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.

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 cues 4.

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.

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 patient

Click on image for larger view.

Organoids generation from primary tissues and pluripotent stem cells and their applications

Figure 1: 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
  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
  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
  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
  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
  hAdSc Establishment: TGFβ inhibitors, Noggin, R-Spondin 1, WNT3A, EGF, FGF10, Nicotinamide
Differentiation: Not reported
Epithelial ductal cells 23
  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
  hPSC Endoderm induction: Activin A
Forget endoderm differentiation: BMP, TGF-β and Wnt inhibitors
Ventral lung airway progenitors: Wnt, BMP, FGF, RA activators
Lung organoids: Wnt, FGF, cAMP and glucocorticoids
Mesenchymal and lung epithelial cells 33
  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
  hPSC Intermediate mesoderm induction: Wnt, GSK3α inhibitor
Organoid formation: GSK3α inhibitor, FGF9
Nephrons and endothelial cells 11,34

Organoid Cell Culture Products


Part Number Description
CLS431751 70 μM Cell Strainer (Corning)
CLS431752 100 μM Cell Strainer (Corning)
CLS3473 24-well ultra-low adhesion plates (Corning)
SCNY00100 Steriflip 100 μm Nylon Net, 25/pk
SCNY00060 Steriflip 60 μm Nylon Net, 25/pk
SCNY00040 Steriflip 40 μm Nylon Net, 25/pk


Part Number Description
SCR103 Collagenase Type I
C6885 Collagenase Type II
C5138 Collagenase Type IV
10269638001 Collagenase/Dispase
D4693 Dispase II
G9020 Gastrin I human
A6964 Accutase
4716728001 Recombinant DNase I, RNase Free

Organoid Matrices

Part Number Description
3532-001-02 Cultrex® BME, Type 2 PathClear®
3533-001-02 Cultrex® Reduced Growth Factor BME, PathClear®
E1270 ECM Gel from Engelbreth-Holm-Swarm murine sarcoma
E6909 ECM Gel, Growth Factor Reduced

Cell Lines

Part Number Description
SCC111 R-Spondin1 expressing 293T Cell Line

Media and Culture Reagents

Part Number Description
D8537 Phosphate Buffered Saline (PBS)
A2058 Bovine Serum Albumin
E9884 Ethylenediaminetetraacetic acid (EDTA)
D5796 DMEM, High Glucose
P4333 Penicillin-Streptomycin
G8541 Stabilized Glutamine (200 mM, solution)
A9165 N-Acetylcysteine
N3376 Nicotinamide
A4403 L-Ascorbic acid
SCM012 NDiff Neuro-2 Medium Supplement (200x)
G0800 GS21™ Supplement (50X)
SCM110 Human ES/iPS Neural Induction Medium
SCM018 Embryoid Body (EB) Formation Medium

Growth Factors and Cytokines

Part Number Description
SRP3196 Recombinant Murine EGF
E9644 Recombinant Human EGF
SRP3227 Recombinant Murine Noggin
SRP4675 Recombinant Human Noggin
SRP3292 Recombinant Human R-Spondin-1 (CHO cells)
SRP6487 Recombinant Human R-Spondin-1 (HEK293 cells)
GF154 Recombinant Mouse Wnt3a
F8924 Recombinant FGF-10
SRP4037 Recombinant FGF-2

Small Molecules

Part Number Description
Y0503 Y-27632 (ROCK inhibitor)
S7076 SB202190 ( p38 MAP kinase inhibitor)
SML1046 CHIR99021 (Glycogen synthase kinase 3 inhibitor)
SML0788 A83-01 (TGFβ kinase/activin receptor-like kinase (ALK 5) inhibitor)

Histology Reagents

Part Number Description
47608 Paraformaldehyde (PFA) 36% in H2O
34860 Methanol
P2287 TWEEN® 20 viscous liquid, cell culture tested
T8787 Triton™ X-100
F6057 Fluoroshield™ with DAPI, histology mounting medium
HPA012530 Anti-LGR5 antibody



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