Throughout the past decades, most of the research has been focused on 2D cultured cell lines. These rather classical cell lines are cheap, easy to handle, and amenable to numerous experimental techniques and methods [1,2].
However, their initial establishment is inefficient and involves extended genetic and phenotypic adaptation to culture conditions. As a result, cell lines are consistently derived from tumours or have acquired oncogenic potential in vitro. Also, when they are used to represent tumours or diseased cells, matching normal cells is lacking. Another drawback is the absence of differentiated cell types that are normally present in the original tissue. Altogether, these drawbacks or limitations make them less suited to study tissue physiology that involves multiple differentiated cell types [1,3].
Based on the reasons mentioned, using alternative model systems became vital over time. For instance, in cancer research, patient-derived xenografts (PDXs) are used. PDXs are better to retain the complexity and heterogeneity of the parental tumour when compared to cell lines. Still, the establishment is inefficient and high-throughput analyses are expensive. Due to the disadvantages of the previous methods, organoid cultures have been developed; and they lack many of the disadvantages associated with cell lines [4].
Some model organisms such as yeast, fruitflies and mice have been used commonly for a long time in traditional research. The establishment of other model systems has become crucial for our understanding—these model systems being the worm and the zebrafish [4]. The worm (Caenorhabditis elegans) was widely used as a model in the 1970s, meanwhile, the genetic conduction of the zebrafish (Danio rerio) has taken place in the 1990s. Currently, the most commonly used model organisms are S. cerevisiae, C. elegans, D. melanogaster, D. rerio and the common house mouse [4]. Even though these systems are extensive, they were still not enough to correctly reflect human physiology. Here is a table comparing the seven methods:

Among animal models, mice are the most relevant for investigating disease-related processes in terms of compatible physiology, large numbers of genetic tools, small size and fast reproductive cycle. However, between species, even the basics of such principles can differ. Species-specific differences exist in many ways and can differ greatly among species. For example, brain development has vastly different time scales in different species. Cortical neurons in the mouse brain reach maturity after five weeks meanwhile in the macaque brain, the maturation time is observed to reach up to four months [2,4]. Even further, in humans, synaptic formation and dendritic development happen in a time frame of months and years in humans; synaptic elimination continues into the third decade of life. Differences between species also exist in the susceptibility to infectious diseases. For instance, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the origin of the COVID-19 pandemic does not cause disease in wild-type mice. This matter made it compulsory for us to generate human models to understand human development and disease successfully. For instance, certain genetic diseases in humans such as neurodevelopmental disorders need more successful therapy & drug development; thus marking the importance of human models [2,4,5].
The term ‘organoid’ has been used to refer to all 3D organotypic cultures derived from primary tissues, embryonic stem cells (ESCs) and induced pluripotent stem cells (IPSCs). In vitro, 3D cellular cluster derived exclusively from primary tissue, ESCs or IPSCs, capable of proliferation and self-organization; exhibiting similar organ functionality as the tissue of origin. Most of the organoid cultures documented contain functional tissue units that lack mesenchymal, stromal, immune and neural cells that intersperse the tissue in vivo. They rely on artificial extracellular matrices to facilitate their self-organization into structures that resemble the native tissue architecture. Used within both basic and translational research, the implementation of organoid-based technologies within academia and industry is a testament to their importance [1,3,6].

The self-organizing traits of mammalian cells have been known and harnessed to generate 3D cultures from primary tissues throughout history. Developing the intestinal organoid culture system in 2009 brought major advantages to the stem cell field [2,7].
These are some of the advantages of organoids that we can list:
- Can be expanded indefinitely even with limited starting material,
- Can be preserved as biobanks and are easily manipulated,
- Similar to primary tissue in both their composition & architecture,
- More physiologically relevant than monolayer culture models, and are more open to manipulation of niche components, signalling pathways and genome editing when compared to in vivo models,
- Adult stem cells can be propagated in organoids,
- Can be propagated for years without any alterations,
- Amenable to numerous experimental techniques,
- Can be derived from multiple sources like adult & foetal tissues, ESCs and IPSCs,
- Human-specific diseases that are hard to model in animals can be studied with patient-derived organoids which are more physiologically relevant than standard 2D culture models [1].
Unlike the previous methods, this new system could put our knowledge the use to deliver a stable culture system that is capable of sustaining the long-term growth of near-physiological epithelia from purified stem cells or isolated crypts [1,4].
The culture system is unexpectedly simple and is commonly referred to as the R-Spandin Method. The R-Spandin Method is used to create 3D structures with distinct crypt and villus-like domains bordering a central lumen that contains dead cells from the constantly renewing epithelial layer. Subsequently, these organoids reflected the in vivo tissue architecture and contained the full complement of stem, progenitor and differentiated cell types. Later on, this system was adopted for generating human intestinal organoids along with the organoids from other organs harbouring Lgr5+ stem cells, including the colon, stomach and liver [3,4,8].
Human Organoid Manufacturing and Their Application
As we have mentioned, organoids can be developed from pluripotent stem cells (PSCs), which can be embryonic (EPSCs), induced (IPSCs), and adult stem cells (ASCs).
Since the organoids derived from patients are identical to each patient’s genotype, they are currently being used for ‘personalized medicine’. Researchers have been successfully producing different types of organoids and have demonstrated their application in numerous fields such as disease modelling, drug screening, pathogenesis and regenerative medicine [9].
Types of organoids developed include intestinal organoids, cerebral organoids, kidney organoids, hepatic organoids, retinal organoids, pancreatic organoids, lung organoids, colonic organoids, gastric organoids, cardiac organoids, thyroid organoids, prostate organoids, saliva-secreting organoids, mammary gland organoids, lingual organoids and spinal organoids. In a study that took place in 2008, scientist [10] created a 3D cerebral cortex tissue from pluripotent stem cells using the SFEBq technique. This development of the culture system created a breakthrough in the organoid field. The study also mentioned that organoids developed from a crypt or a single stem cell are indistinguishable [6,9].
Although the starting cell type and system conditions, physical characteristics of the culture environment and the endogenous and exogenous signals influence the successful formation of organoids in vitro; at a larger scale, production of organoids still needs to be improved. Here are some of the limitations and challenges researchers come across:
- Limited reproducibility of organoids can become a major hurdle if organoids are used for toxicity testing or other high throughput testing,
- The issue of vascularization, although it is thought that this could be solved by either using a spinning bioreactor or by co-culturing with endothelial cells,
- An inefficient supply of nutrients and oxygen exchange to the centre of the organoids affects tissue patterning,
- Manufacturing organoids is tissue maturation: In most of the cases observed, the early stages of organoids are well developed, but the tissues fail to functionally evolve into mature organs,
- Lack blood cells, stroma and immune cells,
- Size of the organoids is a vital factor and it restricts organoid technology and application from reaching its own potential [4].
As mentioned earlier, there are many advantages of organoids but certain drawbacks and challenges make it compulsory for researchers to improve organoids manufacturing. Technologies such as bioengineering tools combined with 3D cell culture techniques most certainly help in improving the size of the organoids formed, which eliminates some of the challenges currently faced [4,6].
References:
- Fatehullah A, Tan SH, Barker N. Organoids as an in vitro model of human development and disease. Nature Cell Biology 2016 18:3. 2016;18(3):246-254. doi:10.1038/ncb3312 (Main,Sections 1,2,3,4)
- Corsini NS, Knoblich JA. Leading Edge Human organoids: New strategies and methods for analyzing human development and disease. Cell. 2022;185:2756-2769. doi:10.1016/j.cell.2022.06.051 (Sections 1,2,3)
- Ashok A, Choudhury D, Fang Y, Hunziker W. Towards manufacturing of human organoids. Biotechnol Adv. 2020;39:107460. doi:10.1016/J.BIOTECHADV.2019.107460 (Sections 2,3,4,6,7)
- Kim J, Koo BK, Knoblich JA. Human organoids: model systems for human biology and medicine. Nature Reviews Molecular Cell Biology 2020 21:10. 2020;21(10):571-584. doi:10.1038/s41580-020-0259-3 (Introduction,Sections 1,2,4)
- Tang XY, Wu S, Wang D, et al. Human organoids in basic research and clinical applications. Signal Transduction and Targeted Therapy 2022 7:1. 2022;7(1):1-17. doi:10.1038/s41392-022-01024-9 (Sections,1,11,12)
- Shankaran A, Prasad K, Chaudhari S, Brand A, Satyamoorthy K. Advances in development and application of human organoids. 3 Biotech 2021 11:6. 2021;11(6):1-22. doi:10.1007/S13205-021-02815-7 (Sections 1,2,5)
- Lehmann R, Lee CM, Shugart EC, et al. Human organoids: a new dimension in cell biology. https://doi.org/101091/mbcE19-03-0135. 2019;30(10):1129-1137. doi:10.1091/MBC.E19-03-0135 (Sections 1,2,3)
- Lancaster MA, Huch M. Disease modelling in human organoids. DMM Disease Models and Mechanisms. 2019;12(7). doi:10.1242/DMM.039347/3370 (Section 1)
- Ashok A, Choudhury D, Fang Y, Hunziker W. Towards manufacturing of human organoids. Biotechnol Adv. 2020;39:107460. doi:10.1016/J.BIOTECHADV.2019.107460 (Sections 2,3,4,6,7)
- Eiraku M, Watanabe K, Matsuo-Takasaki M, cell MKC stem, 2008 undefined. Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. cell.comM Eiraku, K Watanabe, M Matsuo-Takasaki, M Kawada, S Yonemura, M MatsumuraCell stem cell, 2008•cell.com. Accessed August 30, 2023. https://www.cell.com/fulltext/S1934-5909(08)00455-4 (Section 2,3,4)
Figure references:
- Kim J, Koo BK, Knoblich JA. Human organoids: model systems for human biology and medicine. Nature Reviews Molecular Cell Biology 2020 21:10. 2020;21(10):571-584. doi:10.1038/s41580-020-0259-3 (Introduction,Sections 1,2,4)
- Organoids: A new window into disease, development and discovery | Harvard Stem Cell Institute (HSCI). Accessed August 30, 2023. https://hsci.harvard.edu/organoids
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