The rapidly developing organoids provide new models for disease research and clinical application, and their technical optimization and multidisciplinary integration will further explore their application prospects.
The human body is an organic collection from single cells to tissues and then to organs. For scientific research at the organ level, efforts should be made to establish a model more in line with the cellular composition, biological structure and function of human tissues and organs. Organoid technology was born. In 2021, the world 16 countries more than 60 experts made clear the definition of organoids, and consensus: organoids are stem cells, precursor cells and / or differentiated cells between cells and cell-matrix interaction and spontaneous tissue formation in vitro three-dimensional structure, can reproduce in multiple aspects of the structure and function of the corresponding tissues or organs in the body[1]。
Organoids have the characteristics of self-renewal, self-organization, maximum simulation of the structure and function of internal organs, and long-term and stable subculture. Compared to conventional 2 D cell cultures, organoids are closer to the physiological state of the organ. In 2013, organoid technology was rated as one of the top ten technologies of the year by Science (Science), the 2017 method by Nature Methods (Nature Methods) in early 2018, and once again rated as an excellent preclinical disease model by the New England Journal of Medicine (The New England Journal of Medicine) in 2019. In the past 10 years, organoid technology has developed rapidly, and its application involves many aspects, including organ development research, disease occurrence and development analysis, precision medicine, drug research and development, etc.
Development history
The origin of organoids dates back to 1907, when researchers found that mechanically isolated sponge cells could self-organize into new sponge organisms with normal function, revealing for the first time that certain dedifferentiated cells in invertebrates are capable of self-organizing. This finding laid the foundation for the generation of organoid techniques and the proposal of the theory of cell self-organization. In 1960, chicken embryo-derived kidney, liver and skin were found to also have the ability to self-organize and form the corresponding organ structure and function after transplantation in vivo, and this potential seems to come from the cells themselves. In addition, human embryonic stem cells were first isolated in 1998, which triggered a boom in stem cell research and clinical application. In 2006, Professor Yamanaka successfully produced human induced pluripotent stem cells to further address the source and ethical controversy of human tissue samples. The rapid advances in stem cell research also provides the basis for organoid research.
In 2007, Cleaves (H. Clevers) Bowintestinal stem cells were found to be Lgr 5-positive cells. In 2009, his team used matrix gel to conduct 3D culture of Lgr 5 positive stem cells in a single mouse small intestine, and obtained the first intestinal organoids with intestinal crypt-villus structure formed by the expansion of single cells, which opened the chapter of organoid research. So far, the researchers have successfully constructed intestinal organoids, retinal organoids, brain organoids, liver organoids, pancreatic organoids, prostate organs, lung organoids, breast organoids, fallopian tubes, hippocampus organoids, placental organs, skin organoids and corresponding organs[2]。
formation mechanism
Organoids are derived from the directed differentiation of human pluripotent stem cells, tissue-specific adult stem cells, or induced pluripotent stem cells, whose formation is essentially an in vitro recapitulation of tissue-organ development or regeneration. Its three-dimensional structure and function depends on the cell potential and auxiliary environment: the formation of organoids and continuation rely on active stem cell group and their progeny, can repeatedly expand its cell culture, and the external environment through the influence on cell behavior and decision organoid three-dimensional structure and the maintenance of stable growth.
In order to create organoids, researchers need to explore the organoid-initiating (stem) cells, including stem cell identification of tissues and organs, lineage analysis of growth and differentiation, which is the basis for understanding the self-organization of cells as organoids. On the other hand, attention should be paid to the culture environment, such as using the extracellular matrix as an extracellular scaffold to support the three-dimensional structure and specific growth factors and proteins that mimic the in vivo environment to maintain cell proliferation and differentiation. Commonused scaffolds, such as matrix glue, can polymerize to form a three-dimensional matrix at room temperature, which can simulate the structure, components, physical properties and function of the cell basement membrane in the body[3]. With the development of interdisciplinary disciplines such as biomaterials and organ chips, it also provides a new scaffold system for the development of organoids, supplemented by special growth factors and other culture conditions, to help the formation of organoids.
In addition to the cell self-organization mechanism, the "adhesion difference hypothesis" points out that the adhesion protein-mediated cell binding energy reduces the free energy, further enhances the binding strength, reduces the entropy of aggregated cells, and maintains the thermodynamically stable state to maintain the stability of organoids[4]. In addition, symmetry breaking occurs continuously during in vivo development or in vitro organoid formation and is the formation mechanism of organoid substructures. Finally, the differentiation and spatial limitation of different cell types may affect cell rearrangements and are key in determining the structure of organoids.
Organoid application
Organoid research is a hot spot in scientific research and clinical application. The innovation of more and more culture technologies promotes the successful construction of more corresponding organoids of tissues and organs; the development of stem cell technology lays a solid foundation for the cell potential screening of organoids and theoretical development; the cross-integration of frontier technologies such as biological materials and 3D bio-printing has promoted the long-term development and broad application of organoids.
Modeling through organoids can provide new entry points for disease research. In terms of oncology precision medicine and drug development, in 2015, the Cleaves team demonstrated with 22 colorectal cancer organoid libraries. In 2018, other researchers successfully used primary gastric cancer organoids to simulate different molecular subtypes and stages of disease progression of gastric cancer in vitro.2023, both Wnt-dependent and non-independent forms of human lung adenocarcinoma were revealed using the lung cancer organoid library. In terms of genetic mutant disease research, microcephaly is simulated by RNA interference (RNAi) technology and patient-derived iPSCs to demonstrate premature neuronal differentiation in patient organoids, and this defect may help elucidate the disease phenotype[5]。
Providing new in vitro models for human development through organoid modeling, combined with genetic techniques such as gene editing, contributes to an insight into the genetic mechanisms of organisms and provides the basis for disease treatment. In 2021, through the successful establishment of pig and monkey colon organoids, the author's team promoted the mechanism research of animal intestinal homeostasis regulation, and assisted in drug development and toxicity research. Another researcher used tissue organoids established by fetal alveolar epithelial progenitor cells in 2023 to model key mechanisms of cell lineage determination. Moreover, studying functional human microglial phenotypes in health and disease through brain organoids may also provide experimental evidence of specific immune responses in patients with giant malformation and autism. In 2023, KLroves team successfully screened potential new screening targets for the treatment of fatty liver by establishing a CRISPR screening platform based on human fatty liver organoids.
The efficacy and side effects of drugs can be assessed by the organoid model. In 2020, Hua Guoqiang's team performed X-ray, 5-fluorouracil and Elitecan stimulation of locally advanced rectal cancer organoids, and confirmed that the consistency between clinical response and drug sensitivity of rectal cancer organoids reached 84.43%. In 2023, the consistency of drug sensitivity and clinical response reached 83.3%. Currently, Hubrecht Organoid Technology hubrirector (HUB) has established living biobanks, cultured from tissue from patients with cystic fibrosis, to test the therapeutic effect of the new drug ivacato on patients.
Transplantation through organoid expansion can also be used to repair and reconstruct human tissues and damaged organs. Stem cell-derived organoids can avoid ethical issues and rejection and can be used as an ideal organ model for transplantation. In 2013, organoids were shown to be safely transplanted into animals, such as small intestinal organoids that can still retain some important features. In 2022, the Japanese research team transplanted organoids cultured from the patient's own healthy intestinal mucosal stem cells into a patient with refractory ulcerative colitis, the first attempt in the world. Although there are few reports on reconstruction and organ transplantation, it is still the goal and prospect for the future development and application of organoid technology.
The application of organoids has been deeply involved in all aspects of the field of life. It is believed that with the development of science and technology, its application value and application paradigm will be further reflected[6]。
Challenges and prospects
Organoid technology is booming, and although it has many advantages over traditional two-dimensional cell models or animal models and is widely used, it also faces many challenges.
The currently constructed organoids only contain the main cell types of corresponding tissues and organs, and lack blood vessels, nerves and immune system, so they can only partially reproduce the functions and characteristics of tissues and organs. Therefore, constructing functional vascular networks is a difficult problem in the organoid field. In 2019, the researchers successfully induced directed differentiation of endothelial cells, and then their self-organization with neuroectodermal cells formed a partially accessible vascular network[7]. At the same time, organoids cocultured with immune environment are also being actively constructed. In 2018, the co-culture of autologous tumor organoids and peripheral blood lymphocytes was shown to induce the specific immune response of T cells. These works lay the foundation for further construction of organoids with more complex structure and function.
In addition to building multicellular organoids, building multi-organ reciprocal organoid systems are also difficult. In recent years, technologies such as organ chips have also provided solutions for this purpose. In 2017, we successfully constructed a three-tissue organ chip system, and demonstrated the value of multi-tissue integration in vitro research on drug efficacy and side effects[8]. In 2021, Qin's team first used the organoid chip to establish a liver-islet stem cell derived organoid interaction system, providing new strategies and technologies for the research of complex metabolic diseases such as diabetes and the discovery of new drugs. In recent years, the combination of organoid and organ chip technology provides new solutions for constructing a multi-organ interaction system.
Organoid-specific cell arrangement and fine structure, potentially achieved through 3D bioprinting, design structures with more cell-specific and well-separated properties, more finely support the growth and maturation of different cell types, and maintain their overall cellular diversity. In 2019, it successfully used a 3D biological printer with human collagen as raw material to print a functioning heart tissue with a precision of up to 20 microns[9]. In 2020, organoids, biomaterials and chip technologies were successfully integrated to achieve breakthroughs in vitro large-scale organoids and simulation of complex structures of intestinal epithelium. Biological tissue printing technology is expected to create human organ substitutes.
Multicellular specific spatial rearrangement technology promotes the structural and functional organ remodeling in vitro. In 2020, the bladder "class assembly" was successfully established, or multi-layer micro organ, through three-dimensional remodeling of stem cells with epithelial cells, matrix cells, immune cells and muscle cells in the tissue matrix, so as to accurately simulate human tissue and be used in drug development[10]. The assistance and fusion of these techniques compensate for the lack of organoids, but their stability, repeatability and consistency need to be further improved. The field still needs efforts to build up more standardized and highly simulated organoids.
Looking, the research of organoids is promising. Because of its heterogeneity and high efficiency, it has great potential in precision tumor treatment, and also provides effective model research in drug research and development. In addition, new research opportunities for more than 7,000 rare diseases worldwide. In 2021, organoids were included in one of the six key projects of the 14th Five-Year Plan of national Key Research and development released by the Ministry of Science and Technology. The Drug Evaluation Center of the National Drug Administration also included organoids in the guiding principles of gene therapy and gene-modified cell therapy products, and the application of organoids ushered in a period of favorable policies. In conclusion, the rapid development of organoid technology provides important model research tools for scientific research, and although still facing many challenges, we can still foresee its huge application prospect.
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