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Geometric engineering of organoid culture for enhanced organogenesis in a dish

Nature Methods volume 19, pages 1449–1460 (2022)Cite this article

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Abstract

Here, we introduce a facile, scalable engineering approach to enable long-term development and maturation of organoids. We have redesigned the configuration of conventional organoid culture to develop a platform that converts single injections of stem cell suspensions to radial arrays of organoids that can be maintained for extended periods without the need for passaging. Using this system, we demonstrate accelerated production of intestinal organoids with significantly enhanced structural and functional maturity, and their continuous development for over 4 weeks. Furthermore, we present a patient-derived organoid model of inflammatory bowel disease (IBD) and its interrogation using single-cell RNA sequencing to demonstrate its ability to reproduce key pathological features of IBD. Finally, we describe the extension of our approach to engineer vascularized, perfusable human enteroids, which can be used to model innate immune responses in IBD. This work provides an immediately deployable platform technology toward engineering more realistic organ-like structures in a dish.

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Fig. 1: Geometric engineering of conventional organoid culture using OCTOPUS.
Fig. 2: Effects of long-term culture on the maturation of intestinal organoids in OCTOPUS.
Fig. 3: Prolonged culture of human intestinal organoids in OCTOPUS.
Fig. 4: scRNA-seq of human enteroids in OCTOPUS.
Fig. 5: Organoid-based model of human IBD in OCTOPUS.
Fig. 6: Microengineering of vascularized human enteroids in OCTOPUS-EVO.

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Data availability

All the numeric data used in this study are included in the Source data files provided with this paper. The human enteroids scRNA-seq dataset analyzed during the current study is available at the NCBI Gene Expression Omnibus, under accession number GSE203380. The raw images are too large for public deposit and are available from the corresponding author on reasonable request.

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Acknowledgements

We thank R. Wells, W. Yang and G. Al for their input. This work was supported by the National Institutes of Health (NIH) (grant nos. 1DP2HL127720-01, 1UG3DK122644) (D.D.H.); NIH grant no. R01DK124369 (K.E.H.); Children’s Hospital of Philadelphia Institutional Development Funds (K.E.H.); the National Science Foundation (grant no. CMMI:15-48571) (D.D.H.); the Ministry of Trade, Industry & Energy of the Republic of Korea (D.D.H.); the Kwanjeong Educational Foundation (S.E.P.); the Children’s Hospital of Philadelphia Gastrointestinal Epithelium Modeling Program (K.E.H.); and the University of Pennsylvania (D.D.H.). D.D.H. is a recipient of the NIH Director’s New Innovator Award and the Cancer Research Institute Technology Impact Award.

Author information

Author notes

  1. Andrei Georgescu

    Present address: Vivodyne, Inc., Philadelphia, PA, USA

Authors and Affiliations

  1. Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA

    Sunghee Estelle Park, Shawn Kang, Jungwook Paek, Andrei Georgescu, Jeehan Chang, Alex Yoon Yi & Dan Dongeun Huh

  2. NSF Science and Technology Center for Engineering Mechanobiology, University of Pennsylvania, Philadelphia, PA, USA

    Sunghee Estelle Park, Jeehan Chang & Dan Dongeun Huh

  3. Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA, USA

    Benjamin J. Wilkins

  4. Division of Gastroenterology, Hepatology, and Nutrition, Children’s Hospital of Philadelphia, Philadelphia, PA, USA

    Tatiana A. Karakasheva & Kathryn E. Hamilton

  5. Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Kathryn E. Hamilton

  6. Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Kathryn E. Hamilton & Dan Dongeun Huh

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Contributions

S.E.P. designed the research, performed the experiments, analyzed the data and wrote the manuscript. S.K. performed experiments, analyzed data and created figures. J.P., A.G., J.C. and A.Y.Y. provided assistance in the experiments and the preparation of the manuscript. B.J.W. provided assistance in the analysis of the histological data. T.A.K. and K.E.H. provided materials and assistance in the experiments and analyzed the data. D.D.H. designed the research, analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to Dan Dongeun Huh.

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Competing interests

D.D.H. is a cofounder of Vivodyne Inc. and holds equity in Vivodyne Inc. and Emulate Inc. The remaining authors declare no competing interests.

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Nature Methods thanks Maxime Mahe and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Madhura Mukhopadhyay, in collaboration with the Nature Methods team.

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Extended data

Extended Data Fig. 1 Co-culture of organoids in OCTOPUS.

Co-culture of mouse liver and intestinal organoids in OCTOPUS. The OCTOPUS insert shown in this example contains two juxtaposed spiral chambers with independent access ports. Scale bars, 300 μm (left image) and 100 μm (right images).

Extended Data Fig. 2 Diffusion of soluble factors in OCTOPUS.

a, b. Visualization of 70 kDa FITC-dextran diffusion into the inner and outer regions of the hydrogel scaffolds in Matrigel drop (a) and OCTOPUS (b). The organoids in the inner and outer regions were located 600 μm (OCTOPUS)/2400 μm (Matrigel drop) and 400 μm (both groups) from the hydrogel surface, respectively. Scale bars, 100 μm. c. Temporal profiles of mean fluorescence intensity (MFI) due to dextran diffusion. d, e. Visualization of temporal changes in oxygen concentration in the inner and outer regions of Matrigel scaffolds in conventional drop culture (d) and OCTOPUS (e). Quenching of blue fluorescence shown in the micrographs was caused by an increase in the level of oxygen. f. Temporal profiles of normalized fluorescence intensity due to oxygen diffusion into Matrigel (n=3 independent experiments).

Extended Data Fig. 3 Culture of mouse liver organoids in OCTOPUS.

a, b. Formation and extended culture of mouse liver organoids in OCTOPUS and conventional drop culture. Scale bars, 100 μm (n=5 biologically independent experiments). c. Confocal micro-graphs showing the expression of albumin (ALB) in mouse liver organoids. Scale bars, 10 μm. ELISA analysis of (d) albumin and (e) urea in conditioned media (n=3 biologically independent experiments). All data are presented as mean ± SEM and P values are from unpaired, two-sided t test.

Source data

Extended Data Fig. 4 Maturation of intestinal organoids in OCTOPUS.

a. Comparison of bud length. Confocal micrographs show the cross-section of buds extending from the main body of the organoids. In each image, the white dashed lines indicate the approximated positions of the base and tip of a bud. Organoids in OCTOPUS generate more elongated buds. Scale bars, 20 μm. b. Comparison of the number and size of organoid buds in vitro to those of mouse intestinal crypts in vivo. c, d. Quantification of Ki67, Lgr5, and EdU expression in mouse intestinal organoids (n = 5 and 3 biologically independent experiments for (c) and (d), respectively). e. Organoids developing in OCTOPUS exhibit increased expression of Hnf4α, a marker of mature intestinal epithelial cells, compared to the control group in Matrigel drop. Scale bars, 10 μm. f. Quantification of the fraction of Hnf4α+ cells and the level of Hnf4α expression. Immunofluorescence of Hnf4α was normalized with respect to the number of cells (n = 4). g. The graph shows the cellular composition of the intestinal epithelium in OCTOPUS. Quantification was performed by measuring the immunofluorescence of cell type-specific markers described in Fig. 2m. Undifferentiated cells were identified by positive DAPI staining without the expression of differentiation markers. The data indicate the abundance of enterocytes (79% of the differentiated cell population at day 14) (n = 3). All data are presented as mean ± SEM and P values are from unpaired, two-sided t test.

Source data

Extended Data Fig. 5 Effect of co-culture on organoid development in OCTOPUS.

a. Co-culture of intestinal organoids and fibroblasts to mimic the epithelial-stromal unit of the intestine. Scale bar, 300 μm. b, c. In comparison to monoculture, the co-culture organoids are larger and express higher levels of Hnf4α expression. Scale bars, 100 μm (b), and 10 μm (c). All data are presented as mean ± SEM and P values are from unpaired, two-sided t test (n = 5 biologically independent experiments).

Source data

Extended Data Fig. 6 Functional characterization of intestinal organoids in OCTOPUS.

a. Immunofluorescence analysis of PEPT1, a nutrient transporter responsible for intestinal uptake of peptides. In this analysis, organoids in Matrigel drops at the maximum duration of culture (7 days) were compared to those maintained in OCTOPUS for 14 days to examine the contribution of extended culture. Organoids in OCTOPUS exhibit robust immunostaining of PEPT1 predominantly in the villus domain of the organoids. PEPT1 expression in this region is restricted to the apical surface of the epithelium facing the organoid lumen (L), matching the localized distribution of PEPT1 on the brush border membrane of the native intestinal epithelium. This polarized expression of PEPT1 is also observed in the villus surface of the organoids in Matrigel drop culture but the level of expression is significantly lower than that in OCTOPUS. Scale bars, 20 μm. b. Comparison of GLUT immunofluorescence in the villus domain of organoids. GLUT is ex-pressed on both the apical and basal surfaces of the villus epithelium in OCTOPUS. This trans-porter is also present in drop culture but its immunofluorescence is much weaker. Scale bars, 20 μm (n = 3 biologically independent experiments). c. Imaging and quantification of intracellular calcium signaling in intestinal organoids treated with 100 μM ATP. In the plots of relative intensity, the time between organoid stimulation and maximum fluorescence intensity is shaded in pink. Data were normalized to fluorescence intensity at the initial time point (t = 0 min). Scale bars, 100 μm (n = 3). d. Fraction of responsive organoids. The fraction of organoids that respond to ATP and glucose stimulation is larger in OCTOPUS. e. ELISA analysis of an active form of GLP-1 and MUC2 secreted by intestinal organoids. Both analytes are produced in significantly higher concentrations in OCTOPUS, which also continue to increase over time during extended culture (n = 3 biologically independent experiments). All data are presented as mean ± SEM and P values are from unpaired, two-sided t test.

Extended Data Fig. 7 Passaging and expansion of intestinal organoids in OCTOPUS.

a. Experimental procedure for passaging organoids in OCTOPUS. Subculture and expansion of (b) mouse and (c) human intestinal organoids grown in OCTOPUS and Matrigel drop. For comparison, culture conditions (for example, seeding density, hydrogel volume, media composition) were kept the same between the two systems. After 7 days of culture, organoids at a given passage number (Passage N) were physically dissociated and then transferred to new OCTOPUS devices or Matrigel drops (Passage N+1) at densities of 200 crypts/100 μl and 100 crypts/60 μl for mouse and human intestinal organoids, respectively. White solid lines in the micrographs show the outline of culture chambers in the OCTOPUS device or a sessile drop of Matrigel. Scale bars, 1 mm.

Extended Data Fig. 8 IBD enteroids cultured in Matrigel drops.

a. When cultured in Matrigel drops, IBD enteroids show properly polarized epithelial cells (top right) that resemble those in the epithelium of normal enteroids. In comparison to IBD enteroids in OCTOPUS, they also retain the structural integrity of the epithelium as visualized by ZO-1 expression (bottom right). Scale bars, 5 μm. b. UMAP plots showing the expression of two representative LINC genes by IBD and normal enteroids cultured in Matrigel drops. c. UMAP plots showing the expression of 4 representative IBD-associated genes by IBD and normal enteroids cultured in Matrigel drops.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–23, Tables 1 and 2, and captions for videos.

Reporting Summary

Supplementary Video 1

How to use OCTOPUS. OCTOPUS can be easily and quickly set up in a standard multi-well plate. In the first step demonstrated in this video, the OCTOPUS inserts are placed in a 12-well plate. Next, a stem cell-containing ECM hydrogel precursor solution is injected manually into each device through an open access port at the center of the device using a 200-μl pipette tip—this step can be performed by an automated fluid handling system as shown in the lower inset of Fig. 1m. After gelation, organoid media can be pipetted directly into the OCTOPUS-containing wells to initiate organoid culture (not shown in the video).

Supplementary Video 2

Injection and distribution of hydrogel solution. This video shows a fluorescence view of hydrogel injection into OCTOPUS. The ECM solution is injected into the loading chamber without leakage, which is immediately followed by the flow and rapid distribution of the injected solution into the radial culture chambers. For the purpose of visualization, a total volume of 100 μl of Matrigel was mixed with 1-μm fluorescent (red) microbeads. The entire culture chambers were filled within 5 s (not shown in the video).

Supplementary Video 3

Diffusion of FITC-dextran into hydrogel scaffolds. The video shows direct comparison of FITC-dextran (70 kDa) diffusion into the inner regions of hydrogel scaffolds in OCTOPUS and Matrigel drop. In OCTOPUS, fluorescence becomes visible throughout the scaffold within 30 min, illustrating rapid and uniform diffusive transport.

Source data

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Park, S.E., Kang, S., Paek, J. et al. Geometric engineering of organoid culture for enhanced organogenesis in a dish. Nat Methods 19, 1449–1460 (2022). https://doi.org/10.1038/s41592-022-01643-8

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