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  2. Organoids: from traditional 2D cell Culture to 3D culture models
Organoids: from traditional 2D cell Culture to 3D culture models

In recent years, organoid culture technology is developing rapidly. Organoids has been awarded 'Method of the Year 2017' by Nature Methods, for their immense potential as tools to study human biology in health and disease.

Previously, the term ‘organoid’ has been used to encompass all 3D organotypic cultures derived from primary tissues, pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), established cell lines, as well as whole or segmented organs such as organ explants consisting of multiple tissue types. The ‘organoid’ was defined by Fatehullah et al. As an in vitro 3D cellular cluster derived exclusively from primary tissue, ESCs or iPSCs, capable of self-renewal and self-organization, and exhibiting similar organ function as the tissue of origin[1-3].

The development of organoids

Organoid technology is built upon the foundation of stem cell technologies, classical developmental biology and cell-mixing experiments. In the early 20th century, Wilson (1907) demonstrated that dissociated sponge cells can self-organize to regenerate a whole organism. Stem cell research began to thrive when murine ESCs (mESCs) were first isolated and established in 1981 [4-6]. In 2009, Hans Clevers lab established a long-term primary culture to generate the intestinal organoid culture system. It was an outstanding technological leap for the stem cell field[5][7].

Lately, organoids have been successfully generated for an increasing variety of organs, including but not limited to gut, stomach, lung, kidney, liver, pancreas, mammary glands, prostate, thyroid, retina, inner ear, taste bud and brain [8].

Organoid model is a major technological breakthrough that acts as a valuable model for the study of tissue development, disease modeling, drug screening, personalized medicine and cell therapy[1].

Figure 1. Diverse applications of organoid technology
Figure 1. Diverse applications of organoid technology [5].

Recently, organoids have been widely used in many areas, including developmental biology, disease modeling, precision medicine, regenerative medicine, toxicology, drug discovery studies, host-microbiome interactions, gene editing, multi-omics, and phylogenetic studies[5].

Figure 2. Generation of Reproducible Kidney Organoids
Figure 2. Generation of Reproducible Kidney Organoids [9].
Mouse kidney cells are suspended in Matrigel liquid precursor and fabricated organoid beads by microfluid machine and 3D printer. Organoid beads are cultured supplement with Noggin, R-spondin 1, FGF-4 , FGF-basic, SB-431542, CHIR-99021. The size, shape, and composition of the kidney organoids are highly reproducible.
Advantages of organoid model

Organoids represent an important bridge between 2D cultures and in vivo mouse/human models. They are more physiologically relevant than monolayer culture models and are far more amenable to manipulation of niche components, signaling pathways and genome editing than in vivo models [1][10]. Some of the advantages of the organoid models are;

1) Compared to traditional two-dimensional (2D) cell culture, organoids are similar to primary tissue in both their composition and architecture, harboring small populations of genomically stable, self-renewing stem cells that give rise to fully differentiated progeny comprising all major cell lineages at frequencies similar to those in living tissues[11].

2) Organoids can be expanded enormously, cryopreserved as biobanks, and easily manipulated using techniques similar to those established for 2D monolayer culture[11].

3) Primary-tissue-derived organoids lack mesenchyme/stroma that provides a separate system for studying a specific tissue of interest without being influenced by the local microenvironment[1].

Figure 3. Comparison of Organoid Cultures with Two-Dimensional Cell Cultures and Studies in Animals
Figure 3. Comparison of Organoid Cultures with Two-Dimensional Cell Cultures and Studies in Animals[10].

Compared with the traditional patient-derived cancer cell line (PDC) and patient-derived xenograft (PDX) model, the PDO model has unparalleled advantages. In the screening of drugs for tumor therapy, tumor organoid models derived from patient tumors have higher sensitivity, heterogeneity, and stability and can restore the genuine attributes of tumors more effectively. In addition, tumor organoids can be preserved, resuscitated, passed infinitely, and mechanically cultured on a chip for drug screening[13]. Therefore, organoid technology exerts enormous potential in evaluation of efficacy and toxicity of drugs, regenerative medicine, and precision medicine. Organoids have been established successfully for multiple cancer types, including but not limited to stomach cancer, colorectal cancer, liver cancer, pancreatic cancer[14].

Figure 4. Establishment of patient-derived organoids as in vitro tumor models for colorectal cancer
Figure 4. Establishment of patient-derived organoids as in vitro tumor models for colorectal cancer [15].

Many pathogenic viruses that infect humans display species specificity and animal models can’t be used to study those viral infections. Hence, studying viral biology and identifying potential treatments benefits by developing in vitro cell systems (organoids) that closely mimic human physiology. In the current COVID-19 pandemic, organoids have emerged as powerful tools for SARS-CoV-2 research, bridging the gap between cell lines and in vivo animal models[16-17].

The "Magic" in organoid culture medium

Organoids can be generated from tissue-resident adult stem cells (ASCs) or from PSCs. Under appropriate conditions, supplementation of proper culture medium, growth factors and small molecules, stem cells embedded in Matrigel can undergo continuous self-renewal and differentiation, and self-organize into 3D-structures. The methods of culturing different organoids are similar[17].

1) Multiple sources:

1.1). ASCs-derived organoids: Primary tissue that is dissociated into functional sub-tissue units containing stem cells. These functional units are further digested into single cells and FACS-sorted to enrich for stem cells.

1.2). ESCs/iPSCs-derived organoids: stem cells undergo directed differentiation towards the desired germ lineage, eventually generating floating spheroids that are subsequently embedded in extracellular matrix (ECM) to initiate organoid culture[1].

Figure 5. Organoid generation and culture from primary tissue and ESCs/iPSCs
Figure 5. Organoid generation and culture from primary tissue and ESCs/iPSCs[2].

2) Manipulability of niche components:

Organoids are typically cultured in an ECM surrounded by culture media supplemented with specific niche factors (different from air-liquid interface (ALI) method which is introduced recently)[18]. Organoids can either differentiate spontaneously or be induced to differentiate towards desired lineages or cell types by adding suitable differentiation factors and/or withdrawing factors that promote stemness. Common niche and ECM factors include R-spondin, EGF, Noggin, Activin A, and Collagen. Specific small molecules are added such as TGF-β inhibitor A-83-01, GSK3β inhibitor CHIR99021, and ROCK inhibitor Y27632[1].

Stem cells are maintained and perpetuated in organoids, continually giving rise to differentiated progeny. In addition, organoids can be dissociated and plated onto membrane supports coated with Matrigel or Collagen to form 2D monolayer organoid models[17].

MedChemExpress offers a variety of high-quality recombinant proteins and small molecules for organoid culture.

Related products

Cytokines

Human EGF

A well-known growth factor for epithelial tissues; binding to EGF receptors, induces hyperplasic changes.

EGF can be used for the generation of Gastrointestinal tract, liver, thyroid, brain organoids.

Human FGF-2/4/9/10

FGFs play crucial roles in a wide variety of cellular functions, including cell proliferation, survival, metabolism, morphogenesis, and differentiation,

as well as in tissue repair and regeneration. In a 3D extracellular matrix, FGF-2, FGF-7, FGF-9, and FGF-10 promote lung organoid formation.

Human HGF

HGF is a known hepatocyte mitogen that can be used for the liver organoid culture.

Human Wnt3a

Wnt is a master regulator in regulation of cell development, proliferation, differentiation, adhesion, and polarity.

Wnt3a is an essential niche component for maintaining the proliferation of Lgr5-positive stem cells in various organoids such as the small intestine,

large intestine, stomach, pancreas and liver.

Human BMP-4

BMPs play crucial roles in embryogenesis and development, and also in maintenance of adult tissue homeostasis.  

BMP-2 and BMP-4 are widely used in in vitro protocols of generation of hepatic cells from induced pluripotent stem cells (iPS) and from embryonic stem cells (ESC). 

Human Noggin

Noggin is an inhibitor of bone morphogenetic proteins that modulates cellular differentiation, proliferation, and apoptosis.

Noggin is one of the most important components of organoid media are growth factors.

Human DKK-1

DKK-1 is a canonical WNT inhibitor that can induce retinal progenitors to self-organize.

Small-molecule Inhibitor

Y-27632 dihydrochloride 

Y-27632 is a Rho Kinase (ROCK) inhibitor; Has been used to increase the proliferation and reduce apoptosis of progenitor cells grown in vitro.   

A 83-01

A 83-01 is an inhibitor of TGF-β type I receptor ALK5, the Activin/Nodal receptor ALK4, and the nodal receptor ALK7.

References:
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[2] Marina Simian, Mina J Bissell. Organoids: A historical perspective of thinking in three dimensions. J Cell Biol. 2017 Jan 2;216(1):31-40.
[3] HansClevers. Modeling Development and Disease with Organoids. Cell. 2016 Jun 16;165(7):1586-1597.
[4] Madeline A Lancaster, Juergen A. Knoblich. Organogenesis in a dish: modeling development and disease using organoid technologies. Science. 2014 Jul 18;345(6194):1247125.
[5] Claudia Corrò, Vivian S.W. Li, et al. A brief history of organoids. Am J Physiol Cell Physiol. 2020 Jul 1;319(1):C151-C165.
[6] G R Martin. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7634-8.
[7] Sato T, Vries RG, et al. (2009). “Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.” Nature 459(7244): 262–265.
[8] Elisa Suarez Martinez , Amancio Carnero, et al. 3D and organoid culture in research: physiology, hereditary genetic diseases and cancer. Cell Biosci. 2022; 12: 39.
[9] Chengyong He, Shaohua Ma, Zhenghong Zuo, et al. Black Phosphorus Quantum Dots Cause Nephrotoxicity in Organoids, Mice, and Human Cells. Small. 2020 Jun;16(22):e2001371.
[10] Mo Li, Juan C Izpisua Belmonte. Organoids-Preclinical Models of Human Disease. N Engl J Med. 2019 Feb 7;380(6):569-579.
[11] Mariangela Scalise, Fabiola Marino, Daniele Torella, et al. From Spheroids to Organoids: The Next Generation of Model Systems of Human Cardiac Regeneration in a Dish. Int J Mol Sci. 2021 Dec; 22(24): 13180.
[12] Xialin Nie, Zhixing Liang, Linsen Ye, Yang Yang, et al. Novel organoid model in drug screening: Past, present, and future. Liver Research 5 (2021) 72-78.
[13] Chen Liu , Chaoyang Sun , et al. Drug screening model meets cancer organoid technology. Transl Oncol. 2020 Nov; 13(11): 100840.
[14] Hanxiao Xu, Kongming Wu, et al. Organoid technology and applications in cancer research. J Hematol Oncol 11, 116 (2018).
[15] Lisi Zeng, Shuzhong Cui, Shengwei Jiang, et al. Raltitrexed as a synergistic hyperthermia chemotherapy drug screened in patient-derived colorectal cancer organoids. Cancer Biol Med. 2021 Mar 12;18(3):750-762.
[16] Maarten H.Geurts, Jeltevan der Vaart, HansClevers, et al. The Organoid Platform: Promises and Challenges as Tools in the Fight against COVID-19. Volume 16, Issue 3, 9 March 2021, Pages 412-418.
[17] Jelte van der Vaart, Mart M. Lamers, Hans Clevers, et al. Advancing lung organoids for COVID-19 research. Dis Model Mech. 2021 Jun 1; 14(6): dmm049060.
[18] Soumya K Kar, et al. Organoids: a promising new in vitro platform in livestock and veterinary research. Vet Res. 2021 Mar 10;52(1):43.
[19] Yaqi Li, Guoqiang Hua, et al. Organoid based personalized medicine: from bench to bedside. Cell Regen. 2020 Dec; 9: 21.