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Synthetic dynamic hydrogels promote degradation-independent in vitro organogenesis

Nature Materials volume 21, pages 479–487 (2022)Cite this article

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Abstract

Epithelial organoids are most efficiently grown from mouse-tumour-derived, reconstituted extracellular matrix hydrogels, whose poorly defined composition, batch-to-batch variability and immunogenicity limit clinical applications. Efforts to replace such ill-defined matrices for organoid culture have largely focused on non-adaptable hydrogels composed of covalently crosslinked hydrophilic macromolecules. However, the excessive forces caused by tissue expansion in such elastic gels severely restrict organoid growth and morphogenesis. Chemical or enzymatic degradation schemes can partially alleviate this problem, but due to their irreversibility, long-term applicability is limited. Here we report a family of synthetic hydrogels that promote extensive organoid morphogenesis through dynamic rearrangements mediated by reversible hydrogen bonding. These tunable matrices are stress relaxing and thus promote efficient crypt budding in intestinal stem-cell epithelia through increased symmetry breaking and Paneth cell formation dependent on yes-associated protein 1. As such, these well-defined gels provide promising versatile matrices for fostering elaborate in vitro morphogenesis.

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Fig. 1: Synthesis and design of PEG-cytosine50.
Fig. 2: Synthesis and mechanical characterization of Hybrid50 hydrogel.
Fig. 3: Hybrid50 gels support mouse intestinal stem-cell-derived organoid formation from single cells.
Fig. 4: Development of mouse intestinal organoids in synthetic niche with different mechanical properties.
Fig. 5: Extending hybrid hydrogel application for long-term hSI organoid culture.

Data availability

The datasets and the statistical analysis results that support the findings of this study are available in Zenodo with the identifier https://doi.org/10.5281/zenodo.5243103.

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Acknowledgements

We thank M. Meyer for help with stem-cell colony and organoid quantification, S. Gerber and B. Muriel for advice on the synthesis of hybrid gels, O. Mitrofanova for expanding hSI organoids and A. Manfrin for the advice on qPCR primer design. We thank M. Zenobi-Wong and D. Fercher for sortase production and the Université de Lausanne (UNIL) facility for peptide production for peptide synthesis. We acknowledge support from the Ecole Polytechnique Fédérale de Lausanne Bio Imaging & Optics Core Facility for image analysis, and from D. Pioletti and the DLL-Engineering facility at Ecole Polytechnique Fédérale de Lausanne for rheometer use. We thank H.-A. Klok for FTIR and DLS use and the Ecole Polytechnique Fédérale de Lausanne Gene Expression Core Facility for qPCR instrument use. We thank G. Schwank for providing the third hSI organoid line. This work was funded by the National Center of Competence in Research Bio-Inspired Materials for A.C. and D.B., the Swiss 3R Competence Center for S.R. and N.B., the EU Horizon 2020 research programme INTENS (http://www.intens.info/; no. 668294-2), the Personalized Health and Related Technologies Initiative from the Eidgenössische Technische Hochschule (ETH) Board and the Swiss National Science Foundation research grant no. 310030_179447.

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Author notes

  1. These authors contributed equally: Antonius Chrisnandy, Delphine Blondel.

Authors and Affiliations

  1. Laboratory of Stem Cell Bioengineering, Institute of Bioengineering, School of Life Sciences (SV) and School of Engineering (STI), Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland

    Antonius Chrisnandy, Delphine Blondel, Saba Rezakhani, Nicolas Broguiere & Matthias P. Lutolf

  2. Institute of Chemical Sciences and Engineering, School of Basic Science (SB), EPFL, Lausanne, Switzerland

    Matthias P. Lutolf

  3. Roche Institute for Translational Bioengineering (ITB), Pharma Research and Early Development (pRED), F. Hoffmann-La Roche Ltd, Basel, Switzerland

    Matthias P. Lutolf

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  1. Antonius Chrisnandy
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Contributions

M.P.L. and D.B. conceived the initial idea. A.C., D.B. and M.P.L. designed experiments, analysed data and interpreted results. A.C. performed all key experiments for revisions. S.R. was involved in the human organoid culture. N.B. was involved in designing dithiol sortase peptide, sortase synthesis and the force displacement field measurement analysis. All authors read and provided feedback on the manuscript.

Corresponding author

Correspondence to Matthias P. Lutolf.

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The authors declare no competing interests.

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Peer review information Nature Materials thanks Eric Appel, Sina Bartfeld, Melissa Little and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Oscillatory rheological measurement of hydrogels.

a, Evolution of shear moduli over time for Hybrid50 gel at 37 °C (5% strain and 1 Hz) at different PEG content. Data were shown as mean ± S.D (shaded area) from (n = 3) independently prepared gels. b, Determination of gel point for Matrigel and Hybrid50 gel (3% w/v) defined as the time when G’ and G” crossed also phase angle below 45°, indicated by black arrow. The data was representative of (n = 3) independent measurements (5% strain and 1 Hz). c,d, Amplitude sweep (c) and frequency sweep (d) measurement for Matrigel, Hybrid50 and covalent gels at three different stiffnesses at 37 °C. Amplitude sweeps were done at 1 Hz from 1% to 300% strain. Frequency sweeps were performed at 5% strain from 0.01 to 10 Hz. Dotted line in amplitude sweep data (a) indicated the strain used for frequency sweep, still within linear viscoelastic region. Data were shown as mean ± S.D (shaded area) from (n = 3) independently prepared gels. Covalent and hybrid50 gels were swollen prior to measurement while Matrigel was measured subsequent to in-situ gelation. e, Post normalization analysis of frequency sweep results, revealing the influence of different response of hydrogels upon deformation at different time-scale. Data were shown as mean from (n = 3) independently prepared gels.

Extended Data Fig. 2 Stress relaxation curve fitting to generalized Maxwell-Wiechert 3 elements model.

a, Generalized Maxwell-Wiechert 3 elements model fitted perfectly in most of experimental data, determined by coefficient of determination (R2). Data shown as individual data points (Round grey) and curve fit of experimental data mean (triangle black) from (n = 3) independently prepared gels. b, Example of fitted experimental data and curve fit in various hydrogels system.

Extended Data Fig. 3 Stress relaxation curve fitting parameters.

a, Determination of curve fitting parameters for elastic component (σ0) and decay half-time (Td1/2). b-g, Elastic components and decay half-time for Matrigel (b), different cytosine functionalization (Hybrid25 and Hybrid65 gels) (c), different PEG precursors (Aniline50 and COOH50 gels) (d), Cytidine treatment of Hybrid50 gels (e), covalent gels at different stiffnesses (f) and hybrid50 gels at different stiffnesses (g). Data shown as individual data points (Round grey) and curve fit of experimental data mean (triangle black) from (n = 3) independently prepared gels. Statistical analysis was evaluated with one-ways ANOVA followed by Tukey post-hoc analysis (b-f) and unpaired two-tailed student t-test (g), P-values were presented above the data points.

Extended Data Fig. 4 Effect of RGD on mechanical properties of hydrogels.

a, A schematic illustrating functionalization the hydrogel with RGD motif through Michael-type addition between VS and cysteine. b, Effect of RGD to the bulk hydrogel stiffness for Covalent and Hybrid50 gels. We noticed drop of stiffness due to less crosslinking VS-TH at the given polymer content for both covalent and Hybrid50 gels. Data shown as individual data points and line represented mean ± S.D from (n = 3) independently prepared gels from frequency sweep measurement (5% strain and 0.01-10 Hz). c, Stress relaxation profile of the hydrogels with addition of RGD motif. Data were shown as mean ± S.D (shaded area) from (n = 3) independently prepared gels. d, Curve fitting parameters of stress relaxation profile. The stress relaxation (10% strain) of covalent hydrogel remained identical with and without RGD, Hybrid50 gels demonstrated more stress relaxation from 10% to 15% stress relaxed because of addition of 8-PEG-Cytosine50 precursors to satisfy 0.5 mM excess of VS group, but did not alter decay characteristic time. Data shown as individual data points (Round grey) and curve fit of experimental data mean (triangle black) from (n = 3) independently prepared gels. 0.5 mM of RGDSP were used on all experiments. Statistical analysis was evaluated with two-ways ANOVA followed by Tukey (b) and Bonferroni post-hoc analysis (d), P-values were presented above the data points.

Extended Data Fig. 5 3D displacement field of fluorophore beads during mouse intestinal organoid culture in covalent and Hybrid50 gels.

a, A schematic illustrating how the physical remodelling of the hybrid gel during the onset of budding was monitored using fluorescent beads. b, Representative images of displacement fields induced during the collapse and budding of cystic colonies. Orange arrows show a colony collapse before bud formation, and red arrows point to a collapsed colony breaking symmetry and extending an initial bud / bulge. c, The xy components of the displacement field in this plane were used for analysis of the displacement against distance decay curves. Various characteristic events were captured such as collapsing organoid, growth and bud extension. The yellow arrows indicated the direction of bead displacement around the mouse intestinal organoids. The brightfield images represented two different time points earlier and later respectively. The experiments were repeated (n = 2) independent experiments. Scale bar, 50 µm.

Extended Data Fig. 6 Organoids sampling grown in various hydrogels from day 03 to 07.

a, Representative images of sampled organoids from grown in various hydrogels to analyze the presence of Paneth cells (Lyz) and enterocyst (AldoB), also to perform morphological classification. b, Visual quantification of Lyz + (color-coded) over 5 days and clustering of morphologically different population at day 07. The numbers next to dot plot is number of Lyz+ organoids over total number of samples except for enterocyst. The presented data were taken from(n = 3 for day 07 and n = 2 for day 03-06) independent experiment. Scale bar, 50 µm.

Extended Data Fig. 7 Quantitative real-time PCR data of mouse intestinal organoids in synthetic hydrogels.

Heatmap of quantitative real-time PCR data showing the relative expression of mouse intestinal organoids grown in covalent and hybrid50 hydrogels from single cells over four days for Yap1 target genes (Ctgf and Cyr61) and symmetry breaking (Dll1, Atoh1 and Lyz1). The average fold changes were calculated relative to mISCs organoids in covalent gels, derived from (n = 3) independent experiments. Expression level was normalized to Gapdh and relative mRNA expression levels were shown as logarithmic scale (Log2).

Extended Data Fig. 8 Long-term human small intestinal organoids culture.

a, Representative images of hSI organoids (hSI Line 3) over five passages cultured in Hybrid50 hydrogels and Matrigels at day 06 of each passage. b, Quantification of cell number fold changes between day 00 and day 06 at each passage for three different hSI organoids lines expanded in Hybrid50 gels and Matrigel. Data shown as line represented mean ± S.D from (n = 3 hSI lines) with individual data points represented by symbols, hSI line 1 (Circles), hSI line 2 (Squares) and hSI line 3 (Triangles). Scale bar, 50 μm.

Extended Data Fig. 9 Quantitative real-time PCR data of three hSI organoids lines grown in SS-Hybrid50 hydrogels.

Quantitative real-time PCR data showing the relative expression of hSI organoids grown in SS-Hybrid50 gels for differentiated cells markers, stem cells (OLFM4, LGR5 and SOX9), Paneth cells (ATOH1, LYZ, REG3A and DEFA6), Goblet cells (MUC2 and SPINK4), Enteroendocrine cells (CHGA) and Enterocytes (ALDOB, FABP1, FABP2 and VIL1). The data were shown as mean (red line) relative expression to hSI organoids grown in Matrigel mean ± S.E.M (error bars) from three (n = 3) independent experiment (by circles, squares and triangles). Expression level was normalized to GAPDH and relative mRNA expression levels were shown as logarithmic scale (Log2). Statistical analysis was evaluated with two-ways ANOVA followed by Bonferroni post-hoc analysis and P-values were available above the data, statistically significant results were highlighted in red color (below 0.05).

Supplementary information

Supplementary Information

Supplementary Figs. 1–14, Tables 1–3 and descriptions of Videos 1–3.

Reporting Summary

Supplementary Video 1

Representative images of the budding process of mouse intestinal organoids cultured in the hybrid gel. There are two organoids in the video (left and middle). The left organoid first collapsed before bud generation. The organoid in the middle, which had already collapsed, demonstrated a more advanced budding structure.

Supplementary Video 2

Representative images of the budding structure of mouse intestinal organoid cultured in the hybrid gel. This organoid already had a budding structure, which is in extension.

Supplementary Video 3

Representative images of the mouse intestinal organoid cultured in the covalent gel. Three colonies are present in the frame. All of the colonies collapsed but failed to form any budding structure.

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Chrisnandy, A., Blondel, D., Rezakhani, S. et al. Synthetic dynamic hydrogels promote degradation-independent in vitro organogenesis. Nat. Mater. 21, 479–487 (2022). https://doi.org/10.1038/s41563-021-01136-7

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