Attention:

Certain features of Sigma-Aldrich.com will be down for maintenance the evening of Friday August 18th starting at 8:00 pm CDT until Saturday August 19th at 12:01 pm CDT.   Please note that you still have telephone and email access to our local offices. We apologize for any inconvenience.

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
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 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
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

Products for Organoid Cell Culture

Cultureware

Part Number Description
CLS431751 70 μM Cell Strainer (Corning)
CLS431752 100 μM Cell Strainer (Corning)
CLS3473 24-well ultra-low adhesion plates (Corning)


Enzymes

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
H3375 HEPES
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

 

 References

  1. Shanks, N., Greek, R., and Greek, J. (2009) Are animal models predictive for humans? Philos. Ethics Humanit. Med. PEHM 4, 2.
  2. Yin, X., Mead, B. E., Safaee, H., Langer, R., Karp, J. M., and Levy, O. (2016) Engineering Stem Cell Organoids. Cell Stem Cell 18, 25–38.
  3. Lancaster, M. A., and Knoblich, J. A. (2014) Organogenesis in a dish: modeling development and disease using organoid technologies. Science 345, 1247125.
  4. Clevers, H. (2016) Modeling Development and Disease with Organoids. Cell 165, 1586–1597.
  5. Eiraku, M., and Sasai, Y. (2012) Self-formation of layered neural structures in three-dimensional culture of ES cells. Curr. Opin. Neurobiol. 22, 768–777.
  6. Nakano, T., Ando, S., Takata, N., Kawada, M., Muguruma, K., Sekiguchi, K., Saito, K., Yonemura, S., Eiraku, M., and Sasai, Y. (2012) Self-formation of optic cups and storable stratified neural retina from human ESCs. Cell Stem Cell 10, 771–785.
  7. McCracken, 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., and Wells, J. M. (2014) Modelling human development and disease in pluripotent stem-cell-derived gastric organoids. Nature 516, 400–404.
  8. Wong, A. P., Bear, C. E., Chin, S., Pasceri, P., Thompson, T. O., Huan, L.-J., Ratjen, F., Ellis, J., and Rossant, J. (2012) Directed differentiation of human pluripotent stem cells into mature airway epithelia expressing functional CFTR protein. Nat. Biotechnol. 30, 876–882.
  9. Huang, S. X. L., Islam, M. N., O’Neill, J., Hu, Z., Yang, Y.-G., Chen, Y.-W., Mumau, M., Green, M. D., Vunjak-Novakovic, G., Bhattacharya, J., and Snoeck, H.-W. (2014) Efficient generation of lung and airway epithelial cells from human pluripotent stem cells. Nat. Biotechnol. 32, 84–91.
  10. Takebe, T., Sekine, K., Enomura, M., Koike, H., Kimura, M., Ogaeri, T., Zhang, R.-R., Ueno, Y., Zheng, Y.-W., Koike, N., Aoyama, S., Adachi, Y., and Taniguchi, H. (2013) Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature 499, 481–484.
  11. Takasato, M., Er, P. X., Chiu, H. S., Maier, B., Baillie, G. J., Ferguson, C., Parton, R. G., Wolvetang, E. J., Roost, M. S., Chuva de Sousa Lopes, S. M., and Little, M. H. (2015) Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis. Nature 526, 564–568.
  12. Sato, T., and Clevers, H. (2013) Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications. Science 340, 1190–1194.
  13. Bartfeld, S., Bayram, T., van de Wetering, M., Huch, M., Begthel, H., Kujala, P., Vries, R., Peters, P. J., and Clevers, H. (2015) In vitro expansion of human gastric epithelial stem cells and their responses to bacterial infection. Gastroenterology 148, 126–136.e6.
  14. Huch, M., Gehart, H., van Boxtel, R., Hamer, K., Blokzijl, F., Verstegen, M. M. A., Ellis, E., van Wenum, M., Fuchs, S. A., de Ligt, J., van de Wetering, M., Sasaki, N., Boers, S. J., Kemperman, H., de Jonge, J., Ijzermans, J. N. M., Nieuwenhuis, E. E. S., Hoekstra, R., Strom, S., Vries, R. R. G., van der Laan, L. J. W., Cuppen, E., and Clevers, H. (2015) Long-term culture of genome-stable bipotent stem cells from adult human liver. Cell 160, 299–312.
  15. Huch, M., Dorrell, C., Boj, S. F., van Es, J. H., Li, V. S. W., van de Wetering, M., Sato, T., Hamer, K., Sasaki, N., Finegold, M. J., Haft, A., Vries, R. G., Grompe, M., and Clevers, H. (2013) In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature 494, 247–250.
  16. Huch, M., Bonfanti, P., Boj, S. F., Sato, T., Loomans, C. J. M., van de Wetering, M., Sojoodi, M., Li, V. S. W., Schuijers, J., Gracanin, A., Ringnalda, F., Begthel, H., Hamer, K., Mulder, J., van Es, J. H., de Koning, E., Vries, R. G. J., Heimberg, H., and Clevers, H. (2013) Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis. EMBO J. 32, 2708–2721.
  17. Karthaus, W. R., Iaquinta, P. J., Drost, J., Gracanin, A., van Boxtel, R., Wongvipat, J., Dowling, C. M., Gao, D., Begthel, H., Sachs, N., Vries, R. G. J., Cuppen, E., Chen, Y., Sawyers, C. L., and Clevers, H. C. (2014) Identification of multipotent luminal progenitor cells in human prostate organoid cultures. Cell 159, 163–175.
  18. Linnemann, J. R., Miura, H., Meixner, L. K., Irmler, M., Kloos, U. J., Hirschi, B., Bartsch, H. S., Sass, S., Beckers, J., Theis, F. J., Gabka, C., Sotlar, K., and Scheel, C. H. (2015) Quantification of regenerative potential in primary human mammary epithelial cells. Dev. Camb. Engl. 142, 3239–3251.
  19. Maimets, M., Rocchi, C., Bron, R., Pringle, S., Kuipers, J., Giepmans, B. N. G., Vries, R. G. J., Clevers, H., de Haan, G., van Os, R., and Coppes, R. P. (2016) Long-Term In Vitro Expansion of Salivary Gland Stem Cells Driven by Wnt Signals. Stem Cell Rep. 6, 150–162.
  20. Nanduri, L. S. Y., Baanstra, M., Faber, H., Rocchi, C., Zwart, E., de Haan, G., van Os, R., and Coppes, R. P. (2014) Purification and ex vivo expansion of fully functional salivary gland stem cells. Stem Cell Rep. 3, 957–964.
  21. DeWard, A. D., Cramer, J., and Lagasse, E. (2014) Cellular heterogeneity in the mouse esophagus implicates the presence of a nonquiescent epithelial stem cell population. Cell Rep. 9, 701–711.
  22. Mondrinos, M. J., Jones, P. L., Finck, C. M., and Lelkes, P. I. (2014) Engineering de novo assembly of fetal pulmonary organoids. Tissue Eng. Part A 20, 2892–2907.
  23. Boj, S. F., Hwang, C.-I., Baker, L. A., Chio, I. I. C., Engle, D. D., Corbo, V., Jager, M., Ponz-Sarvise, M., Tiriac, H., Spector, M. S., Gracanin, A., Oni, T., Yu, K. H., van Boxtel, R., Huch, M., Rivera, K. D., Wilson, J. P., Feigin, M. E., Öhlund, D., Handly-Santana, A., Ardito-Abraham, C. M., Ludwig, M., Elyada, E., Alagesan, B., Biffi, G., Yordanov, G. N., Delcuze, B., Creighton, B., Wright, K., Park, Y., Morsink, F. H. M., Molenaar, I. Q., Borel Rinkes, I. H., Cuppen, E., Hao, Y., Jin, Y., Nijman, I. J., Iacobuzio-Donahue, C., Leach, S. D., Pappin, D. J., Hammell, M., Klimstra, D. S., Basturk, O., Hruban, R. H., Offerhaus, G. J., Vries, R. G. J., Clevers, H., and Tuveson, D. A. (2015) Organoid models of human and mouse ductal pancreatic cancer. Cell 160, 324–338.
  24. Lancaster, M. A., Renner, M., Martin, C.-A., Wenzel, D., Bicknell, L. S., Hurles, M. E., Homfray, T., Penninger, J. M., Jackson, A. P., and Knoblich, J. A. (2013) Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379.
  25. Greggio, C., De Franceschi, F., Figueiredo-Larsen, M., Gobaa, S., Ranga, A., Semb, H., Lutolf, M., and Grapin-Botton, A. (2013) Artificial three-dimensional niches deconstruct pancreas development in vitro. Dev. Camb. Engl. 140, 4452–4462.
  26. Ciancanelli, M. J., Huang, S. X. L., Luthra, P., Garner, H., Itan, Y., Volpi, S., Lafaille, F. G., Trouillet, C., Schmolke, M., Albrecht, R. A., Israelsson, E., Lim, H. K., Casadio, M., Hermesh, T., Lorenzo, L., Leung, L. W., Pedergnana, V., Boisson, B., Okada, S., Picard, C., Ringuier, B., Troussier, F., Chaussabel, D., Abel, L., Pellier, I., Notarangelo, L. D., García-Sastre, A., Basler, C. F., Geissmann, F., Zhang, S.-Y., Snoeck, H.-W., and Casanova, J.-L. (2015) Infectious disease. Life-threatening influenza and impaired interferon amplification in human IRF7 deficiency. Science 348, 448–453.
  27. Qian, X., Nguyen, H. N., Song, M. M., Hadiono, C., Ogden, S. C., Hammack, C., Yao, B., Hamersky, G. R., Jacob, F., Zhong, C., Yoon, K.-J., Jeang, W., Lin, L., Li, Y., Thakor, J., Berg, D. A., Zhang, C., Kang, E., Chickering, M., Nauen, D., Ho, C.-Y., Wen, Z., Christian, K. M., Shi, P.-Y., Maher, B. J., Wu, H., Jin, P., Tang, H., Song, H., and Ming, G.-L. (2016) Brain-Region-Specific Organoids Using Mini-bioreactors for Modeling ZIKV Exposure. Cell 165, 1238–1254.
  28. Fukuda, M., Mizutani, T., Mochizuki, W., Matsumoto, T., Nozaki, K., Sakamaki, Y., Ichinose, S., Okada, Y., Tanaka, T., Watanabe, M., and Nakamura, T. (2014) Small intestinal stem cell identity is maintained with functional Paneth cells in heterotopically grafted epithelium onto the colon. Genes Dev. 28, 1752–1757.
  29. Dekkers, J. F., Wiegerinck, C. L., de Jonge, H. R., Bronsveld, I., Janssens, H. M., de Winter-de Groot, K. M., Brandsma, A. M., de Jong, N. W. M., Bijvelds, M. J. C., Scholte, B. J., Nieuwenhuis, E. E. S., van den Brink, S., Clevers, H., van der Ent, C. K., Middendorp, S., and Beekman, J. M. (2013) A functional CFTR assay using primary cystic fibrosis intestinal organoids. Nat. Med. 19, 939–945.
  30. Spence, 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., Shroyer, N. F., and Wells, J. M. (2011) Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470, 105–109.
  31. Sato, 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., and Clevers, H. (2011) Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 141, 1762–1772.
  32. Si-Tayeb, K., Noto, F. K., Nagaoka, M., Li, J., Battle, M. A., Duris, C., North, P. E., Dalton, S., and Duncan, S. A. (2010) Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatol. Baltim. Md 51, 297–305.
  33. Dye, 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., White, E. S., Deutsch, G. H., and Spence, J. R. (2015) In vitro generation of human pluripotent stem cell derived lung organoids. eLife 4.
  34. Takasato, M., Er, P. X., Chiu, H. S., and Little, M. H. (2016) Generation of kidney organoids from human pluripotent stem cells. Nat. Protoc. 11, 1681–1692.