[Organoid Technology Introduction]

Small intestinal cell models with crypt and villi structures in vitro were successfully grown in 2009 by Hans Clevers' team at the Hubrecht Institute in the Netherlands by using three-dimensional adult stem cell culture. This technique of culturing adult stem cells or pluripotent stem cells in vitro in three dimensions (3D) is known as "organoid" technology (Organoids), because the cultured tissue analogs can mimic some of the functions of actual organs and have a specific spatial structure.

Figure 1: Intestinal organoids

Organoids can be subcultured steadily for a long time, so their in vitro culture is currently the best technology for simulating the structure and function of in vivo tissues, compared to traditional two-dimensional cell culture. Organoid manipulation is also easier and better suited for studies like high-throughput screening and studies on the causes and progression of diseases than using animal models. After more than ten years of intensive research and development, this ground-breaking technology has succeeded in cultivating numerous tissue-like organs with important physiological features, including the kidney, liver, lung, intestine, brain, prostate, pancreas, and retina. Therefore, organoid technology has broad application prospects in disease modeling, drug screening, precision medicine, organ development, regenerative medicine and other fields.

Figure 2: Timeline of key technological breakthroughs in organoid development [1].

[Prospects for the development of organoids]

The number of organoid-related research articles published significantly increased from 42 in 2010 to 2,097 in 2020 after the Hans Clevers team successfully cultivated small intestinal organoids, indicating that research in this field has become a hotspot in just a decade. As a result, the "Science" magazine listed organoids as one of the top ten technologies in 2013, and "Nature Methods" named them the 2017 annual method in early 2018. They continued to be a hot topic in prestigious journals and magazines in 2019 and were once again featured on the cover of the special issue of "Science" magazine.

At the beginning of 2021, "Organoid-based malignant tumor disease model" was listed as one of the first national key R&D tasks to be launched in the "Notice on Collecting Opinions for the 2021 Project Declaration Guidelines for 6 Key Special Projects during the 14th Five-Year Plan Period" issued by the Ministry of Science and Technology. The organoid was a significant technological advance, according to the National Key R&D Plan of the "14th Five Year Plan," and it was used to create disease models, study the variation and heterogeneity of stem cells and the mechanism such the variation and heterogeneity under pathological conditions, tap new targets for disease diagnosis and treatment, and investigate novel diagnostic and therapeutic approaches. Organoid technology will have great application value and development prospect in the future, which can be told from its increasing presence in top journals and in the documents issued by the Ministry of Science and Technology. 

Figure 3: Notice on Key National R&D Projects during the 14th Five-Year Plan Period issued by the Ministry of Science and Technology in 2021

[Organoid construction method]

The two main categories of organoids currently available aretissue-derived organoids (e.g., tumor-derived organoids) and pluripotent stem cell-derived organoids (e.g., retina-derived organoids, brain-derived organoids).

Figure 4: Tumor glioblastoma organoids Figure 5: Brain organoids

The construction of organoids is a systematic project that requires a strong material basis and a specific culture environment. In general, 3D scaffolds, culture media containing different growth factors and induction supplements, stem cells, supporting cells or tissues, etc., make up the required material basis for organoid construction. The specific culture environment includes the regulation of oxygen, temperature, carbon dioxide, and even some mechanics and other parameters that resemble the growth environment of cells in vivo.

The organoid culture process is essentially a method that provides specialized culture conditions to mimic the micro-environment of cells in vivo. Careful and precise control on this micro-environment for cell growth is crucial for the success. The building of spatial structure, self-organization of cell types, and cell proliferation are all supported by the 3D scaffold. Different types of cells will travel to different areas on the 3D scaffold under the influence of cell clusters because they release different cell adhesion factors, allowing for the separation of cell based on cell types. The spatial constraints result in "genealogical orientation", or "cell fate determination due to spatial constraints". The corresponding culture media and inducers either activate or inhibit the specific signal pathways involved in the formation of organoids and the maintenance of homeostasis. Therefore, these cells are further differentiated by the space constraint and the stimulation of the original cell secretion or an additional inducer, and an organoid subsequently is formed.

 Figure 6: Various techniques for organoid preparation[2]

The traditional method of preparing organoids involves culturing the isolated stem cells on 3D scaffolds (e.g. matrix gel Matrigel), spreading them out in culture dishes or culture well plates, and supplementing them with different growth factors, such as FGF and EGF, to promote the formation of organoids. 

Since the organoid technology came out, numerous culture models have been created. This is because, as shown in Figure 6, the preparation of various organoids necessitates the use of various additive combinations, inducer addition strategies, and initial cell states.

① Different specific physical environments may include static culture by using natural extracellular matrix (ECM, e.g., small intestine organoids or stomach organoids) or synthetic polymer (e.g., small intestine organoids) 3D scaffolds, 3D suspension stirred culture with no extracellular matrix scaffold support (e.g., brain organoids) or proteins containing extracellular matrix (e.g., retina organoids), or cell cluster culture with air-liquid interface (e.g., kidney organoids).

②Different inducer addition strategies include all-endogenous induction of autonomous suits (e.g. retina organoids), exogenous induction of differentiation-endogeneity derived tissues (e.g. kidney organoids) or all-exogenous induction of autonomous suits (e.g. stomach organoids).

③The initial cell state can be single cells (such as small intestine organoid), homogeneous cell cluster (such as retina organoid) or multicellular cell cluster (such as hepatocyte-like cells).

As seen from the examples above,

organoids are expected to become more crucial as an ideal model for various research applications in human health as science advances and technology keeps innovating.

The culture conditions for organoids from various tissue and organ sources must be mapped out based on various experimental conditions before they can be successfully grown. Rapid screening of suitable habitats, induction methods, and initial cell states are the key to cutting the organoid development process and accelerating the development of organoid technologies.

Increasingly more scientists are now starting to investigate novel techniques for cultivating organoids. New solutions are no longer limited to flat static culture methods. For example, dynamic shear force culture methods like the stirring flask method start to be used, which has a profound impact on the research and development of organoid technology.

On November 11, the innovative 3D FloTrix® microSPIN 6-Channel Micro-bioreactor, capable of delivering dynamic shear forces, will be launched by CytoNiche Biotechnology Co. Ltd. The new bioreactor provides a highly anticipated novel culture solution for the research of organoids, 3D cells, and gene therapy. We'll offer a more in-depth analysis of the dynamic culture of organoids in the next issue.  

Coming up next:

Application of stirred reactor in organoid culture

References

[1] Corrò, C., Novellasdemunt, L., & Li, V. S. W. (2020). A brief history of organoids. American Journal of Physiology-Cell Physiology. doi:10.1152/ajpcell.00120.2020

[2] Rossi, G., Manfrin, A., & Lutolf, M. P. (2018). Progress and potential in organoid research. Nature Reviews Genetics. doi:10.1038/s41576-018-0051-9

【CytoNiche】

Beijing CytoNiche Biotechnology Co., Ltd. was established by the research team of Professor Du Yanan from Tsinghua University School of Medicine, and was jointly established by Tsinghua University through equity participation. The core technologies were derived from the transformation of scientific and technological achievements of Tsinghua University. CytoNiche focuses on building an original 3D cell "smart manufacturing" platform, as well as providing overall solutions for the 3D microcarrier-based customized cell amplification process.

CytoNiche's core product, 3D TableTrix® Microcarrier Tablet (Microcarrier), is an independent innovation and the first pharmaceutical excipient grade microcarrier that can be used for cell drug development. It has obtained the certificate of analysis from relevant authoritative institutions such as National Institutes for Food and Drug Control, and obtained 2 qualifications for pharmaceutical excipients from the National Medical Products Administration (CDE approval registration number: F20210000003, F20200000496). Moreover, the product has obtained the DMF qualification for pharmaceutical excipients from U.S. FDA (DMF: 35481). 

Products and services of CytoNiche can be widely used in the upstream process development of gene and cell therapy, extracellular vesicles, vaccines, and protein products. At the same time, it also has broad prospects for applications in the fields of regenerative medicine, organoids, and food technology (cell-cultured meat, etc.).

Our company has a R&D and transformation platform of 5,000 square meters, including a CDMO platform of more than 1,000 square meters, a GMP production platform of 4,000 square meters, and a new 1200 L microcarrier production line. The relevant technologies have obtained more than 100 patents and more than 30 articles about the technologies in international journals have been published. The core technology projects have obtained a number of national-level project support and applications.