An Introduction to Organoids, Organoid Creation, Culture and Applications

Organoids have revolutionized the fields of biological and medical research, and the technology has proven to be an excellent substitute for animal models in preclinical studies. Here, we discuss what organoids are and how they have been developed over the years to become popular research tools. A detailed insight into the techniques for culturing, and characteristics and applications of various organoids that resemble tumors and body structures such as the brain, lungs, intestines, liver, kidneys and retina are provided.

Organoids are three-dimensional (3D) cell culture systems that can be called “mini-organs” as they mimic some of the key multicellular, anatomical and even functional characteristics of real organs.

The term organoid refers to a 3D culture of different cell types derived from tissue explants, tumors, stem cells or other progenitor cells, that self-organize under controlled conditions and differentiate into functional cell types to acquire the complexity, anatomy and physiology of an organ or body structure. A tumor-like organoid derived from primary tumors is referred to as a tumoroid.¹

In 1907, it was proposed that complex organs/tissue structures could be recreated in vitro after Henry Van Peters Wilson showed that sponge cells in culture could self-organize and regenerate into fully-grown organisms. It was thought that this self-organizing and regenerating potential of cells in culture could probably be utilized to recapitulate parts of complete tissues or organs. Standard two-dimensional (2D) cultures comprise growing and maintaining monolayers of individual cell types on the surface of flasks or Petri dishes. However, the challenge was to attain the 3D spatial conformations of cells as they are in their natural environment.²

Subsequent attempts to achieve 3D culture were seen in the 1950s when Ehrmann and Gey cultivated various human cell lineages using collagen isolated from rat tails as a scaffold. Several attempts were made to model in vivo organ systems during the 1980s and-90s using dissociation-reaggregation experiments: for example, cells isolated from chick embryos or mouse fetal lungs were dissociated and allowed to reaggregate in culture. Since 1975, scientists have started to focus on developing scaffolds that facilitated cell–matrix interactions along with cell–cell interactions required for in vitro cell differentiation, just like in tissues and organs, and thus brought on a major leap shift from 2D cultures to 3D organoids. Floating gels or matrices rich in collagen and laminin were used as scaffolds for culturing fragments of tissues or cells isolated from dissociated organs in a 3D environment.^1, 2

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Landmarks in the development of organoids came with the first successful isolation of pluripotent embryonic stem cells (ESCs) from mice in 1981 and human blastocytes in 1998. In 2006, Kazutoshi Takahashi and Shinya Yamanaka genetically modified mouse fibroblasts to develop murine pluripotent stem cells (PSCs). Induced or embryonic PSCs (iPSCs or ESCs) have effective self-renewal and self-organizing capacities that are lacking in primary cells isolated from tissues or organs. PSCs are organ progenitors that differentiate in culture to give rise to multiple cell types that self-organize to form organ-like complex structures in culture, closely resembling the process of organ formation in embryos until birth. The discovery of stem cells improved organoid technology by eliminating the need for isolation and co-culturing of several cell types and reduced the need for expensive differentiating media and complicated scaffolds. Thereafter, the establishment of human induced pluripotent stem cells (hiPSCs) in 2007 revolutionized organoid technology to facilitate the development of organoids from single individuals that could be used for diverse in vitro and preclinical studies.^3, 4

Landmark events in the development of organoids over the years are shown in Figure 1.

Figure 1: A timeline illustrating the remarkable achievements in research and development leading to the generation of organoid technology. Abbreviations: ECM, extracellular matrix; ESC, embryonic stem cell; iPSCs, induced pluripotent stem cells. Credit: Technology Networks.

In the early 21st century, organoids were developed that did not require the use of gels or scaffolds. Cells were cultured either in hanging droplets of medium using surface tension or in medium with nanoparticles levitated by magnetic fields. However, the scaffold-free methods did not gain popularity and did not emerge further. Extracellular matrix (ECM) plays an important role in the in vivo microenvironment and architecture of organs, and natural or synthetic matrix continued to impact organoid improvements significantly. From 2012 onwards, air–liquid interface (ALI) was used to initiate organoids in which cells were cultured in gels immersed in the medium such that the upper layers of the cells were exposed to air, facilitating their polarization and differentiation. ALI models were used to construct organoids from cell lines, cells differentiated from ESCs or primary cells isolated from the skin. Advanced bioengineering and bioprinting techniques have also improved organoid culture techniques in the last decade by fabricating complex microarchitectures of organs that act as scaffolds on which different cell lineages can be seeded and cultured in an enrichment medium to generate an “organ-on-chip” model. Multiorgan-on-a-chip models contain different types of organoids on specially designed scaffolds/compartments on a single chip to allow cross-organ studies and multi-organ modeling of diseases.^2, 5

ESCs, iPSCs, somatic stem cells and cancer cells can all be used to create organoids. Suspensions of cells or fragments can be obtained from tissues, organs or cryopreserved organoids using mechanical or enzymatic dissociations. These cells or tissue fragments are first seeded in differentiating medium on low-attachment plates or culture vessels for 5–7 days to generate 3D cellular aggregates called spheroids. The spheroid formation can also be performed using normal suspension culture, mini bioreactors or roller bottles. These spheroids are then planted into liquid ECM, matrigels or agarose-based gels. The protocols are adapted according to the progenitor cells used and the organ/tissue structure to be recapitulated: in some cases, the cells obtained from dissociated tissues or organoids are suspended and seeded directly into liquid ECM, skipping the differentiation step. The gels are polymerized at 37 ℃ to obtain a 3D culture, which is supplemented with organoid-specific expansion and maturation medium to stimulate the growth of the desired cell types, resulting in organ-like structures. For example, a medium supplemented with the growth factor EGF stimulates the growth of epithelial and tumor organoids; whereas fibroblast growth factor 10 is needed in stomach, breast and liver cancer tumoroids.^{2, 6}

Generally, fully-grown, functional organoids are generated after 30–60 days (maybe even longer) in the maturation medium, replenished twice per week. This mature organoid can be maintained for about 100 days (sometimes up to a year) by changing the maturation medium once or twice every 10 days and passaging the organoids. For passaging, the maturation medium is removed and organoids are gently broken into smaller fragments using mild cell dissociation reagents for 15-20 minutes, followed by vigorous pipetting. After a few centrifugation and washing steps, the organoid pellets finally obtained are resuspended in liquid ECM to repeat the whole process of organoid generation. Organoids can be cryopreserved and fresh organoids can be generated by the complete dissociation of these frozen organoids.^{2, 6} The protocol is highly adaptable to the target organ or body structure to be recreated and a broad range of specialized media and media supplements, as well as other materials such as transwells, well-plates or matrices, are available commercially to uplift the various systems of organoid technology.

A schematic diagram of the protocol for organoid generation and culture is provided in the Figure 2.

Figure 2: Schematic representation of the generation of organoids from human embryos, organs or tumors. Abbreviations: ECM, extracellular matrix; ESC, embryonic stem cell; iPSCs, induced pluripotent stem cells; SC, stem cell; 3D, three-dimensional. Credit: Technology Networks.

Both organoids and spheroids are 3D multicellular culture techniques using tissue explants or co-cultures of cells. Spheroids are spherical-shaped, free-floating aggregates of cells formed spontaneously in culture. Organoids are an advanced and more complex form of spheroids with structural units largely similar to an organ.²

Although the terms organoids and spheroids are interchangeably used, there are subtle differences between organoids and spheroids^{1, 3} that are listed in the table below:

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The advantages of organoids over standard cell culture methods or spheroids are discussed below.

• Conservation of organ-like characteristics: The main advantage of organoids over standard cell culture methods is the 3D spatial arrangements of the cells in culture, which mimic their natural morphologies. How the progenitor cells differentiate and assemble into organ-like structures during organoid development follows mechanisms similar to the natural formation of organs as embryos transform eventually into infants. Therefore, organoids preserve the cellular and molecular mechanisms of the cells in comparison to standard cell cultures or even spheroids. These properties make organoids excellent models to study organ generation and functions for whole organs as well as specific regions.²
  
  
• Intercellular and cell–matrix interactions: ECM provides the main structural organization and allows cellular functions and communications for the proper functioning of biological tissues and organs. Organoids allow interactions among multiple cell types and resemble organ-like complex structures, which is impossible to achieve by any standard monolayer cultures, spheroids or even co-cultures of two or more cell types. Organoids provide an opportunity to study cell-to-ECM interactions as well as cell-to-cell interactions on a 3D level.^{1, 2}
  
  
• Specific representation of species and replacement of animal models: The use of human organoids eliminates species-specific mechanisms and offers effective alternatives to animal models to study human organs and hence provides an opportunity to perform mechanistic studies within the “human model” system.²
  
  
• Modeling on an individual level: Organoids are generated from single individuals or patients to provide cellular, morphological and other scientific data on an individual level. Even tumoroids preserve the histopathological as well as genetic features of the original tumors/cancers from individual patients. Tumoroids, multiple organoids specific for individual organs or multiorgan-on-a-chip allow disease pathogenesis or collective treatment responses of an individual to be studied.^{1, 2, 7}
  
  
• High-throughput and efficient preclinical model: Having more resemblance to in vivo systems compared to spheroids or standard cell cultures, organoids can be employed efficiently to model mechanisms of diseases and test the efficacy of drug candidates and other therapeutic interventions for diseases. Numerous organoids can be generated from limited human materials and therefore several tests can be performed simultaneously on these organoids. Consequently, the timeline of drug screening and testing is reduced and the differences between in vitro testing and actual patient responses are minimized, which in turn eases the economic burden due to production, preclinical and clinical trials before the drugs are released into the market.^{1, 7} 

Pioneering works in the last decade have led to the establishment of human-derived organoids that model tumors, embryos and a broad range of tissues and organs, some of which are highlighted below.

Brain organoids

Organoids derived from human PSCs comprising several cell lineages can recapitulate cellular and regional interactions as well as the formation of blood vessels in the brain. Brain organoids resembling specific regions of the brain or combinations of multiple brain regions and/or cell lineages, serve as representative models to study the development, function and dysfunction of the human brain. Human PSC-derived brain organoid models have been used to study gene mutations in microcephaly, the abnormal inhibitory neurons in autism spectrum disease and epilepsy, the pathogenesis of Parkinson’s disease and mechanisms of Zika virus infections in the brain.^{1, 8, 9}

Lung organoids

Human airway organoids have been employed successfully to study the infectious mechanisms of the respiratory syncytial virus and influenza virus.² Additionally, they served as a representative experimental model for studying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection with subsequent drug screening. Several other alveolar organoids derived from different cell types that mimic specific areas or a combination of multiple areas in the lungs have been developed to study SARS-CoV-2 pathogenesis and facilitate the development of new therapies. More than 1,000 drugs, including 25-hydroxycholesterol, remdesivir and camostat were tested for their effectiveness against SARS-CoV-2 in lung organoid models before they were administered to humans.^{2, 10}

Intestinal organoids

Complex asymmetric structures of intestines, including the crypts and villi, are recreated in organoids from single intestinal stem cells. The highly specialized epithelial barrier plays a major role in the functioning and homeostasis of the intestines. Organoids have allowed the successful identification of druggable biological targets as well as small molecule drugs as regulators in the composition and operation of the barrier epithelia. The intestinal epithelia for both the small intestine and colon recreated using organoids serve as promising models for studying the infectivity of the Omicron variant of SARS-CoV-2.^11-13

Liver organoids

Multicellular liver organoids from human primary hepatic stellate and iPSCs have been applied effectively to model liver fibrosis, lipid metabolism and genetic stability in non-alcoholic fatty liver disease and non-alcoholic steatohepatitis. Liver organoids have been employed to differentiate progenitor cells or PSCs into hepatocytes and liver buds. These could be transplanted with high engraftment efficiency in mice for the generation of mature hepatocytes or even vascularized into fully functional livers in vivo. These findings imply a potential role of organoids in liver transplantations in cases of organ damage or organ failure.^{2, 14}

Kidney organoids

Improved bioengineering techniques have achieved highly complex kidney organoids in recent years that have allowed several diseases to be modeled including polycystic kidney disease, cystic fibrosis, renal cell carcinoma and viral infections. In clinical trials, 19% of drugs fail because of nephrotoxicity. Consequently, several common medications such as cisplatin, gentamicin, aspirin and penicillin G, have recently been tested in kidney organoids for preselection. The therapeutic effects of human recombinant soluble angiotensin-converting enzyme 2 were tested in SARS-CoV-2-infected kidney organoids. Organoids derived from patient-derived iPSCs are also employed to investigate gene mutations and personalized therapies in congenital nephrotic syndrome.¹⁵

Tumor organoids

Recent improvements in organoid technology have recapitulated the tumor microenvironment utilizing primary cells or cancer cell lines for the lung, liver, kidney and heart in culture. Organoids from numerous cancer types can be frozen or cultured for a long time, maintaining their morphologies, histopathologies, epigenetic mechanisms and genetic profile to study cancer progression and clinical features of the tumors. Biobanking of patient-derived organoids (PDOs) for brain, breast, cervical, colorectal, gastric, head and neck, kidney, liver, ovarian and pancreatic cancers have been performed on a large scale to facilitate immunotherapies and precision medicine according to the clinical heterogeneity in cancer patients.^{7, 16}

Retinal organoids

Retinal organoids have been developed successfully from retinal stem cells and independently from ESCs that are capable of largely mimicking the visual cycle and responses to light in healthy eyes. Gene mutations involved in the pathogenesis of the retinal disorders retinitis pigmentosa, Leber congenital amaurosis, retinoblastoma and glaucoma have been modeled effectively using retinal organoids. Transplantation of hiPSC-derived pigmented photoreceptor cells into human retinas could potentially improve vision in age-related macular degeneration.²

Considering the advantages mentioned above, organoids offer potential applications and advancements in the fields of basic research, developmental biology, tissue engineering, drug testing and screening.^{1, 7} Patient-derived organoids have potential implications in precision medicine and cell therapy applications in regenerative medicine.⁷

Drug discovery

Organoids serve as fast and reliable methods for drug toxicity testing, drug discovery, screening and validation. Toxicity of 238 marketed drugs in liver organoids, 25 cardioactive drugs in cardiac organoids, cisplatin and gentamicin in kidney organoids, 39 marketed diarrheagenic drugs in intestinal organoids and vincristine and rotenone in brain organoids have been tested and reported so far.¹⁷ Organoid technology played an important role in the discovery of therapies and therapeutic targets during the COVID-19 pandemic: the efficiency and toxicity of potential drug candidates for the treatment of COVID-19 could be tested on multiple organs simultaneously using multiorgan-on-a-chip organoids.^{17, 18}

Disease modeling

Human iPSC-based organoids are susceptible to pathogens and provide a potential means of modeling host–pathogen interactions during bacterial or viral infections. Cerebral and neural organoids have been employed to model the Zika virus infection. Intestinal organoids have successfully modeled rotavirus pathobiology and gastric organoids have been used to model bacterial infection by Helicobacter pylori. Human PSC-derived lung organoids have shed light on the various aspects of SARS-CoV-2 infection including cell tropism, how genetic profile influences susceptibility to virus infection, mechanisms of viral entry and replication in the host, host cell responses and cellular and metabolic changes after infection.^{19, 20}

Pathological mutations could be introduced into healthy organoids by CRISPR-Cas9 or other gene editing methods to study the effect of these mutations in cancer development. Colorectal adenoma-carcinoma can be recreated by introducing driver mutations into the genes APC, KRAS, TP53, SMAD4 and PIK3CA of healthy organoids, whereas pancreatic ductal adenocarcinoma can be developed by mutating the driver genes KRAS, CDKN2A, SMAD4 and TP53 in healthy organoids.^{2, 19}

Developmental biology

Organoids can be used to mimic morphogenetic events to provide detailed insights into the development and functioning of tissues and organs. Organoids made up of ESCs from mice and humans self-organize and gastrulate to form embryos in culture, providing novel insights into early mammalian development. Also, organoids offer model systems to study the mechanisms of self-organization under different conditions, increasing our understanding of self-organization and controlling of complex multicellular behaviors in vitro.⁷

In addition to ESCs, iPSCs and PSCs from specific tissues are also crucial to study the developmental biology of particular tissues and organs. Organoids recreating the different parts of the female reproductive system greatly improve our understanding of the reproductive organs and open avenues for personalized care for females with complications. In 2019, skin organoids with layers of skin containing pigmented hair follicles were successfully established, which could play an important role in skin regenerative therapies.^{2, 21-23}

Personalized medicine

Organoids serve as the best models to study person-to-person variability in the pathogenesis of diseases, individual responses to therapies and contribute to treatments tailored to individual needs.¹ Organoids offer the potential to study various shared or unique mechanisms in the development and functioning of organs, disease progression or infections/allergies that might differ from the mechanisms involved in immortalized cell lines or animal models.⁷ Screening of the organoid biobank showed correlations between genetic profiles and drug responses for colorectal, breast, prostate and liver cancers. The clinical decision to treat patients with the neoadjuvant chemotherapy regimen FOLFOX, led by organoid-based data, ameliorated liver cancer in a patient. Consistently, a study of patient-derived organoids provided 88% accuracy in predicting response and 100% accuracy in predicting no-response to drug treatments of patients with metastatic gastrointestinal cancer.^{6, 21, 24}

Considering the enormous potential of organoids, current research extensively focuses on improving organoids for personalized medicine for various diseases and developmental conditions.^{24, 25}

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References

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