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Fabrication of 3D condensed tissues incorporating M1 macrophages. (A) Schematic of 3D tissue formation protocol, consisting of aCM, cfb, and monocyte preculture for 3 days (d-10), tissue seeding in 96 well plates (d-7) subsequent culture and M1 maturation for 7 days (d-7 to d0) and activation of M1 (d0). (B) Brightfield image of the injection molded pillar device used for seeding tissues. (C) Annotated representative brightfield image of condensed 3D tissue consisting of aCM+cfb. 9 D) Sharp electrode recordings of single cell aCMs in 3D tissues consisting of aCM+cfb (n=17/N=4) compared to 2D monolayer of aCM for (n=31/N=3), demonstrating significant increase in upstroke velocity for 3D tissues (Mann-Whitney test). (E) IF staining for cardiac marker <t>cTnT</t> and cardiac tissue resident macrophage marker CX3CR1 in aCM+cfb+M1 tissue. Zoomed-in image showing area marked by white rectangle, white arrows indicate macrophages. ****P<0.0001.
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ctnt reafinity conjugated fitc  (Miltenyi Biotec)
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Fabrication of 3D condensed tissues incorporating M1 macrophages. (A) Schematic of 3D tissue formation protocol, consisting of aCM, cfb, and monocyte preculture for 3 days (d-10), tissue seeding in 96 well plates (d-7) subsequent culture and M1 maturation for 7 days (d-7 to d0) and activation of M1 (d0). (B) Brightfield image of the injection molded pillar device used for seeding tissues. (C) Annotated representative brightfield image of condensed 3D tissue consisting of aCM+cfb. 9 D) Sharp electrode recordings of single cell aCMs in 3D tissues consisting of aCM+cfb (n=17/N=4) compared to 2D monolayer of aCM for (n=31/N=3), demonstrating significant increase in upstroke velocity for 3D tissues (Mann-Whitney test). (E) IF staining for cardiac marker <t>cTnT</t> and cardiac tissue resident macrophage marker CX3CR1 in aCM+cfb+M1 tissue. Zoomed-in image showing area marked by white rectangle, white arrows indicate macrophages. ****P<0.0001.
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Fabrication of 3D condensed tissues incorporating M1 macrophages. (A) Schematic of 3D tissue formation protocol, consisting of aCM, cfb, and monocyte preculture for 3 days (d-10), tissue seeding in 96 well plates (d-7) subsequent culture and M1 maturation for 7 days (d-7 to d0) and activation of M1 (d0). (B) Brightfield image of the injection molded pillar device used for seeding tissues. (C) Annotated representative brightfield image of condensed 3D tissue consisting of aCM+cfb. 9 D) Sharp electrode recordings of single cell aCMs in 3D tissues consisting of aCM+cfb (n=17/N=4) compared to 2D monolayer of aCM for (n=31/N=3), demonstrating significant increase in upstroke velocity for 3D tissues (Mann-Whitney test). (E) IF staining for cardiac marker <t>cTnT</t> and cardiac tissue resident macrophage marker CX3CR1 in aCM+cfb+M1 tissue. Zoomed-in image showing area marked by white rectangle, white arrows indicate macrophages. ****P<0.0001.
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Fabrication of 3D condensed tissues incorporating M1 macrophages. (A) Schematic of 3D tissue formation protocol, consisting of aCM, cfb, and monocyte preculture for 3 days (d-10), tissue seeding in 96 well plates (d-7) subsequent culture and M1 maturation for 7 days (d-7 to d0) and activation of M1 (d0). (B) Brightfield image of the injection molded pillar device used for seeding tissues. (C) Annotated representative brightfield image of condensed 3D tissue consisting of aCM+cfb. 9 D) Sharp electrode recordings of single cell aCMs in 3D tissues consisting of aCM+cfb (n=17/N=4) compared to 2D monolayer of aCM for (n=31/N=3), demonstrating significant increase in upstroke velocity for 3D tissues (Mann-Whitney test). (E) IF staining for cardiac marker <t>cTnT</t> and cardiac tissue resident macrophage marker CX3CR1 in aCM+cfb+M1 tissue. Zoomed-in image showing area marked by white rectangle, white arrows indicate macrophages. ****P<0.0001.
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Fabrication of 3D condensed tissues incorporating M1 macrophages. (A) Schematic of 3D tissue formation protocol, consisting of aCM, cfb, and monocyte preculture for 3 days (d-10), tissue seeding in 96 well plates (d-7) subsequent culture and M1 maturation for 7 days (d-7 to d0) and activation of M1 (d0). (B) Brightfield image of the injection molded pillar device used for seeding tissues. (C) Annotated representative brightfield image of condensed 3D tissue consisting of aCM+cfb. 9 D) Sharp electrode recordings of single cell aCMs in 3D tissues consisting of aCM+cfb (n=17/N=4) compared to 2D monolayer of aCM for (n=31/N=3), demonstrating significant increase in upstroke velocity for 3D tissues (Mann-Whitney test). (E) IF staining for cardiac marker <t>cTnT</t> and cardiac tissue resident macrophage marker CX3CR1 in aCM+cfb+M1 tissue. Zoomed-in image showing area marked by white rectangle, white arrows indicate macrophages. ****P<0.0001.
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Fabrication of 3D condensed tissues incorporating M1 macrophages. (A) Schematic of 3D tissue formation protocol, consisting of aCM, cfb, and monocyte preculture for 3 days (d-10), tissue seeding in 96 well plates (d-7) subsequent culture and M1 maturation for 7 days (d-7 to d0) and activation of M1 (d0). (B) Brightfield image of the injection molded pillar device used for seeding tissues. (C) Annotated representative brightfield image of condensed 3D tissue consisting of aCM+cfb. 9 D) Sharp electrode recordings of single cell aCMs in 3D tissues consisting of aCM+cfb (n=17/N=4) compared to 2D monolayer of aCM for (n=31/N=3), demonstrating significant increase in upstroke velocity for 3D tissues (Mann-Whitney test). (E) IF staining for cardiac marker <t>cTnT</t> and cardiac tissue resident macrophage marker CX3CR1 in aCM+cfb+M1 tissue. Zoomed-in image showing area marked by white rectangle, white arrows indicate macrophages. ****P<0.0001.
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Fabrication of 3D condensed tissues incorporating M1 macrophages. (A) Schematic of 3D tissue formation protocol, consisting of aCM, cfb, and monocyte preculture for 3 days (d-10), tissue seeding in 96 well plates (d-7) subsequent culture and M1 maturation for 7 days (d-7 to d0) and activation of M1 (d0). (B) Brightfield image of the injection molded pillar device used for seeding tissues. (C) Annotated representative brightfield image of condensed 3D tissue consisting of aCM+cfb. 9 D) Sharp electrode recordings of single cell aCMs in 3D tissues consisting of aCM+cfb (n=17/N=4) compared to 2D monolayer of aCM for (n=31/N=3), demonstrating significant increase in upstroke velocity for 3D tissues (Mann-Whitney test). (E) IF staining for cardiac marker <t>cTnT</t> and cardiac tissue resident macrophage marker CX3CR1 in aCM+cfb+M1 tissue. Zoomed-in image showing area marked by white rectangle, white arrows indicate macrophages. ****P<0.0001.
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Fabrication of 3D condensed tissues incorporating M1 macrophages. (A) Schematic of 3D tissue formation protocol, consisting of aCM, cfb, and monocyte preculture for 3 days (d-10), tissue seeding in 96 well plates (d-7) subsequent culture and M1 maturation for 7 days (d-7 to d0) and activation of M1 (d0). (B) Brightfield image of the injection molded pillar device used for seeding tissues. (C) Annotated representative brightfield image of condensed 3D tissue consisting of aCM+cfb. 9 D) Sharp electrode recordings of single cell aCMs in 3D tissues consisting of aCM+cfb (n=17/N=4) compared to 2D monolayer of aCM for (n=31/N=3), demonstrating significant increase in upstroke velocity for 3D tissues (Mann-Whitney test). (E) IF staining for cardiac marker cTnT and cardiac tissue resident macrophage marker CX3CR1 in aCM+cfb+M1 tissue. Zoomed-in image showing area marked by white rectangle, white arrows indicate macrophages. ****P<0.0001.

Journal: Frontiers in Immunology

Article Title: GJA5 and ATP1A1 perturbations recapitulate inflammation-related beat irregularities in iPSC-based atrial myocardium tissue model

doi: 10.3389/fimmu.2025.1719392

Figure Lengend Snippet: Fabrication of 3D condensed tissues incorporating M1 macrophages. (A) Schematic of 3D tissue formation protocol, consisting of aCM, cfb, and monocyte preculture for 3 days (d-10), tissue seeding in 96 well plates (d-7) subsequent culture and M1 maturation for 7 days (d-7 to d0) and activation of M1 (d0). (B) Brightfield image of the injection molded pillar device used for seeding tissues. (C) Annotated representative brightfield image of condensed 3D tissue consisting of aCM+cfb. 9 D) Sharp electrode recordings of single cell aCMs in 3D tissues consisting of aCM+cfb (n=17/N=4) compared to 2D monolayer of aCM for (n=31/N=3), demonstrating significant increase in upstroke velocity for 3D tissues (Mann-Whitney test). (E) IF staining for cardiac marker cTnT and cardiac tissue resident macrophage marker CX3CR1 in aCM+cfb+M1 tissue. Zoomed-in image showing area marked by white rectangle, white arrows indicate macrophages. ****P<0.0001.

Article Snippet: Fixed aCM in suspension were co-stained with MLC2a REAfinityTM conjugated with APC (1:10, Miltenyi Biotec) and cTnT REAfinityTM conjugated with FITC (1:10, Miltenyi Biotec) antibodies and incubated for 15 min at RT.

Techniques: Activation Assay, Injection, Single Cell, MANN-WHITNEY, Staining, Marker

Fabrication of 3D condensed tissues incorporating M1 macrophages. (A) Schematic of 3D tissue formation protocol, consisting of aCM, cfb, and monocyte preculture for 3 days (d-10), tissue seeding in 96 well plates (d-7) subsequent culture and M1 maturation for 7 days (d-7 to d0) and activation of M1 (d0). (B) Brightfield image of the injection molded pillar device used for seeding tissues. (C) Annotated representative brightfield image of condensed 3D tissue consisting of aCM+cfb. 9 D) Sharp electrode recordings of single cell aCMs in 3D tissues consisting of aCM+cfb (n=17/N=4) compared to 2D monolayer of aCM for (n=31/N=3), demonstrating significant increase in upstroke velocity for 3D tissues (Mann-Whitney test). (E) IF staining for cardiac marker cTnT and cardiac tissue resident macrophage marker CX3CR1 in aCM+cfb+M1 tissue. Zoomed-in image showing area marked by white rectangle, white arrows indicate macrophages. ****P<0.0001.

Journal: Frontiers in Immunology

Article Title: GJA5 and ATP1A1 perturbations recapitulate inflammation-related beat irregularities in iPSC-based atrial myocardium tissue model

doi: 10.3389/fimmu.2025.1719392

Figure Lengend Snippet: Fabrication of 3D condensed tissues incorporating M1 macrophages. (A) Schematic of 3D tissue formation protocol, consisting of aCM, cfb, and monocyte preculture for 3 days (d-10), tissue seeding in 96 well plates (d-7) subsequent culture and M1 maturation for 7 days (d-7 to d0) and activation of M1 (d0). (B) Brightfield image of the injection molded pillar device used for seeding tissues. (C) Annotated representative brightfield image of condensed 3D tissue consisting of aCM+cfb. 9 D) Sharp electrode recordings of single cell aCMs in 3D tissues consisting of aCM+cfb (n=17/N=4) compared to 2D monolayer of aCM for (n=31/N=3), demonstrating significant increase in upstroke velocity for 3D tissues (Mann-Whitney test). (E) IF staining for cardiac marker cTnT and cardiac tissue resident macrophage marker CX3CR1 in aCM+cfb+M1 tissue. Zoomed-in image showing area marked by white rectangle, white arrows indicate macrophages. ****P<0.0001.

Article Snippet: Tissues were incubated with primary antibodies (cTnT REAfinity conjugated FITC (1:100, Miltenyi Biotec)) and CX3CR1 rabbit anti-human (1H14L7) (1:250, Invitrogen) or Collagen I Polyclonal Antibody (1:100, ThermoFisher Scientific) overnight at 4 °C on a plate shaker.

Techniques: Activation Assay, Injection, Single Cell, MANN-WHITNEY, Staining, Marker