3D Organoid Culture: New In Vitro Models of Development and Disease

2D vs. 3D Cell Model Systems

Model systems drive biological research by recapitulating body processes and functions from the molecular to whole organism level. The human body is composed of both cellular and non-cellular material organized in a highly specialized manner. It is difficult to mimic all aspects of human biology with one in vitro model system. 3D cell culture models are a more accurate representation of the natural environment experienced by cells in the living organism as opposed to growing cells on 2D flat surfaces.

Limitations of Existing Cell Model System

Animal Models	2D Cell Monolayers	3D Cell Aggregates
Differences in human and animal biology Limited usability in imaging and high-throughput studies1 High cost	Cells lose their phenotype Lack cell-cell and cell-matrix interactions Could not mimic cellular functions and signaling pathways as in in-vivo conditions	Transiently resemble cell organization and interactions Difficult to maintain long term cultures Lack potency for self-renewal and differentiation2

What are Organoids?

Organoids are in-vitro derived 3D cell aggregates derived from primary tissue or stem cells that are capable of self-renewal, self-organization and exhibit organ functionality.³ Organoids address the limitations of existing model systems by providing:

• Similar composition and architecture to primary tissue: Organoids harbor small population of self-renewing stem cells that can differentiate into cells of all major cell lineages, with similar frequency as in physiological condition.
• Relevant models of in-vivo conditions: Organoids are more biologically relevant to any model system and are amenable to manipulate niche components and gene sequence.
• Stable system for extended cultivation: Organoids can be cryopreserved as biobanks and expanded indefinitely by leveraging self-renewal, differentiation capability of stem cell and intrinsic ability to self-organize.

Figure 1. Mouse Intestinal Epithelial Organoids. 3D Organoids were generated from adult mouse intestinal tissue following the protocol outlined by Clevers et al. Science. 2013. Organoid cells begin to form lumens and bud structures at around day 3-5 in culture and form complex crypt-like structures around day 7-10. These crypt-like domains are functionally similar to those of the adult intestine, where dividing LGR5+ intestinal stem cells are intercalated with Paneth cells located at the crypt base.

Organoids vs. Spheroids

Organoids and spheroids are both cells cultured in 3 dimensions. Spheroids are often formed from cancer cell lines or tumor biopsies as freely floating cell aggregates in ultra-low attachment plates whereas organoids are derived from tissue stem cells embedded within an ECM hydrogel matrix such as Matrigel. Organoids are highly complex and are more in vivo-like when compared to spheroids. Recently, tumor organoids have shown to predict how well patients respond to cancer drugs to aid in personalized medicine.

Figure 2. Organoids vs. Spheroids. Stem cell derived organoids have more in vivo-like phenotypes with higher order tissue complexity compared to tumor spheroids.

How are Organoids Generated?

Organoids are generated either from primary tissues or pluripotent stem cells (induced pluripotent stem cells (iPSC) or embryonic stem cells (ESCs)) by providing appropriate physical and biochemical cues⁴.

Physical cues: Provide support for cell attachment and survival. Examples include collagen, fibronectin, entactin and laminin.

Biochemical cues: Modulate signaling pathways, thereby influencing proliferation, differentiation and self-renewal. Examples include EGF, FGF10, HGF, R-spondin, WNT3A, Retinoic acid, GSK3β inhibitors, TGF-β inhibitors, HDAC inhibitors, ROCK inhibitors, Noggin, Activin A, p38 inhibitors and Gastrin.

Organoid Qualified Reagents

Applications of Organoids

Organoids are physiologically relevant and amenable to molecular and cell biological analyses, holding great promise in both basic research and translational applications.

Developmental biology: Organoids derived from ESC, iPSCs retain features of their developmental stage and help in studying the process of embryonic development, lineage specification and tissue homeostasis. It also shed light on development of stem cells and their niche.

• Development of organs such as brain²⁴, pancreas²⁵ and stomach⁷ was studied through sequential differentiation steps inducted by modulating Wnt, BMP and FGF signaling pathways

Disease pathology of infectious disease: Organoids represents all components of organ and are suited to study infectious diseases affecting specialized human cell types.

• Lung organoids derived from iPSCs from healthy child carrying null alleles of interferon regulatory factor- 7 gene employed to study influenza virus replication ²⁶
  
• Forebrain organoids derived from human iPSC was employed to study infection of zika virus on neural progenitors²⁷

Regenerative medicine: Transplantation of organoids derived from the adult stem cells aid in replacing the damaged organ or tissue. In addition, feasibility for gene correction using CRISPR/Cas9 technology can be used in treating monogenic hereditary diseases.

• Small intestine organoids retained characteristics of small intestine, such as villus formation and presence of paneth when transplanted in mouse models²⁸

Drug toxicity and efficacy testing: The possibility to test efficacy and toxicity of drugs against representative targets/organs (gut, liver and kidney) could potentially limit the ethical issues associated with animal usage.

• Hyman kidney organoids were employed to demonstrate the nephrotoxicity of cisplatin ¹¹

Personalized medicine: Organoids derived from adult stem cell of individual patients allows ex-vivo testing of drug response.

• Colon organoids were employed to identify treatment options for patients with rare CFTR mutations²⁹
  
• Tumor organoids can be employed to assess the drug response at the level of individual patie

Figure 3. Organoids generation from primary tissues and pluripotent stem cells and their applications

Table 1 Summary of growth factors and biochemical used in the development of various organoids cell types.

Organoids	Source	Culture conditions	Cell types in organoids	Reference
Stomach
	hPSCs	Endoderm induction: Rock inhibitor (Y-27632), Activin A, BMP5 Spheroid generation: WNT, FGF, Noggin, Retinoic acid Organoid formation: Noggin, Retinoic acid, EGF Maturation: EGF	LGR5+ cells, mucous cells, gastric endocrine cells	7
	hAdSC	EGF, Rspondin, Noggin, FGF10, WNT, Gastrin, Nicotinamide and TGFβ inhibitor	LGR5+ cells, pit mucous cells, gland mucous cells, chief cells and enteroendocrine cells	13
Intestine
	hPSC	Endoderm induction: Activin A, BMP4 Hindgut differentiation (spheroid generation): FGF4, WNT3A Organoid formation: FGF4, WNT3A Maturation: RSpondin1, Noggin, EGF, FGF4, WNT	Enterocytes, Goblet, Paneth and enteroendocrine cells	30
	hAdSC	Establishment : EGF, Rspondin, Noggin, WNT3A, Nicotinamide, Gastrin, TGFβinhibitor, p38 inhibitor Differentiation : Without WNT3A, p38 MAP kinase inhibitor and nicotinamide	Intestinal epithelial derivatives and stem cells	31
Colon
	hAdSC	Establishment: EGF, Rspondin, Noggin, WNT3A, Nicotinamide, Gastrin, TGFβinhibitor, p38 inhibitor Differentiation : Without WNT3A, p38 MAP kinase inhibitor and nicotinamide	Epithelial cells and mesenchymal derivatives	31
Liver
	hAdSC	Establishment: Noggin, WNT, ROCK inhibitor Differentiation: Gastrin, EGF, Rspondin, FGF10, hepatocyte growth factor, nicotinamide, TGFβinhibitor, Forskolin	functional hepatocyte cells	14
	hiPSC	Endoderm Induction: Activin A Hepatic specification: BMP4, FGF2, hepatocyte growth factor Maturation: Oncostatin M	Functional hepatocyte cells	32
Pancreas
	hAdSc	Establishment: TGFβ inhibitors, Noggin, R-Spondin 1, WNT3A, EGF, FGF10, Nicotinamide Differentiation: Not reported	Epithelial ductal cells	23
Prostate
	hAdSc	EGF, R-Spondin1, Noggin, TGF-β inhibitor, p38 MAP kinase inhibitor, FGF10, FGF2, PGE2, Nicotinamide and DHT	Differentiated CK5+ basal and CK8+ luminal cells	17
Lung
	hPSC	DHT	Mesenchymal and lung epithelial cells	33
Brain
	hPSC	Neural induction: N2 supplement, NEAA and heparin Differentiation: N2 supplement, 2-mercaptoethanol, insulin Maturation: Vitamin A, retinoic acid	Progenitor populations which produce mature cortical neurons	24
Kidney
	hPSC	Intermediate mesoderm induction: Wnt, GSK3α inhibitor Organoid formation: GSK3α inhibitor, FGF9	Nephrons and endothelial cells	11, 34

Related Organoid Cell Culture Products

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