Organoid technology began in 2009, and the Hans Clevers team at the Hubrecht Institute in the Netherlands successfully cultured adult stem cells into crypts and villus structures of the small intestine.

　　Table of contents

　　1. The concept of organoids

　　2. History of organoid development

　　3. Cultivation of organoids

　　4. Application of organoids

　　5. The industrial chain of organoids

　　6. The market structure of organoids

　　7. The challenge of organoids

　　8. The future development of organoids

　　-01-

　　The concept of organoids

　　Organoids refer to tissue analogs with a certain spatial structure formed by using adult stem cells or pluripotent stem cells for in vitro three-dimensional (3D) culture. Although organoids are not real human organs, they can simulate real organs in structure and function, simulate tissue structure and functions in the body to the greatest extent, and can be passed down and cultivated for a long time and stably for a long time.

　　Currently, organoids are divided into two categories: tissue-derived organoids and pluripotent stem cells-derived organoids.

　　Compared with traditional two-dimensional culture models, organoids represent an innovative technology that can summarize the physiological processes of the entire organism, with advantages such as closer to physiological cell composition and behavior, a more stable genome, and more suitable for biological transfection and high-throughput screening. Compared with animal models, the operation of the organoid model is simpler and can also be used to study mechanisms such as disease occurrence and development. Therefore, it has extensive application prospects in the fields of organ development, precision medicine, regenerative medicine, drug screening, gene editing, disease modeling, etc. In 2013, organoids were rated as one of the top ten technologies of the year by Science magazine. In early 2018, organoids were rated as the 2017 Methods by Nature Methods.

　　Search for the entry "organoids" on Pubmed, the articles related to organoids have jumped from 42 articles in 2010 to 2097 articles in just ten years. Organoids have become a hot research topic with a very rapid trend.

　　

　　-02-

　　History of organoid development

　　The origin of organoids can be traced back to 1907. H. V. Wilson, a 44-year-old professor at the University of Becrolena in the United States, discovered that mechanically separated sponge cells can regroup and self-organize into new sponge organisms with normal functions. His research results were published in 1910. Wilson's research demonstrates that adult organisms have complete information and can successfully develop into new organisms without external help and without starting from a specific anatomical stage.

　　For organoid technology, another very critical opportunity is the development of stem cell technology. I believe everyone is familiar with stem cells. Many people’s first impression of stem cells comes from the media’s introduction to hematopoietic stem cell transplantation therapy for blood diseases. The hot stem cell research in recent years mainly began at the end of the last century. In 1987, A. J. Friedenstein discovered mesenchymal stem cells (MSCs). In 1998, American biologist James Thomson first isolated human embryonic stem cells. In 2007, Professor Shinya Yamaakashi successfully produced induced Pluripotent Stem Cells (iPSCs). Today, most types of non-tumor-derived human organoids can develop from MSCs or iPSCs, and the rapid progress of stem cell research has brought new vitality to organoid research.

　　Major events in organoid development

　

　　Source of data: Am J Physiol Cell Physiol Magazine

　　The development achievements of contemporary organoids have been mainly concentrated in the past decade.

　　In 2007, Hans Clevers' laboratory found that the stem cells of the small intestine and colon were Lgr5+ cells through lineage tracing test.

　　In 2009, Hans Clevers' laboratory used single murine LGR5+ intestinal stem cells to self-organize in vitro to form intestinal organoids with intestinal crypt-villi structure.

　　In 2011, intestinal organoids developed from human pluripotent stem cells and primary adult stem cells were successfully made. In the same year, the retinal organoids cultivated from mouse embryonic stem cells were successfully cultivated for the first time.

　　In 2012, retinal organoids developed from human pluripotent stem cells were successfully cultivated.

　　In 2013, brain organoids developed from human pluripotent stem cells were successfully cultivated. The liver, kidney and pancreatic organoids were successfully cultivated.

　　In 2014, prostate and lung organoids were successfully cultivated.

　　In 2015, breast, fallopian tubes, and hippocampus organoids were successfully cultivated.

　　In 2020, snake venomous organoids were successfully cultivated.

　　The hot topic of research on organoids is the patient-derived organoids (PDOs), which are often called tumor organoids because they are usually used for disease modeling, research and drug screening in tumor patients. The development of tumor organoids began in 2013 and has been on an upward trend since then. Tumor organoids are organoids obtained by biopsy, puncture or surgical removal of tissue in matrigel for several weeks. While tumor organoids highly maintain the heterogeneity of the source tumor and the heterogeneity between patients, the morphology and scale between individual organoids remain basically uniform, providing a fast and excellent technical platform for the fields of tumor pathogenesis research, drug screening, personalized precision medicine, regenerative medicine and other fields.

　　At present, 3D organoid culture technology has successfully cultivated a large number of tissue-like organs with some key physiological structures and functions, such as: kidney, liver, lung, intestine, brain, prostate, pancreas and retina.

　

　　-03-

　　Cultivation of organoids

　　The culture of organoids can use somatic cells, adult stem cells (including progenitor cells) or pluripotent stem cells. In 2009, intestinal organ simulation technology was the first to make breakthroughs, and researchers found that adult intestinal stem cells can proliferate and spontaneously organize in vitro. It is characterized by the ability to express LGR5, a gene encoding the Wnt agonist R-spondin receptor, while requiring specific molecules to surround each other (such as Wnt, KGF and noggin). Based on this as a theoretical basis, researchers have developed a three-dimensional culture system that can reconstruct the appropriate environment of intestinal stem cells in vitro and differentiate from intestinal epithelial cells or single LGR5+ stem cells to organoids with self-renewal ability and maintain the villous structure of the intestinal glands. The model can be continuously amplified for up to 3 months, and the stable genome guarantees the advantages of purification and production amplification. This method has since been used to culture various organoids from epithelial tissues of other major organs.

　　Adult stem cell-derived organoid culture is usually established by embedding isolated adult stem cells or single-cell suspensions of required organ culture into an extracellular matrix (ECM) hydrogel. For example, intestinal organs, crypt cells isolated from the small intestine or colon are sufficient to cultivate organoids, and organoids can also be cultivated from isolated adult intestinal stem cells.

　　Epithelial organoid culture medium is based on culture medium with added growth factors related to the organ. Therefore, organelles extracted from other tissues (such as respiratory tract, liver, pancreas, skin, bladder, brain, heart) require supplementation of relevant growth factors in culture. For example, the brain organoid culture process is shown below: Inducing pluripotent stem cell (iPSC)-derived cells to grow in nerve-induced media, produce neuroectoderm, embed in Matrigel, and grow in a rotating bioreactor or orbital shaker to better spread and obtain three-dimensional brain organs. When exposed to retinoic acid, brain organs form self-assembly and tissues through self-assembly modes, forming different populations of neural progenitor cells, including radial glial cells, which expand to form brain structures.

　　

　　In addition to adult stem cells, pluripotent stem cells (including inducible stem cells and embryonic stem cells) can also use their self-renewal and differentiation capabilities to prepare organoids. Since organoids extracted from pluripotent stem cells are formed by homogeneous population orientation differentiation, tissue-specific cell types and their microenvironment must be recreated in a dynamic process of awakening embryogenesis. Therefore, pluripotent stem cell organoid culture must provide appropriate niche signals during differentiation. Because this process is more complex, pluripotent stem cell organoids often contain cell types different from model organs, complicating the signaling environment and self-organization of the target tissue.

　　1. Organoid culture in scientific research

　　1.1 Study on epithelial organoid interactions

　　Epithelial cells appear at all boundaries in contact with the outside world, such as the skin, respiratory tract, lungs, and digestive tract. They are the first barrier for the body to fight against pathogen infection and the first cell to respond to pathogen infection. To maintain homeostasis and provide a rapid response to infection, epithelial cells work closely with immune cells. Therefore, in the epithelial area, it is also the area with the highest concentration of immune cells in the system. Establishing a model of the interaction between epithelial cells and immune cells is an important means to study anti-infection immunity and post-injury immunity. The establishment of epithelial organoids can accurately simulate the body's epithelial environment and have also been developed to study the interaction between epithelial and immune cells.

　　

　　There are three co-cultivation modes

　　1. Treat organoids with recombinant cytokines present in extracellular matrix to evaluate the effect of immune cell-derived cytokines on epithelial cells. For example, IL-4 and IL-13 promote clumped cell differentiation, while IL-22 supports stem cell proliferation and survival.

　　2. Dige the organ into a single cell, and then grow in the presence of immune cells. Used to evaluate the effect of immune cells and immune cell-derived cytokines (soluble or membrane binding) on organ growth and differentiation, as well as the effect of epithelial cells on immune cell phenotype.

　　3. Add (activated) immune cells, such as T cells or innate lymphoid cells (ILCs) to the intact organoids in ECM or growth medium (suspended culture) to evaluate the interaction between immune cells and epithelial cells. Reads for these assays usually include organs formed by digestion and subsequent transcription. Single-cell RNA sequencing or quantitative PCR (QPCR), imaging and/or flow cytometry to evaluate epithelial and/or immune cell phenotypes. The immune cells used in these co-cultures are either classified directly from mouse tissues (e.g., spleen cells, intestinal intraepithelial lymphocytes, lamina propria T cells, lamina propria ILCs or lung ILC2s), either directly from human peripheral blood, or are first differentiated in vitro.

　　

　　1.2 Study on T cell development in thymus organoids

　　The thymus is the central part of T-cell maturation and differentiation from progenitor cells to mature naive lymphocytes. Bone marrow-derived T cell progenitor cells are positively selected in the thymic cortex and then negatively selected in the thymic medulla. These regions of the thymus are composed of two different epithelial cells: cortical thymic epithelial cells (CTECs) and medulla thymic epithelial cells (MTECs). 3D reconstruction of the thymus has been shown to be the key to simulate its function, several thymus organoids produced: these organ cultures are usually established from humans or fetal rat or neonatal thymus tissue, but there are also reports of in vitro differentiation of TEC-like cells and human embryonic stem cells, and these cultures produce thymus-like structures. Living T cells are produced in vitro and play a role when transplanted onto nude mice. Interestingly, although long-term thymus-like cultures are possible (ex-vitro cultures for up to 56 days), cells lose their colony-forming ability during continuous passage. Importantly, although a dual-energy TEC precursor has been found in adult mice, thymus organs containing cTECs and mTECs have not yet been generated from single stem cells. Furthermore, given that growth factors of certain dual-energy TEC precursors in mice have been found (e.g., BMP 4 and IL-22), it can be tried whether these factors can be used to maintain progenitor-derived organoids of TEC.

　　2. Tumor microenvironment

　　Two main methods are used (taking non-small cell lung cancer as an example)

　　In the holistic method (left), tumor biopsy tissue is cultured in the environment between the gas-liquid interface, and cell suspensions of all tumor cell types, including endogenous immune cells and other non-epithelial cell types, promote the growth of tumor-specific T cells.

　　

　　In the reduction simulation method (right), epithelial organoids are grown from tumor biopsy tissue and are then co-cultured with autoimmune cells from the peripheral blood of the same patient to promote the continuous expansion of tumor-responsive cells.

　　Although the overall method allows the culture of tumor materials including the entire tumor microenvironment and is therefore very similar to the situation in vivo, it is not easy to maintain for a long time, while the reduction simulation method allows long-term culture and expansion of tumor epithelium, which makes wider and longer-term research possible.

　　3. Drug screening

　　3.1 Screening animal models for promoting cardiomyocyte growth compounds, mainly mice, are widely used to study heart disease and provide valuable results. However, due to the wide variation of many species of functional and biological properties, their inferences about human heart disease and drug safety are poor. Human pluripotent stem cells (hPSCs) can provide unlimited sources of human cardiomyocytes for biomedical and drug research, potentially bridging this gap. However, hPSC-derived cardiomyocytes in traditional 2D cultures lack functional maturation, which in some cases hinders their ability to accurately predict human biology and pathophysiology. Multicellular 3D human organoids provide a more accurate model and are a potential solution to this problem. Scientists from the Department of Biomedical Sciences at the University of Queensland, Australia published an article at Cell Stem Cell, explaining their new process of cultivating cardiac myocardial organoids based on 96-well plates and then screening compounds.

　　The specific process is as follows:

　　Based on organoids, 5,000 compounds were screened for different functional compounds.

　　3.2 Applications for specific neural organoid culture and drug screening, first of all, neurospheres are cultured on different differentiation mediums (see the figure below for specific inducing factors and growth factors), to produce different brain organoids such as the brain, midbrain, hippocampus, and forebrain.

　　

　　(Source: Stem Cells Say)

　　-04-

　　Application of organoids

　　As an emerging technology, organoids have great potential in the field of scientific research, including developmental biology, disease pathology, cell biology, regeneration mechanisms, precision medicine, and drug toxicity and efficacy tests. Organoid culture provides a platform for studying human development without ethical restrictions, provides a new platform for drug screening, and is also a complement to the high amount of information of existing 2D culture methods and animal model systems. In addition, organoids are used for cell therapy in order to obtain cells closer to natural human development.

　　By replacing damaged or diseased tissues through the reproduction of organoid stem cells, organoids provide feasibility for autologous and allogeneic cell therapies. This technology also has great potential in the field of regenerative medicine in the future. Using this technology, CRISPR/Cas9 can correct genetic abnormalities in vitro and can return healthy transgenic cells to the patient's body and integrate them into the tissue later. In precision medicine applications, patient-derived organoids have also proven to be valuable diagnostic tools. Prior to treatment, in vitro drug responses were screened using organoids from patient samples from patients to provide guidance and predict treatment outcomes for patients with cancer and cystic fibrosis. With the continuous development of organoid culture systems and their experimental development technologies, organoids have been applied to major research fields.

　　

　　1. Modeling development and disease through organoids. Researchers can simulate human development and disease through organoids, because organoids grow from induced pluripotent stem cells produced by human stem cells or adult cells. Their composition and structure are similar to the primary tissue and are easy to manipulate and cryopreserve. This means that organoids can be used to study human tissues derived from stem cells and are difficult to simulate by animal models, and researchers need only a small amount of starting substance to cultivate organoids.

　　Because it has similar spatial tissue to the corresponding organ, maintains some key characteristics and can reproduce some physiological functions, it is considered a new model for detecting human biology and diseases. Compared with traditional research models such as cell lines, genetically engineered mice and human xenografts (PDX), the organoid models (mouse-derived organoids, MDO and human organoid patients-derived organoids, PDO) can not only be taken from tumor tissues at all stages of normal tissue and tissue cancer, but also their culture system is simple and easy to operate, with low time and money costs, and has high efficiency. Therefore, it has been loved by researchers and was rated as the 2017 annual technology in the field of life sciences by Nature Methods.

　　Comparison of main research models

　　Source: Science Magazine

　　

　　Currently, most of the samples used by cancer alliance organizations such as the International Cancer Genome Consortium (ICGC) and the Cancer Genome Atlas (TCGA) are taken from primary tumors. In contrast, PDO can be taken from any stage in the tumor development process, and only a small portion of the tumor tissue is required to be cultured and amplified in vitro. Researchers have obtained biopsies through puncture in liver cancer, pancreatic cancer and colon cancer liver metastatic tissues and successfully conducted organoid culture in vitro. It can even be cultured into organoids in vitro by circulating tumor cells in prostate cancer patients. Researchers have also successfully cultured organoids from normal cells of the urine tract and bronchial lavage, but it is not clear whether these culture methods can be suitable for tumor organoid cultures from these tissues.

　　2. Stem cell organoid engineering The advancement of stem cell bioengineering technology has improved the ability to control cell types, tissues and interactions, and organoid engineering is requiring manipulation of each structural layer by directly modifying stem cells or controlling the microenvironment. Now, scientists have developed a more precise synthetic environment that can better control stem cell activity by modifying the bioinert region of the matrix with signaling proteins. Organoid engineering technology is particularly useful for developmental research with complex environmental components in vivo and requires precise modeling.

　　3. Organoids and precision medical organoid technology is becoming a tool for individualized treatment. Using organoid technology for individualized treatment refers to formulating therapeutic drugs and methods suitable for this individual through drug screening and genotyping of organoids in vitro. Up to now, the responses of different tumor PDOs to traditional and under development are diverse. Research on currently limited resources has found that the treatment response shown by most PDOs is consistent with the corresponding patient's initial response to treatment. PDOs can also be used to develop new drugs against passive or acquired tolerance. More importantly, PDOs are highly sensitive to drugs with cytotoxicity, so they can better predict the clinical response of patients after use. Next, the researchers integrated the drug response data of a large number of individual PDOs to find common characteristics, and then conducted biomarker development research on a class of similar patients.

　　3.1 Drug Screening Organoid cultures can be used for drug screening, which can correlate the genetic background of the tumor with drug response. The establishment of organoids from healthy tissues of the same patient provides the opportunity to develop less toxic drugs by screening compounds that selectively kill tumor cells without damaging healthy cells. Self-renewing hepatocyte organoid cultures can be used to test the hepatotoxicity of potential new drugs (one of the causes of drug failure in clinical trials).

　　

　　Comparison of drug screening models

　　3.1.1 The advantages of organoid drug screening are fast: the success rate of organoid construction and the culture speed are high. Conventionally, a medicine screen can be performed after one week of organoid culture. The entire process from sample collection to drug sensitivity results can be well controlled within 2 weeks.

　　High flux: In terms of screenable drug flux, organoids can not only screen multiple drugs on the well plate, but each drug can also test different concentrations, and multiple experiments are carried out in parallel.

　　Strong clinical relevance: The clinical relevance and predictive effectiveness of organoids used in cancer drug screening have been fully confirmed in many studies. Vlachogiannis G team published a milestone study on in vitro drug sensitivity testing for tumor organoids in Science, and 110 tissues were extracted from 71 metastatic gastrointestinal cancers to build organoids, and a total of 55 anti-cancer drugs were tested. The research results show that organoid drug screens achieved 93% specificity, 100% sensitivity, 88% positive prediction rate and 100% negative prediction rate, showing extremely high clinical correlation.

　　3.1.2 Process of organoid screening

　　The drug screening process includes three aspects: organoid construction, evaluation, and drug sensitivity testing:

　　Organoid construction: The sample source of organoids is usually malignant effusion such as tumor tissue or ascites. The mainstream culture methods include the more commonly used positive gel drop method, the inverted gel drop method suitable for tumor and testicular organoid culture, the gas-liquid interface method suitable for gas-contact mucosal organoids (intestinal, respiratory tract) culture, and the bioreactor method that requires large amplification (brain organoids). First, the tumor sample tissue from the patient is mechanically cut to obtain tumor cell mass, and then the cell mass enzyme is digested into single cells. After separation and digestion, cells were embedded in matrix gel and seeded on 96/384 well plates, and then covered with culture medium and cytokine culture. Cell pellets of several hundred microns in diameter can be used for drug screening. Important reagents unique to organoid culture include digestive fluid, culture medium (such as Wnt, R-Spondin, Noggin and other cytokines), and matrix gel (Matrigel, etc.).

　　

　　Organoid evaluation: After cultivating organoids, the evaluation and verification of organoids is also crucial. Organoids were identified from multiple dimensions such as morphology, histopathology and molecular genetics through gene sequencing, immunofluorescence, HE staining and other methods. The purpose of the evaluation is to determine the consistency between the organoid and the protumor, which is also a prerequisite for subsequent drug screening.

　　Drug detection: The types of drugs that organoids can currently screen include chemotherapeutic drugs, small-molecular targeted drugs, antibody drugs, etc. The core detection indicators of drug screens are usually IC50 and cell inhibition rate. According to these indicators, the drugs with the best effect on tumor suppression are selected among the screened drugs. In China's registered clinical practice, organoids use sensitivity detection of chemotherapeutic drugs as the mainstream application, and organoids have great room and application potential for detection of sensitivity of targeted drugs and immunotherapy in the future.

　　3.1.3 Development direction of organoid medicine screening

　　Organoid drug screening and second-generation sequencing are used as combination products. The two can be organically combined in clinical practice and complement each other well. Second-generation sequencing detects the patient's target mutations and potential drug-sensitive targets from the genetic level, providing doctors and patients with preliminary drug choices, but the results of second-generation sequencing alone cannot guarantee complete clinical efficacy. Some research reports and clinical cases point out that the potential targeted drugs screened by second-generation sequencing have not shown effectiveness in actual clinical practice, and the uncertainty in this part can be easily investigated through organoids.

　　3.2 Genotype analysis

　　The growth of organoids from different healthy organs and the cultures are then whole-genome sequencing, which can analyze the organ-specific mutation spectrum. By growing organoids from different regions of the same tumor, it can be used to study intratumoral heterogeneity. Region-specific mutation profiles can be revealed by whole-genome sequencing of organoids. Using similar methods as described above, organoids can be used to study the effects of specific compounds on mutation profiles of healthy cells and tumor cells.

　

　　Cancer is caused by the gradual accumulation of mutations in pathogenic genes. Therefore, it is important to understand the mutation processes active during tissue homeostasis and tumorigenesis. (Source: Zhongjun Yatai, Stem Cells Say)

　　-05-

　　Organoid industry chain

　　The upstream companies in the organoid industry are mainly focused on providing reagents and material raw materials for 3D cell culture, including providing cell scaffold materials, extracellular matrix, cell growth factors, culture media and bioreactors. Among them are relatively old reagent manufacturers ThermoFisher, Sigma-Aldrich (now acquired by Merck), and a group of entrepreneurial companies that have achieved great success. InSphero, Switzerland, which provides scaffold-free three-dimensional cell culture products, STEMCELL Technologies, Canada's largest biotech company, provides professional cell culture medium and cell sorting products, and American company Xcell Biosciences, which provides in vivo microenvironment simulation systems, etc.

　　

　　Main 3D cell culture technology

　　According to the data from Meticulous Research Analysis, in 2016, 3D tissue culture accounted for about 9.3% of the global cell analysis and detection market. The market was worth US$818.1 million in 2017, and it is expected to grow at an annual compound growth rate of 8.7% during the period, reaching US$1.242.6 billion in 2022. Among them, the United States contributed about 34.8% of the global 3D cell market, ranking first in the world. In 2016, China's share of the global 3D cell market was far less than that of the United States, but it is expected that China will grow at an annual compound growth rate of 11.8% in the next five years, becoming the country with the highest annual compound growth rate and has market development potential.

　　The revenue points of the midstream and downstream companies in the organoid industry are mainly to provide in vitro drug trial plans and disease models to major new drug testing companies, namely clinical trial outsourcing services. For example, Emulate, a biotech company established by Harvard Wyss School of Bioengineering in 2013, Emulate and FDA will cooperate to evaluate and identify the possibility of using Emulate's "Organs-on-Chips" technology as a platform for toxicology testing, and have successively claimed to form strategic cooperation with AstraZeneca and Roche; Dutch biotech company Mimetas has developed a chip kidney and reached application cooperation agreements with several pharmaceutical companies to use it for drug screening; listed company Organovo has claimed to develop the first 3D printer for biological organs and develop artificial liver and artificial kidney. In October 2017, it announced a cooperation with Viscient Biosciences to further study liver disease.

　　The organoid industry has not yet formed a centralized industrial cluster in the country. In addition to traditional foreign cell culture reagents and raw material agents, mid- and downstream companies are relatively sparse. Several startup companies have emerged in Beijing, Shanghai and Guangzhou.

　　The organoid industry is developing rapidly in European countries, which is indispensable to the earliest start and accumulation of European organoid scientific research. Hubrecht Organoid Technology (HUB), founded by Hans Clevers, the leader of organoids, is the earliest R&D center for organoids. HUB technology license has promoted the emergence of a number of organoid companies including Epistem, Cellesce, Crown Biosciences, and STEMCELL Technologies. In the field of organoids, China has actually shown a significant increase in the number of scientific research in recent years, especially in the two years from 2019 to 2020, and the number of published documents has jumped from sixth place (2009-2019) to second place (2020), second only to the United States. Referring to the European organoid development model, it can be expected that the improvement of China's basic scientific research accumulation will accelerate organoid production.

　　

　　-06-

　　Market competition for organoids

　　Organoids have shown their strong development potential worldwide, and a certain market competition pattern has been formed abroad. Many companies are developing rapidly, such as AIVITA Biomedical, System1 Biosciences, JangoBio, etc. But there has not yet been a real competitive market in China.

　

　　Some domestic and foreign organoid companies

　　Data source: Zhongjun Yatai compiled based on public information

　　-07-

　　The challenge of organoids

　　Organoid culture technology is currently in a stage of technological explosion and scientific research results. The industry has great prospects for development, but it also faces great challenges. For example, how to use the stem cells of human embryos to establish a lasting and stable in vitro model; how to more realistically simulate and reduce the human microenvironment; how to achieve mass production of scientific research-related products, how to convert them into clinical products, etc.

　　As a new drug screen model, although the cost of organoids is lower than that of PDX, it is still much higher than that of cell lines. The cost of organoids is high, including the matrigel used for cultivation. The commonly used matrigel is Matrigel® from BD Biosciences, a company in the United States. It is in a relatively monopoly position in the industry and has a higher price. Matrigel can produce bioactive matrix materials similar to the basement membrane of mammalian cells, helping multiple types of cells to achieve attachment and differentiation. Matrigel is derived from mouse sarcoma cell lines. In addition to the high cost problem, there is also a certain variability between batches. And since it is an animal source, there are limitations on the detection of organic drugs. Considering that mouse-derived extracellular matrix has certain interference with drug screening experiment results, the engineering technology development of matrix for the synthesis of non-animal-derived matrix gels with small exogenous differences will be one of the key issues that need to be solved in the industrialization of organoids. In addition to matrix gel, culture also involves the use of a combination of multiple cytokines, and cell growth factors are usually expensive. Choosing better-effect cytokines and trying to reduce the number of cytokines used can also bring room for cost reduction.

　　At present, most organoids do not have a vascularized structure. Therefore, as the volume of the organoid increases, the organoids are limited by the loss of oxygen and the increase in metabolic waste, which may lead to tissue necrosis. There have been research on constructing tumor organoids in the microenvironment of vascular endothelial cells, and co-culture organoid tumor cells and vascular endothelial cells on Matrigel to generate vascular structures in order to solve the problem of organoid vascularization deletion.

　　Difficulties beyond vascularization also include simulating the interaction between tumor and the immune environment. In 2019, the journal Nature Protocol published related protocols co-cultured by tumor organoids and immune cells, which can reflect and simulate some of the characteristics of the tumor microenvironment. As an example, the interaction between organoids and immune cells can be reshape the interaction between organoids and immune cells by adding activated immune cells to the culture medium, growing with immune cells after the tissue is digested into single cells, and adding recombinant cytokines in ECM.

　　

　　Compared with a single organoid, the construction of an organoid system can make a more comprehensive assessment of the efficacy and potential toxicity of the drug. At present, organoids can only detect the inhibitory effect of drugs on tumors, and cannot predict whether there are other side effects and safety risks in other organs and tissues. To solve this problem, in 2017, Skardal et al. constructed an organoid system composed of the heart, lungs and liver integrated into the closed circulation concern body to achieve the purpose of comprehensively revealing the toxicity and efficacy of drugs to different organs.

　　Repeatability and consistency are also major bottlenecks in organoid development, which is largely due to the lack of process control and the gap in industry standards. Excessive participation of human factors and low degree of automation in the process of organoid culture leads to large errors caused by accidental system. At the same time, organoid detection methods are very scarce. Live observation is mainly focused on morphological observation, and breakpoint observation is focused on the detection of various indicators based on fluorescence. Optical, electrochemical and other means that can detect various indicators of organoids in real time are still lacking. At present, many researchers are committed to creating newer organoids and making organoids that have not been made before. We can make organoids of the hippocampus, pituitary gland, gland, spleen, and kidney, but it is difficult to determine that an organoid that meets the requirements needs to meet the statistical indicators of those individuals such as size, shape, gene expression, etc., and populations such as variance between organoids. This will limit the efficient research of organoids and the transformation into clinical research.

　　Engineering control during organoid culture is also an urgent problem to be solved. Currently, organoid cultures mostly use Matrigel hydrogel as the culture medium, a colloidal mixture of Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells produced by Corning Life Sciences. Matrigel is difficult to apply in many treatment scenarios in humans because it contains exogenous ingredients. On the other hand, although there have been some examples of combining organoids and microfluidic control technology, it is still immature to use microfluidic chips to simulate the fluid environment in which organoids survive. How to use microfluidic control and other technologies to control the fluid microenvironment during organoid culture is an urgent problem to be solved. At the same time, the diameter of existing organoids is about 100-500μm. Although it has a certain degree of scale effect, it is still difficult to simulate scenes of real tissues and organs. If organoids of a larger scale are to be created, the vascularization of organoids is also a very important issue.

　

　　-08-

　　The future development of organoids

　　1. Policy promotion

　　On January 28, 2021, the Ministry of Science and Technology issued the "Notice on Soliciting Opinions on the 2021 Annual Project Application Guide for Six Key Special Projects of the National Key R&D Plan for the 14th Five-Year Plan", and listed the "organoid-based malignant tumor disease model" as the first batch of key special tasks to be launched in the "14th Five-Year Plan" national key R&D plan. In addition, the "14th Five-Year Plan" National Key R&D Plan pointed out that organoids, as a major technological breakthrough, are used in the establishment of disease models, and can be used to study stem cell mutation, heterogeneity and its mechanism in pathological states, explore new targets for disease diagnosis and treatment, and explore new diagnostic and treatment strategies. In addition, in 2022, the U.S. Senate and House of Representatives approved the U.S. Food and Drug Administration Modernization Act 2.0, canceling the mandatory requirement for animal experiments for new drugs before clinical practice, and recommending some non-animal detection methods, including organoids, cell models, organ chips and microphysiological systems. Organoid technology will have great application value and development prospects in the future.

　　

　　Data source: Ministry of Science and Technology

　　2. Development of organoid technology

　　Traditional preparation method is a 3D culture technology that relies on growth factors and has its limitations (such as insufficient control of organoids and local environment). In addition, traditional preparation methods cannot replicate the complex and dynamic microenvironment in the development of organs well, and this microenvironment is a favorable factor in organ formation, which makes it difficult to obtain a more complete organoid process similar to the development of the internal organs.

　　The most cutting-edge at present is the "organoid chip" technology composed of organ chip technology and instruments. Organoids are not the only ones, the integration of multiple models is the king, and research based on in situ tissue and animal models will still be the gold standard for biomedical research.

　　Organoids have become an attractive and easy-to-replicate human tissue model for humans, but taking brain organoids as an example, there are still many differences between them and in situ cell types, which may confuse people's understanding of endogenous brain function and may propose misleading hypotheses about neuropathology, which may ultimately mislead treatments. This requires in-depth understanding and characterization of each cellular properties in in situ tissue, including cell types characterized by methods such as transcriptome, epigenome, and protein abundance; cell structural tissues including spatial tissue, morphological and physical connectivity parameters; and cell and tissue functional indicators including metabolic status and electrophysiology.

　　Today, organoids cannot stand on their own, and in the future, organoids do not need to stand on their own. The integration of multiple models is the best solution for research. Looking ahead, there is great prospects for organoid research. The highly simulated organoid disease model is expected to continue to make new progress in the fields of precision medicine, regenerative medicine, etc. At the same time, "organoid +" is expected to bring new growth points to organoid research. Organoid technology combined with live real-time imaging technology is expected to allow people to observe early human development for the first time; combined with biological 3D printing, it is expected to realize functional organoid-based treatment; combined with the "Human Cell Atlas (HCA)" technology, the organoid cell map will promote disease-centered research including rare genetic diseases, complex multi-factor diseases, and precise tumor treatment.