Tahani Baakdhah provides an overview of organoids and gives us an insight into her own work on bioreactors for retinal organoids.
A new era of personalized medicine is on the horizon thanks to modern technology, which allows scientists to use patients’ stem cells to make different three-dimensional (3D) mini organs called ‘organoids’.
Inside these organoids, cells assume organization similar to that found in the original organ they represent. This self-organization is the result of a multitude of instructive cues originating from the extracellular matrix and the surrounding culture media, as well as signals from other cell types within the organoid [101, 102, 1]. Scientists have successfully created many organoids including brain, lungs, kidney, liver, intestine, heart, thyroid, thymus, salivary gland, prostate, cancer and more [2, 103, 3, 5, 6, 7, 8, 9]. These organoids are created from stem cells, which can be derived from adult tissue or pluripotent stem cells.
Healthy organoid models offer unique perspectives on studying embryonic organ development by allowing researchers to observe the interactions between cells in the developing organ as they happen. In contrast, disease model organoids are used to clarify organ-specific molecular and cellular mechanisms of different disease pathologies, such as brain organoids in the case of schizophrenia, autism and the Zika virus among others.
Another important use of such disease models is testing the pharmacological effects of certain drugs on tissues derived from patient cells; this facilitates the development of patient-specific platforms for drug-testing and toxicology studies, allowing for the optimization of treatments for diseases such as cystic fibrosis and diabetes, as well as microbial infections. It also enables the identification of drug-sensitivity biomarkers, bringing the promise of personalized medicine closer to fulfillment [10, 11, 12].
Organoid models play a versatile role in a wide range of regenerative medicine applications. In 2018, Nie showed that liver endothelial cells derived from liver organoids were able to replace lost cells leading to improved mouse survival after transplantation into an acute liver failure model .
In another example of their versatility, a drug called forskolin was used to search for treatable cystic fibrosis mutation in affected patients . This disease is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an anion channel expressed on the surface of epithelial cells. CFTR is responsible for fluid and electrolyte homeostasis.
Mutations in CFTR result in accumulation of viscous mucus in the respiratory and gastrointestinal tracts due to defective epithelial ion transport. Forskolin, which activates CFTR through increasing intracellular cyclic AMP levels, induces rapid swelling of intestinal organoids derived from healthy tissue. By contrast, swelling is diminished or absent in intestinal organoids derived from cystic fibrosis patients, identifying patients who would benefit from the drug. When the CFTR mutation was restored using CRISPR, the organoids showed swelling in response to forskolin.
Organoids can also be used to assess the cytotoxicity of patient’s own immune cells. Co-culturing donor-derived T-cells with tumor-derived organoids is used to predict the cytotoxicity of these T-cells towards patient-derived tumor organoids. T-cells that demonstrate in vitro cytotoxicity can subsequently be expanded in vitro and administrated to the patient to induce a stronger and more effective anti-tumor immune response .
Utilizing retinal organoids in my research
As a researcher working on the retina, organoids are particularly interesting to me. In our lab, we isolate retinal stem cells from mouse and human eyes and increase their number using stirring bioreactor cultures, before differentiating them into different retinal cell types for transplantation.
In my recently published paper , I showed that cells cultured on cell microcarriers in hypoxic conditions expanded 10x more compared to our regular static culture. Microcarriers are small spherical beads (diameters between 90 — 300Î¼m) made of different substrates and suspended in growth medium; different coatings provide adherent surfaces for the cells to attach to, grow and expand . The static culture method comes with a number of disadvantages and limitations including the inability to precisely control most of the culture conditions — such as oxygen, pH and temperature — and the inability to monitor the interaction between different retinal cell types owing to the nature of 2D culture. There is also a lack of proper cellular organization, and mechanical and chemical signaling dynamics, in comparison to the 3D environment found in vivo.
The retina is the inner most layer of the eye that absorbs then translates light into electrochemical signals, which the brain’s visual cortex can understand. There are different types of retinal cells that make this process successful — retinal pigment epithelial cells, rod and cone photoreceptors, bipolar cells, ganglion cells, horizontal and amacrine cells — and degenerative eye diseases lead to the loss of one or more of these retinal cell types resulting in partial or complete loss of vision.
In current retinal disease models and clinical trials, the lost cells are replaced by cells derived from adult or pluripotent stem cells grown in 2D cultures. Most differentiation protocols involve growing retinal organoids from pluripotent stem cells, such as embryonic stem cells or induced pluripotent stem cells [15, 16].
Pluripotent stem cells are grown on Matrigel matrix in 2D culture to produce embryoid bodies (EB), 3D aggregations of cells that will later form the three embryonic layers: ectoderm, mesoderm and endoderm. EB bodies are then harvested and supplemented with neural and retinal induction media. When the optic vesicle become visible, usually by day 7 of EB differentiation, they get dissected and maintained in a culture flask or in a suspension stirring bioreactor. Then, they will be maintained in retinal differentiation media until mature differentiation is achieved. Bioreactors control culture conditions and support better cell survival within retinal stem cells spheres and, by optimization bioreactor culture protocols, many hope that we will be able to grow mature retinal organoids in the near future .
The hurdles for retinal organoids
The continued development and utility of lab-produced retinal organoids has the potential to effectively alleviate a lot of problems inherent to the conventional techniques being used. Retinal organoid systems display cell—cell and cell—matrix interactions, cellular heterogeneity, and physiological responses reflective of human biology, thus, are much more accurate models for studying retinal pathology as well as normal physiology [17, 18, 19]. The currently used development protocols, unfortunately, remain a major hurdle standing in the way of fully harnessing the application of organoids as, using these protocols, organoids will pass through many developmental stages but are never able to progress to the full mature retina [20, 21, 15, 19] .
Advance RNA-seq technologies and transcriptome analysis identified divergent regulatory dynamics between developing retinas in vivo and in organoids. Organoid-derived retinal cells displayed temporal dysregulation of specific signaling pathways and reduced expression of genes involved in photoreceptor function and survival, which can be contributed to lack of natural instructive signals available in vivo. Brooks  showed that adding docosahexaenoic acid or FGF1, the two components missing in organoid cultures, facilitated photoreceptor differentiation and maturation. The advancement of new protocols is of paramount importance and is currently under way in different laboratories around the world.
Despite their potential, organoids are a relatively new technology in the field of regenerative medicine, and in an Instagram poll I posted in late October 2019, only 38% of responders (majority of my followers are scientists/grad students) said that they had heard the term ‘retinal organoid’, compared with 62% who said they had never heard about it before.
As in any other new technology, there are still many issues that need to be addressed: the functional maturation of differentiated cells, the lack of certain essential cell types (e.g. microglia), the lack of optimal physiological interplay between the various retinal cell types, as well as the characteristic lack of vascularization. However, researchers remain enthusiastic and are trying a host of methods to overcome these [19, 4].
In an effort to address one of these problems, researchers are currently trying to improve the perfusion of organoids by incorporating innovative vascularization solutions. Some of the methods include creating microphysiological systems — specifically organ-on-a-chip platforms that will allow embedding vasculatures into organoids microenvironment — and utilizing stirring suspension bioreactors to enhance nutrient and oxygen distribution/perfusion and diffusion within the organoid .
Organoids as an emerging system are showing a lot of promise; promise not only limited to deepening our understanding of organ development and pathology. By offering a practical platform to develop personalized organoids from patients’ own cells, they will open the doors wider than ever before for drug testing, gene editing and tissue transplantation, paving the way for achieving more tangible results in the field of regenerative medicine.
Have any additional questions about this story? Ask us in the comments, below.
Find out more in these top picks from the Editor:
- Panel discussion on organoids
- Organoids: ask the experts
- The organoid revolution: an interview with Amritha Jaishankar
- Takebe T, Wells J. Organoids by design. Science, 364, 956—959 (2019).
- Nie Y, Zheng Y, 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).
- StrÃ¸nen E, Toebes M, Kelderman S, et al. Targeting of cancer neoantigens with donor-derived T cell receptor repertoires. Science, 352, 6291, 1337—1341 (2016).
- Achberger K, Probst C, Haderspeck J, et al. Merging organoid and organ-on-a-chip technology to generate complex multi-layer tissue models in a human retina-on-a-chip platform. ELife, 8:e46188 (2019).
- Nugraha B, Buono M, Boehmer L, Hoerstrup S, Emmert M. Human cardiac organoids for disease modeling. Clin. Pharm. Therap. 105, 79—85 (2018).
- Puca L, Bareja R, Prandi D, et al. Patient derived organoids to model rare prostate cancer phenotypes. Nat Commun. 9, 2404, (2018).
- Saito Y, Onishi N, Takami H, et al. Development of a functional thyroid model based on an organoid culture system. Biochem. Biophys. Res. 497, 2, 783—789 (2018).
- Tajima A, Pradhan I, Geng X, Trucco M, Fan Y. Construction of thymus organoids from decellularized thymus scaffolds in: Methods in molecular biology. Turksen K (ed). Volume 1576 (2016)
- Wallach T, Bayrer J. Intestinal organoids: new frontiers in the study of intestinal disease and physiology. J. Pediatr. Gastroenterolo. Nutr. 64, 2, 180—185 (2017).
- Dekkers J, Berkers G, Kruisselbrink E, et al. Characterizing responses to CFTR-modulating drugs using rectal organoids derived from subjects with cystic fibrosis. Sci. Transl. Med. 8, 344ra84 (2016).
- Huch M, Knoblich J, Lutolf M, Martinez-Arias A. The hope and the hype of organoid research. Development, 144, 6, 938—941 (2017).
- Xu H, Jiao Y, Qin S, Zhao W, Chu Q, Wu K. Organoid technology in disease modelling, drug development, personalized treatment and regeneration medicine. Exp. Hematol. Oncol. 7, 30 (2018)
- Baakdhah T, van der Kooy D. Expansion of retinal stem cells and their progeny using cell microcarriers in a bioreactor. Biotechnol. Prog. 35, 3 (2019)
- Hewitt C, Lee K, Nienow A, Thomas R, Smith M, Thomas C. Expansion of human mesenchymal stem cells on microcarriers. Biotechnol. Lett. 33, 11, 2325—2335 (2011)
- Fligor C, Langer K, Sridhar A, et al. Three-dimensional retinal organoids facilitate the investigation of retinal ganglion cell development, organization and neurite outgrowth from human pluripotent stem cells. Sci. Rep. 8, 1 (2018).
- VÃ¶lkner M, Kurth T, Karl M. The mouse retinal organoid trisection recipe: efficient generation of 3D retinal tissue from mouse embryonic stem cells in: Methods in molecular biology. Weber B, Langmann T (eds). Volume 1834, 119—141 (2019).
- Aparicio J, Shayler D, Cobrinik D. Retinal organoids: an emerging technology for retinal disease research and therapy in: Cellular therapies for retinal disease. Schwartz S, Nagiel A, Lanza R (eds). 117—138 (2017).
- Cora V, Haderspeck J, Antkowiak L, et al. A cleared view on retinal organoids. Cells, 8, 5, 391 (2019).
- Llonch, S., Carido, M., & Ader, M. Organoid technology for retinal repair. Dev. Biol. 433, 132—143 (2018).
- Brooks M, Chen H, Kelley R, et al. (2019). Improved retinal organoid differentiation by modulating signaling pathways revealed by comparative transcriptome analyses with development in vivo. Stem Cell Reports. 13, 5, 891-905 (2019).
- Capowski E, Samimi K, Mayerl S, et al. Reproducibility and staging of 3D human retinal organoids across multiple pluripotent stem cell lines. Development, 146:dev171686 (2019).
101) Zeng Y, Win-Shwe T, Ito T, Sone H. Organoid – an overview.
ScienceDirect Topics (2018)
(Accessed 10 November 2019)
102) Barbuzano J, Organoids: a new window into disease, development and discovery,
Harvard stem cell institute news, 7 November (2017).
(Accessed 10 November 2019)
103) Kwon D, Organoids don’t accurately model human brain development.
The scientist magazine, 23 October (2019)
(Accessed 10 November 2019)