Title: Engineering Stem Cell Self-organization to Build Better Organoids
Abstract: Organoids form through self-organization processes in which initially homogeneous populations of stem cells spontaneously break symmetry and undergo in-vivo-like pattern formation and morphogenesis, though the processes controlling this are poorly characterized. While these in vitro self-organized tissues far exceed the microscopic and functional complexity obtained by current tissue engineering technologies, they are non-physiological in shape and size and have limited function and lifespan. Here, we discuss how engineering efforts for guiding stem-cell-based development at multiple stages can form the basis for the assembly of highly complex and rationally designed self-organizing multicellular systems with increased robustness and physiological relevance. Organoids form through self-organization processes in which initially homogeneous populations of stem cells spontaneously break symmetry and undergo in-vivo-like pattern formation and morphogenesis, though the processes controlling this are poorly characterized. While these in vitro self-organized tissues far exceed the microscopic and functional complexity obtained by current tissue engineering technologies, they are non-physiological in shape and size and have limited function and lifespan. Here, we discuss how engineering efforts for guiding stem-cell-based development at multiple stages can form the basis for the assembly of highly complex and rationally designed self-organizing multicellular systems with increased robustness and physiological relevance. Tightly regulated cellular self-organization programs orchestrate dynamic interactions between cells and their environments, ensuring the robustness of tissue and organ development, homeostasis, and regeneration in multicellular organisms. Because self-organization is an emergent property of an integrated multicellular system, complex patterning events cannot be simply explained by causal links between genes and the phenotypes at the level of single cells but rather must be understood at the population level through sequential iterations of local interactions between individual cells (or subsets of cells) and their environment. As such, self-organization depends on the intrinsic capacity of cells to sense, integrate, and respond to various systemic and local cues, such as morphogen gradients (Sagner and Briscoe, 2017Sagner A. Briscoe J. Morphogen interpretation: concentration, time, competence, and signaling dynamics.Wiley Interdiscip. Rev. Dev. 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This implies that by providing an optimal and permissive environment and by adding key spatiotemporal cues to drive multicellular responses, it should also be possible to harness the cell’s self-organization potential outside of an organism, i.e., in vitro. Indeed, when cultured in a three-dimensional (3D) environment that provides permissive growth conditions but no external patterning blueprint, initially uniform groups of cells can display emergent behaviors akin to aspects of morphogenesis and organogenesis. In marked contrast to 2D mammalian cell cultures that normally acquire a flattened morphology, cells in 3D environments form more physiologically relevant multicellular structures. For example, mammary epithelial cell aggregates spontaneously self-organize into lumenized spherical structures, which can generate lactating mammary acini (Barcellos-Hoff et al., 1989Barcellos-Hoff M.H. Aggeler J. Ram T.G. Bissell M.J. Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane.Development. 1989; 105: 223-235Crossref PubMed Google Scholar) and even undergo branching morphogenesis (Nelson et al., 2006Nelson C.M. Vanduijn M.M. Inman J.L. Fletcher D.A. Bissell M.J. Tissue geometry determines sites of mammary branching morphogenesis in organotypic cultures.Science. 2006; 314: 298-300Crossref PubMed Scopus (380) Google Scholar). 3D aggregates of salivary epithelial cells can likewise engage in budding and clefting processes, similar to those driving salivary gland development (Wei et al., 2007Wei C. Larsen M. Hoffman M.P. Yamada K.M. Self-organization and branching morphogenesis of primary salivary epithelial cells.Tissue Eng. 2007; 13: 721-735Crossref PubMed Scopus (0) Google Scholar), and epithelial cysts constructed of canine kidney cells undergo tubulogenesis when uniformly exposed to hepatocyte growth factor (O’Brien et al., 2002O’Brien L.E. Zegers M.M.P. Mostov K.E. Opinion: Building epithelial architecture: insights from three-dimensional culture models.Nat. Rev. Mol. Cell Biol. 2002; 3: 531-537Crossref PubMed Scopus (444) Google Scholar). The unique property of stem cells that allows them to both self-renew and differentiate into cell types from multiple lineages adds a layer of complexity to their behavior that has been harnessed to emulate features of organogenesis in cell culture. Seminal studies from the laboratories of Yoshiki Sasai (Eiraku et al., 2011Eiraku M. Takata N. Ishibashi H. Kawada M. Sakakura E. Okuda S. Sekiguchi K. Adachi T. Sasai Y. Self-organizing optic-cup morphogenesis in three-dimensional culture.Nature. 2011; 472: 51-56Crossref PubMed Scopus (855) Google Scholar) and Hans Clevers (Sato et al., 2009Sato T. Vries R.G. Snippert H.J. van de Wetering M. Barker N. Stange D.E. van Es J.H. Abo A. Kujala P. Peters P.J. Clevers H. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.Nature. 2009; 459: 262-265Crossref PubMed Scopus (2165) Google Scholar) revealed that, aside from directing stem cells to commit toward cell types of various lineages, these cells and their progeny seem to follow their innate developmental programs and self-organize into “organoids”—structures that mimic multiple histological and functional aspects of real tissues and organs, including preserving niches containing self-renewing stem cells. For instance, Sasai and colleagues recapitulated aspects of eye development in vitro, showing that mouse and human pluripotent stem cells (PSCs) can self-organize into a bilayered optic-cup-like structure when cultured in 3D (Eiraku et al., 2011Eiraku M. Takata N. Ishibashi H. Kawada M. Sakakura E. Okuda S. Sekiguchi K. Adachi T. Sasai Y. Self-organizing optic-cup morphogenesis in three-dimensional culture.Nature. 2011; 472: 51-56Crossref PubMed Scopus (855) Google Scholar, Nakano et al., 2012Nakano T. Ando S. Takata N. Kawada M. Muguruma K. Sekiguchi K. Saito K. Yonemura S. Eiraku M. Sasai Y. Self-formation of optic cups and storable stratified neural retina from human ESCs.Cell Stem Cell. 2012; 10: 771-785Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar). Since the pioneering work of Sasai and colleagues, researchers have developed protocols for transforming aggregates of PSCs into organoid versions of multiple organs, including the brain, intestine, stomach, lung, liver, and kidney (Clevers, 2016Clevers H. Modeling Development and Disease with Organoids.Cell. 2016; 165: 1586-1597Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar, Lancaster and Knoblich, 2014Lancaster M.A. Knoblich J.A. Organogenesis in a dish: modeling development and disease using organoid technologies.Science. 2014; 345 (Published online July 18, 2014)https://doi.org/10.1126/science.1247125Crossref Scopus (578) Google Scholar, McCauley and Wells, 2017McCauley H.A. Wells J.M. Pluripotent stem cell-derived organoids: using principles of developmental biology to grow human tissues in a dish.Development. 2017; 144: 958-962Crossref PubMed Scopus (37) Google Scholar, Rossi et al., 2018Rossi G. Manfrin A. Lutolf M.P. Progress and potential in organoid research.Nat. Rev. Genet. 2018; 19: 671-687Crossref PubMed Scopus (10) Google Scholar). Perhaps the archetypal organoid system has been described by Sato, Clevers, and colleagues. They demonstrated that adult Lgr5+ intestinal stem cells (ISCs) embedded in 3D Matrigel and provided with uniform niche signals, including R-spondin1, Noggin, and EGF, not only survive and proliferate to produce ISC colonies, but subsequently undergo morphogenesis to form structures that approximate the adult small intestinal mucosa: crypt-like projections that radiate outward from a spherical epithelial structure surrounding a central lumen (Sato et al., 2009Sato T. Vries R.G. Snippert H.J. van de Wetering M. Barker N. Stange D.E. van Es J.H. Abo A. Kujala P. Peters P.J. Clevers H. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.Nature. 2009; 459: 262-265Crossref PubMed Scopus (2165) Google Scholar, Sato et al., 2011aSato T. van Es J.H. Snippert H.J. Stange D.E. Vries R.G. van den Born M. Barker N. Shroyer N.F. van de Wetering M. Clevers H. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts.Nature. 2011; 469: 415-418Crossref PubMed Scopus (1134) Google Scholar). Importantly, these structures reconstitute the principal geometrical, architectural, and cellular hallmarks of the native epithelium. Notably, around the same time, Kuo and colleagues reported an air-liquid interface approach for the long-term 3D culture of intestinal organoids, comprising myofibroblasts that provided key niche signals (Ootani et al., 2009Ootani A. Li X. Sangiorgi E. Ho Q.T. Ueno H. Toda S. Sugihara H. Fujimoto K. Weissman I.L. Capecchi M.R. Kuo C.J. Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche.Nat. Med. 2009; 15: 701-706Crossref PubMed Scopus (374) Google Scholar). Intestinal organoids, like other gastrointestinal organoids such as those derived from the colon (Jung et al., 2011Jung P. Sato T. Merlos-Suárez A. Barriga F.M. Iglesias M. Rossell D. Auer H. Gallardo M. Blasco M.A. Sancho E. et al.Isolation and in vitro expansion of human colonic stem cells.Nat. Med. 2011; 17: 1225-1227Crossref PubMed Scopus (317) Google Scholar, Sato et al., 2011bSato T. Stange D.E. Ferrante M. Vries R.G.J. Van Es J.H. Van den Brink S. Van Houdt W.J. Pronk A. Van Gorp J. Siersema P.D. Clevers H. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium.Gastroenterology. 2011; 141: 1762-1772Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar) or stomach (Barker et al., 2010Barker N. Huch M. Kujala P. van de Wetering M. Snippert H.J. van Es J.H. Sato T. Stange D.E. Begthel H. van den Born M. et al.Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro.Cell Stem Cell. 2010; 6: 25-36Abstract Full Text Full Text PDF PubMed Scopus (749) Google Scholar), are powerful models for the study of basic adult stem cell (ASC) biology, tissue homeostasis and regeneration, and patient-specific diseases. These organoids could also serve as sources of tissue for autologous transplants. The self-organizing processes that build organoids—requiring only minimal external cues and spatially homogeneous cocktails of growth factors—can generate impressive levels of tissue organization and functionality that cannot be matched by classical tissue engineering approaches relying primarily on a scaffold. At the same time, their full reliance on cell-autonomous self-organization lacking in pre-defined extrinsic patterning instructions may be their main weakness. This invariably introduces stochasticity in organoid formation and leads to heterogeneities in size, shape, and cell-type composition. Moreover, current organoid culture technology limits their size to the millimeter scale, precluding their widespread adoption for a wealth of applications, such as regenerative medicine. In this review, we argue that self-organization can be engineered by spatiotemporally controlling cell-cell and cell-extracellular matrix (ECM) interactions and that this approach should allow researchers to push the limits of existing in vitro organogenesis. To bio-fabricate more functional and size-relevant tissues, the main challenges relate to controlling and scaling-up stem cell self-organization. We propose that this could be achieved by focusing on four major stages during the in vitro developmental process that are summarized in Figure 1: (1) controlling initial culture conditions, (2) directing symmetry breaking, (3) imposing boundary conditions to guide cellular self-patterning, and (4) rationally engineered scale-up. The inherent self-organization capacity of stem cells does not mean that elaborate tissues can be formed in any condition. Rather, it emphasizes the importance of the environment in steering the cellular development in a highly context-dependent manner. Indeed, what is generally referred to as self-organization in organoids may already be considered “directed” self-organization as, for example, mouse embryonic stem cell (ESC) aggregates can be cultured to form both optic cups (Eiraku et al., 2011Eiraku M. Takata N. Ishibashi H. Kawada M. Sakakura E. Okuda S. Sekiguchi K. Adachi T. Sasai Y. Self-organizing optic-cup morphogenesis in three-dimensional culture.Nature. 2011; 472: 51-56Crossref PubMed Scopus (855) Google Scholar) or gastruloids (Beccari et al., 2018Beccari L. Moris N. Girgin M. Turner D.A. Baillie-Johnson P. Cossy A.C. Lutolf M.P. Duboule D. Arias A.M. Multi-axial self-organization properties of mouse embryonic stem cells into gastruloids.Nature. 2018; 562: 272-276Crossref PubMed Scopus (4) Google Scholar) depending on the physicochemical cues provided. Small variations in the initial conditions are bound to have dramatic influences on the final patterning and morphogenesis outcome, since organogenesis and organoid formation are characteristically non-linear deterministic systems (Dahl-Jensen and Grapin-Botton, 2017Dahl-Jensen S. Grapin-Botton A. The physics of organoids: a biophysical approach to understanding organogenesis.Development. 2017; 144: 946-951Crossref PubMed Scopus (2) Google Scholar). Consequently, controlling the initial conditions to improve organoid reproducibility and designing in vitro developmental trajectories toward desired patterning outcomes are of paramount importance. Indeed, because self-organization is iterative and cumulative, small deviations from the initial optimal condition can steer development away from a desired emergent behavior. This feature has been termed “stigmergy,” which refers to an outcome of a “stimulation” that is dependent on the history of previous “stimulations” (Sasai, 2013aSasai Y. Cytosystems dynamics in self-organization of tissue architecture.Nature. 2013; 493: 318-326Crossref PubMed Scopus (188) Google Scholar). We believe this can be countered by identifying the optimal conditions for robust and elaborate self-organization by exploring the effect of the initial cell number and density (i.e., size of a stem cell aggregate), geometry, and microenvironment (Figure 2). The self-organization potential of a stem cell depends on its origin and stage of development (Rossi et al., 2018Rossi G. Manfrin A. Lutolf M.P. Progress and potential in organoid research.Nat. Rev. Genet. 2018; 19: 671-687Crossref PubMed Scopus (10) Google Scholar), thus representing an obvious first important consideration for organoid-based tissue engineering (Figure 2A). PSC-derived organoids are obtained by mimicking the presumptive sequential signaling interactions operating during in vivo development, whereas those derived from ASCs are obtained by providing signaling cues that operate in the respective adult tissues. PSCs possess a broader potency that allows the coordinated generation of cells from multiple germ layers. One striking example is the development of PSC-derived intestinal (Spence et al., 2011Spence J.R. Mayhew C.N. Rankin S.A. Kuhar M.F. Vallance J.E. Tolle K. Hoskins E.E. Kalinichenko V.V. Wells S.I. Zorn A.M. et al.Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro.Nature. 2011; 470: 105-109Crossref PubMed Scopus (690) Google Scholar), gastric (McCracken et al., 2014McCracken K.W. Catá E.M. Crawford C.M. Sinagoga K.L. Schumacher M. Rockich B.E. Tsai Y.H. Mayhew C.N. Spence J.R. Zavros Y. Wells J.M. Modelling human development and disease in pluripotent stem-cell-derived gastric organoids.Nature. 2014; 516: 400-404Crossref PubMed Scopus (309) Google Scholar), or liver organoids (Dye et al., 2015Dye B.R. Hill D.R. Ferguson M.A.H. Tsai Y.H. Nagy M.S. Dyal R. Wells J.M. Mayhew C.N. Nattiv R. Klein O.D. et al.In vitro generation of human pluripotent stem cell derived lung organoids.eLife. 2015; (Published online March 24, 2015)https://doi.org/10.7554/eLife.05098Crossref Scopus (186) Google Scholar) composed of an epithelial layer covered by mesenchymal cells that co-evolve during organoid formation. Indeed, exploiting tissue-tissue crosstalk during organoid development may be a powerful strategy for increasing the complexity of organoids. However, despite their exciting potential, PSC-derived organoids have yet to be predictably developed, as exemplified by the fact that ESC aggregates can produce a seemingly random number of vesicles during optic cup formation (Eiraku et al., 2011Eiraku M. Takata N. Ishibashi H. Kawada M. Sakakura E. Okuda S. Sekiguchi K. Adachi T. Sasai Y. Self-organizing optic-cup morphogenesis in three-dimensional culture.Nature. 2011; 472: 51-56Crossref PubMed Scopus (855) Google Scholar) and that gastric organoids are formed within a larger, uncontrolled tissue aggregate (Noguchi et al., 2015Noguchi T.K. Ninomiya N. Sekine M. Komazaki S. Wang P.C. Asashima M. Kurisaki A. Generation of stomach tissue from mouse embryonic stem cells.Nat. Cell Biol. 2015; 17: 984-993Crossref PubMed Scopus (38) Google Scholar). In contrast, the potency of ASCs has already been restricted and is linked to their tissue of origin, so guiding ASC-organoid formation generally does not require extensive sequential switching through different lineage-specifying culture conditions. ASC-based organoids more closely recapitulate the homeostatic conditions and regenerative processes of the corresponding tissues, with a microscopic architecture closer to that of adult tissue (Clevers, 2016Clevers H. Modeling Development and Disease with Organoids.Cell. 2016; 165: 1586-1597Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). Unfortunately, this restricted potential compared to PSCs means that ASCs lack the necessary tissue-tissue interactions to promote organ-level complexity, such as in the formation of organ buds derived from reciprocal epithelial and mesenchymal interactions (Ikeda et al., 2019Ikeda E. Ogawa M. Takeo M. Tsuji T. Functional ectodermal organ regeneration as the next generation of organ replacement therapy.Open Biol. 2019; 9: 190010Crossref PubMed Scopus (0) Google Scholar). As another example, vilification of the small intestine is believed to be initiated by mesenchymal clustering beneath the intestinal epithelium and subsequent modification of the mechanobiological environment around the epithelium (Wells and Spence, 2014Wells J.M. Spence J.R. How to make an intestine.Development. 2014; 141: 752-760Crossref PubMed Scopus (71) Google Scholar). Consequently, increasing the tissue complexity of ASC-derived organoids will require positioning multiple cell types in an environment in which the tissues can interact to properly self-organize. Of note, organoids have also been derived from mouse embryonic pancreatic progenitors (Greggio et al., 2013Greggio C. De Franceschi F. Figueiredo-Larsen M. Gobaa S. Ranga A. Semb H. Lutolf M. Grapin-Botton A. Artificial three-dimensional niches deconstruct pancreas development in vitro.Development. 2013; 140: 4452-4462Crossref PubMed Scopus (99) Google Scholar) and mouse fetal intestinal progenitors (Fordham et al., 2013Fordham R.P. Yui S. Hannan N.R.F. Soendergaard C. Madgwick A. Schweiger P.J. Nielsen O.H. Vallier L. Pedersen R.A. Nakamura T. et al.Transplantation of expanded fetal intestinal progenitors contributes to colon regeneration after injury.Cell Stem Cell. 2013; 13: 734-744Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, Navis et al., 2019Navis M. Martins Garcia T. Renes I.B. Vermeulen J.L. Meisner S. Wildenberg M.E. van den Brink G.R. van Elburg R.M. Muncan V. Mouse fetal intestinal organoids: new model to study epithelial maturation from suckling to weaning.EMBO Rep. 2019; 20 (Published online February 2019)https://doi.org/10.15252/embr.201846221Crossref PubMed Scopus (1) Google Scholar, Yui et al., 2018Yui S. Azzolin L. Maimets M. Pedersen M.T. Fordham R.P. Hansen S.L. Larsen H.L. Guiu J. Alves M.R.P. Rundsten C.F. et al.YAP/TAZ-Dependent Reprogramming of Colonic Epithelium Links ECM Remodeling to Tissue Regeneration.Cell Stem Cell. 2018; 22: 35-49Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). In addition to the advantages and disadvantages seen in ASC-derived organoids, such fetal organoids can be employed for example as models of developmental maturation (Navis et al., 2019Navis M. Martins Garcia T. Renes I.B. Vermeulen J.L. Meisner S. Wildenberg M.E. van den Brink G.R. van Elburg R.M. Muncan V. Mouse fetal intestinal organoids: new model to study epithelial maturation from suckling to weaning.EMBO Rep. 2019; 20 (Published online February 2019)https://doi.org/10.15252/embr.201846221Crossref PubMed Scopus (1) Google Scholar). An in-between approach is to partially differentiate PSCs toward a specific progenitor cell fate before aggregating them with cells of other lineages (Takebe et al., 2013Takebe T. Sekine K. Enomura M. Koike H. Kimura M. Ogaeri T. Zhang R.R. Ueno Y. Zheng Y.W. Koike N. et al.Vascularized and functional human liver from an iPSC-derived organ bud transplant.Nature. 2013; 499: 481-484Crossref PubMed Scopus (755) Google Scholar). Such “primed” stem/progenitor cells have the advantage of being still proliferative with a high differentiation potential, while their partial commitment could prevent the development of non-desired tissue structures. However, one important aspect to keep in mind in such a setting is that this “primed” cell population may be highly heterogeneous, which could compromise the robustness and reproducibility of the approach. Indeed, since multicellular responses depend on previous culture conditions (Sasai, 2013aSasai Y. Cytosystems dynamics in self-organization of tissue architecture.Nature. 2013; 493: 318-326Crossref PubMed Scopus (188) Google Scholar), it is particularly important to carefully characterize the starting cell population, making use of technologies such as single-cell RNA sequencing (RNA-seq) (Camp et al., 2018Camp J.G. Wollny D. Treutlein B. Single-cell genomics to guide human stem cell and tissue engineering.Nat. Methods. 2018; 15: 661-667Crossref PubMed Scopus (5) Google Scholar). One of the principal requirements for organoid culture is an environment that both confers the key biophysical and biochemical input signals while remaining permissive to self-organization. Most organoids developed to date have relied on ECMs derived from animals, such as Matrigel or collagens. However, the poorly defined composition, heterogeneous nature, and batch-to-batch variability of such matrices hinder the establishment of robust processes. Alternative approaches have focused on engineering synthetic stem cell microenvironments that can mimic key features of natural ECMs (Figure 2B). Particularly, 3D screening approaches can be used to synthesize and test hydrogels of varying stiffnesses, degradabilities, and bioactivities for their influence on stem cell fate (Ranga et al., 2014Ranga A. Gobaa S. Okawa Y. Mosiewicz K. Negro A. Lutolf M.P. 3D niche microarrays for systems-level analyses of cell fate.Nat. Commun. 2014; 5: 4324Crossref PubMed Scopus (107) Google Scholar). This methodology has also been applied to stem-cell-based morphogenesis, where the study of poly(ethylene glycol) (PEG)-based hydrogel formulations decoupled the effect of parameters such as stiffness and ECM composition on neuroepithelial cyst patterning (Meinhardt et al., 2014Meinhardt A. Eberle D. Tazaki A. Ranga A. Niesche M. Wilsch-Bräuninger M. Stec A. Schackert G. Lutolf M. Tanaka E.M. 3D reconstitution of the patterned neural tube from embryonic stem cells.Stem Cell Reports. 2014; 3: 987-999Abstract Full Text Full Text PDF PubMed Google Scholar, Ranga et al., 2016Ranga A. Girgin M. Meinhardt A. Eberle D. Caiazzo M. Tanaka E.M. Lutolf M.P. Neural tube morphogenesis in synthetic 3D microenvironments.Proc. Natl. Acad. Sci. USA. 2016; 113: E6831-E6839Crossref PubMed Scopus (0) Google Scholar). Synthetic or semi-synthetic matrices have also been used to grow pancreatic organoids (Greggio et al., 2013Greggio C. De Franceschi F. Figueiredo-Larsen M. Gobaa S. Ranga A. Semb H. Lutolf M. Grapin-Botton A. Artificial three-dimensional niches deconstruct pancreas development in vitro.Development. 2013; 140: 4452-4462Crossref PubMed Scopus (99) Google Scholar) as well as intestinal organoids derived from PSCs (Cruz-Acuña et al., 2017Cruz-Acuña R. Quirós M. Farkas A.E. Dedhia P.H. Huang S. Siuda D. García-Hernández V. Miller A.J. Spence J.R. Nusrat A. García A.J. Synthetic hydrogels for human intestinal organoid generation and colonic wound repair.Nat. Cell Biol. 2017; 19: 1326-1335Crossref PubMed Scopus (49) Google Scholar) and ASCs (Gjorevski et al., 2016Gjorevski N. Sachs N. Manfrin A. Giger S. Bragina M.E. Ordóñez-Morán P. Clevers H. Lutolf M.P. Designer matrices for intestinal stem cell and organoid culture.Nature. 2016; 539: 560-564Crossref PubMed Scopus (230) Google Scholar). Of note, the fabrication of defined matrices derived from naturally occurring materials, such as alginate (Capeling et al., 2019Capeling M.M. Czerwinski M. Huang S. Tsai Y.H. Wu A. Nagy M.S. Juliar B. Sundaram N. Song Y. Han W.M. et al.Nonadhesive Alginate Hydrogels Support Growth of Pluripotent Stem Cell-Derived Intestinal Organoids.Stem Cell Reports. 2019; 12: 381-394Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar), fibrin (Broguiere et al., 2018Broguiere N. Isenmann L. Hirt C. Ringel T. Placzek S. Cavalli E. Ringnalda F. Villiger L. Züllig R. Lehmann R. et al.Growth of Epithelial Organoids in a Defined Hydrogel.Adv. Mater. 2018; 30: e1801621Crossref PubMed Scopus (3) Google Scholar), or recombinantly engineered ECMs (DiMarco et al., 2015DiMarco R.L. Dewi R.E. Bernal G. Kuo C. Heilshorn S.C. Protein-engineered scaffolds for in vitro 3D culture of primary adult intestinal organoids.Biomater. Sci. 2015; 3: 1376-1385Crossref PubMed Google Scholar) is another promising strategy to improve the translational relevance of organoids. The optimal conditions for the growth and patterning of mouse intestinal organoids were interrogated using synthetic matrices with a modular ligand composition, stiffness, and degradability and were found to vary, with an intermediate stiffness promoting efficient stem cell expansion and a lower stiffness promoting crypt formation (Gjorevski et al., 2016Gjorevski N. Sachs N. Manfrin A. Giger S. Bragina M.E. Ordóñez-Morán P. Clevers H. Lutolf M.P. Designer matrices for intestinal stem cell and organoid culture.Nature. 2016; 539: 560-564Crossref PubMed Scopus (230) Google Scholar). Of note, the covalent cross-linking of currently used synthetic hydrogels is not permissive to the growth of larger (i.e., millimeter-sized) tissues and generally restricts those morphogenetic processes that require extensive matrix displacement or remodeling (Blondel and Lutolf, 2019Blondel D. Lutolf M.P. Bioinspired Hydrogels for 3D Organoid Culture.Chimia (Aarau). 2019; 73: 81-85Crossref PubMed Scopus (1) Google Scholar). Intriguingly, fine-tuning the dynamical softening of PEG hydrogels in mouse intestinal organoids could satisfy the requirements for both initial optimal stem cell expansion and subsequent organoid patterning (Gjorevski et al., 2016Gjorevski N. Sachs N. Manfrin A. Giger S. Bragina M.E. Ordóñez-Morán P. Clevers H. Lutolf M.P. Designer matrices for intestinal stem cell and organoid culture.Nature. 2016; 539: 560-564Crossref PubMed Scopus (230) Google Scholar), but this might limit long-term culture because of the irreversible nature of the chemical degradation process. We thus postulate that ideal synthetic environments for organoid culture and larger-scale tissue engineering, apart from fulfilling the key signaling functions, should prevent the accumulation of excessive compressive forces in response to tissue growth and morphogenesis. Hydrogels that have been partially or completely physically crosslinked more realistically capture the viscoelasticity and dynamics of native ECMs. They should thus be well suited to relax in response to tissue-induced compressive stresses by breaking and subsequently rearranging their network, permitting cellular remodeling without compromising the stability of the material over time (McKinnon et al., 2014McKinnon D.D. Domaille D.W. Cha J.N. Anseth K.S. Biophysically defined and cytocompatible covalently adaptable networks as viscoelastic 3D cell culture systems.Adv. Mater. 2014; 26: 865-872Crossref PubMed Scopus (129) Google Scholar). Although they have not yet been used in