Journal: Scientific Reports

Article Title: An exploratory in vitro co-culture of enteric neurons and smooth muscle cells demonstrates neuronal contribution to muscle layer formation

doi: 10.1038/s41598-026-39409-3

Figure Lengend Snippet: Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells). SMCs alone, with direct contact and without direct contact to isolated myenteric plexus cells.

Article Snippet: Fig. 8 Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells).

Techniques: Co-Culture Assay, Isolation

Journal: Scientific Reports

Article Title: An exploratory in vitro co-culture of enteric neurons and smooth muscle cells demonstrates neuronal contribution to muscle layer formation

doi: 10.1038/s41598-026-39409-3

Figure Lengend Snippet: Immunofluorescence staining of enteric nervous system (ENS) cells co-cultured with smooth muscle cells in a three-dimensional HyStem-C hydrogel, showing ( a ) glial fibrillary acidic protein (GFAP)–positive glial fibers (magenta), ( b ) smooth muscle cells stained for smooth muscle actin (SMA, cyan), ( c ) neuronal fibers labeled with βIII-tubulin (Tuj1, green), and ( d ) merged image. Cell nuclei are counterstained with DAPI (blue). Non-specific staining observed in the SMA channel indicates the presence of additional cell types, likely fibroblasts. Scale bar 50 μm. ( e ) higher-magnification image of a GFAP-positive glial cell (magenta). Scale bar 100 μm.

Article Snippet: Fig. 8 Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells).

Techniques: Immunofluorescence, Staining, Cell Culture, Labeling

Journal: Scientific Reports

Article Title: An exploratory in vitro co-culture of enteric neurons and smooth muscle cells demonstrates neuronal contribution to muscle layer formation

doi: 10.1038/s41598-026-39409-3

Figure Lengend Snippet: Contracting muscle fibers in the live cultures of muscle cells together with ENS cells in HyStem-C Hydrogel (real time): ( a ) thin muscle fibers, ( b ) thick muscle fibers. Light microscopy.

Article Snippet: Fig. 8 Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells).

Techniques: Light Microscopy

Journal: Scientific Reports

Article Title: An exploratory in vitro co-culture of enteric neurons and smooth muscle cells demonstrates neuronal contribution to muscle layer formation

doi: 10.1038/s41598-026-39409-3

Figure Lengend Snippet: Smooth muscle and ENS cells in 3D scaffolds (14 days) co-cultured in 3D scaffold, showing neurons in green (ß-Tubulin III), muscle cells in red (smooth muscle actin (SMA)) and nuclei in blue (DRAQ5) ( a ) Confocal microscopy lower Magnification, ( b ) Confocal microscopy 3D structure in merged channels view from the side in higher magnification (Scale bars 50 μm) and ( c ) Confocal microscopy, separated channels view from above in higher magnification, ( d ) Electron microscopy of two muscle cells in close contact within the thickness of the three-dimensional matrix. The cells have large nuclei, an elongated shape, and actin microfilaments typical for muscle cells. Scale bar 2 μm, ( e ) Electron microscopy of plasma membrane of a muscle cell, with arrows indicating caveolae. Scale bar 200 nm.

Article Snippet: Fig. 8 Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells).

Techniques: Cell Culture, Confocal Microscopy, Electron Microscopy, Clinical Proteomics, Membrane

Journal: Scientific Reports

Article Title: An exploratory in vitro co-culture of enteric neurons and smooth muscle cells demonstrates neuronal contribution to muscle layer formation

doi: 10.1038/s41598-026-39409-3

Figure Lengend Snippet: Images of 3 weeks old co-culture of ENS cells and muscle cells in different layouts: ( a ) SMCs alone, ( b ) paracrine interaction between SMCs and ENS cells, ( c ) direct contact between SMCs and ENS cells Scale bars 100 μm, ( d ) paracrine interaction between SMCs and ENS in higher magnification, ( e ) Direct contact between smooth muscle and ENS cells in higher magnification.– muscle cells Scale bars 20 μm, ( f ) 3D reconstruction of muscle fibers in confocal microscopy by the direct contact between SMCs and ENS, ( j ) neurons, which are intercommunicated in the neuronal net within the muscle layer by the direct contact between SMCs and ENS. Confocal microscopy, Green (ß-Tubulin III) – neurons, red (smooth muscle actin (SMA)) – muscle cells. Scale bars 50 μm.

Article Snippet: Fig. 8 Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells).

Techniques: Co-Culture Assay, Confocal Microscopy

Journal: Scientific Reports

Article Title: An exploratory in vitro co-culture of enteric neurons and smooth muscle cells demonstrates neuronal contribution to muscle layer formation

doi: 10.1038/s41598-026-39409-3

Figure Lengend Snippet: Imaging of the whole thickness of the 3D scaffolds after 3 weeks of ENS cells and muscle cells co-culture in different layouts: ( a ) SMCs without ENS cells, ( b ) paracrine interaction between SMCs and ENS cells, ( c ) direct contact between SMCs and ENS cells. Confocal microscopy, Green (ß-Tubulin III) – neurons, red (smooth muscle actin (SMA)) – muscle cells.

Article Snippet: Fig. 8 Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells).

Techniques: Imaging, Co-Culture Assay, Confocal Microscopy

Journal: Scientific Reports

Article Title: An exploratory in vitro co-culture of enteric neurons and smooth muscle cells demonstrates neuronal contribution to muscle layer formation

doi: 10.1038/s41598-026-39409-3

Figure Lengend Snippet: Confocal microscopy images of smooth muscle and ENS cells co-cultured in 3D scaffolds (14 days of culture). Secretory vesicles and synapses are labelled in green (Synaptobrevin 2), while muscle cells in red (smooth muscle actin (SMA)). Scale bars 20 μm.

Article Snippet: Fig. 8 Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells).

Techniques: Confocal Microscopy, Cell Culture

Journal: Scientific Reports

Article Title: An exploratory in vitro co-culture of enteric neurons and smooth muscle cells demonstrates neuronal contribution to muscle layer formation

doi: 10.1038/s41598-026-39409-3

Figure Lengend Snippet: Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells). SMCs alone, with direct contact and without direct contact to isolated myenteric plexus cells.

Article Snippet: Fig. 8 Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells).

Techniques: Co-Culture Assay, Isolation

Journal: Scientific Reports

Article Title: An exploratory in vitro co-culture of enteric neurons and smooth muscle cells demonstrates neuronal contribution to muscle layer formation

doi: 10.1038/s41598-026-39409-3

Figure Lengend Snippet: Images of 3 weeks old co-culture of ENS cells and muscle cells in different layouts: ( a ) SMCs alone, ( b ) paracrine interaction between SMCs and ENS cells, ( c ) direct contact between SMCs and ENS cells Scale bars 100 μm, ( d ) paracrine interaction between SMCs and ENS in higher magnification, ( e ) Direct contact between smooth muscle and ENS cells in higher magnification.– muscle cells Scale bars 20 μm, ( f ) 3D reconstruction of muscle fibers in confocal microscopy by the direct contact between SMCs and ENS, ( j ) neurons, which are intercommunicated in the neuronal net within the muscle layer by the direct contact between SMCs and ENS. Confocal microscopy, Green (ß-Tubulin III) – neurons, red (smooth muscle actin (SMA)) – muscle cells. Scale bars 50 μm.

Article Snippet: Fig. 8 Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells).

Techniques: Co-Culture Assay, Confocal Microscopy

Journal: Scientific Reports

Article Title: An exploratory in vitro co-culture of enteric neurons and smooth muscle cells demonstrates neuronal contribution to muscle layer formation

doi: 10.1038/s41598-026-39409-3

Figure Lengend Snippet: Imaging of the whole thickness of the 3D scaffolds after 3 weeks of ENS cells and muscle cells co-culture in different layouts: ( a ) SMCs without ENS cells, ( b ) paracrine interaction between SMCs and ENS cells, ( c ) direct contact between SMCs and ENS cells. Confocal microscopy, Green (ß-Tubulin III) – neurons, red (smooth muscle actin (SMA)) – muscle cells.

Article Snippet: Fig. 8 Schematic representation of plates with inserts and different layouts of muscle and ENS cells in a three-dimensional matrix: ( a ) plates with insert, which allows the medium to surround and support the co-culture from all sides, ( b ) SMCs without ENS cells in the 3D-matrix, ( c ) SMCs and ENS cells equally distributed in 3D-matrix, ( d ) SMCs distributed in upper and lower layers, ENS cells - in the middle layer of the 3D-matrix. ( e ) SMCs distributed in upper and lower layers, ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix. ( f ) SMCs and ENS cells distributed in the middle layer of the 3D-matrix. ( g ) mixed SMCs and ENS cells densely arranged in the same plane of the middle layer of the 3D-matrix, ( h ) different layouts between SMCs and isolated myenteric plexus (ENS cells).

Techniques: Imaging, Co-Culture Assay, Confocal Microscopy