, Abingdon) were rendered hydrophilic with 70% ethanol.

Techniques: Cell Culture, Staining, Microscopy, Software, Immunofluorescence

Journal: bioRxiv

Article Title: Inflammatory Synovial Fibroblast Culture in 3D Systems: A Comparative Transcriptomic and Functional Study

doi: 10.1101/2022.12.21.521283

Figure Lengend Snippet: Differentially expressed genes in CIA SFs compared to healthy controls in not cultured cells and cells cultured in 2D, 3D rigid scaffolds and FNPEG hydrogels were compared using the bioinformatic tool Metascape. A) Circos plot representing significantly up-regulated genes overlapping in not cultured cells, 2D cultured cells and cells in 3D rigid scaffolds and FNPEG hydrogels. (Groups represented on the arc outside; red: 2D, blue: Alvetex ® scaffold, green: FNPEG hydrogels). B) Enriched ontology clusters in CIA SFs compared to healthy cells in all systems tested. Clusters were hierarchically clustered into a tree based on Kappa-statistical similarities among their gene memberships. C) TRRUST (Transcriptional Regulatory Relationships Unraveled by Sentence-based Text mining, https://www.grnpedia.org/trrust/ ) was used to identify activation of Transcription Factor-specific pathways in CIA SFs based on literature curation. The heatmap cells are colored by their p-values, white cells indicate the lack of enrichment for that term in the corresponding gene list. All results were generated with the bioinformatic tool Metascape.

Article Snippet: Alvetex ® scaffolds (AMS Biotechnology (Europe) Lt, Abingdon) were rendered hydrophilic with 70% ethanol.

Techniques: Cell Culture, Activation Assay, Generated

Journal: bioRxiv

Article Title: Inflammatory Synovial Fibroblast Culture in 3D Systems: A Comparative Transcriptomic and Functional Study

doi: 10.1101/2022.12.21.521283

Figure Lengend Snippet: SFs cultured in 2D conditions, and cells subsequentially transferred to Alvetex ® scaffolds and FNPEG hygrogels were stimulated with IL-1β (10 ng/ml, 6 hours), when RNA was extracted to conduct RNA-Seq experiments (n=3, 75 bp paired-end, 30 M reads). A) Differential expression (DE) of genes, plotted as a volcano plot where x = log2 Fold Change in CIA cells, y = log10 padj value. Genes that passed a threshold of padj < 0.01 and |log2foldChange| > 2 in the arthritic (CIA) SFs are colored in blue [downregulated] and red [upregulated]. Tables show the top-50 up-regulated genes in response to IL-1β for each condition. Significantly enriched KEGG pathways are shown. B) Circos plot show how IL-1β upregulated genes overlap in 2D and 3D systems (On the arc outside, red: 2D, blue: Alvetex ® scaffold, green: FNPEG hydrogels). On the arc inside, each gene is assigned a spot in the arc, dark orange = genes shared in multiple lists, light orange = gene unique to that list. Purple lines link the same gene that are shared by multiple gene lists. C) Enriched ontology clusters in 2D and 3D systems upon IL-1β stimulation were identified and hierarchically clustered into a tree based on Kappa-statistical similarities among their gene memberships. D) DE expressed genes upon IL1β were applied to TRRUST (Transcriptional Regulatory Relationships Unraveled by Sentence-based Text mining, https://www.grnpedia.org/trrust/ ) analysis to identify activation of Transcription Factorspecific pathways based on literature curation. In heatmaps, cells are colored by their p-values, white cells indicate the lack of enrichment for that term in the corresponding gene list. All results were generated with the bioinformatic tool Metascape.

Article Snippet: Alvetex ® scaffolds (AMS Biotechnology (Europe) Lt, Abingdon) were rendered hydrophilic with 70% ethanol.

Techniques: Cell Culture, RNA Sequencing Assay, Expressing, Activation Assay, Generated