normal human dermal fibroblasts nhdf  (PromoCell)

Journal: Aging Cell

Article Title: Overactivation of Cdc42 GTPase Impairs the Cytotoxic Function of NK Cells From Old Individuals Towards Senescent Fibroblasts

doi: 10.1111/acel.70398

Figure Lengend Snippet: Natural killer cells from old adults reveal reduced cytotoxicity towards senescent fibroblasts. (A) Graphical illustration of the NK cell mediated target cell cytotoxicity assay. Target cells were first stained with calcein acetomethoxymethyl (AM), a vital fluorescent dye. Calcein AM is a non‐fluorescent compound that pass the intact cell membrane into the cytoplasm. Hydrolysis of calcein AM by intracellular esterases in live cells generates calcein, a hydrophilic, intensely fluorescent molecule which reliably stays in the cytoplasm. The stained target cells were next co‐cultured with NK cells isolated from young or old human or mice. NK cells exert their cytotoxicity towards target cells through the release of perforin and granzyme B. Upon lysis of target cells, the calcein dye is released and the loss of the dye is measured as a shift in fluorescence intensity by flow cytometry. Dead cells will appear to the left of the histogram, while alive cells on the right side. The percentage of dead cells can then simply be calculated and presented. (B) Graphical scheme depicts the experimental groups: Co‐cultures of NK cells from young adults with senescent human dermal fibroblasts in the top row and NK cells from old adults with senescent HDF in the bottom row. (C) Histogram (bi‐exponential scale) showing cytotoxicity of NK cells from young and old adults on different senescent HDF. RS, replicative senescent HDF, DIS, doxorubicin induced senescent HDF, IR, ionizing radiation induced senescence, CA, chronologically aged HDF (~75 years). The peak in the left part of the histogram showing the dead cell population and the percentage of dead cells. (D) The graph depicts the percentage of senescent HDF death ( y ‐axis) by NK cells isolated from young and old human adult. Data were represented as mean (Percentage of senescent fibroblast death) ± SEM. N = 5. Two tailed Student's t ‐test was used to assess the significance between young and old groups for each of senescence model. (E) Illustration of the experimental design showing cytotoxic activity of NK cells derived from bone marrow and spleen of young and old mice against aged murine dermal fibroblasts (MDF). (F) Histogram depicting cytotoxicity of NK cells from young and old mice on old MDF. The peak in the left part of the histogram showing the dead cell population and the percentage of dead cells. (G) The graph depicts the percentage of senescent MDF death ( y ‐axis) by NK cells isolated from young and old mice. Data were represented as mean (Percentage of senescent fibroblast death) ± SEM. N = 3. Each mouse NK cell sample used in the cytotoxicity assay was the pool of NK cells isolated from three different mice. One‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups.

Article Snippet: Human dermal fibroblast , ATCC , Cat. #ATCC‐PCS‐201‐010.

Techniques: Cytotoxicity Assay, Staining, Membrane, Cell Culture, Isolation, Lysis, Fluorescence, Flow Cytometry, Two Tailed Test, Activity Assay, Derivative Assay, Comparison

Journal: Aging Cell

Article Title: Overactivation of Cdc42 GTPase Impairs the Cytotoxic Function of NK Cells From Old Individuals Towards Senescent Fibroblasts

doi: 10.1111/acel.70398

Figure Lengend Snippet: CASIN restores impairment of conjugation, degranulation and mitochondrial ATP generation in old Natural killer cells. (A) Synapse formation with conjugation of NK cells with the target senescent fibroblasts and the tubulin network pulling the perforin and granzyme B containing vesicles in the direction of the synapse. (B) Fusion of the NK cell derived secretory granules with the presynaptic membrane of NK cells and concomitant exposure of CD107a at the cell membrane and the release of perforin and granzyme B into the synaptic cleft towards the target cell. (C & D) Percentage of NK cell conjugation with senescent HDF when co‐cultured for (C) 60 and (D) 90 min at an effector to target (E:T) cell ratio of 1:1. Data were represented as mean (percentage of cell conjugation) ± SEM, N = 3. (E) Degranulation of NK cells when co‐cultured with senescent HDF for 7 h at an effector to target (E:T) cell ratio of 10:1. Data were represented as mean (mean fluorescence intensity) ± SEM, N = 6. One‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups in C, D and E. (F) Seahorse flux analysis showed quantification of ATP generated by NK cells treated with vehicle from young donors and from NK cells treated with either vehicle or CASIN from old donors. The ATP generation either by glycolysis or by oxidative phosphorylation and total ATP was assessed. Data were represented as mean (ATP level) ± SEM, N = 7. Two‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups. (G) Mitochondrial structure showing the chemical structure of the mitochondrial fluorescent probe JC‐1 that can form J‐aggregates (red) and J‐monomers (green) indicating high and low mitochondrial membrane potential, respectively. (H) Flow cytometry analysis of J‐aggregates (red) and J‐monomers (green) of young NK cells treated with vehicle, old NK cells treated with either vehicle or CASIN. (I) The graph depicts the percentage of J‐aggregates (Q2 population of figure H) of young NK treated with vehicle, and old NK cells treated with either vehicle or CASIN. Data were represented as mean (percentage of cell with J‐aggregate) ± SEM, N = 6. (J) Quantification of the ratio of J‐aggregates to J‐monomers from young and old NK treated with vehicle and old NK cells treated with CASIN. Data were represented as mean (ratio) ± SEM, N = 5. One‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups in I and J.

Article Snippet: Human dermal fibroblast , ATCC , Cat. #ATCC‐PCS‐201‐010.

Techniques: Conjugation Assay, Derivative Assay, Membrane, Cell Culture, Fluorescence, Comparison, Generated, Phospho-proteomics, Flow Cytometry

Journal: Aging Cell

Article Title: Overactivation of Cdc42 GTPase Impairs the Cytotoxic Function of NK Cells From Old Individuals Towards Senescent Fibroblasts

doi: 10.1111/acel.70398

Figure Lengend Snippet: CASIN treatment improves the cytotoxic ability of Natural killer cells from old humans and mice. (A) Graphical illustration of experimental plan, where young NK cells treated with vehicle and old NK cells treated with either vehicle or CASIN for 8 h and thereafter subjected to co‐culture with target senescent HDF exerting their differential killing ability. (B) Representative histograms depicting the killing ability of different experimental groups as measured by flow cytometry. Peak at the left side of histogram, showing the dead senescent HDF population with percentage of dead cells. (C) Quantification of the percentage of target senescent HDF death executed by young NK cells treated with vehicle, and old NK cells treated with either vehicle or CASIN. Data were represented as mean (Percentage of senescent fibroblast death) ± SEM. N = 4. (D) Representative histograms show the distribution of K562 killing by young NK cells treated with vehicle, and old NK cells treated with either vehicle or CASIN. Peak at the left side of histogram, showing the dead K562 population with percentage of dead cells. (E) Graph shows the percentage of target cell (K562) death mediated either by young NK cells treated with vehicle or by old NK cells treated with either vehicle or CASIN. Data were represented as mean (Percentage of K562 lysis) ± SEM. N = 4. One‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups in C and E. (F) Illustration of the experimental design for treatment of young mice (average age 120 days) treated with vehicle and old mice (average age 650 days) treated with either vehicle or CASIN. Following treatment, NK cells were isolated from spleen and bone marrow and subjected to co‐cultures with murine dermal fibroblasts (MDF) derived from old mice (average age 650 days). (G) Flow cytometry with representative histograms depicting old/senescent MDF killing by NK cells isolated from bone marrow (left panel) and spleen (right panel) of vehicle and CASIN treated old mice. Peak at the left side of histogram, showing the dead old MDF population with percentage of dead cells. (H) Quantification of the percentage of old/senescent MDF killing by NK cells isolated from bone marrow and spleen of young and old mice treated with vehicle and old mice treated with CASIN. Data were represented as mean (percentage of old/senescent MDF lysis) ± SEM, N = 4, where each group contains pool of NK cells isolated from 4 different mice of same treatment group. Two‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups. (I) Graphical summary. Unrestrained Cdc42 activity causes failure of old NK cells to kill senescent fibroblasts. Unrestrained Cdc42 activity disrupts the microtubular network and impaired mitochondrial ATP resulting in reduced conjugation, and impaired degranulation of lytic vesicles into the synaptic cleft with reduced cytotoxicity. CASIN can attenuate all these steps and in part attenuate the killing of senescent fibroblasts (senescent HDF).

Article Snippet: Human dermal fibroblast , ATCC , Cat. #ATCC‐PCS‐201‐010.

Techniques: Co-Culture Assay, Flow Cytometry, Lysis, Comparison, Isolation, Derivative Assay, Activity Assay, Conjugation Assay

Journal: Bioactive Materials

Article Title: Multifactorial bioengineering in the tendon context to maintain tenocyte phenotype and to direct dermal fibroblasts towards tenogenic lineage

doi: 10.1016/j.bioactmat.2025.11.008

Figure Lengend Snippet: Immunofluorescence intensity analysis for COL I, III, IV, V, VI, COL I/COL III ratio, TNMD and aSMA in hTCs and hDFs after 4, 7 and 10 days of culture under PLLA, +TGFB2, +MMC and +MMC + TGFB2 conditions. (∗p < 0.05 vs respective PLLA group at the same time point; ‡p < 0.05 vs respective + TGFB2 group at the same time point; #p < 0.05 vs. respective + MMC group at the same time point; §p < 0.05 between cell types at the same time point and condition; &p < 0.05 vs. respective condition at day 4). N = 3.

Article Snippet: Cryopreserved hDFs (PCS-201-012) were purchased from the American Type Culture Collection (ATCC, United Kingdom).

Techniques: Immunofluorescence

Journal: Bioactive Materials

Article Title: Multifactorial bioengineering in the tendon context to maintain tenocyte phenotype and to direct dermal fibroblasts towards tenogenic lineage

doi: 10.1016/j.bioactmat.2025.11.008

Figure Lengend Snippet: Principal component analysis ( A ) of the quantitative proteomic data obtained from hTCs and hDFs after 10 days of culture under TCP, PLLA, +TGFB2, +MMC and +MMC + TGFB2 conditions, as well as from TT samples. Venn diagram representing the top 100 protein hits in TT group vs. hTCs ( B ) and hDFs ( C ) cultured under PS, PLLA, +TGFB2, +MMC and +MMC + TGFB2 conditions. Gene ontology analysis ( D ) of the top 100 protein hits in TT group vs. hTCs and hDFs cultured under PS, PLLA, +TGFB2, +MMC and +MMC + TGFB2 conditions. N = 3.

Article Snippet: Cryopreserved hDFs (PCS-201-012) were purchased from the American Type Culture Collection (ATCC, United Kingdom).

Techniques: Cell Culture

Journal: Bioactive Materials

Article Title: Multifactorial bioengineering in the tendon context to maintain tenocyte phenotype and to direct dermal fibroblasts towards tenogenic lineage

doi: 10.1016/j.bioactmat.2025.11.008

Figure Lengend Snippet: Gene set enrichment analysis of the quantitative proteomic profile of hDFs cultured for 10 days under PLLA ( A ), +TGFB2 ( B ), +MMC ( C ), and +MMC + TGFB2 ( D ) conditions. All contrasts were performed against hDFs cultured for 10 days under TCP conditions. A gene set containing all protein hits in TT group vs hDFs TCP group (tendon signature) was used for all analyses. Venn diagram summarizing the different core enrichment proteins identified in PLLA, +TGFB2, +MMC and +MMC + TGFB2 groups by means of gene set enrichment analysis ( E ). GO analysis of the core enrichment proteins identified in +MMC + TGFB2 group by means of gene set enrichment analysis ( F ). (NES: normalised enrichment score). N = 3.

Article Snippet: Cryopreserved hDFs (PCS-201-012) were purchased from the American Type Culture Collection (ATCC, United Kingdom).

Techniques: Cell Culture

Journal: Bioactive Materials

Article Title: Multifactorial bioengineering in the tendon context to maintain tenocyte phenotype and to direct dermal fibroblasts towards tenogenic lineage

doi: 10.1016/j.bioactmat.2025.11.008

Figure Lengend Snippet: Heatmap displaying the quantitative matrisome of hTCs and hDFs cultured under PLLA, +TGFB2, +MMC and +MMC + TGFB2 conditions for 10 days. Hierarchical clustering was applied to all samples. The heatmap displays the z-scores for every matrisome protein identified, being these grouped into the different matrisome categories. Both hTCs and hDFs clustered first in relation to TGFB2 treatment (alone or in combination with MMC), and ultimately in relation with the experimental conditions to which these were subjected. The + MMC + TGFB2 experimental condition exerted the highest changes into the quantitative matrisome of hTCs and hDFs, increasing ECM protein levels throughout all the categories of the matrisome. N = 3.

Article Snippet: Cryopreserved hDFs (PCS-201-012) were purchased from the American Type Culture Collection (ATCC, United Kingdom).

Techniques: Cell Culture

Journal: Frontiers in Medical Technology

Article Title: Evaluation of liposome - encapsulated Centella asiatica ethanolic extract for enhanced in vitro and in vivo wound healing

doi: 10.3389/fmedt.2026.1740835

Figure Lengend Snippet: Effects of liposome-encapsulated C.asiatica extract on fibroblast viability and migration. (A) Cell viability of normal human dermal fibroblasts (NHDFs) treated with EE, blank liposome, LEC, or vitamin E at concentrations of 6.5–100 µg/mL, as determined by the MTT assay. (B) Fibroblast migration assessed by scratch-wound assay following treatment with the same formulations and concentrations. Results are expressed as relative values normalized to the untreated control (set to 100%) and presented as mean ± SD ( n = 3). Statistical analysis was performed using two-way ANOVA followed by Tukey's multiple-comparison test. Different letters above bars indicate statistically significant differences between treatment groups at the same concentration ( p < 0.05 ).

Article Snippet: Mouse macrophage cells (RAW 264.7, ATCC TIB-71) were used for anti-inflammatory assays, while normal human dermal fibroblasts (NHDF; ATCC PCS-201-012TM) were employed to model skin wound healing.

Techniques: Migration, MTT Assay, Scratch Wound Assay Assay, Control, Comparison, Concentration Assay

Journal: Frontiers in Medical Technology

Article Title: Evaluation of liposome - encapsulated Centella asiatica ethanolic extract for enhanced in vitro and in vivo wound healing

doi: 10.3389/fmedt.2026.1740835

Figure Lengend Snippet: Representative hematoxylin and eosin (H&E)–stained sections of excision wound tissues on Day 12. ( A ) Liposome-encapsulated C.asiatica extract (LEC)–treated wounds, ( B ) vitamin E–treated wounds, ( C ) blank liposome control group, and ( D ) normal saline group. Black arrows indicate re-epithelialization and restoration of epidermal continuity, yellow arrows indicate fibroblast proliferation and granulation tissue formation, and red arrows indicate neovascularization. The LEC-treated group demonstrates complete re-epithelialization, dense fibroblast proliferation, and enhanced neovascularization compared with control groups. Scale bar = 400 µM.

Article Snippet: Mouse macrophage cells (RAW 264.7, ATCC TIB-71) were used for anti-inflammatory assays, while normal human dermal fibroblasts (NHDF; ATCC PCS-201-012TM) were employed to model skin wound healing.

Techniques: Staining, Control, Saline

Journal: Frontiers in Medical Technology

Article Title: Evaluation of liposome - encapsulated Centella asiatica ethanolic extract for enhanced in vitro and in vivo wound healing

doi: 10.3389/fmedt.2026.1740835

Figure Lengend Snippet: Histological evaluation and collagen deposition in excision wounds on Day 12. ( A ) Collagen content area (%) quantified from Masson's trichrome–stained sections in wounds treated with normal saline (NS), blank liposome ( B ), liposome-encapsulated C. asiatica extract (LEC), or vitamin E (VE) ( B ) Semi-quantitative histological scores for re-epithelialization, fibroblast/granulation tissue formation, and neovascularization, assessed using a standardized ordinal scale ranging from 0 (absent) to 3 (marked) Data are presented as mean ± SD ( n = 5 rats per group). Statistical analysis for collagen area fraction was performed using one-way ANOVA followed by Tukey's multiple-comparison test, while histological scores were analyzed using the Kruskal–Wallis test followed by Dunn's multiple-comparison test. Different letters above bars indicate statistically significant differences among treatment groups within the same parameter ( p < 0.05).

Article Snippet: Mouse macrophage cells (RAW 264.7, ATCC TIB-71) were used for anti-inflammatory assays, while normal human dermal fibroblasts (NHDF; ATCC PCS-201-012TM) were employed to model skin wound healing.

Techniques: Staining, Saline, Comparison