Partner, Neighbor, Housekeeper and Dimension: 3D versus 2D Glomerular Co-Cultures Reveal Drawbacks of Currently Used Cell Culture Models

Abstract

Signaling-pathway analyses and the investigation of gene responses to different stimuli are usually performed in 2D monocultures. However, within the glomerulus, cells grow in 3D and are involved in direct and paracrine interactions with different glomerular cell types. Thus, the results from 2D monoculture experiments must be taken with caution. We cultured glomerular endothelial cells, podocytes and mesangial cells in 2D/3D monocultures and 2D/3D co-cultures and analyzed cell survival, self-assembly, gene expression, cell-cell interaction, and gene pathways using live/dead assay, time-lapse analysis, bulk-RNA sequencing, qPCR, and immunofluorescence staining. Without any need for scaffolds, 3D glomerular co-cultures self-organized into spheroids. Podocyte- and glomerular endothelial cell-specific markers and the extracellular matrix were increased in 3D co-cultures compared to 2D co-cultures. Housekeeping genes must be chosen wisely, as many genes used for the normalization of gene expression were themselves affected in 3D culture conditions. The transport of podocyte-derived VEGFA to glomerular endothelial cells confirmed intercellular crosstalk in the 3D co-culture models. The enhanced expression of genes important for glomerular function in 3D, compared to 2D, questions the reliability of currently used 2D monocultures. Hence, glomerular 3D co-cultures might be more suitable in the study of intercellular communication, disease modelling and drug screening ex vivo.

Keywords: 3D co-culture; bulk-RNA sequencing; cell culture model; cell matrix; cell–cell communication; glomerular spheroids.

Figure 1

Schematic illustration of the generation of 3D spheroids by self-assembly of the cells in agarose micro-wells.

Figure 2

Mono- and co-cultures of differentiated, conditionally immortalized human podocytes (a); primary human glomerular endothelial cells (GEC) (b); primary human mesangial cells (MC) (c); and co-culture of all three glomerular cell types (PEM) (d) in 2D (a–d) or 3D (a′–d′) in agarose micro-wells display different morphology. Survival assay of spheroids (a″–d″). Living cells appear in green fluorescence and dead cells in red. Scale bar of (a–d,a′–d′) represents 750 µm and 100 µm for (a″–d″).

Figure 3

Self-organization of the 3D glomerular co-culture. (A) Self-assembly of 3D glomerular co-cultures: (a) HE stained section of the 3D glomerular co-culture. Scale bar represents 50 µm; (b) top-down view onto a three-dimensionally reconstructed multiphoton microscopy image stack of the glomerular 3D co-culture. Podocytes are labeled in blue, glomerular endothelial cells in magenta and mesangial cells in green. Scale bar represents 50 µm. An animated video is provided in Supplementary Video S2; and (c) confocal image of 3D glomerular co-culture. Podocytes (blue) cluster in the center and glomerular tdTomato-Farnesyl endothelial reporter cells (red) developed a reticular structure with mesangial cells (green) in-between. The fluorescent cell types are shown individually and as an overlay image. Scale bar represents 200 µm. (d) immunofluorescent picture shows that multiple spheroids can be produced at the same time in a high throughput fashion. Scale bar represents 500 µm. (B) Ultrastructural characterization of the cellular morphology of the 3D glomerular co-culture by SEM: (a) overview image of the whole 3D spheroid. Scale bar represents 50 µm; (b) glomerular endothelial cell with fenestrae-like pores (red boxes). Scale bar represents 2 µm; (c) cell–cell interactions of protrusions connecting cells (white arrowheads). Scale bar represents 2 µm; and (d) microvilli-like protrusions located on the cell surfaces (white arrowheads). Scale bar represents 2 µm.

Figure 3

Self-organization of the 3D glomerular co-culture. (A) Self-assembly of 3D glomerular co-cultures: (a) HE stained section of the 3D glomerular co-culture. Scale bar represents 50 µm; (b) top-down view onto a three-dimensionally reconstructed multiphoton microscopy image stack of the glomerular 3D co-culture. Podocytes are labeled in blue, glomerular endothelial cells in magenta and mesangial cells in green. Scale bar represents 50 µm. An animated video is provided in Supplementary Video S2; and (c) confocal image of 3D glomerular co-culture. Podocytes (blue) cluster in the center and glomerular tdTomato-Farnesyl endothelial reporter cells (red) developed a reticular structure with mesangial cells (green) in-between. The fluorescent cell types are shown individually and as an overlay image. Scale bar represents 200 µm. (d) immunofluorescent picture shows that multiple spheroids can be produced at the same time in a high throughput fashion. Scale bar represents 500 µm. (B) Ultrastructural characterization of the cellular morphology of the 3D glomerular co-culture by SEM: (a) overview image of the whole 3D spheroid. Scale bar represents 50 µm; (b) glomerular endothelial cell with fenestrae-like pores (red boxes). Scale bar represents 2 µm; (c) cell–cell interactions of protrusions connecting cells (white arrowheads). Scale bar represents 2 µm; and (d) microvilli-like protrusions located on the cell surfaces (white arrowheads). Scale bar represents 2 µm.

Figure 4

Comparison of transcriptomic data from glomerular monocultures and co-cultures between 2D and 3D culture conditions displays differences regarding cell type-specific marker, extracellular matrix and signaling components: (a) principal component analysis of glomerular cell monoculture and co-culture in 2D and 3D. Each condition was analyzed as a biological triplicate. Glomerular endothelial cell (GEC), mesangial cells (MC), co-culture of differentiated conditionally immortalized human podocytes, mesangial cells and glomerular endothelial cells (PEM); and (b–d) heatmaps representing gene expression of extracellular matrix components, cell type-specific markers, adhesion and signaling molecules of glomerular endothelial cell (GEC) monoculture (b), mesangial cell (MC) monoculture (c), and glomerular co-culture of podocytes, mesangial cells and glomerular endothelial cells (PEM) (d) in 2D and 3D. Each condition was analyzed as a biological triplicate.

Figure 5

Bulk-RNA sequencing of glomerular monocultures and co-cultures in 2D and 3D exhibits differences in genes involved in hypoxia and housekeeping genes: (a) heatmap demonstrating expression of genes involved in hypoxia response in 2D and 3D monoculture of glomerular endothelial cells (GEC) and mesangial cells (MC) as well as 2D and 3D co-cultures of GEC, MC and differentiated, conditionally immortalized human podocytes (PEM); and (b) heatmap representing alterations of typically used housekeeping genes in 2D compared to 3D monoculture of GEC and MC as well as 2D and 3D glomerular co-cultures.

Figure 6

Pathway analysis of bulk-RNA sequencing data from 2D and 3D glomerular cultures. Pathways analysis was performed for monoculture of glomerular endothelial cells (GEC) (a), monoculture of mesangial cells (MC) (b) and co-culture of podocytes, GECs and MCs (c) in 3D or rather 2D. Pathways that were more active in 3D cultures and less active in 2D cultures are colored in light blue. In contrast, pathways that were more active in 2D cultures and less active in 3D cultures were colored in dark blue.

Figure 6

Pathway analysis of bulk-RNA sequencing data from 2D and 3D glomerular cultures. Pathways analysis was performed for monoculture of glomerular endothelial cells (GEC) (a), monoculture of mesangial cells (MC) (b) and co-culture of podocytes, GECs and MCs (c) in 3D or rather 2D. Pathways that were more active in 3D cultures and less active in 2D cultures are colored in light blue. In contrast, pathways that were more active in 2D cultures and less active in 3D cultures were colored in dark blue.

Figure 7

qPCR for cell type-specific markers, extracellular matrix and cellular response to hypoxia of 2D and 3D glomerular cultures. (A) qPCR for cell type-specific maker expression and cellular signaling: (a) comparison of glomerular endothelial cell (GEC) markers in 3D versus 2D monoculture of GECs; (b) Comparison of mRNA expression of mesangial cell (MC) markers in 3D versus 2D monoculture of MCs; (c) comparison of mRNA expression of podocyte, MC markers and GEC markers in 3D versus 2D glomerular co-cultures of podocytes, GECs and MCs (PEM). (B) qPCR for expression of extracellular matrix: (a) comparison of 3D versus 2D monoculture of GECs; (b) comparison of mRNA expression of extracellular matrix in 3D versus 2D monoculture of MCs; (c) comparison of mRNA expression of extracellular matrix in 3D versus 2D glomerular co-cultures. (C) qPCR for expression of hypoxia-related genes: (a) comparison between 3D and 2D monoculture of a: GECs and (b) mesangial cells; and (c) comparison of 3D versus 2D glomerular co-cultures. n = 3 for each experiment. Data are given as mean ± SEM, * p < 0.05, ** p < 0.01.

Figure 8

Immunofluorescence staining showing the production of extracellular matrix and the localization of podocyte-specific and glomerular endothelial cell-specific marker proteins in 3D glomerular co-cultures. (A) 3D reconstructions of multiphoton microscopy image-stacks of glomerular spheroids showing collagen IV (red) and laminin (green) expression in glomerular endothelial cell (GEC) monoculture spheroids, mesangial cell (MC) monoculture spheroids and glomerular co-culture (PEM). Scale bar represents 50 µm. (B) Confocal microscopy images of glomerular co-culture spheroids (PEM) demonstrating CD31 (green), synaptopodin (red) and merged images. Nuclear staining is shown in blue. Scale bar represents 50 µm.

Figure 9

Podocyte-derived VEGFA is transported to glomerular endothelial cells in 3D glomerular co-cultures. Confocal microscopy of 3D glomerular co-culture displays green fluorescent podocyte-derived VEGFA, mesangial cells labeled in blue with eBioscience™ Cell Proliferation Dye eFluor™ 450 and red fluorescent tdTomato-Farnesyl glomerular endothelial cell reporter cells. Podocytes were electroporated with a plasmid carrying human VEGFA sequence coupled with the GFP sequence, resulting in the expression of green, fluorescent VEGFA prior to co-culture. Scale bar 50 µm.