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Cellular extrusion bioprinting improves kidney organoid reproducibility and conformation


Directed differentiation of human pluripotent stem cells to kidney organoids brings the prospect of drug screening, disease modelling and the generation of tissue for renal replacement. Currently, these applications are hampered by organoid variability, nephron immaturity, low throughput and limited scale. Here, we apply extrusion-based three-dimensional cellular bioprinting to deliver rapid and high-throughput generation of kidney organoids with highly reproducible cell number and viability. We demonstrate that manual organoid generation can be replaced by 6- or 96-well organoid bioprinting and evaluate the relative toxicity of aminoglycosides as a proof of concept for drug testing. In addition, three-dimensional bioprinting enables precise manipulation of biophysical properties, including organoid size, cell number and conformation, with modification of organoid conformation substantially increasing nephron yield per starting cell number. This facilitates the manufacture of uniformly patterned kidney tissue sheets with functional proximal tubular segments. Hence, automated extrusion-based bioprinting for kidney organoid production delivers improvements in throughput, quality control, scale and structure, facilitating in vitro and in vivo applications of stem cell-derived human kidney tissue.

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Fig. 1: Generation of highly reproducible iPSC-derived kidney organoids by extrusion-based cellular bioprinting of day 7 intermediate mesoderm cell paste.
Fig. 2: Application of bioprinted organoids for compound testing in 96-well format.
Fig. 3: Use of extrusion bioprinting to alter organoid conformation.
Fig. 4: Changing organoid conformation reduces unpatterned tissue and increases nephron number and maturation (also refer to Extended Data Fig. 4).
Fig. 5: scRNAseq comparison of manual organoids, bioprinted R0 ‘dots’ and bioprinted R40 ‘lines’.
Fig. 6: Generation of a kidney tissue patch using 3D extrusion cellular bioprinting.

Data availability

All transcriptional profiling datasets have been submitted to GEO. These include scRNAseq from manual and two bioprinted organoid conformations (GEO GSE152014) and bulk-RNAseq data comparing bioprinted organoids of different conformations (GEO GSE138733). The image data used for quantification of morphology in different bioprinted organoid confirmations is available at

Code availability

Image and single cell RNAseq analysis scripts are available at


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Download references


We thank M. Le Moing and the Murdoch Children’s Research Institute Translational Genomics Unit for 10x single cell and hash-tag oligo library preparation and sequencing and bulk-RNAseq. We thank M. Burton and the Murdoch Children’s Research Institute Microscopy Core for imaging support. M.H.L. is a Senior Principal Research Fellow of the National Health and Medical Research Council, Australia (APP1136085). This work was supported by Organovo Inc., the Methuselah Foundation, California’s Stem Cell Agency (EDUC2-08388), NHMRC (GNT1100970, GNT1098654), the National Institutes of Health (UH3DK107344) and a Medical Research Future Fund Kidney Disease Team grant.

Author information

Authors and Affiliations



K.L., J.M.V., J.W.H., B.S., S.P., S.C.P., A.E.C. and M.H.L. contributed to the experimental design and planning. K.L., J.M.V., J.W.H., A.C., K.B., D.A., P.X.E., S.W., S.H., K.S.T., F.L. and L.J.H. developed methods and reagents, and performed and analysed experiments. All authors contributed to the interpretation of data. K.L., J.M.V., J.W.H., A.E.C. and M.H.L. contributed to the writing of the manuscript.

Corresponding author

Correspondence to Melissa H. Little.

Ethics declarations

Competing interests

M.H.L. is an inventor on a patent related to kidney organoid generation. Access to the bioprinter was facilitated by the Methuselah Foundation. M.H.L. received contract research funding from Organovo Inc.

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Extended data

Extended Data Fig. 1 Histology of bioprinted kidney organoids.

a. Histological cross section of an entire day 7 + 18 bioprinted kidney organoid showing clear evidence of an interconnecting epithelium (arrowheads) from which nephrons arise. b. Immunostaining of a bioprinted kidney organoid section showing a GATA3 + ECAD + connecting segment / collecting duct with multiple attached ECAD + GATA3- nephrons. c. Immunostaining of bioprinted kidney organoid section showing ECAD + nephrons attached to MAFB + glomeruli. d. Brightfield, histological and immunofluorescence comparisons of kidney organoids generated manually (5 × 105 cells per organoid), using dry cell paste controlled for organoid diameter versus dry cell paste controlled for cell number versus wet cell paste. All image panels are representative of at least n = 3 organoids from multiple experiments.

Extended Data Fig. 2 Patterning of kidney structures in bioprinted organoids generated from varying starting cell numbers.

a. Immunofluorescence of organoids from a single starting differentiation used to generate manual organoids (5 × 105 cells) versus bioprinted organoids generated from as few as 4,000 cells. Representative images from n = 3 organoids stained. Scale bars represent 200 µm. b. Differentiation timecourse of bioprinted organoids generated using the MAFBmTagBFP2 reporter iPSC line. c. MAFBmTagBFP2 bioprinted organoids on the same Transwell filter with 4 K, 50 K or 100 K of cells per organoid showing fluorescence reporter imaging (blue) and staining for differentiation (ECAD,green; LTL, blue; GATA3, red; NPHS1, purple). Images are representative of at least n = 3 organoids. Scale bars represent 200 µm. d. MAFBmTagBFP2 bioprinted organoids on the same Transwell filter all generated using 100 K of cells per organoid showing live fluorescence imaging (blue) and staining for differentiation (ECAD,green; LTL, blue; GATA3, red; NPHS1, purple). Scale bars represent 200 µm. Representative wells from at least n = 3 are shown, with representative stained organoids alongside.

Extended Data Fig. 3 Quantification of bead density and MAFBmTagBFP2 reporter signal in organoids with varied conformations.

a. Representative image of fluorescent bead signal (greyscale) at D7 + 0 across an entire print pattern showing all 5 conformations, from left to right: ratio 0 (3 replicates), ratio 40, ratio 30, ratio 20, ratio 10. b. Composite image of each conformation at D7 + 12 showing mTagBFP2 reporter expression (cyan) and bead signal (red). Note images are placed on a black background. Scale bar is 1 mm for A and B. c. Quantification of total organoid area (refer to Methods) and mTagBFP2 area in replicate organoids (compare to Fig. 3g). d. Table of organoid numbers by replicate plate and ratio used for quantification in C and Fig. 3g. e. Example of 9 replicate organoids produced using ratio 20. Organoids are consistent between 3 organoids from separate wells on each plate, and between plates. f. Representative images (from total n = 27 organoids from 2 independent experiments) of sparse labelling with CellTrace Far Red dye to quantify organoid height at D7 + 0 (Fig. 3d). XY and orthogonal view are shown. g. Schematic of the scoring method used for quantification, described fully in Supplementary Methods.

Extended Data Fig. 4 MAFBmTagBFP2 reporter expression in organoids correlates to total nephron number.

a,b. Examples of low resolution, high throughput imaging used to quantify MAFB+ area as a proxy for nephron volume in organoids. Brightfield and MAFBmTagBFP2 signal was captured for each organoid using a low NA 4x objective with a spinning disk system, enabling fast capture of many samples. With a large axial depth of field, these images capture the majority of signal within each organoid in a single plane. Given the similarity in thickness (E,F, Fig. 3) this planar area is approximately proportional to total MAFB + glomerular volume and hence correlates to nephron number. A portion of an example image used for quantification of R0 (a) and R40 (b) organoids at D7 + 12 is shown. Note R40 organoids are much longer and were captured by stitching multiple image fields. Only a small portion of the organoid is shown. c,d. Samples were fixed and stained at D7 + 12 for MAFBmTagBFP2 reporter (Cyan), mature podocyte marker NPHS1 (Red) and atypical protein kinase C (aPKC, Green), a marker of the apical cell membrane. Each nephron consists of a rounded glomerular structure containing podocytes (examples highlighted by white arrows) connected to other tubular segments that are marked by aPKC but lack NPHS1. Nephrons are seen throughout the field and are packed together so that individual nephrons cannot be easily separated visually. MAFBmTagBFP2 reporter is expressed specifically in NPHS1 expressing podocytes, but is absent from other nephron segments (aPKC+, NPHS1 regions) or from other cell types. Images are maximum projections (50 µm span). e,f. Both conditions have a similar axial morphology in nephron-containing regions when viewed as an orthogonal slice (ie along the imaging Z-axis). A single orthogonal slice rendered from a 3D stack is shown. g,h. Cropped high-resolution fields showing a single glomerulus for each condition confirm co-expression of MAFBmtagBFP2 reporter and NPHS1 in podocytes. A single confocal slice is shown. All images are representative of at least n = 3 stained samples.

Extended Data Fig. 5 Quantification of large image data sets associated with organoids used for single cell RNA seq.

Line organoids are approximately 12 mm long. a. Representative images from 3 separate wells across replicates and conditions. b. Quantification of MAFB-mTagBFP2 reporter area by set and condition. Data is as in Fig. 5b, but here is separated by set. c. Quantification of GATA3-mCherry reporter area. Note that Y-axis scale differs between B and c, as GATA3 area represents a substantially smaller proportion of the organoid in most cases. d. GATA3 area as a proportion of total measured reporter area (MAFB + GATA3), highlighting a shift in R0 toward a more distalised fate. E. The total number of individual organoids used for quantification, by set and condition.

Extended Data Fig. 6 Analysis of single cell RNA datasets.

a. Variability within the datasets represented as a UMAP plot, coloured by transcriptional cluster, predicted cell cycle phase, main cell type and organoid conformation (clockwise from top left). b. Marker genes of main cells type, WT1 and PAX2 (nephron), PDGFRA (stroma) and SOX17 (endothelial). c. Proportion of each cell type in replicate conditions. P value (one-way ANOVA) indicated if p < 0.2. D. UMAP representation of nephron cells after re-transformation and clustering at higher resolution. Plots are coloured by transcriptional cluster, predicted cell cycle phase and organoid conformation. Cluster identities are stated. e. Marker genes identifying each cluster: GATA3 (distal), HNF1B (pre-tubule), CUBN (proximal), HNF4A (proximal), FOXC2 (pre-podocyte), MAFB (pre-podocyte / podocyte), PODXL (podocyte), SIX2 (progenitor), EYA1 (progenitor). f. Stromal UMAP coloured by transcriptional cluster, predicted cell cycle phase and organoid conformation (top to bottom). g. Markers of specific stromal clusters; SIX2, LYPD1, FOXC2, HOXA11 (Cluster 3, nephron progenitor-like), WNT5A, LHX9 (Cluster 7) and ZIC1 and ZIC4 (Cluster 10). h. Heatmap of scaled log counts per million of pseudo bulk counts from scRNAseq sets for the top 100 most significantly expressed genes identified in bulk RNAseq analysis (Fig. 4). Each column represents a single cluster from a single replicate (for example R40, Nephron, Set 1). Hierarchical clustering of the limited gene set indicates that bulk-RNAseq changes are largely driven by changes in the nephrons and endothelial cells.

Extended Data Fig. 7 The spatial distribution of stromal markers by wholemount immunoflouresence.

a–c. Immunofluorescence staining for markers of organoid stromal populations based on scRNA profiling. R0 organoids consist of a nephron containing area (Nephrons), a central role (Core) where nephrons are largely absent, and a thin edge (Thin edge) of monolayer cells that are typically not observed in brightfield imaging. R40 line organoids are primarily composed of a dense nephron-containing region and a thin monolayer edge, with no central core. Stromal population markers (A) MEIS1/2/3, (B) SIX1 and (C) SOX9 are present in the areas surrounding nephrons, and within the thin monolayer sheet at the edge of each organoid, but are largely absent from the central core of R0 organoids. Representative images from n = 3 organoids stained per condition are shown. Images are maximum projections spanning the full volume of the organoid. d. UMAP plots representing stromal cells in scRNA datasets, colour coded to show expression of MEIS1, MEIS2, SIX1 and SOX9. These combined markers include most of the cells in the dataset, suggesting that the absence of staining in the central core observed in (E) may indicate low overall cellularity in that region.

Extended Data Fig. 8 Direct comparison between kidney organoids and human fetal kidney confirms improved maturation of proximal tubules within R40 bioprinted lines.

a. UMAP plots comparing transcriptional identity based on unbiased clustering in Seurat (left) and prediction using the scPred method where cells are according to their similarity to a human fetal kidney (HFK) dataset (right). Identity assignment is based the most similar human fetal kidney cell type. b. The proportion of cells assigned to each cell type identity across replicates. Points show individual replicate values colour coded by replicate barcode (where HTO-1 is Set 1). Bars show SEM. P-values based on one-way ANOVA indicate a significant difference in the number of cells predicted to be Pre-Pod cells, with greatest abundance in the R40 datasets. Bioprinted conditions (R40 and R0) have more cells predicted to be podocytes, and less distal and pre-tubule cells. However, these changes were not significant. These results support the trends presented in Fig. 5. c. The distribution of maximum similarity scores for the classification of each cell across conformations, plotted by cell type predicted. Most cells show a high similarity to the predicted fetal kidney cell type. d. Genes identified as significantly increased in R40 versus Manual organoids (SLC51B, FABP3 and SULT1E1) are expressed in the mature proximal tubule cells of human fetal kidney, confirming their association with a more mature cell type. A gene that was significantly decreased in R40 vs Manual organoids (SPP1) is expressed selectively in less mature cell types, supporting increased maturity in R40 proximal cells. UMAP shows transcriptional identity in human fetal kidney data. Top left plot is colour coded by human fetal kidney cell types specific to developing (renal vesicle and comma shaped body [RV_CSB], blue; proximal early nephron [PEN], red) and mature proximal tubule (PT, green). Lower left plot shows a ‘dot plot’ style representation of selected gene where size indicates the percentage of HFK cells expressing the gene and colour indicates normalised expression level. Normalised expression of each gene per cell is indicated on individual UMAP plots where expression is colour coded.

Supplementary information

Supplementary Information

Tables 1 and 6, Methods and references.

Reporting Summary

Supplementary Data 1

Differentially expressed genes between conditions, within clusters for nephron and stromal subsets of scRNA data. Data for each comparison are contained within separate tabs, with a guide to set naming in the first tab.

Supplementary Table 2

Differentially expressed genes identified between ratio 40 and ratio 0 organoids, identified in bulk-RNAseq. Positive log fold-change indicates increased expression in R40

Supplementary Table 3

Whole dataset cluster markers for integrated scRNAseq dataset

Supplementary Table 4

Nephron cluster markers for integrated scRNAseq dataset

Supplemetnary Table 5

Stromal cluster markers for integrated scRNAseq dataset

Supplementary Video 1

Novogen 3D bioprinter generating kidney organoids.

Supplementary Video 2

3D rendering of a bioprinted dot organoid from Fig. 4d.

Supplementary Video 2

3D rendering of a bioprinted line organoid from Fig. 4..

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Lawlor, K.T., Vanslambrouck, J.M., Higgins, J.W. et al. Cellular extrusion bioprinting improves kidney organoid reproducibility and conformation. Nat. Mater. 20, 260–271 (2021).

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