Over the past few decades, scientists put great efforts into trying to ‘crack’ the great mystery of cell communication and tissue assembly.

Although in vitro 2D cell cultures have been widely used and serve as an important research tool, the lack of tissue architecture and the complexity of such models do not fully reflect the true biological processes in vivo.

The way to tackle these obstacles took shape in three-dimensional (3D) models referred to as ‘Organoids’. These 3D models aim to recapitulate the cellular heterogeneity, structure and functions of the primary tissues.

What is an Organoid?

The term Organoid refers to cells growing in a 3D environment that form mini-clusters and differentiate into functional cell types. These clusters recapitulate the function of an organ in vivo, and thereby are called by some – ‘mini-organs’.

Organoids are usually derived from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), neonatal stem cells or adult stem cells (ASCs).(1)

Traveling in time…

Back in 1907, Henry Van Peters Wilson described the first attempt of in vitro organism regeneration, by demonstrating that dissociated sponge cells can self-organize to regenerate a whole organism.(2)

Since then, and over the years, many research groups have tried to conquer that mountain, starting with dissociating and re-aggregating amphibian pronephros and chick embryos and gaining more understanding of the dissociation and differentiation processes. (1)

An important milestone in stem cell research occurred in 1981 when pluripotent stem cells (PSCs) were first isolated from mouse embryos(3). But only in 1998 embryonic stem cells, derived from human blastocysts were isolated and cultured for the first time. Following that was the establishment of iPSCs by the reprogramming of mouse and human fibroblasts, and ongoing improvement of cell culture conditions by simulating the in vivo microenvironment. (1)

The breakthrough came with the discovery that single leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5) expressing adult intestinal stem cells can form 3D intestinal organoids in Matrigel that self-organize and differentiate into crypt-villus structures. This was the first report on establishing 3D organoid culture derived from a single ASC (4), which set the scene for many subsequent organoid works in other systems, including mesendoderm[RM1] [BO2]  (e.g., stomach, liver, pancreas, lung, and kidney) and neuroectoderm (brain and retina) using either ASCs or PSCs. (Figure 1) (1)

Timeline for the development of organoid cultures

Figure 1: A Timeline for the development of organoid cultures. A summary of key landmark studies and breakthroughs leading to the establishment of various organoid technologies. (1)

Organoid Applications – An abundance of possibilities

Drug discovery

The progress towards solving the riddle of mimicking tissue and organ function has opened a worldwide of new possibilities in a wide range of therapeutic applications.

Reducing the number of animals required for cytotoxicity studies, replacing animals with human Organoids and thus allowing better biological relevance of drug functionality or toxicity for humans – those are just two of the multiple advantages of using organoids for drug discovery and cytotoxicity trials.

Another hopeful aplication for drug discovery lies in   human liver organoids as they capture the unique metabolic profile of the human liver – A critical organ involved in drug metabolism.

Regenerative Medicine

The shortage of matched donor tissues and complications of life-long immunosuppression are major challenges of organ transplantation. The recent organoid technology characterized by high expansion capacity and genetically stable features suggests that primary patient derived organoids (PDOs) could potentially be explored as alternative treatment strategies to organ transplantation. (1)

It has already been demonstrated that mouse colonic organoids, expanded in vitro from a single adult Lgr5+ stem cell, could indeed be engrafted into damaged mouse colon and form functional crypt units.(5) Similar results were observed using fetal progenitor-derived small intestinal organoids.(6)

Besides intestinal organoids, mouse adult liver organoids have also been shown to rescue liver failure and restore the hepatic functions in several trials. (7)(8)

Organoid tissue repair approaches have also been suggested as a means of correcting genetic defects. Here, genome-editing technology (e.g. CRISPR/Cas9) could be used to correct gene-related organ issues on patient iPSCs ex vivo. (9) The gene-corrected cells would then be induced to form organoid tissue for subsequent transplantation.

Disease Modeling

Patient-derived iPSCs can also be targeted for building organoid models of tissue diseases, genetic disorders, infectious diseases, degenerative diseases and cancer. (10-13) These organoids can then be used to create disease-specific research models to uncover a better understanding of disease pathologies or as an additional approach to evaluate potential treatments and therapies in vitro during the drug discovery process.

In addition to all the proposed uses that have been mentioned there are many more therapeutic applications explored, using organoid models, including Biobanking and Precision Medicine, Developmental Biology and more. (Figure 2)

Key Organoid applications

Figure 2: Key Organoid applications. The variety of applications span over multiple research and biomedical different areas. (1)

Deciding which culture conditions to use

Different kinds of organoids are grown in different culture conditions (Figure 3). The most common matrix is solid Extra-cellular Matrix (ECMs) that support cell growth and to which cells can adhere (e.g Matrigel or other animal-derived hydrogels). The advantage of these natural matrices is the presence of ECM components and growth factors, which makes cell growth and differentiation very efficient. However, this complexity and the variability in composition make it more difficult to control the culture environment and achieve consistent and stable outcomes.

For these reasons, chemically defined hydrogels have been recently introduced as a substitute for the less well-defined natural matrices. These hydrogels allow the biochemistry and mechanics of the culture environment to be controlled but are less bioactive and need to be customized to match the specific requirements of different organoids. (14)

Another strategy is culturing 3D cell aggregates in suspension. This way of culturing organoids has been used to grow optic cup, cerebral, cerebellar and hippocampal organoids.

The third platform for growing organoids is an air-liquid interface method in which cells are cultured in the form of a pellet on a thin, microporous membrane, with cell culture medium only on the basal side of the membrane. One of the uses of this method was generating kidney organoids. (14) (Figure 3)

Physical features of the culture environment of organoids

Figure 3: The main types of culture conditions are shown, together with examples of organoids that rely on them. (14)

At this point in time, choosing a specific protocol for deriving a specific organoid is largely driven by empirical considerations. (14)

Starting cell type and initial culture conditions

The steps of cell assembly towards forming an organoid depend crucially on the initial culture conditions.

When organoids are derived from a single cell type (e.g. small intestine organoids), self-organization of the cell population necessarily involves symmetry-breaking and subsequent patterning to generate spatially separated domains of different cell types. (Figure 4). In general, when an organoid derivation protocol starts from a single cell, an early step of cell expansion is required before self-organization can occur.

On the opposite side, some protocols involve the co-culturing of cell types that have been separately pre-differentiated (e.g. PSC-derived liver organoids). In these protocols, most of the disparate cell identities are already established, therefore self-organization primarily involves cell sorting and subsequent architectural rearrangements.(Figure 4) (14) The initial conditions of the cell population will also affect the range of applicability of an organoid as a biological model system.

Figure 4: Organoids can be formed from cell populations with different starting conditions. Examples of an organoid protocol starting from a single cell (intestinal organoids), a homogeneous multicellular ensemble (optic cup organoids) and a heterogeneous co-culture of different cell types (liver organoids) are shown. (14)

Summary

Organoids hold a promise for the future for a range of biological and biomedical implementations – from drug discovery to developmental biology. The ability to study human development and disease, without the constraints of invasive procedures to access human or animal tissues is an exciting prospect and holds many advantages.  However, translating organoid technology into real-life preclinical and clinical applications is considerably more challenging.

Although the ability of stem cells to self –organize under 3D conditions has been revealed and groundbreaking studies have been published, there are still many limitations regarding this issue. Major challenges such as regulating self-organization to generate organoids that develop to physiologically relevant shapes and sizes, prolonging organoid lifespan to create mature, functional tissues that reach homeostasis and recapitulating multi-factorial pathologies by incorporating additional key tissue compartments of native organs (such as the vasculature or immune system) – these challenges still need to be overcome using a multidisciplinary approach, and lessons from the field. (14)

An important factor in organoid formation is the quality and capacity of its foundation cells. Cell health is a key factor for any successful downstream application. Choosing and using a reliable, high quality culturing media is an essential step in the formation of a well-functioning, complex, intact organoid.

 

References

  1. Corrò C, Novellasdemunt L and Li V.S.W; A brief history of organoids.  Stem Cell and Cancer Biology Laboratory, The Francis Crick Institute, London United Kingdom; Am J Physiol Cell Physiol 319: C151–C165, 2020.
  2. Wilson HV. A new method by which sponges may be artificially reared. Science 25: 912–915, 1907.
  3. Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci USA 78: 7634–7638, 1981.
  4. Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, van Es JH, Abo A, Kujala P, Peters PJ, Clevers H. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459: 262–265, 2009.
  5. Yui S, Nakamura T, Sato T, Nemoto Y, Mizutani T, Zheng X, Ichinose S, Nagaishi T, Okamoto R, Tsuchiya K, Clevers H, Watanabe M. Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5+ stem cell. Nat Med 18: 618–623, 2012.
  6. Fordham RPYS, Yui S, Hannan NR, Soendergaard C, Madgwick A, Schweiger PJ, Nielsen OH, Vallier L, Pedersen RA, Nakamura T, Watanabe M, Jensen KB. Transplantation of expanded fetal intestinal progenitors contributes to colon regeneration after injury. Cell Stem Cell 13: 734–744, 2013.
  7. Nie YZZY, Zheng YW, Ogawa M, Miyagi E, Taniguchi H. Human liver organoids generated with single donor-derived multiple cells rescue mice from acute liver failure. Stem Cell Res Ther 9: 5, 2018.
  8. Hu H, Gehart H, Artegiani B, LÖpez-Iglesias C, Dekkers F, Basak O, van Es J, Chuva de Sousa Lopes SM, Begthel H, Korving J, van den Born M, Zou C, Quirk C, Chiriboga L, Rice CM, Ma S, Rios A, Peters PJ, de Jong YP, Clevers H. Long-term expansion of functional mouse and human hepatocytes as 3D organoids. Cell 175: 1591–1606, 2018.
  9. Wen-Li D, Mei-Ling G, Xin-Lan L, Ji-Neng L, Huan Z, Kai-Wen H, Xi-Xi X, Ling-Yun L,Yu-Chen C, Yan-Ping L, Deng P, Tian X and Zi-Bing J; Gene Correction Reverses Ciliopathy and Photoreceptor Loss in iPSC-Derived Retinal Organoids from Retinitis Pigmentosa Patients. Stem Cell Reports 10: 1267–1281, 2018.
  10. Kunishige O, Masako O, Ochiai K, Orihashi S, Yoshitaka H. Genetic reconstitution of tumorigenesis in primary intestinal cells. Proc Natl Acad Sci. 110: 11127–11132, 2013.
  11. Dekkers JF, Wiegerinck CL, De Jonge HR, Bronsveld I, Janssens HM,  De Winter-de Groot KM, Brandsma AM, De Jong NWM, Bijvelds MLC,  ScholteBJ, Nieuwenhuis EES, Van den Brink S, Clevers H, Van der Ent CK, Middendorp S & Beekman JM. A functional CFTR assay using primary cystic fibrosis intestinal organoids. Nat. Med. 19: 939–945, 2013.
  12. Bigorgne, A. E. et al. TTC7A mutations disrupt intestinal epithelial apicobasal polarity. J. Clin. Invest. 124: 328–337, 2014.
  13. Maliszewska-Olejniczak K, Brodaczewska K. K, Bielecka Z. F. & Czarnecka A. M. Three-Dimensional Cell Culture Model Utilization in Renal Carcinoma; Methods in molecular biology. 1817: 47-66, 2018
  14. Rossi G, Manfrin A and Lutolf MP; Progress and potential in organoid research. Nature Reviews Genetics 19: 671-687, 2018