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Expected cellular responses of human articular chondrocytes under dynamic culture and IL‑1β‐induced inflammatory conditions. Representative results illustrate chondrocyte morphology, viability, extracellular matrix (ECM) expression, metabolic activity, and inflammatory responses under gravity‐driven flow conditions. (A) Representative z‐projection fluorescence images of chondrocytes cultured for 21 days in microchannels coated with a fibronectin–collagen type IV mixture. Cells form aggregates and microtissue‐like structures across regions I–III corresponding to increasing fluid shear stress levels. Cells are stained for F‐actin (red) and nuclei (DAPI, blue). (B) Representative Live/Dead fluorescence images showing time‐dependent viability and proliferation of chondrocytes cultured under dynamic conditions on days 3, 7, 14, and 21 (control), compared with IL‑1β‐stimulated cultures on day 21 (1 and 10 ng/ml, applied from day 15 to day 21). Control cultures show progressive formation of compact microtissue‐like aggregates (white arrows), whereas IL‑1β stimulation results in disrupted aggregates (yellow arrows). Green: calcein‐AM (live cells); red: ethidium homodimer‐1 (dead cells). (C) Representative z‐projection immunofluorescence images of ECM marker expression in region I after 21 days of culture, comparing control and IL‑1β‐stimulated samples. Images show merged fluorescence channels for collagen type II (green), aggrecan (red), and nuclei (DAPI, blue). (D–E) Quantification of corrected total cell fluorescence (CTCF) for collagen type II (D) and aggrecan (E) across regions I–III for control and IL‑1β‐stimulated cultures at day 21 (n = 44 cells per group). (F) Cell metabolic activity measured using a PrestoBlue assay for control and IL‑1β‐stimulated samples at day 21 ( n = 5 independent samples per group). (G–H) Quantification of inflammatory mediators in culture supernatant collected at day 21 using multiplex ELISA, including matrix metalloproteinases (MMP‐1, MMP‐2, MMP‐3, MMP‐7, MMP‐9, MMP‐13) and cytokines (IL‑1β, IL‑6, IL‑8) ( n = 3 independent samples per group). Values below the lower limit of quantification are indicated as <LLOQ. (I) Quantification of collagen type II fragments released into the culture medium at day 21, measured by ELISA ( n = 3 independent samples per group). All fluorescence images include a 100 µm scale bar, and white dashed lines indicate microchannel boundaries. Statistical analysis was performed using the Kruskal–Wallis test with Dunn's post hoc comparison (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n.s., not significant). Conditions include control (no <t>cytokine)</t> and IL‑1β stimulation (1 and 10 ng/ml). Note: Panels are adapted from da Silva et al. , licensed under CC BY 3.0.
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Expected cellular responses of human articular chondrocytes under dynamic culture and IL‑1β‐induced inflammatory conditions. Representative results illustrate chondrocyte morphology, viability, extracellular matrix (ECM) expression, metabolic activity, and inflammatory responses under gravity‐driven flow conditions. (A) Representative z‐projection fluorescence images of chondrocytes cultured for 21 days in microchannels coated with a fibronectin–collagen type IV mixture. Cells form aggregates and microtissue‐like structures across regions I–III corresponding to increasing fluid shear stress levels. Cells are stained for F‐actin (red) and nuclei (DAPI, blue). (B) Representative Live/Dead fluorescence images showing time‐dependent viability and proliferation of chondrocytes cultured under dynamic conditions on days 3, 7, 14, and 21 (control), compared with IL‑1β‐stimulated cultures on day 21 (1 and 10 ng/ml, applied from day 15 to day 21). Control cultures show progressive formation of compact microtissue‐like aggregates (white arrows), whereas IL‑1β stimulation results in disrupted aggregates (yellow arrows). Green: calcein‐AM (live cells); red: ethidium homodimer‐1 (dead cells). (C) Representative z‐projection immunofluorescence images of ECM marker expression in region I after 21 days of culture, comparing control and IL‑1β‐stimulated samples. Images show merged fluorescence channels for collagen type II (green), aggrecan (red), and nuclei (DAPI, blue). (D–E) Quantification of corrected total cell fluorescence (CTCF) for collagen type II (D) and aggrecan (E) across regions I–III for control and IL‑1β‐stimulated cultures at day 21 (n = 44 cells per group). (F) Cell metabolic activity measured using a PrestoBlue assay for control and IL‑1β‐stimulated samples at day 21 ( n = 5 independent samples per group). (G–H) Quantification of inflammatory mediators in culture supernatant collected at day 21 using multiplex ELISA, including matrix metalloproteinases (MMP‐1, MMP‐2, MMP‐3, MMP‐7, MMP‐9, MMP‐13) and cytokines (IL‑1β, IL‑6, IL‑8) ( n = 3 independent samples per group). Values below the lower limit of quantification are indicated as <LLOQ. (I) Quantification of collagen type II fragments released into the culture medium at day 21, measured by ELISA ( n = 3 independent samples per group). All fluorescence images include a 100 µm scale bar, and white dashed lines indicate microchannel boundaries. Statistical analysis was performed using the Kruskal–Wallis test with Dunn's post hoc comparison (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n.s., not significant). Conditions include control (no <t>cytokine)</t> and IL‑1β stimulation (1 and 10 ng/ml). Note: Panels are adapted from da Silva et al. , licensed under CC BY 3.0.
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Expected cellular responses of human articular chondrocytes under dynamic culture and IL‑1β‐induced inflammatory conditions. Representative results illustrate chondrocyte morphology, viability, extracellular matrix (ECM) expression, metabolic activity, and inflammatory responses under gravity‐driven flow conditions. (A) Representative z‐projection fluorescence images of chondrocytes cultured for 21 days in microchannels coated with a fibronectin–collagen type IV mixture. Cells form aggregates and microtissue‐like structures across regions I–III corresponding to increasing fluid shear stress levels. Cells are stained for F‐actin (red) and nuclei (DAPI, blue). (B) Representative Live/Dead fluorescence images showing time‐dependent viability and proliferation of chondrocytes cultured under dynamic conditions on days 3, 7, 14, and 21 (control), compared with IL‑1β‐stimulated cultures on day 21 (1 and 10 ng/ml, applied from day 15 to day 21). Control cultures show progressive formation of compact microtissue‐like aggregates (white arrows), whereas IL‑1β stimulation results in disrupted aggregates (yellow arrows). Green: calcein‐AM (live cells); red: ethidium homodimer‐1 (dead cells). (C) Representative z‐projection immunofluorescence images of ECM marker expression in region I after 21 days of culture, comparing control and IL‑1β‐stimulated samples. Images show merged fluorescence channels for collagen type II (green), aggrecan (red), and nuclei (DAPI, blue). (D–E) Quantification of corrected total cell fluorescence (CTCF) for collagen type II (D) and aggrecan (E) across regions I–III for control and IL‑1β‐stimulated cultures at day 21 (n = 44 cells per group). (F) Cell metabolic activity measured using a PrestoBlue assay for control and IL‑1β‐stimulated samples at day 21 ( n = 5 independent samples per group). (G–H) Quantification of inflammatory mediators in culture supernatant collected at day 21 using multiplex ELISA, including matrix metalloproteinases (MMP‐1, MMP‐2, MMP‐3, MMP‐7, MMP‐9, MMP‐13) and cytokines (IL‑1β, IL‑6, IL‑8) ( n = 3 independent samples per group). Values below the lower limit of quantification are indicated as <LLOQ. (I) Quantification of collagen type II fragments released into the culture medium at day 21, measured by ELISA ( n = 3 independent samples per group). All fluorescence images include a 100 µm scale bar, and white dashed lines indicate microchannel boundaries. Statistical analysis was performed using the Kruskal–Wallis test with Dunn's post hoc comparison (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n.s., not significant). Conditions include control (no <t>cytokine)</t> and IL‑1β stimulation (1 and 10 ng/ml). Note: Panels are adapted from da Silva et al. , licensed under CC BY 3.0.
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Expected cellular responses of human articular chondrocytes under dynamic culture and IL‑1β‐induced inflammatory conditions. Representative results illustrate chondrocyte morphology, viability, extracellular matrix (ECM) expression, metabolic activity, and inflammatory responses under gravity‐driven flow conditions. (A) Representative z‐projection fluorescence images of chondrocytes cultured for 21 days in microchannels coated with a fibronectin–collagen type IV mixture. Cells form aggregates and microtissue‐like structures across regions I–III corresponding to increasing fluid shear stress levels. Cells are stained for F‐actin (red) and nuclei (DAPI, blue). (B) Representative Live/Dead fluorescence images showing time‐dependent viability and proliferation of chondrocytes cultured under dynamic conditions on days 3, 7, 14, and 21 (control), compared with IL‑1β‐stimulated cultures on day 21 (1 and 10 ng/ml, applied from day 15 to day 21). Control cultures show progressive formation of compact microtissue‐like aggregates (white arrows), whereas IL‑1β stimulation results in disrupted aggregates (yellow arrows). Green: calcein‐AM (live cells); red: ethidium homodimer‐1 (dead cells). (C) Representative z‐projection immunofluorescence images of ECM marker expression in region I after 21 days of culture, comparing control and IL‑1β‐stimulated samples. Images show merged fluorescence channels for collagen type II (green), aggrecan (red), and nuclei (DAPI, blue). (D–E) Quantification of corrected total cell fluorescence (CTCF) for collagen type II (D) and aggrecan (E) across regions I–III for control and IL‑1β‐stimulated cultures at day 21 (n = 44 cells per group). (F) Cell metabolic activity measured using a PrestoBlue assay for control and IL‑1β‐stimulated samples at day 21 ( n = 5 independent samples per group). (G–H) Quantification of inflammatory mediators in culture supernatant collected at day 21 using multiplex ELISA, including matrix metalloproteinases (MMP‐1, MMP‐2, MMP‐3, MMP‐7, MMP‐9, MMP‐13) and cytokines (IL‑1β, IL‑6, IL‑8) ( n = 3 independent samples per group). Values below the lower limit of quantification are indicated as <LLOQ. (I) Quantification of collagen type II fragments released into the culture medium at day 21, measured by ELISA ( n = 3 independent samples per group). All fluorescence images include a 100 µm scale bar, and white dashed lines indicate microchannel boundaries. Statistical analysis was performed using the Kruskal–Wallis test with Dunn's post hoc comparison (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n.s., not significant). Conditions include control (no <t>cytokine)</t> and IL‑1β stimulation (1 and 10 ng/ml). Note: Panels are adapted from da Silva et al. , licensed under CC BY 3.0.
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Expected cellular responses of human articular chondrocytes under dynamic culture and IL‑1β‐induced inflammatory conditions. Representative results illustrate chondrocyte morphology, viability, extracellular matrix (ECM) expression, metabolic activity, and inflammatory responses under gravity‐driven flow conditions. (A) Representative z‐projection fluorescence images of chondrocytes cultured for 21 days in microchannels coated with a fibronectin–collagen type IV mixture. Cells form aggregates and microtissue‐like structures across regions I–III corresponding to increasing fluid shear stress levels. Cells are stained for F‐actin (red) and nuclei (DAPI, blue). (B) Representative Live/Dead fluorescence images showing time‐dependent viability and proliferation of chondrocytes cultured under dynamic conditions on days 3, 7, 14, and 21 (control), compared with IL‑1β‐stimulated cultures on day 21 (1 and 10 ng/ml, applied from day 15 to day 21). Control cultures show progressive formation of compact microtissue‐like aggregates (white arrows), whereas IL‑1β stimulation results in disrupted aggregates (yellow arrows). Green: calcein‐AM (live cells); red: ethidium homodimer‐1 (dead cells). (C) Representative z‐projection immunofluorescence images of ECM marker expression in region I after 21 days of culture, comparing control and IL‑1β‐stimulated samples. Images show merged fluorescence channels for collagen type II (green), aggrecan (red), and nuclei (DAPI, blue). (D–E) Quantification of corrected total cell fluorescence (CTCF) for collagen type II (D) and aggrecan (E) across regions I–III for control and IL‑1β‐stimulated cultures at day 21 (n = 44 cells per group). (F) Cell metabolic activity measured using a PrestoBlue assay for control and IL‑1β‐stimulated samples at day 21 ( n = 5 independent samples per group). (G–H) Quantification of inflammatory mediators in culture supernatant collected at day 21 using multiplex ELISA, including matrix metalloproteinases (MMP‐1, MMP‐2, MMP‐3, MMP‐7, MMP‐9, MMP‐13) and cytokines (IL‑1β, IL‑6, IL‑8) ( n = 3 independent samples per group). Values below the lower limit of quantification are indicated as <LLOQ. (I) Quantification of collagen type II fragments released into the culture medium at day 21, measured by ELISA ( n = 3 independent samples per group). All fluorescence images include a 100 µm scale bar, and white dashed lines indicate microchannel boundaries. Statistical analysis was performed using the Kruskal–Wallis test with Dunn's post hoc comparison (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n.s., not significant). Conditions include control (no <t>cytokine)</t> and IL‑1β stimulation (1 and 10 ng/ml). Note: Panels are adapted from da Silva et al. , licensed under CC BY 3.0.
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Expected cellular responses of human articular chondrocytes under dynamic culture and IL‑1β‐induced inflammatory conditions. Representative results illustrate chondrocyte morphology, viability, extracellular matrix (ECM) expression, metabolic activity, and inflammatory responses under gravity‐driven flow conditions. (A) Representative z‐projection fluorescence images of chondrocytes cultured for 21 days in microchannels coated with a fibronectin–collagen type IV mixture. Cells form aggregates and microtissue‐like structures across regions I–III corresponding to increasing fluid shear stress levels. Cells are stained for F‐actin (red) and nuclei (DAPI, blue). (B) Representative Live/Dead fluorescence images showing time‐dependent viability and proliferation of chondrocytes cultured under dynamic conditions on days 3, 7, 14, and 21 (control), compared with IL‑1β‐stimulated cultures on day 21 (1 and 10 ng/ml, applied from day 15 to day 21). Control cultures show progressive formation of compact microtissue‐like aggregates (white arrows), whereas IL‑1β stimulation results in disrupted aggregates (yellow arrows). Green: calcein‐AM (live cells); red: ethidium homodimer‐1 (dead cells). (C) Representative z‐projection immunofluorescence images of ECM marker expression in region I after 21 days of culture, comparing control and IL‑1β‐stimulated samples. Images show merged fluorescence channels for collagen type II (green), aggrecan (red), and nuclei (DAPI, blue). (D–E) Quantification of corrected total cell fluorescence (CTCF) for collagen type II (D) and aggrecan (E) across regions I–III for control and IL‑1β‐stimulated cultures at day 21 (n = 44 cells per group). (F) Cell metabolic activity measured using a PrestoBlue assay for control and IL‑1β‐stimulated samples at day 21 ( n = 5 independent samples per group). (G–H) Quantification of inflammatory mediators in culture supernatant collected at day 21 using multiplex ELISA, including matrix metalloproteinases (MMP‐1, MMP‐2, MMP‐3, MMP‐7, MMP‐9, MMP‐13) and cytokines (IL‑1β, IL‑6, IL‑8) ( n = 3 independent samples per group). Values below the lower limit of quantification are indicated as <LLOQ. (I) Quantification of collagen type II fragments released into the culture medium at day 21, measured by ELISA ( n = 3 independent samples per group). All fluorescence images include a 100 µm scale bar, and white dashed lines indicate microchannel boundaries. Statistical analysis was performed using the Kruskal–Wallis test with Dunn's post hoc comparison (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n.s., not significant). Conditions include control (no <t>cytokine)</t> and IL‑1β stimulation (1 and 10 ng/ml). Note: Panels are adapted from da Silva et al. , licensed under CC BY 3.0.
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Expected cellular responses of human articular chondrocytes under dynamic culture and IL‑1β‐induced inflammatory conditions. Representative results illustrate chondrocyte morphology, viability, extracellular matrix (ECM) expression, metabolic activity, and inflammatory responses under gravity‐driven flow conditions. (A) Representative z‐projection fluorescence images of chondrocytes cultured for 21 days in microchannels coated with a fibronectin–collagen type IV mixture. Cells form aggregates and microtissue‐like structures across regions I–III corresponding to increasing fluid shear stress levels. Cells are stained for F‐actin (red) and nuclei (DAPI, blue). (B) Representative Live/Dead fluorescence images showing time‐dependent viability and proliferation of chondrocytes cultured under dynamic conditions on days 3, 7, 14, and 21 (control), compared with IL‑1β‐stimulated cultures on day 21 (1 and 10 ng/ml, applied from day 15 to day 21). Control cultures show progressive formation of compact microtissue‐like aggregates (white arrows), whereas IL‑1β stimulation results in disrupted aggregates (yellow arrows). Green: calcein‐AM (live cells); red: ethidium homodimer‐1 (dead cells). (C) Representative z‐projection immunofluorescence images of ECM marker expression in region I after 21 days of culture, comparing control and IL‑1β‐stimulated samples. Images show merged fluorescence channels for collagen type II (green), aggrecan (red), and nuclei (DAPI, blue). (D–E) Quantification of corrected total cell fluorescence (CTCF) for collagen type II (D) and aggrecan (E) across regions I–III for control and IL‑1β‐stimulated cultures at day 21 (n = 44 cells per group). (F) Cell metabolic activity measured using a PrestoBlue assay for control and IL‑1β‐stimulated samples at day 21 ( n = 5 independent samples per group). (G–H) Quantification of inflammatory mediators in culture supernatant collected at day 21 using multiplex ELISA, including matrix metalloproteinases (MMP‐1, MMP‐2, MMP‐3, MMP‐7, MMP‐9, MMP‐13) and cytokines (IL‑1β, IL‑6, IL‑8) ( n = 3 independent samples per group). Values below the lower limit of quantification are indicated as <LLOQ. (I) Quantification of collagen type II fragments released into the culture medium at day 21, measured by ELISA ( n = 3 independent samples per group). All fluorescence images include a 100 µm scale bar, and white dashed lines indicate microchannel boundaries. Statistical analysis was performed using the Kruskal–Wallis test with Dunn's post hoc comparison (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n.s., not significant). Conditions include control (no <t>cytokine)</t> and IL‑1β stimulation (1 and 10 ng/ml). Note: Panels are adapted from da Silva et al. , licensed under CC BY 3.0.
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Expected cellular responses of human articular chondrocytes under dynamic culture and IL‑1β‐induced inflammatory conditions. Representative results illustrate chondrocyte morphology, viability, extracellular matrix (ECM) expression, metabolic activity, and inflammatory responses under gravity‐driven flow conditions. (A) Representative z‐projection fluorescence images of chondrocytes cultured for 21 days in microchannels coated with a fibronectin–collagen type IV mixture. Cells form aggregates and microtissue‐like structures across regions I–III corresponding to increasing fluid shear stress levels. Cells are stained for F‐actin (red) and nuclei (DAPI, blue). (B) Representative Live/Dead fluorescence images showing time‐dependent viability and proliferation of chondrocytes cultured under dynamic conditions on days 3, 7, 14, and 21 (control), compared with IL‑1β‐stimulated cultures on day 21 (1 and 10 ng/ml, applied from day 15 to day 21). Control cultures show progressive formation of compact microtissue‐like aggregates (white arrows), whereas IL‑1β stimulation results in disrupted aggregates (yellow arrows). Green: calcein‐AM (live cells); red: ethidium homodimer‐1 (dead cells). (C) Representative z‐projection immunofluorescence images of ECM marker expression in region I after 21 days of culture, comparing control and IL‑1β‐stimulated samples. Images show merged fluorescence channels for collagen type II (green), aggrecan (red), and nuclei (DAPI, blue). (D–E) Quantification of corrected total cell fluorescence (CTCF) for collagen type II (D) and aggrecan (E) across regions I–III for control and IL‑1β‐stimulated cultures at day 21 (n = 44 cells per group). (F) Cell metabolic activity measured using a PrestoBlue assay for control and IL‑1β‐stimulated samples at day 21 ( n = 5 independent samples per group). (G–H) Quantification of inflammatory mediators in culture supernatant collected at day 21 using multiplex ELISA, including matrix metalloproteinases (MMP‐1, MMP‐2, MMP‐3, MMP‐7, MMP‐9, MMP‐13) and cytokines (IL‑1β, IL‑6, IL‑8) ( n = 3 independent samples per group). Values below the lower limit of quantification are indicated as <LLOQ. (I) Quantification of collagen type II fragments released into the culture medium at day 21, measured by ELISA ( n = 3 independent samples per group). All fluorescence images include a 100 µm scale bar, and white dashed lines indicate microchannel boundaries. Statistical analysis was performed using the Kruskal–Wallis test with Dunn's post hoc comparison (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n.s., not significant). Conditions include control (no <t>cytokine)</t> and IL‑1β stimulation (1 and 10 ng/ml). Note: Panels are adapted from da Silva et al. , licensed under CC BY 3.0.
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Expected cellular responses of human articular chondrocytes under dynamic culture and IL‑1β‐induced inflammatory conditions. Representative results illustrate chondrocyte morphology, viability, extracellular matrix (ECM) expression, metabolic activity, and inflammatory responses under gravity‐driven flow conditions. (A) Representative z‐projection fluorescence images of chondrocytes cultured for 21 days in microchannels coated with a fibronectin–collagen type IV mixture. Cells form aggregates and microtissue‐like structures across regions I–III corresponding to increasing fluid shear stress levels. Cells are stained for F‐actin (red) and nuclei (DAPI, blue). (B) Representative Live/Dead fluorescence images showing time‐dependent viability and proliferation of chondrocytes cultured under dynamic conditions on days 3, 7, 14, and 21 (control), compared with IL‑1β‐stimulated cultures on day 21 (1 and 10 ng/ml, applied from day 15 to day 21). Control cultures show progressive formation of compact microtissue‐like aggregates (white arrows), whereas IL‑1β stimulation results in disrupted aggregates (yellow arrows). Green: calcein‐AM (live cells); red: ethidium homodimer‐1 (dead cells). (C) Representative z‐projection immunofluorescence images of ECM marker expression in region I after 21 days of culture, comparing control and IL‑1β‐stimulated samples. Images show merged fluorescence channels for collagen type II (green), aggrecan (red), and nuclei (DAPI, blue). (D–E) Quantification of corrected total cell fluorescence (CTCF) for collagen type II (D) and aggrecan (E) across regions I–III for control and IL‑1β‐stimulated cultures at day 21 (n = 44 cells per group). (F) Cell metabolic activity measured using a PrestoBlue assay for control and IL‑1β‐stimulated samples at day 21 ( n = 5 independent samples per group). (G–H) Quantification of inflammatory mediators in culture supernatant collected at day 21 using multiplex ELISA, including matrix metalloproteinases (MMP‐1, MMP‐2, MMP‐3, MMP‐7, MMP‐9, MMP‐13) and cytokines (IL‑1β, IL‑6, IL‑8) ( n = 3 independent samples per group). Values below the lower limit of quantification are indicated as <LLOQ. (I) Quantification of collagen type II fragments released into the culture medium at day 21, measured by ELISA ( n = 3 independent samples per group). All fluorescence images include a 100 µm scale bar, and white dashed lines indicate microchannel boundaries. Statistical analysis was performed using the Kruskal–Wallis test with Dunn's post hoc comparison (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n.s., not significant). Conditions include control (no cytokine) and IL‑1β stimulation (1 and 10 ng/ml). Note: Panels are adapted from da Silva et al. , licensed under CC BY 3.0.

Journal: Current Protocols

Article Title: A Gravity‐driven Microfluidic Platform for High‐throughput Culture and Inflammatory Stimulation of Human Articular Chondrocytes

doi: 10.1002/cpz1.70489

Figure Lengend Snippet: Expected cellular responses of human articular chondrocytes under dynamic culture and IL‑1β‐induced inflammatory conditions. Representative results illustrate chondrocyte morphology, viability, extracellular matrix (ECM) expression, metabolic activity, and inflammatory responses under gravity‐driven flow conditions. (A) Representative z‐projection fluorescence images of chondrocytes cultured for 21 days in microchannels coated with a fibronectin–collagen type IV mixture. Cells form aggregates and microtissue‐like structures across regions I–III corresponding to increasing fluid shear stress levels. Cells are stained for F‐actin (red) and nuclei (DAPI, blue). (B) Representative Live/Dead fluorescence images showing time‐dependent viability and proliferation of chondrocytes cultured under dynamic conditions on days 3, 7, 14, and 21 (control), compared with IL‑1β‐stimulated cultures on day 21 (1 and 10 ng/ml, applied from day 15 to day 21). Control cultures show progressive formation of compact microtissue‐like aggregates (white arrows), whereas IL‑1β stimulation results in disrupted aggregates (yellow arrows). Green: calcein‐AM (live cells); red: ethidium homodimer‐1 (dead cells). (C) Representative z‐projection immunofluorescence images of ECM marker expression in region I after 21 days of culture, comparing control and IL‑1β‐stimulated samples. Images show merged fluorescence channels for collagen type II (green), aggrecan (red), and nuclei (DAPI, blue). (D–E) Quantification of corrected total cell fluorescence (CTCF) for collagen type II (D) and aggrecan (E) across regions I–III for control and IL‑1β‐stimulated cultures at day 21 (n = 44 cells per group). (F) Cell metabolic activity measured using a PrestoBlue assay for control and IL‑1β‐stimulated samples at day 21 ( n = 5 independent samples per group). (G–H) Quantification of inflammatory mediators in culture supernatant collected at day 21 using multiplex ELISA, including matrix metalloproteinases (MMP‐1, MMP‐2, MMP‐3, MMP‐7, MMP‐9, MMP‐13) and cytokines (IL‑1β, IL‑6, IL‑8) ( n = 3 independent samples per group). Values below the lower limit of quantification are indicated as

Article Snippet: Chondrocyte growth medium (HC Growth Medium, Cell Applications, cat. no. 411‐500) DPBS (sterile, Gibco, cat. no. 14190144) Recombinant IL‐1β (R&D Systems, cat. no. 201‐LB‐025) (separately at −20°C) Live/Dead Viability/Cytotoxicity Kit containing calcein‐AM and ethidium homodimer 1 (EthD‐1) (Invitrogen, cat. no. LS224) PrestoBlue Cell Viability Reagent (Invitrogen, cat. no. A13261) 4% paraformaldehyde (PFA) in DPBS (Thermo Scientific, cat. no. J61899.AP) Normal donkey serum Bovine serum albumin (BSA) Triton X‐100 Tetramethylrhodamine (TRITC)‐phalloidin staining (EMD Millipore/Merck, cat. no. 90228) Mouse anti‐Collagen II antibody (NB600‐844, Novus Biologicals) Goat anti‐Aggrecan antibody (AF1220, R&D Systems) Rabbit anti‐Mouse IgG (H+L), FITC‐conjugated secondary antibody (NB7543, Novus Biologicals) Donkey anti‐Goat IgG (H+L), Northern Lights 557 (NL557)‐conjugated secondary antibody (NL001, R&D Systems) 4′,6‐diamidino‐2‐phenylindole (DAPI) Solution (Novus Biologicals, Bio‐Techne, cat. no. NBP2‐31156) Q‐Plex Human MMP 6 Plex kit (340949HU, Quansys Biosciences) Q‐Plex Human Custom Cytokine Plex kit (107749GR, Quansys Biosciences) Human collagen type II alpha 1 ELISA kit (BSKH63293, Bioss Antibodies) Pre‐established chondrocyte cultures generated using Basic Protocol Rocker platform (AKITA rocker, FinnAdvance or equivalent) CO incubator maintained at 37°C and 5% CO Class II biosafety cabinet Wide‐field fluorescence microscope (e.g., Leica DM4B or equivalent) equipped with blue/green and red/green excitation/emission filter sets.

Techniques: Morphology, Expressing, Activity Assay, Fluorescence, Cell Culture, Shear, Staining, Control, Immunofluorescence, Marker, Prestoblue Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Analysis, Comparison