At the end of December 2022, the U.S. House of Representatives approved the FDA Modernization Act 2.0. The FDA no longer requires mandatory animal testing for new and generic drugs. It aims to advocate reducing the use of animal testing and encourage gradual application of alternative models where feasible.

Organoids are a three-dimensional (3D) cell culture system that has proven to be an excellent alternative to animal models in preclinical studies. Driven by FDA policies, we can repeatedly see domestic and foreign organs and organ chips receiving financing. This application direction has become a hot spot in biomedical investment and financing.

In the past 10 years, the organoid market has grown significantly. During the period from 2010 to 2020, the growth rate of new companies was as high as 64%; as of Q4 2022, the cumulative market value of this field was approximately US$1.031 billion.

Number of companies and market share in the organoid market from 2010 to 2022

Organoids are tissue and organ analogues formed by using pluripotent stem cells, adult stem cells, etc. for targeted induction in vitro and 3D culture. They can simulate many aspects of the complex structure and function of tissues in vivo. Organoids induced by stem cells can be used for basic research on human tissue development, regeneration and repair mechanisms, and can also be used for diagnosis, disease modeling, drug screening and evaluation, and clinical regenerative medicine. Previously, Nature Methods evaluated organoid technology: using three-dimensional tissue models induced by stem cells to provide a powerful method for human biological research. So far, literature and other materials have reported many engineering strategies for stem cell induction into organoid culture.

Based on summarizing previous data, this article summarizes the culture methods of stem cells to induce organoids, and briefly discusses the key factors in stem cell culture to induce organoids, such as the source of cells, extracellular matrix and cytokines.

Currently, according to source, organoids are mainly divided into three categories:

① Organoids derived from pluripotent stem cells;

② Organoids derived from adult stem cells;

③ Tumor organoids derived from tumor tissue.

In terms of the source of initial tissue cells for organoid culture, although there have been reports that human kidney cancer, thyroid cancer, colon cancer, liver cancer, lung cancer and kidney cancer tissues were chopped up, stored in liquid nitrogen for more than a year, and then resuscitated and extracted primary cells, and the cells were cultured in 3D, the results showed that tissue cryopreservation had no significant impact on cell vitality and growth rate [3]. However, it is still widely believed that fresh tissue that has not been frozen is the best choice for organoid culture.

The preparation of organoids is a relatively complex project. The classic process of organoid preparation is to culture the isolated stem cells on a 3D scaffold, and add various growth factors such as Epidermal Growth Factor (EGF), fibroblast growth factor (FGF), and other additives to the culture environment to promote the formation and growth of the corresponding organoid. Finally, the organoid must be identified.

Figure 1. Schematic diagram of stem cell-derived organoid culture process

The induction of stem cells into organoids requires specific reagent consumables and other material conditions (such as specific growth factors, 3D scaffolds, stem cell differentiation inducers, etc.) and specific culture conditions (such as appropriate temperature, appropriate oxygen concentration, carbon dioxide concentration and some specific mechanical parameters that simulate in vivo conditions). These are extremely important conditions for the induction of stem cells into organoids to create a complex microenvironment suitable for their growth and development.

1. Various cultivation models of organoids

In view of the crucial importance of different physical environmental conditions for the development of stem cell tissues into organoids, a variety of culture models under different physical environmental conditions have been developed to meet the different culture needs of stem cells to induce organoids, such as using natural extracellular matrix 3D scaffolds for static culture (such as small intestine organoids), using 3D suspension and agitation culture without extracellular matrix scaffold support (such as brain organoids), Use gas-liquid interface cell mass culture (such as nephroid organs), etc.(see Figure 2).

Figure 2. Organoid culture in different physical environments

Among them, extracellular matrix provides a suitable 3D scaffold for the growth of organoid cells. Organoid extracellular matrix is prepared by matrigel. Matrigel accounts for a high proportion of the cost of organoid preparation. The currently commonly used matrigel is Matrigel® from BD Biosciences Company in the United States. On the one hand, the price is high. On the other hand, because it originates from a mouse sarcoma cell line, differences between batches of matrigel may lead to different qualities and complex compositions between different experimental or organoid products. Problems such as unknown biological components and immunogenicity bring certain difficulties to basic research or clinical transformation. Therefore, low-cost, non-animal cell-derived extracellular matrix synthesized in vitro will be an important issue that needs to be solved in the organoid industry. one.

However, under the conditions of static culture systems, it is difficult to supply food and nutrients, especially for large-volume organs. The stirred dynamic culture system mentioned above can be selectively used, which can provide sufficient oxygen exchange and nutrients for larger volumes of organoids to promote the development and maintenance of organoids.

2. The right biochemical microenvironment is crucial

Appropriate biochemical microenvironment plays an important role in the induction of stem cells to produce organoids and maintain the stability of organoids. Various cytokines or some inducers are crucial to maintaining the microenvironment required for stem cell differentiation, organoid development and stability. Depending on the source of stem cells or the type of organoid prepared, the types or combinations of additives or cytokines required are different. Even tissues and organs with very similar structures, The additives or cytokine combinations required are also different. In terms of cytokines, the classic cytokine combination scheme is WENR (abbreviated as WENR is the combination of four cytokines, Wnt- 3a, EGF, Noggin and R-Spondins, respectively, after taking the initials). The increase and decrease combinations of these four cytokines are suitable for most organoid culture experiments.

① The Wnt signaling pathway is a very complex protein interaction system that plays important functions in regulating cell development, proliferation and differentiation, adhesion and self-renewal. The Wnt proteins participating in this signaling pathway constitute a huge family of secreted proteins. Among them, the most representative Wnt3a cytokine is widely distributed in the body and is crucial in regulating cell renewal, proliferation, differentiation and movement. It is one of the most commonly used cytokines in organoid culture.

② EGF is one of the main ligands of EGFR currently discovered. Because EGF ligands bind to extracellular receptors (such as EGF), EGFR dimerization causes the intracellular domain to form tyrosine kinase activation, activating downstream signaling pathways, and through a cascade of signals, the signal is finally transmitted to the cell nucleus, regulating the expression of corresponding genes, and then controls physiological activities such as cell growth, division, differentiation, migration and adhesion. EGF is a cytokine required for the culture of many organoids such as the gastrointestinal tract, thyroid, liver, and brain.

③ The R-Spondin family is an important Wnt activator discovered in recent years. Its family consists of four members, namely R-Spondin-1, R-Spondin-2, R-Spondin-3 and R-Spondin-4. The Wnt-R-Spondin-1 signaling pathway is particularly important in organoid culture. The extracellular Wnt ligand of this signaling pathway needs to bind to Frizzled and Lrp5/6 to be activated. The ubiquitination degradation mechanism in cells degrades Frizzled, causing the Wnt signaling pathway to be inhibited. R-spondin can inhibit the ubiquitination of Frizzled and thereby promote the activation of the Wnt pathway.[6] Therefore, two cytokines, Wnt-3a and R-Spondin, often need to be added at the same time during organoid culture.

④ Noggin is an antagonist of bone morphogenetic proteins (BMPs) and plays an important role in early embryonic development, limb formation and nervous system development. Noggin, as an important regulator of Wnt signaling pathway and BMP signaling, plays an important role in the development of many tissues and organs.

3. Identification of organoids

Since the consistency between organoids induced by stem cells in vitro and their corresponding in vivo tissues is the key to determining the quality of organoids, organoids should be identified. Preliminary identification can be used to observe the morphology using microscope and H&E staining methods to assess whether the organoid and the corresponding tissues and organs in the body have similar histological morphological characteristics; then protein immunoblotting (WB), qPCR, ELISA, flow cytometry, IHC, etc. can be used to detect whether the organoid expresses the expected Biomarker; identification of the gene status of the organoid cells is also necessary. Gene sequencing and transcriptome sequencing can be used to identify whether the organoid has certain gene deletions, mutations and expression profile characteristics; In addition, for some organoids, it is possible to test whether they have expected functions, such as: CFTR activity and function detection of intestinal organoid cells, whether heart organoids can beat spontaneously, etc.