Biotech

Scalable production of uniform and mature organoids in a 3D geometrically-engineered permeable membrane

Time Stamp: October 30, 2024 8:00 PM
Source Node: 707154

Design and fabrication of 3D geometrically-engineered, permeable membrane for organoid culture platform

To generate organoids that are not only uniform but also mature at a scalable level, we first aimed to establish an organoid culture platform. This platform offers both geometrical constraints for precise organoid regulation and an unrestricted supply of soluble factors, ensuring the efficient diffusion and exchange of nutrients, growth factors, and oxygen (Fig. 1a). We named this platform UniMat (Uniform and Mature organoid culture platform), and developed it based on three primary design criteria:

**Fig. 1: Development of UniMat for uniform and mature organoid culture.**

(1) UniMat should efficiently partition individual organoids, providing geometrical constraints that ensure organoid uniformity;

(2) The individual microwells in UniMat should promote cell aggregation, which is essential for the initial formation of cohesive 3D aggregates crucial to organoid development;

(3) UniMat should possess a porous structure, creating a permeable environment that facilitates an unrestricted supply of soluble factors necessary for organoid maturation.

To realize this concept, we engineered a flat nanofiber (NF) membrane into a 3D permeable microwell array, maintaining a porous membrane structure to ensure high permeability to both gases and solutes.

To fabricate the UniMat, we employed the electrospinning process, followed by a subsequent matched-mold thermoforming process, based on our previous work²² (Fig. 1b). In this study, we developed a NF membrane composed of polycaprolactone (PCL) and Pluronic F108 (PF108) NFs. The selection of PCL was based on its wide use in electrospinning and excellent biocompatibility^23,24. PF108, an amphiphilic synthetic polymer, was incorporated to increase the hydrophilicity of the membrane^25,26 (Supplementary Fig. 1a). This hydrophilic membrane reduced light scattering in a liquid cell culture medium by minimizing differences in refractive indices between the NF membrane and the surrounding medium, thus enhancing light transmittance (Supplementary Fig. 1b), which improves cell observation²⁷. The manufactured NF membranes exhibited consistent physical properties, including membrane thickness and NF diameter distribution, across different experimental batches under our defined conditions (Supplementary Figs. 1c, d), paving the way for the standardized production of reliable and uniform UniMat. The matched-mold thermoforming process allowed for precise microstructuring of a thin and flat NF membrane, ranging in thickness from 50 μm to 150 μm, into a 3D shape. This results in improved shape flexibility and replication fidelity without compromising the porous structure of the NF membrane. Leveraging this capability, we successfully engineered an electrospun free-standing NF membrane with a thickness of 50 μm into a 3D microwell array-structured membrane, resulting in a UniMat. Subsequently, the UniMat was incorporated into the bottom opening of a custom-made 24-well insert wall in a free-standing configuration, allowing for compatibility with standard cell culture plates (Fig. 1c). The successful fabrication of UniMat, with its 3D microwell array-structured NF membrane, was confirmed using scanning electron microscopy (SEM) analysis (Fig. 1d).

In this study, we fabricated the UniMat by microstructuring an electrospun NF membrane to form a V-shaped microwell array (Fig. 1e), promoting the collection and aggregation of seeded cells at the bottom regions of microwells. The V-shaped design enhances cell confinement owing to its inclined walls, ensuring a more defined region for cell placement within every microwell. Importantly, by merely modifying the mold geometry used in the matched-mold thermoforming process, the size of the microwells could be tuned to accommodate the growth of organoids of various sizes. We fabricated three different types of UniMats, each with distinct width and depth dimensions: UniMat400 (width: 400 μm, depth: 343 μm), UniMat600 (width: 600 μm, depth: 517 μm), and UniMat800 (width: 800 μm, depth: 691 μm) (Supplementary Fig. 2). The microwell in the UniMat preserved a porous architecture similar to the original NF membrane (Fig. 1f), and possessed a thin wall thickness of approximately 30 μm (Fig. 1g). These characteristics enabled the UniMat to display a high permeability for molecules of varying molecular weights, which was more than twice that of a conventional PET porous membrane (Fig. 1h).

To validate the capability of the UniMat to generate uniform and mature organoids, we selected kidney organoids as a representative lineage of organs. Kidney organoids are complex and hold significant biomedical interest because of their potential applications in disease modeling and drug testing. For the differentiation of kidney organoids, we optimized the Morizane protocol²⁸, to ensure the consistent generation of kidney organoids from human induced pluripotent stem cells (hiPSCs) in 3D culture platforms (Supplementary Notes 1 and 2 and Supplementary Figs. 3 and 4). This refined protocol was then seamlessly incorporated into the UniMat, where nephron progenitor cells (NPCs) derived from hiPSCs were seeded onto the UniMat400, which was coated with a thin layer of agarose hydrogel to enhance low attachment condition at the bottom of UniMat, on day 9. The subsequent differentiation processes within the UniMat400 led to the formation of kidney organoids by day 24-26 (Fig. 1i). Notably, the sharp angle and narrow base of the NF microwell guided cells towards the center of each microwell, thereby enhancing cell-to-cell contact and promoting aggregate formation. This strategic approach successfully addressed the challenges encountered with the previously utilized U-shaped configuration²², where evenly distributed NPCs along the U-well curve resulted in low aggregation efficiency, thus restricting the ability to achieve stable and consistent aggregates crucial for reliably producing uniform organoids (Supplementary Fig. 5). Upon differentiation, the majority of kidney organoids exhibited nephron-like structures, including podocytes (PODXL⁺), proximal tubules (LTL⁺), and distal tubules (CDH1⁺), within the UniMat (Fig. 1j). As a result, approximately 87 ± 5% of all pretubular aggregates were successfully developed into nephron-like kidney organoids, achieving around 5 organoids per mm² within the UniMat400.

UniMat improves uniformity of organoids

Having demonstrated the successful production of kidney organoids in the UniMat400, we utilized it as a model to explore its potential to reduce the variability of organoids. We assessed the morphological consistency in size and structure of kidney organoids in the UniMat platforms (Fig. 2a), which provide geometric guidance from the NPC stage. This was further compared to the uniformity observed in organoids that were exclusively generated on a plate coated with Geltrex hydrogel (hereinafter referred to as the hydrogel layer), without any physical constraints (Fig. 2a). Notably, kidney organoids cultured in each UniMat demonstrated a more consistent size distribution compared to those on the hydrogel layer (Fig. 2b and Supplementary Table 1). Moreover, the size of organoids, including diameter and height, appeared to be regulated through the UniMat-based culture, correlating with the size of the V-shaped microwells in the UniMat (Fig. 2b and Supplementary Fig. 6).

**Fig. 2: Generation of uniform kidney organoids in UniMat.**

To delve deeper into the mechanism by which UniMat controls the organoid size, we examined the initial aggregate size, which is known to influence organogenesis and organoid formation. Given the distinct sizes and arrays of V-shaped microwells in each of the UniMat400, UniMat600, and UniMat800 (Supplementary Fig. 7a), we noted that within 6 h of seeding, aggregates were formed in sizes corresponding to each specific UniMat (Supplementary Fig. 7b). These measured sizes closely matched with the estimated sizes based on the geometrical data of the microwells and the average number of cell sedimentations per microwell in each UniMat (Supplementary Note 3 and Supplementary Fig. 7c-f). Our findings suggest that by adjusting both the microwell size and the cell seeding density in the UniMat, the initial aggregate size can be effectively modulated. Notably, the size variances among the organoids grown in different UniMats were consistent with the trends observed in the initial aggregate sizes, underscoring the pivotal role of UniMat in determining organoid size.

Having established that the UniMat can effectively regulate organoid size, we further investigated the structural variability of kidney organoids. Using confocal imaging rendered with the Imaris 3D surface rendering software, we quantified the presence of glomerular podocytes (PODXL⁺), proximal tubules (LTL⁺), and distal tubules (CDH1⁺) in individual kidney organoids (Fig. 2c). Subsequently, we evaluated the distribution of these key structures by calculating the fraction of each structure’s volume relative to the total volume of each organoid. Notably, while the average fractions of each structure were comparable between organoids cultured on the hydrogel layer and those in the UniMat, kidney organoids cultured in the UniMat exhibited less structural variability compared to those cultured on the hydrogel layer (Fig. 2d and Supplementary Table 2). These findings indicate that the geometrical constraints provided by the UniMat promote the production of organoids with more uniform sizes and structures. Especially in the UniMat800, we observed a greater reduction in structural variability, with a 4-fold decrease in podocytes, a 5-fold decrease in proximal tubules, and a 4-fold decrease in distal tubules, as compared to the organoids cultured on the hydrogel layer (Supplementary Table 2). Based on our data, the UniMat800-cultured kidney organoids exhibited greater uniformity and they had an average size comparable to those cultured on the hydrogel layer. In this regard, UniMat800 was selected as the representative UniMat for further experiments.

To further assess the potential of UniMat in improving the functional uniformity of organoids, we randomly selected 10 organoids from both the hydrogel layer and UniMat. We then performed a quantitative real-time polymerase chain reaction (qRT-PCR) analysis to quantify the expression levels of key genes associated with podocytes (PODXL), proximal tubules (SLC34A1), and distal tubules (CDH1). Intriguingly, in accordance with the reduced structural variability observed in UniMat, the organoids cultured in UniMat exhibited less gene expression heterogeneity compared to those grown on the hydrogel layer (Fig. 2e and Supplementary Table 3). These results underscore the UniMat’s ability to enhance functional uniformity in organoids, demonstrating its potential to yield more precise and consistent experimental results. Moreover, UniMat showed a capacity to improve the uniformity of organoid formation efficiency, which is quantified as the number of organoids formed per mm². In determining this efficiency, the variability in the UniMat-based culture was substantially lower than in the hydrogel layer-based culture (Fig. 2f and Supplementary Table 4). Notably, the formation efficiency was relatively consistent across three independent batches for the UniMat-based cultures (Fig. 2f and Supplementary Table 4). These findings highlight the reproducibility of organoid formation through the UniMat, suggesting that the UniMat provides a consistent and reliable platform for generating uniform organoids in a scalable manner.

UniMat enhances maturity of organoids

After examining organoid uniformity, we investigated if the utilization of the permeable UniMat could promote the maturation of organoids, thereby addressing the limitations associated with conventional impermeable microwell platforms. For this experiment, we employed the AggreWell™800 Microwell Plate (hereinafter referred to as AggreWell), as a representative conventional microwell platform, with a width of 800 µm, similar to the dimensions of the UniMat used in this study. Confocal microscopic analysis revealed the formation of kidney organoids with nephron-like structures, including glomerular podocytes (PODXL⁺), proximal tubules (LTL⁺), and distal tubules (CDH1⁺), in both the AggreWell and UniMat (Fig. 3a and Supplementary Fig. 8a). However structural distribution differed between the two platforms. Specifically, the UniMat-cultured organoids had fewer podocytes but more proximal and distal tubule structures compared to AggreWell-cultured organoids (Fig. 3b). Despite the lower podocyte distribution in UniMat, these podocytes in the UniMat displayed mature characteristics with the basally localized expression of ZO-1 at the edge of cell-cell joints (Fig. 3c), which is a marker associated with tight junction observed within podocyte slit diaphragms in the native kidney tissue²⁹ and mature kidney organoids³⁰. qRT-PCR analysis also highlighted the upregulated expressions of NPHS1 and PODXL (Supplementary Fig. 8b), both essential for podocyte integrity and function, serving as markers for the slit diaphragm in glomerular filtration^31,32. This suggests that our UniMat can generate mature kidney organoids with mature glomerular cells, a contrast to those produced by the AggreWell-based culture. Furthermore, the kidney organoids cultured in UniMat exhibited morphogenesis of tubular structures, as evidenced by polarized proximal tubules with apical enrichment of the brush border marker LTL³³ (Fig. 3d). Consistent with this polarization, there was an upregulation of mRNA expression levels for ABCB1, AQP1, and SLC34A1, which are vital for the proximal tubule’s functions in solute transport and water reabsorption, essential components of the kidney’s filtration and absorption systems^34,35,36. Furthermore, UMOD, which plays a crucial role in urine concentration within the loop of Henle^37,38, and CDH1, reflecting epithelial integrity within the distal tubules³⁹, also showed increased expression levels in the kidney organoids cultured in the UniMat (Supplementary Fig. 8c). This indicates enhanced functional potential compared to the kidney organoids cultured in the AggreWell. To further examine the physiological relevance of the tubular functions of these kidney organoids, we performed an in vitro dextran uptake assay. After a 24-h exposure to 10 kDa dextran, selective uptake of dextran within LTL⁺ proximal tubules confirmed the absorptive functionality of the UniMat-cultured kidney organoids (Fig. 3e). Specifically, these kidney organoids demonstrated the ability to uptake and retain dextran within LTL⁺ proximal tubule epithelial cells (Fig. 3e), indicating their capacity for reabsorption.

**Fig. 3: Generation of mature kidney organoids in UniMat.**

Having identified that kidney organoids formed in the permeable UniMat exhibited greater maturity than those in the impermeable microwells, we next addressed the question of how the unconstrained supply of soluble factors through the 3D geometrically-engineered, permeable membrane in the UniMat influences the maturity of organoids. This question also arose in light of the limitation of conventional impermeable microwell platforms. We initially investigated whether the enhanced maturation of kidney organoids in UniMat was due to its permeable environment affecting the survival of NPCs. Viability tests on day 11 showed no significant difference in NPC survival rates between the UniMat and AggreWell (Supplementary Fig. 9). This led us to conclude that the mature kidney organoids in UniMat result from the facilitated supply of soluble factors during NPC differentiation, rather than differences in survival of NPC aggregates. To further explore the impact of UniMat’s permeability on organoid maturity, we fabricated two different UniMat variants with distinct permeability characteristics. We achieved these characteristics by strategically blocking the pores of the NF membrane through precise adjustments of the matched-mold thermoforming process conditions (Fig. 3f). One variant, named UniMat-LP, was designed to have a lower permeability than the original UniMat, resembling the permeability of a conventional PET membrane (Fig. 3g). The second variant, UniMat-ZP, exhibited zero permeability (Fig. 3g). These modifications allowed for controlled studies on the influence of different levels of UniMat permeability on organoid maturation. Our spatiotemporal numerical simulations predicted active glucose diffusion around a single kidney organoid situated in a microwell of the original UniMat, supplied from the culture medium through its permeable nanofibrous wall within 72 h (Fig. 3h). However, with the reduced permeability, the glucose diffusion level around the kidney organoid decreased, as observed in both UniMat-LP and UniMat-ZP (Fig. 3h). This glucose diffusion simulation highlighted the effectiveness of UniMat’s permeability in facilitating nutrient supply to organoids. Notably, in concordance with the simulation results, mRNA expression levels of NPHS1, PODXL (podocyte), ABCB1, AQP1, SLC34A1 (proximal tubule), UMOD (loop of Henle), and CDH1 (distal tubule) increased in response to the enhanced permeability (Fig. 3i). Therefore, our findings indicated that the unconstrained supply of soluble factors within the UniMat, facilitated by high permeability through the 3D geometrically-engineered, permeable membrane, enables the generation of mature kidney organoids.

UniMat enhances vascularization of organoids

Engineering vascularization of organoids stands as one of the primary objectives in ongoing research efforts to advance organoid technology⁴⁰. In kidney development, vascularization plays a crucial role and is closely linked to the maturation of other cell types. Specifically, the differentiation of endothelial cells in the glomerulus is largely influenced by podocytes-produced VEGFA, which is crucial for attracting endothelial precursors to the glomerulus, a key step in the development of glomerular endothelial cells⁴¹. In turn, endothelial cells secrete PDGFRβ, a factor essential for the glomerular filtration function, which attracts mesangial cells⁴². In a previous study, researchers transplanted kidney organoids generated in the impermeable microwell platform into mice to promote vascularization¹⁵. Our study aimed to determine if our UniMat could promote in vitro vascularization in kidney organoids without relying on an animal host. We observed an increase in vascularization in the kidney organoids when cultured in the UniMat, as evidenced by the increased presence of renal vasculature (PECAM1⁺) (Fig. 4a). To quantify this vascularization, we evaluated confocal z-stack images of kidney organoids using the AngioTool⁴³ (Supplementary Fig. 10a). The vessel percent area of PECAM⁺ vasculature in the UniMat-cultured organoids exhibited an increase of over two-fold compared to the AggreWell-cultured organoids (Fig. 4b). Additionally, we found that the PECAM⁺ vasculature of UniMat-cultured organoids had more than a two-fold increase in both junctional density (i.e., branch points per unit area) and average vessel length (i.e., interjunctional distance) compared to the AggreWell-cultured organoids (Fig. 4b). qRT-PCR analysis further confirmed that UniMat-cultured kidney organoids showed the upregulated expression of PECAM1 (Fig. 4c), a mature endothelial marker⁴⁴, indicating enhanced vascular development in these organoids. To ascertain whether the vascularization induced by a permeable environment extended to the glomerular compartments of organoids, we employed confocal imaging to quantify PODXL⁺ podocyte clusters invaded by PECAM1⁺ vascular structures in the kidney organoids cultured in both AggreWell and UniMat (Fig. 4d and Supplementary Movies 1 and 2). While PECAM1⁺ vascular invasion into the glomerular structures was rarely observed in the AggreWell-cultured organoids (Fig. 4d, e and Supplementary Movie 1), there was a noticeable increase in PECAM1⁺ vascular invasion in the UniMat culture (Fig. 4d, e and Supplementary Movie 2). Enhanced imaging at higher magnification clearly showed the PECAM⁺ vasculature penetrating the podocyte cluster (Fig. 4f). Additionally, we observed lumen-like structures formed by the invaded PECAM1⁺ cells, characterized as open areas without nuclei (DAPI^–) and surrounded by PECAM1⁺ region regions⁴⁴ (Fig. 4f). Intriguingly, our analysis using confocal 3D rendering revealed that the vascular structures in the kidney organoids cultured in the AggreWell displayed the characteristics of endothelial cell precursors⁴⁵, expressing both PECAM1⁺ and PODXL⁺, compared to the kidney organoid in UniMat (Supplementary Fig. 10b), suggesting an immature vascularization of organoids. These findings suggest that the vascular structures within the kidney organoids cultured in the UniMat exhibit greater development compared to those cultured in AggreWell.

**Fig. 4: Generation of enhanced vascularized kidney organoids in UniMat.**

Building on our results, which demonstrated enhanced vascularization in kidney organoids cultured in UniMat, we hypothesized that their functionality within the renin-angiotensin-aldosterone system (RAAS) would also be promoted in this environment^46,47. In kidney tissue, angiotensin-converting enzyme (ACE), a key regulator of RAAS, is highly expressed in the vascular endothelial cells and proximal tubules⁴⁸. ACE is essential for sodium and water homeostasis controlling blood pressure and fluid balance control in the body⁴⁸ and plays pivotal roles in normal kidney development⁴⁷. Using enzyme-linked immunosorbent assay (ELISA)⁴⁹, we measured ACE secretion levels in kidney organoids cultured in both systems (Supplementary Fig. 10c). Notably, ACE protein levels were 8-fold higher in UniMat compared to AggreWell (Supplementary Fig. 10c), suggesting that the UniMat environment, which promotes organoid vascularization, also enhances the functional characteristics of kidney organoids.

UniMat supports long-term stability and functionality of organoids

We extended our investigation to assess the long-term stability and functional integrity of kidney organoids cultured in UniMat over a 50-day period. Our analysis further focused on potential structural loss and the risk of fibrosis, major concerns in long-term cultured organoids^50,51,52. The findings revealed that organoids cultured in UniMat retained their mature nephron structures, as evidenced by the maintenance of PODXL⁺ podocytes, LTL⁺ polarized proximal tubules, and CDH1⁺ distal tubules (Supplementary Fig. 11a). Mature vascular structures, indicated by PECAM1⁺, were also maintained in UniMat-cultured organoids. Additionally, these organoids exhibited higher expression of genes encoding functional markers for podocytes (NPHS1, PODXL), proximal tubules (ABCB1, AQP1, SLC34A1), the loop of Henle (UMOD), distal tubules (CDH1), and endothelial cells (PECAM1) compared to AggreWell-cultured organoids (Supplementary Fig. 11b), demonstrating UniMat’s capability to support the stability and functionality of organoids in long-term cultivation. While α-smooth muscle actin (αSMA), a marker for myofibroblasts elevated in the interstitial areas of fibrotic kidneys⁵³, was expressed in the UniMat-cultured organoids at day 50 (Supplementary Fig. 11c), it was localized separately from nephron structures, maintaining nephron integrity (Supplementary Fig. 11d). In contrast, AggreWell-cultured organoids exhibited αSMA⁺ cells interspersed within nephron structures (Supplementary Figs. 11c, d), indicating potential structural impairment due to fibrotic changes. The low fibrotic changes in UniMat are likely due to the adequate supply of oxygen and growth factors during long-term culture, which mitigates pro-fibrotic pathways driven by cellular senescence and hypoxia^50,51,52.

Single-cell transcriptomic profiling of kidney organoids in UniMat

To investigate the comprehensive capability of UniMat, we performed single-cell RNA sequencing (scRNA-seq) using the 10X Genomics platform and analyzed the data using the Seurat R package. We compared the kidney organoids cultured in UniMat to those cultured in the AggreWell to assess the functionality of UniMat’s permeability in the maturation of kidney organoids. After implementing quality control measures, we isolated 17,412 cells from day-25 kidney organoids in AggreWell and 13,098 cells from those in UniMat. These 30,510 cells were subsequently integrated and visualized using the Uniform Manifold Approximation and Projection (UMAP). The cells were categorized into 16 distinct clusters, each of which was annotated by comparing differentially expressed genes to known markers specific for cell types (Fig. 5a). A dot plot across different clusters exhibited unique transcript expression patterns for each cluster (Fig. 5b). Notably, 11 of these clusters were identified as differentiated kidney cells: podocytes (POD; PODXL, NPHS1, and NPHS2)^54,55, early proximal tubules (EPT; SPP1 and HPN with low LRP2 and SLC3A1 expression)^54,55, proximal tubules (PT; UGT3A1, LRP2, and SLC3A1)⁵⁶, loop of Henle/distal tubules (LOH/DT; MAL, WFDC2, and EPCAM)⁵⁵, endothelial cells (EC; PECAM1, CDH5, and SOX17)⁵⁷, juxtaglomerular cells (JG; REN and PIP5K1B)⁵⁸, and five mesenchymal clusters (Mesen; COL1A1 and COL4A1)^15,55. The proportion of differentiated kidney cells in both platforms was similar, accounting for 78.70% of the cells in UniMat and 74% in AggreWell among whole cells (Fig. 5c). The remaining cells included nephron progenitor cells (NPC), defined by expression of SIX2 and EYA1, along with proliferative premature tubular cells (Tub.pre; MKI67, SPP1, and FXYD2)^54,59, proliferative mesenchymal cells (Prolif.Mesen; CENPF, COL1A1, and COL4A1)⁵⁴, and muscle cells (Muscle; MYPLF and MYOG)⁵⁵ (Fig. 5c).

We further examined the cell-type proportions in each kidney organoid from both AggreWell and UniMat (Supplementary Table 5 and Supplementary Fig. 12). These findings were then juxtaposed with a recently published scRNA-seq dataset for human and mouse nephrons with a focus on core nephron segments such as podocytes, broad proximal tubules (including early proximal tubules), and loop of Henle/distal tubules (Fig. 5d). In the AggreWell samples, podocytes constituted 35.74%, broad proximal tubules 48.58%, and loop of Henle/distal tubules 15.68% (Fig. 5d). This distribution aligns with prior findings using the Morizane protocol^55,60, which showed high percentages of podocytes and proximal tubules. Remarkably, kidney organoids from the UniMat exhibited a 4.5-fold decrease in podocytes (7.97%), but higher proportions of broad proximal tubules (60.93%) and loop of Henle/distal tubules (31.10%). These proportions more closely resemble the cellular composition of natural nephrons in both mice^61,62 and humans^63,64 (Fig. 5d). Significantly, within the broad proximal tubule category, the proportion of early proximal tubules was 18.7% in UniMat, as compared to 62% in AggreWell, highlighting a higher presence of mature proximal tubules in the former. The higher tubule-to-glomerulus ratio observed in UniMat-cultured kidney organoids is strongly associated with enhanced WNT4 expression—a key signaling molecule in kidney development processes such as the mesenchymal-to-epithelial transition and nephron formation^57,65 (Supplementary Fig. 13). This elevated WNT signaling in the UniMat system is likely driven by the efficient delivery of the WNT agonist CHIR99021 (CHIR) during the critical nephron patterning stage on days 9-11 through the permeable membrane. Beyond nephron cells, there was a noticeable difference in the NPC cluster: AggreWell exhibited a fraction 1.7 times higher than that of UniMat (Fig. 5e). This further demonstrated the enhanced maturity of kidney organoids cultivated on the UniMat platform (Fig. 5e and Supplementary Table 5). Given that tissue structure and function hinge on cellular compositions, these results emphasize the greater in vivo relevance of kidney organoids generated using UniMat.

We next examined gene expression profiles of podocytes, proximal tubules, loop of Henle/distal tubules as well as endothelial cell clusters (Fig. 5f and Supplementary Fig. 14). The analysis revealed elevated expression of genes related to differentiation in the podocytes, loop of Henle/distal tubules, and endothelial cells clusters. This suggests a greater degree of maturity in kidney organoids cultivated using the UniMat. Although the gene expression profiles in the proximal tubule cluster appeared similar between UniMat and AggreWell, a low proportion of early proximal tubules in UniMat (Supplementary Fig. 15) indicates a more mature state of these tubules.

To explore the potential pathways and functions that differentiate kidney organoids in UniMat compared to those in AggreWell, we carried out Biological Process Gene Ontology (GO) enrichment analysis on genes that were upregulated in podocytes, proximal tubules, loop of Henle/distal tubules, and endothelial cells clusters within the UniMat (Fig. 5g). In the podocyte cluster, UniMat showed elevated expression of genes associated with glomerulus development (GO:0032835) and glomerular basement membrane development (GO:0032836) (Supplementary Table 6). For both the proximal tubule and the loop of Henle/distal tubule clusters, the most enriched GO terms were associated with the negative regulation of mesenchymal cell apoptosis in metanephros development (GO:1900212), metanephric nephron tubule formation (GO:0072289), and ureter morphogenesis (GO:0072197) (Supplementary Table 7). These observations corroborate the advanced tubular structures observed in the UniMat-cultured kidney organoids. In the loop of Henle/distal tubule cluster, enriched GO terms revealed elevated expression of genes like CLDN3, CLDN19, and CRB3, which are linked to the positive regulation of cell junction assembly (GO:1901890) (Supplementary Table 7). This suggests enhanced epithelial junctions in kidney organoids cultured in UniMat. Additionally, the GO term related to metanephric distal convoluted tubule development (GO:0072221) was enriched in UniMat (Supplementary Table 7), indicating a higher propensity for kidney organoids to differentiate into distal tubules in UniMat as compared to AggreWell, as shown in Fig. 5d. In the endothelial cell cluster, upregulated genes such as SOX17, SOX18, NRP1, and CLIC4, played a role in endothelial cell differentiation (GO:0060956) (Supplementary Table 8). These findings are in line with the improved development of kidney organoids in UniMat.

Potential of UniMat in standardizing organoid-based assays

While earlier researches have utilized kidney organoids to simulate polycystic kidney disease (PKD) and mimic its pathological features, the inconsistent morphology of these organoids often resulted in considerable variability in both size and cystic regions^15,66. Recognizing that UniMat facilitates the generation of kidney organoids with shape and functional consistency and higher maturity in tubular structures across different nephron segments, we employed these organoids for PKD modeling to evaluate UniMat’s potential as a drug testing platform. To induce PKD, we chose forskolin, which serves as a direct activator of adenylyl cyclase (AC), replicating the cyclic adenosine monophosphate (cAMP)-mediated pathophysiological processes central to PKD^67,68,69. We exposed the UniMat-cultured kidney organoids to a 30 µM concentration of forskolin for 48 h (Fig. 6a) to reproduce the elevated-cAMP levels that lead to the abnormal cystic formation characteristic of PKD’s physiology. This enabled us to evaluate the platform’s suitability for generating PKD organoids⁷⁰. In response to the forskolin treatment, we observed both an enlargement and the formation of cysts within organoids. This confirmed the UniMat’s ability to reliably produce PKD organoids on a scalable level (Fig. 6b).

**Fig. 6: Consistency and repeatability of disease modeling and drug testing in UniMat.**

To evaluate if the uniformly mature kidney organoids generated in UniMat enhance the reliability of PKD modeling, we compared the cyst-forming features of organoids cultured in the hydrogel layer, AggreWell, and UniMat. To facilitate closer observation, the kidney organoids cultured in UniMat and AggreWell were transferred to individual ultra-low attachment plates prior to 48-h exposure to forskolin at concentrations of 10 and 30 µM (Fig. 6a). Notably, cysts became evident within 24 h of forskolin exposure, predominantly in the LTL⁺ and CDH1⁺ tubular regions (Supplementary Fig. 16). To quantify cyst dimensions, we measured the areas of individual kidney organoids on day 26, prior to forskolin treatment, and calculated an average area to establish a baseline for the collective pre-treatment state of the organoids. After 48 h of treatment, we measured the areas of individual organoids again. The post-treatment areas were compared to the pre-treatment average, and the change was expressed as a percentage, termed percent area. This method efficiently captured the collective response of the organoid population to the treatment and simplified the quantification process by focusing on collective changes across the group rather than tracking each organoid individually. As expected, a considerable variability in the size and morphology of kidney organoids cultured on the hydrogel layer resulted in inconsistent cyst formation, in terms of both location and dimensions (Fig. 6c). A similar pattern of variability and incomplete cyst formation was observed in organoids cultured in the AggreWell, implying the existence of undifferentiated, non-renal structures unresponsive to forskolin treatment (Fig. 6c). Additionally, cyst formation in kidney organoids from both hydrogel layer and AggreWell was erratic, regardless of forskolin dosage and time of exposure (Fig. 6e). In contrast, organoids in the UniMat exhibited uniform cysts throughout their entire structure, resulting in consistent increases in overall sizes. This trend was also observed when forskolin was directly applied to the kidney organoids within UniMat (Supplementary Fig. 17). This can be attributed to the high and consistent differentiation of nephrons. When comparing the percentage area of individual forskolin-treated PKD organoids to their average size before treatment, we found that clear dose-dependent responses were observed specifically in the UniMat (Fig. 6e).

To explore the impact of uniform disease modeling on drug testing results, we exposed PKD organoids, induced by forskolin (30 µM), to CFTR inhibitor-172 at concentrations of 50 or 100 µM (Fig. 6a). This PKD drug, which targets the cytoplasmic side of CFTR, had previously demonstrated its potential to inhibit cyst growth in PKD mice and in vitro models^71,72. The PKD organoid induced from kidney organoids cultured in UniMat displayed consistent reductions in size (Fig. 6d). The percent area, calculated in the same manner as the cyst dimensions, showed a clear dose-dependent decrease (Fig. 6f). In contrast, the PKD organoids from both hydrogel layer and AggreWell experienced inconsistent changes in cyst dimensions. Our results indicate that, within the UniMat platform, we can validate not only the formation but also the reduction of cysts by simply comparing average size changes across the organoid population, without the need to track individual organoids.

Next, we further validated the consistency and repeatability of UniMat for uniform disease modeling by implementing an acute kidney injury (AKI) model. Previous AKI organoid models have shown expression of kidney injury molecule-1 (KIM-1)^8,73, a selective kidney injury marker significantly upregulated in injured proximal tubules^74,75. However, the variability of renal injury between organoids remains a concern. To assess UniMat’s suitability for uniform kidney injury induction, we exposed kidney organoids to varying concentrations of lipopolysaccharide (LPS), an endotoxin from gram-negative bacteria known to cause AKI, for 24 hours⁷⁶ (Supplementary Fig. 18a). Consistent with previous findings⁷⁷, we observed KIM-1 expression in LTL⁺ proximal tubules of organoids cultured in hydrogel layers, AggreWell, and UniMat in response to LPS (Supplementary Fig. 18c). Comparing the percentage of KIM-1⁺ area relative to the total organoid area, we found considerable variability in hydrogel layers and AggreWell (Supplementary Fig. 18b), with unpredictable KIM-1 expression levels regardless of LPS concentration. In contrast, UniMat-cultured organoids showed stable, dose-dependent KIM-1 expression (Supplementary Fig. 18b). Consistent with our PKD modeling, the variability in differentiated proximal tubular proportion in hydrogel layers and the presence of undifferentiated, non-renal structures in AggreWell complicated renal injury assessment. However, UniMat-cultured organoids, with evenly differentiated proximal tubules, allowed for precise evaluation of proximal tubular injury. In summary, these findings, including consistent cyst changes in PKD organoids, highlight UniMat’s applicability as a consistent and repeatable platform for accurate disease modeling and drug testing by facilitating the uniform and mature differentiation of organoids.

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Source: https://www.nature.com/articles/s41467-024-53073-z

Scalable production of uniform and mature organoids in a 3D geometrically-engineered permeable membrane

Design and fabrication of 3D geometrically-engineered, permeable membrane for organoid culture platform

UniMat improves uniformity of organoids

UniMat enhances maturity of organoids

UniMat enhances vascularization of organoids

UniMat supports long-term stability and functionality of organoids

Single-cell transcriptomic profiling of kidney organoids in UniMat

Potential of UniMat in standardizing organoid-based assays

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