Journal: International Journal of Molecular Sciences

Article Title: Anakinra-Loaded Sphingomyelin Nanosystems Modulate In Vitro IL-1-Dependent Pro-Tumor Inflammation in Pancreatic Cancer

doi: 10.3390/ijms25158085

Figure Lengend Snippet: Preparation of the anakinra-loaded sphingomyelin nanosystems (ANK-SNs). ( A ) Schematic representation of the single-step SN preparation by the ethanol injection method, followed by the dropwise addition of ANK solution in PBS at physiological pH. ( B ) ANK isolation from its pharmaceutical form (Kineret ® ) by using desalting columns. (Created with BioRender.com ).

Article Snippet: The next day, the medium was replaced with IMDM 2% FBS alone or with 20 ng/mL of each recombinant human IL-1α + IL-1β (R&D Systems, Minneapolis, MN, USA).

Techniques: Injection, Isolation

Journal: International Journal of Molecular Sciences

Article Title: Anakinra-Loaded Sphingomyelin Nanosystems Modulate In Vitro IL-1-Dependent Pro-Tumor Inflammation in Pancreatic Cancer

doi: 10.3390/ijms25158085

Figure Lengend Snippet: Physicochemical characterization of sphingomyelin nanosystems (SNs) (VitE/SM/NHS) and anakinra-loaded sphingomyelin nanosystems (ANK-SNs) (VitE/SM/NHS/ANK) using several analytical techniques. ( A ) Size (nm) (black columns) and surface charge (mV) (turquoise columns) of SNs and ANK-SNs, measured by dynamic light scattering (DLS) and dynamic light scattering (LDA). Data are expressed as mean ± SD (at least n = 5). ( B ) Complementary physicochemical characterization by nanoparticle tracking analysis (NTA) of SNs and ANK-SNs (n ± 5) is represented as size vs. light scattering intensity (arbitrary unit a.u.). ( C ) Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) analysis of native ANK (calculated exact mass of 17.260 kDa) and ANK-SNs (calculated mean mass ~20 kDa). ( D ) Representative field emission scanning electron microscopy (FESEM) images of SNs (left) and ANK-SNs (right). SN scale bar: 200 nm. ANK-SN scale bar: 1 μm.

Article Snippet: The next day, the medium was replaced with IMDM 2% FBS alone or with 20 ng/mL of each recombinant human IL-1α + IL-1β (R&D Systems, Minneapolis, MN, USA).

Techniques: Electron Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Anakinra-Loaded Sphingomyelin Nanosystems Modulate In Vitro IL-1-Dependent Pro-Tumor Inflammation in Pancreatic Cancer

doi: 10.3390/ijms25158085

Figure Lengend Snippet: Internalization and cytotoxicity assays of nanosystems using pancreatic ductal adenocarcinoma (PDAC) cells. ( A ) Representative confocal microscopy images after 2 h and 4 h treatment in L3.6p cells. Fluorescent-labeled TopFluor ® sphingomyelin nanosystems (SNs) and TopFluor ® anakinra-loaded sphingomyelin nanosystems (ANK-SNs) are in green. Cell nuclei stained with Hoechst 33342 are in blue. ( B ) Cell viability determined by Alamar Blue TM assay after treatment with SNs and ANK-SNs (0.1 mg/mL to 10 mg/mL) for 4 h and 24 h. Data are expressed as means ± SD. The dotted line is set at 50% of viability.

Article Snippet: The next day, the medium was replaced with IMDM 2% FBS alone or with 20 ng/mL of each recombinant human IL-1α + IL-1β (R&D Systems, Minneapolis, MN, USA).

Techniques: Confocal Microscopy, Labeling, Staining

Journal: International Journal of Molecular Sciences

Article Title: Anakinra-Loaded Sphingomyelin Nanosystems Modulate In Vitro IL-1-Dependent Pro-Tumor Inflammation in Pancreatic Cancer

doi: 10.3390/ijms25158085

Figure Lengend Snippet: Anakinra (ANK) and anakinra-loaded sphingomyelin nanosystems (ANK-SNs) are equivalent in down-modulating cytokine secretion by IL-1-activated cancer-associated fibroblasts (CAFs). ( A ) In vitro experimental model. CAFs were treated with recombinant IL-1α + IL-1β, mimicking tumor-derived IL-1, in the absence or in the presence of ANK, ANK-SNs, or SNs. Secretion of TSLP, IL-8, IL-6, and TGF-β was measured after culture. (Created with BioRender.com ). ( B ) Dose–response curve to detect the best concentrations of ANK for inhibiting TSLP secretion by CAFs (n = 3). ANK was added at the indicated concentrations. Untreated CAFs were used as negative (nt) control; IL-1α + IL-1β-treated CAFs were used as positive (IL-1α + IL-1β) control. ( C – F ) ANK, ANK-SNs, and SNs (VitE/SM/NHS formulation), tested for their capacity to down-modulate cytokine secretion, were added at the indicated concentrations and based on the titration curve shown in ( B ). Negative and positive controls used were as in B. ( C ) TSLP. Left , TSLP secretion with controls (n = 2). Right , TSLP percentage inhibition of cumulative experiments (n = 7). ( D ) IL-8. Left , IL-8 secretion with controls (n = 2). Right , IL-8 percentage inhibition of cumulative experiments (n = 6). ( E ) IL-6. Left , IL-6 secretion with controls (n = 2) . Right , IL-6 percentage inhibition of cumulative experiments (n = 6). ( F ) TGF-β. Left , TGF-β secretion with controls (n = 2). Right , TGF-β percentage inhibition of cumulative experiments (n = 3). Data are mean ± SEM from the indicated number (n) of independent experiments. Significance was calculated by one-way ANOVA test and Newman–Keuls post-test. Values were considered significantly different for * p < 0.05, ** p < 0.01, *** p < 0.001; ns = not significant.

Article Snippet: The next day, the medium was replaced with IMDM 2% FBS alone or with 20 ng/mL of each recombinant human IL-1α + IL-1β (R&D Systems, Minneapolis, MN, USA).

Techniques: In Vitro, Recombinant, Derivative Assay, Control, Formulation, Titration, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Anakinra-Loaded Sphingomyelin Nanosystems Modulate In Vitro IL-1-Dependent Pro-Tumor Inflammation in Pancreatic Cancer

doi: 10.3390/ijms25158085

Figure Lengend Snippet: Anakinra-loaded sphingomyelin nanosystems (ANK-SNs) are superior to free anakinra (ANK) in down-modulating the secretion of IL-17, but not of IFN-γ, by in vitro differentiated Th17 cells. ( A ) In vitro experimental model. Naïve CD4 + T cells were differentiated towards Th17 cells by treatment with anti-CD2/CD3/CD28-coated beads and IL-1β, IL-23, IL-6, and TGF-β, and in the absence or in the presence of ANK, ANK-SNs, or SNs. On day 5, Th17 cells were collected and restimulated with anti-CD2/CD3/CD28-coated beads, and IL-17 and IFN-γ secretion levels were measured by ELISA. (Created with BioRender.com ). ( B ) Dose–response curve to determine the best concentrations of ANK for inhibiting IL-17 secretion in Th17 cells (n = 3). ANK was added at the indicated concentrations. Th0 (i.e., naïve CD4 + T cells activated with anti-CD2/CD3/CD28-coated beads only) were used as a negative control. Th17 cells were used as a positive control. ( C , D ) ANK, ANK-SNs, and SNs (VitE/SM/NHS formulation), tested for their capacity to down-modulate cytokine secretion by Th17 cells, were added at the indicated concentrations and based on the titration curve shown in B. ( C ) IL-17. Left , IL-17 secretion with controls (n = 2). Right , IL-17 percentage inhibition of cumulative experiments (n = 11) ( D ) IFN-γ. Left , IFN-γ secretion with controls (n = 2). Right , IFN-γ percentage inhibition of cumulative experiments (n = 10). ( E ) IL-17 percentage inhibition of cumulative experiments using 5 μg/mL of ANK/ANK-SNs (n = 6). ( F ) IFN-γ percentage inhibition of cumulative experiments using 5 μg/mL of ANK/ANK-SNs (n = 4). Data are mean ± SEM from the indicated number (n) of independent experiments. Significance was calculated by one-way ANOVA test and Newman–Keuls post-test. Values were considered significantly different for * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The next day, the medium was replaced with IMDM 2% FBS alone or with 20 ng/mL of each recombinant human IL-1α + IL-1β (R&D Systems, Minneapolis, MN, USA).

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Negative Control, Positive Control, Formulation, Titration, Inhibition

Journal: bioRxiv

Article Title: Human iPSC-derived pericyte-like cells carrying APP Swedish mutation overproduce beta-amyloid and induce cerebral amyloid angiopathy-like changes

doi: 10.1101/2024.06.07.597867

Figure Lengend Snippet: (A) Representative immunocytochemistry images of day 21 iPLCs from control lines, stained for PDGFRβ, α-SMA, and NG2. Nuclei are stained with DAPI. Scale bars, 100 μm. The experiments were conducted twice for all lines. (B) Relative gene expression levels of PDGFRB, DES, LAMA2, DLC1 , and PDE7B were compared among iPLCs, iECs, iAstrocytes, and iPSCs shown as fold change to GAPDH . Statistical differences to iPLCs are indicated (iPLCs, n = 6 lines; iECs, n = 2 lines; iAstro-cytes, n = 2 lines; iPSCs, n = 3 lines, experiments were repeated three times for pericytes and two times for rest of cell types). (C) Relative gene expression levels of LAMA2, PDE7B, CD248, DES , and ACTA2 were compared across Day 7, 21, 31, and 50 iPLCs shown as fold change to GAPDH . Statistical differences to Day 7 iPLCs are indicated (pericytes, n = 4 lines (for all time points); two batches). (D) Representative images from 2D tube formation assay showcase various culture combinations: iECs alone, iPLCs alone, iECs exposed to angiogenic cocktail or iPLC CM, and iECs co-cultured with iPLCs. Images were taken 6 hours post-replating. Scale bars, 300 μm. (E) The diagram demonstrates master segments (red arrows) and mesh structures (blue polygons). (F) Statistical analysis was conducted to compare the number of master segments, meshes count, and meshes area across various culture conditions, with significance determined compared to iECs alone. The experiments were replicated using two batches, each consisting of seven control lines of iPLCs. (G) A schematic diagram illustrating the setup for measuring permeability. (H) Representative immunocytochemistry images depict iECs and iPLCs on transwell inserts, stained with CD31 and α-SMA, respectively. Nuclei are stained with DAPI. Scale bars, 100 μm. The staining was performed once for two control and two APPswe lines. (I) Permeability of fluorescently labeled dextran was evaluated across different culture conditions, with values presented as fold changes relative to iECs-only wells. “+iECs” or “control iPLCs” denotes cells cultured with iECs on the basolateral side of the inserts. Statistical differences compared to iECs only are highlighted. The experiments were replicated in two batches, involving seven control lines of iPLCs. (J-K) Western blots were performed for Occludin using samples from one batch of various culture conditions, including iECs alone and co-cultured with iECs or iPLCs on the basolateral side of inserts. GAPDH was used as a loading control, and quantification of the blots was normalized to GAPDH levels. The statistics involving seven control lines of iPLCs. The dots represent average values from batches and technical repeats of each line. Only iECs from (F, I, K) dots represent means of technical repeats within each batch. The data are presented as mean ± SD. Statistical analysis utilized one-way ANOVA with Dunnett’s multiple comparison test, with significance de-noted: *p < 0.05, **p < 0.01,***p < 0.001 and ****p < 0.0001.

Article Snippet: The medium was collected from wells pre-stimulated with 20 ng/ml TNFα and 20 ng/ml IL-1β (both from PeproTech) for 24 hours.

Techniques: Immunocytochemistry, Staining, Expressing, Tube Formation Assay, Cell Culture, Permeability, Labeling, Western Blot, Comparison

Journal: bioRxiv

Article Title: Human iPSC-derived pericyte-like cells carrying APP Swedish mutation overproduce beta-amyloid and induce cerebral amyloid angiopathy-like changes

doi: 10.1101/2024.06.07.597867

Figure Lengend Snippet: (A) Volcano plot depicting DEGs between control and APPswe iPLCs (cutoffs: Adjusted p-value <0.05 and absolute log2 fold change >1.5). The analysis included seven control and three APPswe lines. (B-C) Ingenuity Pathway Analysis to identify the top 10 downregulated (C) and upregulated (D) canonical pathways with the highest z-scores (p-value < 0.05) when comparing control pericytes to APPswe pericytes. (D) Genes related to gene risk associated with AD from GWAS, angiogenesis and pericyte contraction process. (cutoffs: Adjusted p-value <0.05 and absolute log2 fold change >1). (E) Genes involved in RHOGDI, Paxillin and Actin Cytoskeleton signaling pathways. (F) Pathview pathway analysis to identify the top 10 affected pathways with the highest enrichment score when comparing APPswe pericytes to control pericytes. (G) Gene ontology (GO) enrichment analysis revealed pathways enriched in Biological Process, Molecular Function, and Cellular Component in APPswe pericyte-like cells relative to controls.

Article Snippet: The medium was collected from wells pre-stimulated with 20 ng/ml TNFα and 20 ng/ml IL-1β (both from PeproTech) for 24 hours.

Techniques:

Journal: bioRxiv

Article Title: Human iPSC-derived pericyte-like cells carrying APP Swedish mutation overproduce beta-amyloid and induce cerebral amyloid angiopathy-like changes

doi: 10.1101/2024.06.07.597867

Figure Lengend Snippet: (A) IL-6, IL-8, MCP-1, RANTES, and VCAM-1 concentrations were measured in iPLCs culture media after 24 hours of TNFα and IL-1β stimulation. Results were normalized to total protein content. Statistical differences between treatment and genotype were indicated. The experiments were replicated with two batches, involving seven control and three APPswe lines. (B-C) 2D tube formation assays involved culturing iECs alongside both control and APPswe iPLCs (B), scale bars, 300 μm. Statistical comparisons were made regarding the number of master segments, number of meshes, and mesh area between co-cultures of control and APPswe iPLCs with iECs (C), highlighting significant differences compared to controls. This analysis was conducted across two experimental batches, including seven control and three APPswe lines. (D) VEGFA levels in lysates from control and APPswe iPLCs were measured and normalized to total protein content. Statistical differences relative to controls are indicated. This analysis included two experimental batches, with six control and three APPswe lines. (E) Permeability to fluorescently labeled dextran was assessed in cultures of iECs alone, iECs co-cultured with iECs, control or APPswe iPLCs on the basolateral side of inserts, using 4 and 70 kDa fluorophore-conjugated dextran. Statistical differences relative to iECs co-cultured with control iPLCs are noted. This evaluation was repeated across two experimental batches, including seven control and three APPswe lines. (F) Representative blots of Occludin from control and APPswe iPLCs used GAPDH as the loading control, with quantification normalized to GAPDH levels. The analysis including two experimental batches with seven control and three APPswe lines. The dots represent the average values obtained from batches and technical repeats of each line. The data are presented as mean ± SD. Statistical analysis was performed using two-way ANOVA with Bonferroni multiple comparison test (A, F and G) or t-test (C, D). The significance levels are denoted: *p < 0.05, **p < 0.01,***p < 0.001 and ****p < 0.0001.

Article Snippet: The medium was collected from wells pre-stimulated with 20 ng/ml TNFα and 20 ng/ml IL-1β (both from PeproTech) for 24 hours.

Techniques: Permeability, Labeling, Cell Culture, Comparison

Journal: bioRxiv

Article Title: Human iPSC-derived pericyte-like cells carrying APP Swedish mutation overproduce beta-amyloid and induce cerebral amyloid angiopathy-like changes

doi: 10.1101/2024.06.07.597867

Figure Lengend Snippet: (A-B) Electrical impedance measurements were employed to evaluate the contractile response of iPLCs to various concentrations of ET-1 and ATP. This analysis was conducted in two experimental batches using two control lines. Response curves were normalized to vehicle control, producing a cell index that depicted the response (A), while the response slope, indicating contraction speed post treatment, was also presented (B). (C) The cell index of the contractile response of control and APPswe iPLCs to ET-1 treatment, across three experimental batches involving five control and three APPswe lines. (D) Statistical analysis compared contraction strength and recovery time to normal size, with significance indicated compared to the controls. The experiments were replicated with three batches, involving five control and three APPswe lines. (E) Gene expression levels of EDNRA and EDNRB were compared between control and APPswe iPLCs, quantified as fold changes relative to GAPDH. The experiments were replicated with two batches, involving six control and three APPswe lines. (F-G) Blots for phosphorylated (p)-Erk and total (t)-Erk from control and APPswe iPLCs were conducted at time intervals of 5, 10, 15, 30, 45, and 60 minutes post-ET-1 treatment, as well as for non-treated (NT) cells, using GAPDH as the loading control (F). Quantification was normalized to GAPDH levels, with each time point further normalized to the NT well of the control line (G). This analysis was carried out one experimental batch, involving one control and one APPswe line. (H-I) Blots for p-Erk and t-Erk from control and APPswe iPLCs were obtained 10 minutes after ET-1 treatment and from NT cells, with GAPDH serving as the loading control (H). Blot quantification was normalized to GAPDH levels, and treatment groups were further normalized to the NT wells of their respective lines (G). This analysis was performed in a single experimental batch, including seven control and three APPswe lines. The dots represent the average values obtained from batches and technical repeats of each line. The data are presented as mean ± SD. Statistical analysis was performed using two-way ANOVA with Bonferroni multiple comparison test (A, F and G) or t-test (C, D). The significance levels are denoted: *p < 0.05, **p < 0.01,***p < 0.001 and ****p < 0.0001.

Article Snippet: The medium was collected from wells pre-stimulated with 20 ng/ml TNFα and 20 ng/ml IL-1β (both from PeproTech) for 24 hours.

Techniques: Expressing, Comparison

Journal: Frontiers in Immunology

Article Title: Skeletal muscle fibers produce B-cell stimulatory factors in chronic myositis

doi: 10.3389/fimmu.2023.1177721

Figure Lengend Snippet: mRNA expression of BAFF and CXCL-12 in human myotubes under pro-inflammatory conditions by quantitative (real-time)-PCR after 24 hours of stimulation: (A) Significant induction of BAFF mRNA expression upon IFN-γ (alone or in combination) and of (B) CXCL-12 upon IL-1β (alone or in combination) in human primary myotube cultures. The results are averages of two independent experiments and the myotubes were obtained from non-myopathic donors. BAFF, B-cell-activating factor of the tumor necrosis family; CXCL-12, CXC-chemokine ligand 12. IFN-γ, Interferon γ; IL-1β, Interleukin 1β; TNFα, Tumor necrosis factor α. Data shown as mean +SD. Statistics by ANOVA with Tukey's multiple comparison test, indicating significance by *P< 0.05; **P< 0.01; ***P< 0.001.

Article Snippet: Well-differentiated myotubes, as revealed by immunocytochemical staining for the muscle marker desmin, were either kept as unstimulated controls in X-Vivo 15 medium (Cambrex Bioscience) and exposed to the cytokines IFN-γ (300 units/ml), TNFα (10 ng/ml), and IL-1β (20 ng/ml) (all from Chemicon International Inc., Temecula, USA) in serum-free X-Vivo 15 medium.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Comparison

Journal: Frontiers in Immunology

Article Title: Skeletal muscle fibers produce B-cell stimulatory factors in chronic myositis

doi: 10.3389/fimmu.2023.1177721

Figure Lengend Snippet: Analysis of BAFF and CXCL-12 expression on protein levels in human myotubes under pro-inflammatory conditions by immunocytochemistry: (A) Exemplary finding of BAFF expression in human myotubes after 48 hours exposure to IFN-γ compared to controls (microphotographs acquired using a 20x objective). Greyscale analysis revealed a tendency of an increased protein production, which did not reach statistical significance (t-test). (B) Exemplary finding of CXCL-12 expression in human myotubes after 48 hours exposure to IL-1β compared to controls (microphotographs acquired using a 20x objective). Greyscale analysis revealed a tendency of an increased protein production, which did not reach statistical significance (t-test). The myotubes were obtained from non-myopathic donors. The results are shown as mean +SD from two independent experiments. BAFF, B-cell-activating factor of the tumor necrosis family; CXCL-12, CXC-chemokine ligand 12. IFN-γ, Interferon γ; IL-1β, Interleukin 1β.

Article Snippet: Well-differentiated myotubes, as revealed by immunocytochemical staining for the muscle marker desmin, were either kept as unstimulated controls in X-Vivo 15 medium (Cambrex Bioscience) and exposed to the cytokines IFN-γ (300 units/ml), TNFα (10 ng/ml), and IL-1β (20 ng/ml) (all from Chemicon International Inc., Temecula, USA) in serum-free X-Vivo 15 medium.

Techniques: Expressing, Immunocytochemistry

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Effect of pro-inflammatory cytokine priming and storage temperature of the mesenchymal stromal cell (MSC) secretome on equine articular chondrocytes

doi: 10.3389/fbioe.2023.1204737

Figure Lengend Snippet: Storage time and temperature affect the CM pro-proliferative effect. CMs from BM-MSCs (P3) were harvested, filtered, aliquoted and stored at different temperatures for 2 days (n = 3), 11 days (n = 3) and 1 month (n = 2). eACs (P2) were seeded in monolayer at 20,000 cells/cm 2 . After 17 h, the culture medium was removed, cells were washed with PBS and treated with CMs. Cultures were monitored for 48 h with an Incucyte ® live imaging system. Each condition was tested in triplicate and experiments were repeated 2 or 3 times. Cells were counted on 3 representative areas of each picture taken after 48 h of treatment and just after addition of CM. Relative cell confluence was determined from the ratio of the number of cells at 48 h to the number of cells at 0 h. Histograms show the impact of CM storage temperature—−80°C; −20°C; 4°C; 20°C; 37°C–over all experiments, independently of the storage time (up to 1 month) (A) . Impact of each storage time on eAC proliferation rate was also analyzed (B) . Mean values are represented as histograms ± standard deviation and analyzed using the Mann-Whitney test; * p < 0.05, ** p < 0.01, *** p < 0.005 significantly different from the control M3D condition. 10% FBS: chondrocyte amplification medium; used as positive control; A.U.: arbitrary unit; BM-MSCs: bone marrow-mesenchymal stromal cells; CM: conditioned medium; eACs: equine articular chondrocytes; FBS: fetal bovine serum; M3D: control medium with 2% FBS; P2: passage 2; P3: passage 3.

Article Snippet: For priming experiments, equine IL-1β (10 and 20 ng/mL), TNF-α (10 and 20 ng/mL) or IFN-γ (50 and 100 ng/mL) (R&D Systems, Bio-Techne; Minneapolis, Minnesota, United States) were added to the preconditioning medium for 24 h. Preconditioned medium with no cytokine supplementation was also used to obtain naive CM.

Techniques: Imaging, Standard Deviation, MANN-WHITNEY, Amplification, Positive Control

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Effect of pro-inflammatory cytokine priming and storage temperature of the mesenchymal stromal cell (MSC) secretome on equine articular chondrocytes

doi: 10.3389/fbioe.2023.1204737

Figure Lengend Snippet: Priming with pro-inflammatory cytokines does not impact the eAC proliferation rate. BM-MSCs (P3) were incubated with IL-1β (20 ng/mL), TNF-α (10 ng/mL) or IFN-γ (100 ng/mL) for 24 h. Then, cells were washed twice with PBS and M3D was added to cultures for 24 or 48 h. CMs from BM-MSCs were harvested, filtered, aliquoted and stored at −80°C. In parallel, eACs (P2) were seeded in monolayer at 20,000 cells/cm 2 in a 96-well plate. After 16 h, the medium was removed, cells were washed with PBS and treated with CMs. Cultures were monitored for 48 h using an Incucyte ® live imaging system. Each condition was tested in triplicate, and cells were counted on 3 representative areas of each image taken just after CM addition and after 48 h of treatment. Relative cell confluence was determined from the ratio of the (number of cells at 48 h to the number of cells at 0 h. Histograms show the impact of cytokine priming on the eAC proliferation rate over all experiments (n = 5). Mean values are shown as histograms ± standard deviation, and were analyzed using the Mann-Whitney test to compare the difference with the M3D condition. BM-MSCs: bone marrow-mesenchymal stromal cells; A.U.: arbitrary unit; BMP2: bone morphogenetic protein 2; CM: conditioned medium; eACs: equine articular chondrocytes; FBS: fetal bovine serum; M3D: control medium with 2% FBS; P2: passage 2; P3: passage 3.

Article Snippet: For priming experiments, equine IL-1β (10 and 20 ng/mL), TNF-α (10 and 20 ng/mL) or IFN-γ (50 and 100 ng/mL) (R&D Systems, Bio-Techne; Minneapolis, Minnesota, United States) were added to the preconditioning medium for 24 h. Preconditioned medium with no cytokine supplementation was also used to obtain naive CM.

Techniques: Incubation, Imaging, Standard Deviation, MANN-WHITNEY

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Effect of pro-inflammatory cytokine priming and storage temperature of the mesenchymal stromal cell (MSC) secretome on equine articular chondrocytes

doi: 10.3389/fbioe.2023.1204737

Figure Lengend Snippet: The effects of CM on gene expression can depend on storage duration. CMs from BM-MSCs were harvested, filtered, aliquoted and stored at different temperatures for 2 days, 11 days or 1 month. eACs (P2) were seeded in collagen sponges at 800,000 cells/sponge and then cultured in hypoxia. After 17 h, eACs were treated with CMs for 14 days. Then, sponges were harvested, washed twice with PBS and stored at −80°C. Total RNA was extracted and RT-qPCRs were carried out to assess gene expression. The expression of target genes was normalized using the reference genes β-ACTIN and PPIA . Experiments were repeated for each storage time with different strains of eACs and BM-MSCs (n = 4). Values are shown as box plots (median, quartiles, extreme values and mean (indicated with a “+”) and were analyzed using the Mann-Whitney test, * p < 0.05 significantly different from the M3D condition or the 2-day storage time for each temperature group (# p < 0.05). BM-MSCs: bone marrow-mesenchymal stromal cells; CM: conditioned medium; eACs: equine articular chondrocytes; M3D: control medium with 2% FBS; P2: passage 2; RT: room temperature; RT-qPCR: reverse transcription-quantitative polymerase chain reaction.

Article Snippet: For priming experiments, equine IL-1β (10 and 20 ng/mL), TNF-α (10 and 20 ng/mL) or IFN-γ (50 and 100 ng/mL) (R&D Systems, Bio-Techne; Minneapolis, Minnesota, United States) were added to the preconditioning medium for 24 h. Preconditioned medium with no cytokine supplementation was also used to obtain naive CM.

Techniques: Expressing, Cell Culture, MANN-WHITNEY, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Effect of pro-inflammatory cytokine priming and storage temperature of the mesenchymal stromal cell (MSC) secretome on equine articular chondrocytes

doi: 10.3389/fbioe.2023.1204737

Figure Lengend Snippet: Effect of cytokine priming on the gene expression of immunomodulatory markers. At 50% confluency, MSC amplification medium with or without IL-1β (10 and 20 ng/mL), TNF-α (10 and 20 ng/mL) or IFN-γ (50 and 100 ng/mL) were added to BM-MSC cultures (P3) for 6 or 24 h (n = 4). After two washes with PBS, media were replaced with M3D for 24 h. Then, media were removed, MSCs were washed twice again with PBS and stored at −80°C until RNA extraction. The gene expression of several immunomodulatory markers was assessed using RT-qPCRs, and normalized using the reference genes β-ACTIN and PPIA . Values are shown as box plots (median, quartiles, extreme values and mean (indicated with a “+”) and were analyzed using the Mann-Whitney test, * p < 0.05, significantly different from the M3D condition within each cytokine priming group. BM-MSCs: bone marrow-mesenchymal stromal cells; M3D: unconditioned medium; CM: conditioned medium; eACs: equine articular chondrocytes; M3D: control medium with 2% FBS; P3: passage 3.

Article Snippet: For priming experiments, equine IL-1β (10 and 20 ng/mL), TNF-α (10 and 20 ng/mL) or IFN-γ (50 and 100 ng/mL) (R&D Systems, Bio-Techne; Minneapolis, Minnesota, United States) were added to the preconditioning medium for 24 h. Preconditioned medium with no cytokine supplementation was also used to obtain naive CM.

Techniques: Expressing, Amplification, RNA Extraction, MANN-WHITNEY

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Effect of pro-inflammatory cytokine priming and storage temperature of the mesenchymal stromal cell (MSC) secretome on equine articular chondrocytes

doi: 10.3389/fbioe.2023.1204737

Figure Lengend Snippet: Priming of BM-MSCs with cytokines leads to an increase in pro-inflammatory molecule concentration in the CM. At 50% confluency, MSC amplification medium only or containing either IL-1β (20 ng/mL), TNF-α (10 ng/mL) or IFN-γ (100 ng/mL) was added to BM-MSC cultures (P3) for 24 h. Media were then removed, cells were washed twice with PBS and M3D was added. After 24 h, media were collected and stored at −80°C. Dosages of cytokines were performed using the MILLIPLEX ® Equine Cytokine/Chemokine Magnetic Bead assay (Merck-Millipore) according to the manufacturer’s recommendations. Measurements were performed in duplicate with the Luminex ® MAGPIX ® CCD imager and processed with the Luminex ® xPONENT software (n = 4). Values are shown as box plots (median, quartiles, extreme values and mean (indicated with a “+”) and were analyzed using the Mann-Whitney test, * p < 0.05 significantly different from the unprimed CM3D condition. eACs: equine articular chondrocytes; BM-MSCs: bone marrow-mesenchymal stromal cells; CM3D: (unprimed) conditioned medium.

Article Snippet: For priming experiments, equine IL-1β (10 and 20 ng/mL), TNF-α (10 and 20 ng/mL) or IFN-γ (50 and 100 ng/mL) (R&D Systems, Bio-Techne; Minneapolis, Minnesota, United States) were added to the preconditioning medium for 24 h. Preconditioned medium with no cytokine supplementation was also used to obtain naive CM.

Techniques: Concentration Assay, Amplification, Luminex, Software, MANN-WHITNEY

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Effect of pro-inflammatory cytokine priming and storage temperature of the mesenchymal stromal cell (MSC) secretome on equine articular chondrocytes

doi: 10.3389/fbioe.2023.1204737

Figure Lengend Snippet: Primed CMs differentially modulate the gene expression of cartilage and OA-associated molecules in eACs. BM-MSCs (P3) were incubated with IL-1β (20 ng/mL), TNF-α (10 ng/mL) or IFN-γ (100 ng/mL) for 24 h. Then, cells were rinsed twice with PBS and M3D was added to cultures for 24 or 48 h. CMs from BM-MSCs were harvested, filtered, aliquoted and stored at −80°C. In parallel, eACs (P2) were seeded in collagen sponges at 800,000 cells/sponge and, after 17 h, were treated with CMs for 14 days. Then, sponges were harvested, washed twice with PBS and stored at −80°C. Total RNA was collected from these cultures and RT-qPCRs were carried out to assess gene expression. The expression of target genes was normalized using the reference genes β-ACTIN and PPIA . The D0 condition corresponds to eACs cultured in monolayer until P2. Experiments were repeated with different strains of eACs and BM-MSCs (n = 5). Values are represented as box plots (median, quartiles, extreme values and mean (indicated with a "+") and were analyzed using the Kruskal-Wallis and the Dunn’s tests. * p < 0.05, ** p < 0.01, *** p < 0.005, significantly different from the M3D condition. # p < 0.05, ## p < 0.01, ### p < 0.005, significantly different from the D0 condition. $ p < 0.05, $$ p < 0.01, $$$ p < 0.005, significantly different from the M3D + B condition Note: BM-MSCs: bone marrow-mesenchymal stromal cells; BMP2: Bone morphogenetic protein 2; CM: conditioned medium; eACs: equine articular chondrocytes; M3D: control medium with 2% FBS; P2: passage 2; P3: passage 3; RT-qPCR: reverse transcription-quantitative polymerase chain reaction.

Article Snippet: For priming experiments, equine IL-1β (10 and 20 ng/mL), TNF-α (10 and 20 ng/mL) or IFN-γ (50 and 100 ng/mL) (R&D Systems, Bio-Techne; Minneapolis, Minnesota, United States) were added to the preconditioning medium for 24 h. Preconditioned medium with no cytokine supplementation was also used to obtain naive CM.

Techniques: Expressing, Incubation, Cell Culture, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Effect of pro-inflammatory cytokine priming and storage temperature of the mesenchymal stromal cell (MSC) secretome on equine articular chondrocytes

doi: 10.3389/fbioe.2023.1204737

Figure Lengend Snippet: Priming of BM-MSCs with pro-inflammatory cytokines modulates protein accumulation of cartilage biomarkers in eAC cultures. BM-MSCs (P3) were incubated with IL-1β (20 ng/mL), TNF-α (10 ng/mL) or IFN-γ (100 ng/mL) for 24 h. Then, cells were rinsed twice with PBS and M3D was added to cultures for 24 (A) or 48 h (B) . CMs from BM-MSCs were harvested, filtered, aliquoted and stored at −80°C. In parallel, eACs (P2) were seeded in collagen sponges at 800,000 cells/sponge and, after 17 h, were treated with CMs for 14 days. Then, sponges were harvested, washed twice with PBS and stored at −80°C. Total proteins were extracted using a dedicated buffer supplemented with protease inhibitors and western blots were run to evaluate protein levels, as shown in the representative images. The D0 condition corresponds to eACs cultured in monolayer until P2. Experiments were repeated with different strains of eACs and BM-MSCs (n = 5). The densitometry of each band was measured thanks to the Image Lab software (Biorad). The relative values (relative to GAPDH) are indicated below each blot. BM-MSCs: bone marrow-mesenchymal stromal cells; CM: conditioned medium; eACs: equine articular chondrocytes; M3D: control medium with 2% FBS; P2: passage 2; P3: passage 3.

Article Snippet: For priming experiments, equine IL-1β (10 and 20 ng/mL), TNF-α (10 and 20 ng/mL) or IFN-γ (50 and 100 ng/mL) (R&D Systems, Bio-Techne; Minneapolis, Minnesota, United States) were added to the preconditioning medium for 24 h. Preconditioned medium with no cytokine supplementation was also used to obtain naive CM.

Techniques: Incubation, Western Blot, Cell Culture, Software

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Effect of pro-inflammatory cytokine priming and storage temperature of the mesenchymal stromal cell (MSC) secretome on equine articular chondrocytes

doi: 10.3389/fbioe.2023.1204737

Figure Lengend Snippet: Priming of BM-MSCs with pro-inflammatory cytokines modulates protein accumulation of cartilage biomarkers in eAC cultures, in the presence of BMP2. BM-MSCs (P3) were incubated with IL-1β (20 ng/mL), TNF-α (10 ng/mL) or IFN-γ (100 ng/mL) for 24 h. Then, cells were rinsed twice with PBS and M3D was added to cultures for 24 (A) or 48 h (B) . CMs from BM-MSCs were harvested, filtered, aliquoted and stored at −80°C. In parallel, eACs (P2) were seeded in collagen sponges at 800,000 cells/sponge and, after 17 h, were treated with CMs with or without BMP2 (50 ng/mL) for 14 days. Then, sponges were harvested, washed twice with PBS and stored at −80°C. Total proteins were extracted using a dedicated buffer supplemented with protease inhibitors and western blots were run to evaluate protein levels, as shown in the representative images. The D0 condition corresponds to eACs cultured in monolayer until P2. Experiments were repeated with different strains of eACs and BM-MSCs (n = 3). The densitometry of each band was measured thanks to the Image Lab software (Biorad). The relative values (relative to GAPDH) are indicated below each blot. BM-MSCs: bone marrow-mesenchymal stromal cells; CM: conditioned medium; eACs: equine articular chondrocytes; M3D: control medium with 2% FBS; P2: passage 2; P3: passage 3.

Article Snippet: For priming experiments, equine IL-1β (10 and 20 ng/mL), TNF-α (10 and 20 ng/mL) or IFN-γ (50 and 100 ng/mL) (R&D Systems, Bio-Techne; Minneapolis, Minnesota, United States) were added to the preconditioning medium for 24 h. Preconditioned medium with no cytokine supplementation was also used to obtain naive CM.

Techniques: Incubation, Western Blot, Cell Culture, Software