neonatal human dermal fibroblasts (hdf n  (ZenBio)

Journal: bioRxiv

Article Title: Identification of potent HSV antivirals using 3D bioprinted human skin equivalents

doi: 10.1101/2024.12.04.626896

Figure Lengend Snippet: (A) Dermis equivalents were 3D printed onto the apical side of transwell inserts using the RegenHU 3D Discovery bioprinter (image courtesy of RegenHU). Keratinocytes were pipetted onto the apical surface of the dermis. In the submerged model, the tissues were infected at the apical surface. In the ALI model, tissues were brought to ALI and then infected at the basolateral surface (created with BioRender.com ). (B) H&E and IHC images of differentiated ALI tissues. K10 (cyan) and K14 (red) identify keratinocytes in the suprabasal and basal layer of the epidermis respectively (scale bar 50µm) (C) Submerged tissues were infected at various MOI and then imaged at specified times. Fibroblasts express tdTomato (orange) while infected cells express GFP (green) (scale bar 1mm). (D) GFP and tdTomato signal at each MOI and timepoint (*** P < 0.001, **** P < 0.0001 by ordinary one-way ANOVA). (E) Maximum projection of infected tissues from the top (column 1) or side (column 2) view. H&E (column 3) and IHC (column 4) staining of infected submerged or ALI models (scale bar 1mm (column 1) or 50µm (column 2, 3, 4).

Article Snippet: Neonatal human dermal fibroblasts (HDF N , Zen Bio DFN-F) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco 11965) supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin streptomycin.

Techniques: Infection, Staining

Journal: bioRxiv

Article Title: Identification of potent HSV antivirals using 3D bioprinted human skin equivalents

doi: 10.1101/2024.12.04.626896

Figure Lengend Snippet: (A) Correlation plot of Max %Activity (maximum reduction in GFP) vs. Max %Viability (maximum reduction in tdTomato) of 106 ‘hits’ tested in dose response. Top candidate antivirals (50% or greater reduction in GFP) that did not kill over 50% of tdTomato transduced fibroblasts are identified by the red shaded box. (B) Schematic illustrating %Activity dose response profiles of different Concentration-Response Curve classes (CRC). (C) Venn diagram showing divergent and coinciding targets for 41 top candidate antivirals in both submerged and ALI models. (D) Schematic of compounds selection from 738 compounds in the primary screen to 106 ‘hits’ tested in dose-response to 41 selected candidates and 11 top candidates selected to move forward. Of the 41 selected candidates, 23 are current or experimental HSV treatments. (E) Dose response curves of candidate antivirals in “ciclovir” family, known to treat HSV-1, in submerged and ALI models.

Article Snippet: Neonatal human dermal fibroblasts (HDF N , Zen Bio DFN-F) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco 11965) supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin streptomycin.

Techniques: Activity Assay, Concentration Assay, Selection

Journal: bioRxiv

Article Title: Identification of potent HSV antivirals using 3D bioprinted human skin equivalents

doi: 10.1101/2024.12.04.626896

Figure Lengend Snippet: (A) Punch biopsies from six donors were collected and dissociated by enzymatic and mechanical processes. Vero cells, keratinocytes (B), and fibroblasts (C) were infected with GFP-expressing HSV-1, and live cell images were taken every two hours. (D) Keratinocytes, fibroblasts, and Vero cells were infected with GFP-expressing HSV-1 and then treated with acyclovir at the specified doses. Representative live cell images were taken at the peak of GFP expression. (Scale bar 500µm). (E) Dose-response curve of acyclovir in keratinocyte cultures compared to Vero cells (grey line). (F) Dose-response curve of acyclovir in fibroblast cultures compared to Vero cells (grey line). (G) IC 50 values for each donor in each cell type (*** P < 0.001, * P < 0.05, linear mixed model).

Article Snippet: Neonatal human dermal fibroblasts (HDF N , Zen Bio DFN-F) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco 11965) supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin streptomycin.

Techniques: Infection, Expressing

Journal: bioRxiv

Article Title: Identification of potent HSV antivirals using 3D bioprinted human skin equivalents

doi: 10.1101/2024.12.04.626896

Figure Lengend Snippet: (A) Dose-response curves for the 11 top candidate antivirals compared to acyclovir (ACV) (keratinocytes blue and grey, respectively; fibroblasts green and black, respectively). (B) IC 50 values for each top candidate antiviral compared between keratinocytes (blue) and fibroblasts (green). Striped bars (FMP, VRD) indicate candidate antivirals that failed to reduce GFP expression by at least 50% consistently. (C) Maximum inhibition for each top candidate antiviral is compared between keratinocytes (blue) and fibroblasts (green). Statistical significance was determined by linear mixed model (*** P < 0.001, ** P < 0.01, * P < 0.05) for (B) and (C) . (D) CC 50 dose-response curves for all twelve candidate antivirals compared to their respective IC 50 to IC 80 dose ranges. Keratinocyte data is from 20HPI (grey), while fibroblast data is from 48HPI (red).

Article Snippet: Neonatal human dermal fibroblasts (HDF N , Zen Bio DFN-F) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco 11965) supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin streptomycin.

Techniques: Expressing, Inhibition

Journal: bioRxiv

Article Title: Identification of potent HSV antivirals using 3D bioprinted human skin equivalents

doi: 10.1101/2024.12.04.626896

Figure Lengend Snippet: (A) Pairwise comparisons of IC 50 values for candidate antivirals in the four models tested. (B) Pairwise comparisons of CC 50 values for candidate antivirals in the four models tested. (C) Fold change was determined by dividing the IC 50 value of each candidate antiviral in keratinocytes by the IC 50 of the same candidate antiviral in submerged models. (D) Fold change was determined by dividing the IC 50 value of each candidate antiviral in fibroblasts by the IC 50 of the same candidate antiviral in ALI models. (E) IC 50 values for each candidate antiviral were pooled (keratinocytes and fibroblasts, submerged and ALI), then IC 50 values for candidate antivirals in 2D were divided by IC 50 values in 3D. (C) (D) (E) Green bars indicate candidate antivirals that were more potent in 3D, while blue bars indicate candidate antivirals that are potent in 2D.

Article Snippet: Neonatal human dermal fibroblasts (HDF N , Zen Bio DFN-F) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco 11965) supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin streptomycin.

Techniques: