Biocompatibility of Hydrogels for Glomerular 3D Co-Culture: A Comparative Analysis

Abstract

Conventional 2D mono-cultures fall short in replicating the complex microenvironment of glomerular tissue, where cell-cell and cell-matrix interactions are critical. To better mimic in vivo conditions, the development of robust 3D co-culture systems is essential. Here, we systematically evaluate five hydrogel matrices-Matrigel, alginate dialdehyde-gelatin (ADA-GEL), fibrin, recombinantly produced spider silk protein eADF4(C16)-RGD, and allyl-modified gelatin (GelAGE)-for their suitability in supporting glomerular 3D co-culture. The hydrogels are assessed for handling properties, cell viability, and the support of physiological cell behavior using bright-field microscopy, live/dead assays, immunofluorescence, and multiphoton imaging. Among the tested hydrogels, GelAGE and eADF4(C16)-RGD demonstrate superior biocompatibility and structural support. Due to its ease of use and comparable biological performance, GelAGE and spider silk protein eADF(C16)-RGD are selected for further mechanical characterization, revealing favorable viscoelastic properties. These findings position both hydrogels as a promising candidate for engineering physiologically relevant 3D glomerular models and advancing kidney tissue research.

Keywords: ADA‐GEL; GelAGE; fibrin gel; glomerular co‐culture; matrigel; spider silk.

FIGURE 1

Matrigel as an ECM‐mimicking environment for glomerular cells. (A) Schematic illustration of Matrigel hydrogel fabrication with encapsulated glomerular cells. (B) Bright‐field microscopy of hMC in Matrigel domes on coverslips after one (upper panel) and four days (middle and lower panels). The middle panel shows the formation of the characteristic growth pattern of hMCs. The lower panel reveals self‐formation of 3D mono‐culture spheroids. Scale bars: left side: 750 µm and right side: 300 µm. (C) Confocal microscopy of 2D hMC (top) and 3D hMC spheroids embedded in Matrigel domes (bottom) after four days depicts cell elongation and cell‐cell contacts. Nuclei were stained with Hoechst 33342 (blue), and cell surface marker WGA (red). Scale bar: 50 µm. ECM: extracellular matrix, hMC: human mesangial cells, WGA: wheat germ agglutinin. Experiments with Matrigel were performed more than three times.

FIGURE 2

ADA‐GEL as an ECM‐mimicking environment for glomerular cells. (A) Schematic illustration of ADA‐GEL hydrogel fabrication with encapsulated glomerular cells. (B) Viability of hMCs grown as 2D culture (upper panel) and 3D mono‐culture (lower panel) in ADA‐GEL using live (green)/dead (red) fluorescence staining after five days imaged by fluorescence microscopy. Scale bars: 300 µm. (C) Morphology of hMCs using confocal microscopy (upper panel: 2D cultivation, lower panel: 3D mono‐culture, blue: Hoechst 33342, red: WGA, right: merge). Scale bar: 50 µm. ADA‐GEL: alginate dialdehyde‐gelatin, ECM: extracellular matrix, hMC: human mesangial cells, WGA: wheat germ agglutinin. Experiments with ADA‐GEL were performed more than three times.

FIGURE 3

Fibrin gel as an ECM‐mimicking environment for glomerular cells. (A) Schematic illustration of fibrin gel fabrication from fibrinogen and thrombin with encapsulated glomerular cells. (B) Viability of hMC spheroids, hGEC spheroids, and hPodocytes in fibrin gels composed of high fibrinogen (left panel) and low fibrinogen (right panel) using live (green)/dead (red) fluorescence staining after one day of culture imaged by fluorescence microscopy. Scale bar: 300 µm. (C) Viability of podocytes in fibrin gels composed of high fibrinogen with (upper panel) and without (lower panel) aprotinin using live (green)/dead (red) fluorescence staining after seven and 14 days of culture imaged by fluorescence microscopy. Scale bar: left side: 750 µm and right side: 300 µm. ECM: extracellular matrix, hMC: human mesangial cells, hGEC: human glomerular endothelial cells, hPodocytes: immortalized human podocytes. Experiments with fibrin were performed more than three times.

FIGURE 4

Spider silk as an ECM‐mimicking environment for glomerular co‐culture. (A) Schematic illustration of recombinantly produced spider silk‐like proteins for hydrogel fabrication with encapsulated glomerular cells. (B) Bright‐field (left panels) and live (green)/dead (red) fluorescence staining (right panels) of hMC, hGEC, and hPodocytes and 3D glomerular co‐culture of hMC, hGEC, and hPodocytes in spider silk‐based hydrogels. Imaged after one day, nine days, and 14 days of culture. Scale bars: Bright‐field: 1,000 µm and live/dead: 300 µm. (C) Bright‐field (left panels) and live (green)/dead (red) fluorescence staining (right panels) of 3D glomerular co‐culture of hMC, hGEC, and hPodocytes with incorporated VEGF‐A coated fibers in spider silk‐based hydrogels. Imaged after one day and seven days of culture. Scale bars: Bright‐field: 1,000 µm and live/dead: 300 µm. (D) Bright‐field (left panels) and live (green)/dead (red) fluorescence staining (right panels) of 3D glomerular co‐culture of hMC, hGEC, and hPodocytes in spider silk collagen IV composite hydrogels. Imaged after one day and seven days of culture. Scale bars: Bright‐field: 1,000 µm and live/dead: 300 µm. (E) Light‐sheet microscopy image (after 14 days) of 3D glomerular co‐culture in spider silk stained with WGA (red) and Phalloidin (green). Scale bar: 500 µm. ECM: extracellular matrix, hMC: human mesangial cells, hGEC: human glomerular endothelial cells, hPodocytes: immortalized human podocytes, VEGF‐A: vascular endothelial growth factor‐A, WGA: wheat germ agglutinin. Experiments with spider silk were performed more than three times.

FIGURE 5

Mechanical characterization of spider silk hydrogel with encapsulated glomerular co‐culture. (A) Testing setup: Rheometer to perform multi‐modal mechanical testing of glued cylindrical spider silk samples in compression and torsional shear under large strains. (B) Cyclic loading behavior of cell‐laden spider silk hydrogels (D0, D7: n = 6, D1, D14: n = 7) during cyclic compression up to a maximum strain of 15% in compression (left), and during cyclic shear up to a maximum shear of 30% in torsional shear (right) over 14 days of cultivation. (C) Corresponding average maximum nominal stresses in compression (left), and torsional shear (right) over 14 days of cultivation. (D) Average normalized stress relaxation behavior of spider silk hydrogels with encapsulated glomerular co‐culture consisting of hMC, hGEC and hPodocytes at a maximum strain of 15% in compression (left), and at a maximum shear of 30% in torsional shear (right) over 14 days of cultivation. (E) Corresponding average normalized stress after 300 s relaxation in compression (left), and torsional shear (right) over 14 days of cultivation. Significances were calculated using one‐way ANOVA tests if all samples were normally distributed and Kruskal‐Wallis tests otherwise, followed by Tukey‐Kramer tests for multiple comparisons. Significance values: ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, **** p < 0.0001. hMC: human mesangial cells, hGEC: human glomerular endothelial cells, hPodocytes: immortalized human podocytes.

FIGURE 6

GelAGE as an ECM‐mimicking environment for glomerular co‐culture. (A) Schematic illustration of GelAGE hydrogel fabrication with encapsulated glomerular cells. (B) Multiphoton microscopy images of 3D glomerular co‐culture in GelAGE labeled with phalloidin after one day, seven days, and 14 days of culture. Scale bar: 200 µm. (C) Bright‐field (left panels) and live (green)/dead (red) fluorescence staining (right panels) of 3D glomerular co‐culture of hMC, hGEC, and hPodocytes in GelAGE‐based hydrogels without VEGF‐A coated fibers (left panel) and with VEGF‐A coated fibers incorporated within the hydrogel (right panel) after one day, seven days and 14 days of culture. Scale bars: Bright‐field: 1,000 µm and live/dead: 300 µm. (D) Cryo‐SEM images of GelAGE hydrogels with encapsulated glomerular co‐culture consisting of hMC, hGEC, and hPodocytes. Left picture: without cells, middle picture: with cells from the co‐culture spheroid after one day of cultivation, and right picture after seven days of cultivation. Encircled structures: cells. Arrows: Area altered by cells. Scale bar: 10 µm. ECM: extracellular matrix, GelAGE: allyl‐modified gelatin, hGEC: human glomerular endothelial cells, hMC: human mesangial cells, hPodocytes: immortalized human podocytes, VEGF‐A: vascular endothelial growth factor‐A. Experiments with GelAGE were performed more than three times.

FIGURE 7

Mechanical characterization of GelAGE hydrogel with encapsulated glomerular co‐culture. (A) Testing setup: Rheometer to perform multi‐modal mechanical testing of glued cylindrical GelAGE samples in compression, tension, and torsional shear under large strains. (B) Cyclic loading behavior of cell‐laden GelAGE hydrogels (n = 5 for each time point) during cyclic compression‐tension up to a maximum strain of 15% in compression, 2.5% in tension (left), and during cyclic shear up to a maximum shear of 30% in torsional shear (right) over 14 days of cultivation. (C) Corresponding average maximum nominal stresses in compression (left), tension (center), and torsional shear (right) over 14 days of cultivation. (D) Average normalized stress relaxation behavior of GelAGE hydrogels with encapsulated glomerular co‐culture consisting of hMC, hGEC and hPodoytes at a maximum strain of 15% in compression (left), 2.5% in tension (center), and at a maximum shear of 30% in torsional shear (right) over 14 days of cultivation. (E) Corresponding average normalized stress after 300s relaxation in compression (left), tension (center), and torsional shear (right) over 14 days of cultivation. Significances were calculated using one‐way ANOVA if all samples were normally distributed and Kruskal‐Wallis tests otherwise, followed by Tukey‐Kramer tests for multiple comparisons. Significance values: ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001.