Comprehensive Guide to Organoid Cultivation

 — A comprehensive article to deepen your understanding of organoids.

Introduction to Organoids

Organoids are three-dimensional structures with physiological functions that mimic the normal (or diseased) state of internal organs or tissues, achieved through 3D cultivation outside the body. In simpler terms, organoids are three-dimensional cell cultures where stem cells are cultured in a matrix gel. Under the influence of chemical inhibitors/activators, cytokines, and culture additives, organoids develop into tissue structures similar to corresponding organs.

Characteristics of Organoids

Organoids possess self-renewal capabilities, maintaining the physiological structure and function of the source tissue. They are often referred to as "micro-organs in a dish." Utilizing the self-renewal, differentiation, and self-organizing abilities of stem cells, organoids can be cryopreserved for use in biobanks and can undergo unlimited expansion. Organoids are highly complex and, compared to 2D cells, more closely resemble the in vivo state.

Figure 1. Organoid cultivation of human colon adenocarcinoma cells [1]

Applications of Organoids

The distinctive feature of organoids is their ability to better simulate the in vivo environment, making them suitable for molecular and cellular biology analyses. Bridging the gap between animal and cellular levels, organoids offer a superior solution for research in areas such as tumor studies, drug screening, regenerative medicine, and more. They have been widely applied in functional tissue induction, disease model establishment, drug screening, anti-inflammatory tests, clinical research, and various other research aspects, showing great potential in both basic research and translational applications.

As organoid cultivation systems and experimental techniques continue to evolve, organoids have been used for various tissues and organs, including the intestines (small intestine/colon), stomach, liver, heart, lungs, prostate, pancreas, kidneys, breasts, brain-like structures, retina, inner ear, and more.

Organoids derived from tumor stem cells have shown significant potential in understanding the mechanisms of tumor occurrence and development, screening drug sensitivity, and promoting precision medicine and personalized diagnosis. Multiple articles from Cell and Science indicate that organoids exhibit high sensitivity and specificity in predicting the effectiveness of anticancer drugs. Recently, tumor organoids have demonstrated their role in predicting patient responses to cancer drugs and assisting in the formulation of personalized treatment plans.

Researching Development Mechanisms: Organoids' differentiation capabilities make them suitable for studying embryonic development processes and mechanisms. The induction processes regulated by signaling pathways such as Wnt and BMP can be used to study the development of organs like the brain, pancreas, and stomach [2][3][4].

Establishing Disease Injury Models: Organoids induced from specific tissues or organs can be used to study models of specific diseases. Teams led by Zhao Bing and Lin Xinhua used human organoid infection models to study the molecular mechanisms of SARS-CoV-2 infection and liver damage, providing crucial tools for researching the pathogenic mechanisms of the virus and subsequent drug development [5]. The research group led by Deng Hongkui at Peking University's School of Life Sciences used small molecules and cytokines to stimulate the construction of a novel intestinal organoid with features of damage regeneration—Hyper Organoid. This organoid can be passaged and amplified for an extended period, maintaining the genome and promoting the repair of colon tissue damage, alleviating the pathological symptoms in an acute colitis animal model, and more [6].

Regenerative Medicine: Stem cell-derived organoids can repair or replace damaged or diseased tissues to restore normal tissue function. They have extensive applications in cell therapy, including for other neurodegenerative diseases, diabetes, cardiovascular diseases, retinal disorders, spinal cord injuries, and more. As a novel treatment in the field of regenerative medicine—DA01, using small molecules like SB-431542, LDN193189, CHIR-99021, Y-27632, and Sonic Hedgehog (Shh) protein, stimulates pluripotent stem cells to differentiate into dopaminergic neurons. These neurons are then transplanted into the injured areas of the brains of late-stage Parkinson's disease patients, providing a new direction and approach for the treatment of the disease [7].

Drug Toxicity and Efficacy Testing: Organoids can be used to verify the pharmacotoxicity of new drugs in specific organs or tissues, providing data support for new drug development. The use of Hyman kidney organoids to verify the renal toxicity of Cisplatin is an example [8].

Drug Screening: Organoids derived from stem cells can be used for in vitro testing of drug reactions, providing theoretical support for drug screening. Colon organoids can be used to study medication plans for patients with CFTR mutations, and tumor organoids can be used to assess individualized medication situations for patients [9].

Developmental History of Organoids

Sources of Organoids

Normal organoids primarily originate from stem cells, including pluripotent stem cells (PSCs) and adult stem cells (ASCs). Pluripotent stem cells include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Compared to pluripotent stem cells, adult stem cells have the advantage of being simpler and faster to model, but the disadvantage of constructing organoid structures that are relatively simpler. Organoid structures constructed from pluripotent stem cells are more complex.

Commonly Used Small Molecules in Organoid Culture (Summary): Super practical, don't forget to bookmark!

❶ Y-27632 (Cat#53006ES, Cat#52604ES): A potent inhibitor of Rock, competitively inhibiting p160ROCK (Ki=140 nM) and ROCK-II (IC50=800 nM) by ATP competition. It also inhibits PRK2 (IC50=600 nM). Typically added during the first seeding in plate culture; subsequent medium changes may not require addition. Treatment of human embryonic stem cells with Y-27632 (10 µM) for 1 h can inhibit apoptosis, increase clone efficiency, and extend cell passages.

Recommended working concentration: 10 μM

❷ SB-202190 (Cat#53005ES): An efficient p38 MAPK kinase inhibitor, targeting p38α/β. SB202190 can induce differentiation of human embryonic stem cells into cardiac muscle cells, promote self-renewal of neural stem cells, and is applicable to the culture of gastrointestinal and mammary gland organoids.

Recommended dissolution concentration: Dissolve 10 mg in 3.018 mL of DMSO to obtain a 10 mM solution; store at -20℃.

Recommended working concentration: 10 μM

❸ CHIR-99021 (Cat#53003ES): An aminopyrimidine derivative, acting as a GSK-3 (GSK3α/β) inhibitor. It induces differentiation of human embryonic stem cells into the endoderm and is used in kidney and retinal organoid cultures. CHIR-99021, when used in combination with other reagents, stimulates somatic cell reprogramming into stem cells.

Recommended dissolution concentration: Dissolve 5 mg in 3.58 mL of DMSO to obtain a 3 mM solution; store at -20℃.

Recommended working concentration: 3 μM

❹ A 83-01 (Cat#53002ES): An Activin/NODAL/TGF-β pathway inhibitor, inhibiting ALK5/4/7 kinase activity. Generally used in the culture of liver, prostate, and mammary gland organoids. It is commonly used to inhibit the differentiation of induced pluripotent stem cells (iPSCs) and maintain the self-renewal of cells in vitro.

Recommended dissolution concentration: Dissolve 5 mg in 5.93 mL of DMSO to obtain a 2 mM solution; store at -20℃. (Note: This product is unstable in solution and is recommended for immediate use after preparation.)

Recommended working concentration: 2 μM

❺ Gastrin I (Cat#53007ES): Gastrin is an endogenous gastrointestinal peptide hormone that stimulates gastric wall cells to secrete gastric acid. It is crucial for studies on gastrointestinal organoids. When culturing intestinal and hepatic organoids, adding gastrin helps prolong the survival time of organoids.

Recommended dissolution concentration: Dissolve 1 mg in 2.38 mL of 1% ammonia solution to obtain a 0.2 mM solution; store at -20℃.

Recommended working concentration: 10 nM

❻ Nicotinamide (Cat#51402ES): Nicotinamide, a B3 vitamin, participates in various enzymatic redox reactions and is used in the culture of gastrointestinal, hepatic, and mammary gland organoids. Nicotinamide, in conjunction with cytokines and other biochemical reagents, exhibits anti-inflammatory properties, promotes the differentiation of mesenchymal stem cells into insulin-producing cells, inhibits sirtuins activity, and is used to promote organoid formation and extend organoid lifespan.

Recommended dissolution concentration: Dissolve 100 mg in 8.19 mL of H2O (or DMSO) to obtain a 100 mM solution; store at -20℃.

Recommended working concentration: 10 mM

❼ Forskolin (Cat#51001ES): Forskolin can activate adenylate cyclase, commonly used to elevate intracellular cAMP levels. Forskolin induces differentiation of various cell types, activates PXR and FXR, and has antiplatelet aggregation and antihypertensive effects. When culturing hepatic organoids, it is essential to add this substance.

Recommended working concentration: 1-10 μM

❽ Prostaglandin E2 (Cat#60810ES): Prostaglandin E2 (PGE2) regulates many physiological systems, mediating cell proliferation and differentiation. It is needed when culturing hepatic and prostatic organoids and is associated with smooth muscle relaxation, inflammation, reproduction, sleep cycle regulation, and gastric mucosal integrity.

Recommended dissolution concentration: Dissolve 1 mg in 0.28 mL of DMSO to obtain a 10 mM solution; store at -20℃.

Recommended working concentration: 500 nM

❾ N-acetyl-L-Cysteine (Cat#50303ES): N-acetyl-L-cysteine (NAC) is a precursor to the antioxidant glutathione, with antioxidant and ROS inhibitory effects. It inhibits neuronal cell apoptosis and is required in the culture process of most organoids.

Recommended dissolution concentration: Dissolve 2 g in 24.51 mL of H2O (or DMSO) to obtain a 500 mM solution; store at -20℃.

Recommended working concentration: 1 mM

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