Challenges to, and prospects for, reverse engineering the gastrointestinal tract using organoids
2022; Elsevier BV; Volume: 40; Issue: 8 Linguagem: Inglês
10.1016/j.tibtech.2022.01.006
ISSN0167-9430
AutoresPanagiota Kakni, Roman Truckenmüller, Pamela Habibović, Stefan Giselbrecht,
Tópico(s)Pluripotent Stem Cells Research
ResumoOrganoids are self-organizing 3D cell culture systems that constitute a unique tool for studying development and disease.Over the past decade, efficient protocols to grow organoids that resemble the different parts of the GIT have been established.Bioengineering approaches have been used to improve organoid cultures.Organoid fusion and on-chip methods are explored to increase complexity of organoids to better recapitulate in vivo tissues. For over a decade, organoids mimicking the development, physiology, and disease of the digestive system have been a topic of broad interest and intense study. Establishing organoid models that recapitulate all distinct regions of the gastrointestinal tract (GIT) has proven challenging since each tissue surrogate requires tailor-made modifications of the original protocol to generate intestinal organoids. In this review, we discuss the challenges and current advances of the GIT organoid models. Moreover, we envision the next-generation GIT organoids as integrated organoid models, able to recapitulate structural and functional characteristics of multiple regions of the digestive tube in a single in vitro model. We discuss these new trends and provide an outlook for the future of GIT in vitro models. For over a decade, organoids mimicking the development, physiology, and disease of the digestive system have been a topic of broad interest and intense study. Establishing organoid models that recapitulate all distinct regions of the gastrointestinal tract (GIT) has proven challenging since each tissue surrogate requires tailor-made modifications of the original protocol to generate intestinal organoids. In this review, we discuss the challenges and current advances of the GIT organoid models. Moreover, we envision the next-generation GIT organoids as integrated organoid models, able to recapitulate structural and functional characteristics of multiple regions of the digestive tube in a single in vitro model. We discuss these new trends and provide an outlook for the future of GIT in vitro models. The emergence of, and rapid progress in, organoid models have contributed significantly to the field of stem cell research. Organoids can be derived from two main stem cell types: pluripotent stem cells (PSCs; see Glossary) or organ-specific adult stem cells (ASCs). For PSC-derived models, both embryonic (ESCs) and induced pluripotent (iPSCs) stem cells can be used. The resulting organoids contain either both epithelial and mesenchymal cell types or only epithelial cells [1.Broutier L. et al.Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation.Nat. 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These self-organizing, 3D models are promising tools to bridge the gap between in vitro monolayer cultures and in vivo studies, because they recapitulate tissue function and structure more accurately than do conventional 2D cell cultures. Although they are characterized by an increased structural and functional complexity, they are amenable to most standard experimental techniques [5.Fatehullah A. et al.Organoids as an in vitro model of human development and disease.Nat. Cell Biol. 2016; 18: 246-254Crossref PubMed Scopus (820) Google Scholar, 6.Min S. et al.Gastrointestinal tract modeling using organoids engineered with cellular and microbiota niches.Exp. Mol. 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In 2009, two pioneering models for growing intestinal organoids from adult mouse tissues were described. The first was developed by Clever's team, who used previously identified Lgr5+ stem cells [9.Barker N. et al.Identification of stem cells in small intestine and colon by marker gene Lgr5.Nature. 2007; 449: 1003-1007Crossref PubMed Scopus (3894) Google Scholar] to generate 3D epithelial organoids that resembled the crypt-villus architecture and expressed all the differentiated cell types present in the intestine in vivo [10.Sato T. et al.Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.Nature. 2009; 459: 262-265Crossref PubMed Scopus (4027) Google Scholar]. The second model was described by Kuo's team, who embedded murine small and/or large intestinal fragments, including stromal cells, in collagen type I gels and placed them in a 3D air–liquid interface (ALI-3D) system [11.Ootani A. et al.Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche.Nat. Med. 2009; 15: 701-706Crossref PubMed Scopus (608) Google Scholar]. A couple of years later, the first PSC-derived intestinal organoid model was described. In this case, a stepwise differentiation method that mimics the embryonic intestinal development was required. Specifically, PSCs were initially differentiated toward the definitive endoderm, then hindgut, and finally toward intestinal tissue [12.Spence J.R. et al.Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro.Nature. 2011; 470: 105-109Crossref PubMed Scopus (1257) Google Scholar]. Apart from the simple columnar epithelium, hPSC-derived intestinal organoids comprise a mesenchymal layer that develops along with the epithelium, thus indicating the importance of epithelial–mesenchymal interactions during intestine specification. Despite the common developmental origin, each part of the digestive system demonstrates a characteristic stem cell niche [13.Fujii M. et al.Modeling human digestive diseases With CRISPR-Cas9-modified organoids.Gastroenterology. 2019; 156: 562-576Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar,14.Date S. Sato T. Mini-gut organoids: reconstitution of the stem cell niche.Annu. Rev. Cell Dev. Biol. 2015; 31: 269-289Crossref PubMed Scopus (131) Google Scholar] and epithelial turnover [3.Günther C. et al.What gastroenterologists and hepatologists should know about organoids in 2019.Dig. Liver Dis. 2019; 51: 753-760Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar]. 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The embryonic development of the GIT is a complex process initiated during gastrulation [83.Zorn A.M. Wells J.M. Vertebrate endoderm development and organ formation.Annu. Rev. Cell Dev. Biol. 2009; 25: 221-251Crossref PubMed Scopus (530) Google Scholar, 84.Montgomery R.K. et al.Development of the human gastrointestinal tract: twenty years of progress.Gastroenterology. 1999; 3: P702-P731Abstract Full Text Full Text PDF Scopus (227) Google Scholar, 85.Wells J.M. Melton D.A. Vertebrate endoderm development.Annu. Rev. Cell Dev. Biol. 1999; 15: 393-410Crossref PubMed Scopus (422) Google Scholar, 86.Gao S. et al.Tracing the temporal-spatial transcriptome landscapes of the human fetal digestive tract using single-cell RNA-sequencing.Nat. Cell Biol. 2018; 20: 721-734Crossref PubMed Scopus (73) Google Scholar, 87.Kiefer J.C. Molecular mechanisms of early gut organogenesis: a primer on development of the digestive tract.Dev. Dyn. 2003; 228: 287-291Crossref PubMed Scopus (23) Google Scholar, 88.Dunn N.R. Hogan B.L.M. The endoderm from a diverse perspective.Development. 2018; 145dev163550Crossref PubMed Scopus (2) Google Scholar]. Gastrulation is a critical early step in developing multicellular organisms because it gives rise to the three primary germ layers: the endoderm, mesoderm, and ectoderm [83.Zorn A.M. Wells J.M. Vertebrate endoderm development and organ formation.Annu. Rev. Cell Dev. Biol. 2009; 25: 221-251Crossref PubMed Scopus (530) Google Scholar, 84.Montgomery R.K. et al.Development of the human gastrointestinal tract: twenty years of progress.Gastroenterology. 1999; 3: P702-P731Abstract Full Text Full Text PDF Scopus (227) Google Scholar, 85.Wells J.M. Melton D.A. Vertebrate endoderm development.Annu. Rev. Cell Dev. Biol. 1999; 15: 393-410Crossref PubMed Scopus (422) Google Scholar,89.Nowotschin S. et al.The endoderm: a divergent cell lineage with many commonalities.Development. 2019; 146dev150920Crossref PubMed Scopus (35) Google Scholar]. Following gastrulation, the naïve endoderm transforms into a primitive gut tube, surrounded by splanchnic mesoderm, through a series of morphogenetic events [83.Zorn A.M. Wells J.M. Vertebrate endoderm development and organ formation.Annu. Rev. Cell Dev. Biol. 2009; 25: 221-251Crossref PubMed Scopus (530) Google Scholar,85.Wells J.M. Melton D.A. Vertebrate endoderm development.Annu. Rev. Cell Dev. Biol. 1999; 15: 393-410Crossref PubMed Scopus (422) Google Scholar,87.Kiefer J.C. Molecular mechanisms of early gut organogenesis: a primer on development of the digestive tract.Dev. Dyn. 2003; 228: 287-291Crossref PubMed Scopus (23) Google Scholar,90.Zorn A.M. Wells J.M. Molecular basis of vertebrate endoderm development.Int. Rev. Cytol. 2007; 259: 49-111Crossref PubMed Scopus (125) Google Scholar]. During this time, three distinct regions, the foregut, midgut, and hindgut, become prominent as the gut tube is patterned along the anterior–posterior (A–P) axis. Epithelial–mesenchymal interactions drive each one of these regions to undergo further patterning to form specific primary organs. The foregut gives rise to the esophagus, trachea, stomach, lungs, thyroid, liver, biliary system, and the pancreas; the midgut gives rise to the small intestine; and the hindgut to the large intestine. As development proceeds, organ buds in conjunction with their surrounding mesenchyme continue to proliferate and differentiate into functional organs that eventually branch from the main tube [83.Zorn A.M. Wells J.M. Vertebrate endoderm development and organ formation.Annu. Rev. Cell Dev. Biol. 2009; 25: 221-251Crossref PubMed Scopus (530) Google Scholar,91.Sheaffer K.L. Kaestner K.H. Transcriptional networks in liver and intestinal development.Cold Spring Harb. Perspect. Biol. 2012; 4a008284Crossref PubMed Scopus (35) Google Scholar]. During gut development, maintenance of regional identity relies on three mechanisms. First, the interplay between numerous signaling pathways, including the FGF, Wnt, Shh, BMP, RA and Notch pathways, tightly coordinates local gene expression throughout development and function. Each region requires different combinations of transcription factors. Extracellular signaling factors also have crucial stage-specific roles. Finally, morphogenetic processes and correct cell positioning are required for proper signaling between neighboring tissues [83.Zorn A.M. Wells J.M. Vertebrate endoderm development and organ formation.Annu. Rev. Cell Dev. Biol. 2009; 25: 221-251Crossref PubMed Scopus (530) Google Scholar,91.Sheaffer K.L. Kaestner K.H. Transcriptional networks in liver and intestinal development.Cold Spring Harb. Perspect. Biol. 2012; 4a008284Crossref PubMed Scopus (35) Google Scholar,92.Grapin-Botton A. Melton D.A. Endoderm development: from patterning to organogenesis.Trends Genet. 2000; 16: 124-130Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar]. The human GIT comprises a series of hollow organs joined in a long muscular tube running from the oral cavity to the anus. These organs are the esophagus, stomach, small intestine, and large intestine. The embryonic development of the GIT is a complex process initiated during gastrulation [83.Zorn A.M. Wells J.M. Vertebrate endoderm development and organ formation.Annu. Rev. Cell Dev. Biol. 2009; 25: 221-251Crossref PubMed Scopus (530) Google Scholar, 84.Montgomery R.K. et al.Development of the human gastrointestinal tract: twenty years of progress.Gastroenterology. 1999; 3: P702-P731Abstract Full Text Full Text PDF Scopus (227) Google Scholar, 85.Wells J.M. Melton D.A. Vertebrate endoderm development.Annu. Rev. Cell Dev. Biol. 1999; 15: 393-410Crossref PubMed Scopus (422) Google Scholar, 86.Gao S. et al.Tracing the temporal-spatial transcriptome landscapes of the human fetal digestive tract using single-cell RNA-sequencing.Nat. Cell Biol. 2018; 20: 721-734Crossref PubMed Scopus (73) Google Scholar, 87.Kiefer J.C. Molecular mechanisms of early gut organogenesis: a primer on development of the digestive tract.Dev. Dyn. 2003; 228: 287-291Crossref PubMed Scopus (23) Google Scholar, 88.Dunn N.R. Hogan B.L.M. The endoderm from a diverse perspective.Development. 2018; 145dev163550Crossref PubMed Scopus (2) Google Scholar]. Gastrulation is a critical early step in developing multicellular organisms because it gives rise to the three primary germ layers: the endoderm, mesoderm, and ectoderm [83.Zorn A.M. Wells J.M. Vertebrate endoderm development and organ formation.Annu. Rev. Cell Dev. Biol. 2009; 25: 221-251Crossref PubMed Scopus (530) Google Scholar, 84.Montgomery R.K. et al.Development of the human gastrointestinal tract: twenty years of progress.Gastroenterology. 1999; 3: P702-P731Abstract Full Text Full Text PDF Scopus (227) Google Scholar, 85.Wells J.M. Melton D.A. Vertebrate endoderm development.Annu. Rev. Cell Dev. Biol. 1999; 15: 393-410Crossref PubMed Scopus (422) Google Scholar,89.Nowotschin S. et al.The endoderm: a divergent cell lineage with many commonalities.Development. 2019; 146dev150920Crossref PubMed Scopus (35) Google Scholar]. Following gastrulation, the naïve endoderm transforms into a primitive gut tube, surrounded by splanchnic mesoderm, through a series of morphogenetic events [83.Zorn A.M. Wells J.M. Vertebrate endoderm development and organ formation.Annu. Rev. Cell Dev. Biol. 2009; 25: 221-251Crossref PubMed Scopus (530) Google Scholar,85.Wells J.M. Melton D.A. Vertebrate endoderm development.Annu. Rev. Cell Dev. Biol. 1999; 15: 393-410Crossref PubMed Scopus (422) Google Scholar,87.Kiefer J.C. Molecular mechanisms of early gut organogenesis: a primer on development of the digestive tract.Dev. Dyn. 2003; 228: 287-291Crossref PubMed Scopus (23) Google Scholar,90.Zorn A.M. Wells J.M. Molecular basis of vertebrate endoderm development.Int. Rev. Cytol. 2007; 259: 49-111Crossref PubMed Scopus (125) Google Scholar]. During this time, three distinct regions, the foregut, midgut, and hindgut, become prominent as the gut tube is patterned along the anterior–posterior (A–P) axis. Epithelial–mesenchymal interactions drive each one of these regions to undergo further patterning to form specific primary organs. The foregut gives rise to the esophagus, trachea, stomach, lungs, thyroid, liver, biliary system, and the pancreas; the midgut gives rise to the small intestine; and the hindgut to the large intestine. As development proceeds, organ buds in conjunction with their surrounding mesenchyme continue to proliferate and differentiate into functional organs that eventually branch from the main tube [83.Zorn A.M. Wells J.M. Vertebrate endoderm development and organ formation.Annu. Rev. Cell Dev. Biol. 2009; 25: 221-251Crossref PubMed Scopus (530) Google Scholar,91.Sheaffer K.L. Kaestner K.H. Transcriptional networks in liver and intestinal development.Cold Spring Harb. Perspect. Biol. 2012; 4a008284Crossref PubMed Scopus (35) Google Scholar]. During gut development, maintenance of regional identity relies on three mechanisms. First, the interplay between numerous signaling pathways, including the FGF, Wnt, Shh, BMP, RA and Notch pathways, tightly coordinates local gene expression throughout development and function. Each region requires different combinations of transcription factors. Extracellular signaling factors also have crucial stage-specific roles. Finally, morphogenetic processes and correct cell positioning are required for proper signaling between neighboring tissues [83.Zorn A.M. Wells J.M. Vertebrate endoderm development and organ formation.Annu. Rev. Cell Dev. Biol. 2009; 25: 221-251Crossref PubMed Scopus (530) Google Scholar,91.Sheaffer K.L. Kaestner K.H. Transcriptional networks in liver and intestinal development.Cold Spring Harb. Perspect. Biol. 2012; 4a008284Crossref PubMed Scopus (35) Google Scholar,92.Grapin-Botton A. Melton D.A. Endoderm development: from patterning to organogenesis.Trends Genet. 2000; 16: 124-130Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar]. Despite tremendous progress in this field, organoids are still far from a perfect system and come with certain limitations and challenges, such as the use of undefined animal tumor-derived extracellular matrix (ECM), lack of complexity and surrounding tissues, low levels of maturation, uncontrolled growth, and many others. In an attempt to improve this system, several approaches have been described focusing either solely on biological or on a combination of biological and engineering inspired methods. Specifically, to increase complexity and achieve further maturation, various co-culture systems, bioreactors, and organoid-on-a-chip methods have been used. Fully defined, tunable, synthetic matrices and bioprinting techniques have also been established to replace poorly defined matrices and to better control organoid architecture. In this review, we provide an overview of methods that have been applied to tackle some of these issues. Mapping the landscape of the GI organoid field is an important step toward the next generation of 3D in vitro models, which are capable of modeling multiregional architectures and functions by integrating multiple distinct regions of tissues or organs in one system. We discuss the role of advanced technologies in this endeavor, including roadmaps based on the compartmentalization strategy of organ-on-chip systems and the recently established organoid fusion techniques. GI organoids have been widely used as an in vitro model to study development, physiology, and disease. However, one limiting factor in extending their applicability in translational and clinical research is the use of poorly defined matrices (e.g., Matrigel, Cultrex, or Geltrex). These ECM substitutes are derived from mouse sarcoma cells. They also show batch-to-batch variability, their physical and chemical properties are difficult to control, and they potentially transfer pathogens [31.Capeling M.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 (107) Google Scholar]. Hence, numerous groups have tried to replace those with other synthetic matrices. Initially, a hydrogel containing a fibronectin-derived, cell-adhesive, extended Arg-Gly-Asp (RGD) amino acid sequence, and an elastin-like structural backbone was able to support the growth of mouse intestinal organoids under ALI-3D conditions [32.DiMarco R.L. et al.Protein-engineered scaffolds for in vitro 3D culture of primary adult intestinal organoids.Biomater. Sci. 2015; 3: 1376-1385Crossref PubMed Google Scholar]. Later on, enriched polyethylene glycol (PEG) hydrogels were found to support the growth and differentiation of mouse intestinal stem cells into organoids [33.Gjorevski N. et al.Designer matrices for intestinal stem cell and organoid culture.Nature. 2016; 539: 560-564Crossref PubMed Scopus (744) Google Scholar]. The same hydrogel was used to culture human ASC-derived organoids, but the efficiency was lower, suggesting that further optimization of the protocol was required [33.Gjorevski N. et al.Designer matrices for intestinal stem cell and organoid culture.Nature. 2016; 539: 560-564Crossref PubMed Scopus (744) Google Scholar]. For the culture of human PSC (hPSC)-derived intestinal organoids, mid/hindgut spheroids were embedded in a PEG-4MAL (maleimide groups at each terminus) hydrogel functionalized with RGD peptides and expanded to mature intestinal organoids with similar efficiency to Matrigel [34.Cruz-Acuña R. et al.PEG-4MAL hydrogels for human organoid generation, culture, and in vivo delivery.Nat. Protoc. 2018; 13: 2102-2119Crossref PubMed Scopus (73) Google Scholar]. Interestingly, these hydrogels were able to guide organoids toward intestinal mucosal wounds upon injection in mice, thus supporting localized engraftment of the organoids and more efficient wound closure [34.Cruz-Acuña R. et al.PEG-4MAL hydrogels for human organoid generation, culture, and in vivo delivery.Nat. Protoc. 2018; 13: 2102-2119Crossref PubMed Scopus (73) Google Scholar,35.Spence J. et al.PEG-4MAL hydrogels for in vitro culture of human organoids and in vivo delivery to sites of injury.Protoc. Exch. 2017; (Published online November 21, 2017)https://doi.org/10.1038/protex.2017.098Crossref Google Scholar]. Unmodified alginate, an algae-derived polysaccharide, has been proven capable of supporting the growth of PSC-derived intestinal organoids both in vitro and in vivo with similar efficiency to Matrigel. By contrast, primary cell-derived organoids lacking mesenchyme failed to grow in this matrix [31.Capeling M.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 (107) Google Scholar]. A plant-based nanocellulose hydrogel functionalized with RGD peptides and glycine supported the culture of mouse intestinal organoids, which were initially formed in Matrigel, but failed to support the formation of organoids from freshly isolated mouse intestinal crypts [36.Curvello R. et al.Engineered Plant-based nanocellulose hydrogel for small intestinal organoid growth.Adv. Sci. 2021; 82002135Crossref Scopus (21) Google Scholar]. All these studies demonstrate the versatility of synthetic matrices with regard to the physical properties (stiffness, degradability, viscoelasticity, etc.) and bioactivity of the organoid environment. Engineered ECM substitutes offer the possibility to tailor the biochemical and mechanical properties according to the organoid type and application. This will be of increasing importance once multiple organoid types need to be cultured and maintained in a single cell culture system. However, these synthetic and highly defined hydrogels are still in their infancy. They can only be used for certain applications and still cannot fully replace the widely used commercially available natural matrices. Another downside of using these ill-defined, solid hydrogel matrices is that they usually hinder the use of organoids in high-throughput and pharmacological studies. Embedding organoids in viscous hydrogels complicates their handling and downstream processing because they are usually randomly distributed in the matrix and their direct accessibility is impaired. Hydrogels also appear to cause more variation in the size and shape of the organoids [37.Kakni P. et al.Intestinal organoid culture in polymer film-based microwell arrays.Adv. Biosyst. 2020; 4e2000126PubMed Google Scholar]. To overcome these issues, platforms that support the growth of organoids without a solid matrix have been described. It has been shown that murine intestinal stem cells cultured in flat-bottom plates require 10% Matrigel to generate organoids, whereas only 1% was sufficient in V-shaped wells of 96-well plates. This set-up also allowed for easier single-organoid tracking during the experiment and was used to study stem cell competition following irradiation [38.Fujimichi Y. et al.An efficient intestinal organoid system of direct sorting to evaluate stem cell competition in vitro.Sci. Rep. 2019; 9: 1-9Crossref PubMed Scopus (11) Google Scholar]. Mouse intestinal organoids, derived from ASCs, have successfully been cultured in thermoformed polymer film-based microwell arrays using 5% Matrigel as a media supplement [37.Kakni P. et al.Intestinal organoid culture in polymer film-based microwell arrays.Adv. Biosyst. 2020; 4e2000126PubMed Google Scholar]. Within this platform, organoids grew in a more controlled environment, which improved the homogeneity of the organoids and allowed for an extended culture period. Furthermore, hydrogel microwell arrays have been shown to facilitate the culture of both mouse and human intestinal organoids with 2% Matrigel, achieving rapid and homogeneous organoid growth and facilitating automated high-throughput analysis. A proof-of-concept drug screening was also performed to demonstrate
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