Engineered organoids for biomedical applications

Abstract

As miniaturized and simplified stem cell-derived 3D organ-like structures, organoids are rapidly emerging as powerful tools for biomedical applications. With their potential for personalized therapeutic interventions and high-throughput drug screening, organoids have gained significant attention recently. In this review, we discuss the latest developments in engineering organoids and using materials engineering, biochemical modifications, and advanced manufacturing technologies to improve organoid culture and replicate vital anatomical structures and functions of human tissues. We then explore the diverse biomedical applications of organoids, including drug development and disease modeling, and highlight the tools and analytical techniques used to investigate organoids and their microenvironments. We also examine the latest clinical trials and patents related to organoids that show promise for future clinical translation. Finally, we discuss the challenges and future perspectives of using organoids to advance biomedical research and potentially transform personalized medicine.

Keywords: Disease modeling; Organoids; Regenerative medicine; Spheroids; Stem cell; Therapies.

Fig 1.

Schematic diagram summarizing the generation and potential applications of organoid models in preclinical research and precision medicine. Organoids mimicking multiple essential patient tissues such as the brain, lung, and liver could be developed. Diverse sources of cells could be harvested from humans and cultured to form organoids. Organoid development could be facilitated by advanced engineering approaches such as induction of self-assembly, hydrogel scaffold formation, microfluidics fabrication, and additive manufacturing. These approaches can revolutionize current research and novel therapeutic strategies in regenerative medicine, drug development, and developmental biology.

Fig. 2.. Examples of stem-cell-derived organoids and organoid differentiation.

Ai. Human crypt ISCs could differentiate into the different cell types of the intestine (top: Alkaline phosphatase for mature enterocytes, middle: Periodic acid–Schiff staining (PAS) for goblet cells, bottom: Synaptophysin for enteroendocrine cells). Aii-iii Immunofluorescence stains using Mucin2 (red) for goblet cells and Chromogranin A (green) for enteroendocrine cells (red arrow and inset) and Lysozyme (Lysz; green) for Paneth cells. Reproduced from [42] with permission from Elsevier 2011. B. Confocal laser scanning microscopy of terminal ductal-lobular unit resembling structures at different passages showing Vimentin (red), E-cadherin(green), integrin-α6(red), and DAPI: blue, Scale bar: 100 μm). Reproduced from [44] with permission from The Company of Biologists 2015. C. Ci, (A) Representative FACS gating strategy for the analysis of ductal cells in the salivary gland. Left panel shows the exclusion of lineage marker-expressing cells. Right panel depicts the distribution of EpCAM^high, EpCAM^med, and EpCAM^neg cells in dissociated adult mouse salivary gland. FSC, forward scatter. (B) Sphere-forming efficiency of EpCAM^high, EpCAM^med, and EpCAM^neg populations (**p < 0.005). Data are expressed as the mean ± SEM of three independent experiments. Cii, Differential interference contrast image of a growing minigland until 9 days (d) of culture. Ciii, Representative example of a salisphere and a minigland originating from single EpCAM^high in 9-day-old culture. Civ, Toluidine blue staining shows uniform lumen formation throughout minigland (arrows). Scale bars represent 100 μm (ii, iii) and 10 μm (iv). Reproduced from [45] with permission from Cell Press, Elsevier 2016.

Fig. 3.. Approaches to improving organoid development.

A. Schematic representation of organoid culture in ECM matrices to recapitulate native tissue. B. Effect of the degradation rate of scaffolds on the hPSCs-derived lung organoid maturation into airway structures as cultured on poly (lactide-co-glycolide) (PLG), PEG, and PCL scaffolds and transplanted into the epididymal fat pad of nonobese-diabetic-severe-combined-immunodeficient (NOD-SCID) IL2Rgnull (NSG) mice. The Black arrows show pseudostratified epithelium duplicating native airway epithelium (degradable scaffolds). Histological examination of organoids seeded on PEG and PCL scaffolds demonstrated intact scaffold (orange arrows) and clusters of cells (no-organized epithelium, blue arrows). Scale bar = 200 μm. Figure adapted with the permission of Elsevier from [84]. C. Schematic representation of 3D bioprinting approach to deposit bioink containing organoids into a specific architecture. D. Bioprinted epithelial organoids and tumoroid in Geltrex, rat-tail collagen (rtCOL), rat mammary ECM (rtMECM), and human mammary ECM (huMECM) hydrogels. Cellular arrays with ~50 cells at a time at 500 μm intervals were printed to recapitulate the architectural features of tumor organoids. Fluorescence image of (i) mammary epithelial cell line (MCF-12A), (ii) human breast cancer cell line (MCF-7), and (iii) MB-MDA-468 printed in a linear pattern within different ECM hydrogels. Large organoids of MCF-12A cells with duct-like luminal morphologies (exceeding 3mm in length) were generated in all ECM substrates. Meanwhile, MCF-7 cells generated grape-like morphology in Geltrex and maintained a spherical shape in other ECM substrates after 14 days of culture. Also, MB-MDA-468 cells formed small clusters in Geltrex and single tumoroid in rtCOL, and rtMECM. In comparison, cells failed to grow appropriately in huMECM. Scale bars 200 μm. Figure reproduced from [120] with permission from Elsivier.

Fig. 4.. Engineering microfluidics for organoid development.

Ai. Schematic representation of brain organoids-on-a-chip. Brain organoids are formed from hiPSCs. Aii, Neurogenesis and tissue morphology of the organoids showing TUJ1 and SOX2 markers in 33-day organoids (indicated by arrows) on a chip. Reproduced from [149] with permission from The Royal Society of Chemistry 2018. B. The brain organoid-on-a-chip model proposed by Wang et al. (Reproduced from [134] by Royal Society of Chemistry 2018) to study neurodevelopment problems caused by prenatal nicotine exposure.

Fig. 5.. Engineering and characterization of Lgr5-EGFP-tdTomato colon organoids.

A. Lgr5⁺ cell depletion in colon tissue after radiation exposure. The black and white dotted lines indicate irradiated areas. Scale bar: 200 μm. B. Strategy for colon organoid transplantation in the mouse model of radiation proctitis. C. Evaluation of colon organoids in vitro treated with 4-hydroxytamoxifen (4-OHT) before and after transplantation. Scale bar: 200 μm. D. Lgr5+ cell expression in colon organoid-engrafted tissue at 1 week and 8 months after transplantation. Scale bars: 200 μm (organoid engrafted tissue), 500 μm (HandE-stained tissue). E. Lgr5, PTK7, Chg A, Muc2, and lysozyme mRNAs were expressed in colon organoids grown in medium with or without Wnt3a for 3 days. F. Representative images of tissues engrafted by colon organoids cultured in medium with and without Wnt3a (bright field and HandE, GFP/Chg A, villin, and Ki67 staining). Scale bars: 100 μm (HandE, EGFP/Chg A, villin, Ki67 staining). G. Areas of proctitis injury 2 weeks after transplantation of colon organoids cultured in medium with Wnt3a or without Wnt3a. Reproduced from [139] with permission from Elsevier 2021.

Fig. 6.. Organoid system integrated with biosensors and high throughput functionality.

A. Schematic representation of integrated multiorgan-on-a-chip platform consisting of micro bioreactors, breadboard, reservoir, bubble trap, physical sensors, and electrochemical biosensors for automated sensing of various biomarkers, data processing, and optimized cell culture condition maintenance. Reproduced from [246] with permission from the National Academy of Sciences 2017. B. Organ-on-an-electronic-chip for electrical interrogations of human electrogenic spheroids. Bi, Cardiac spheroids encapsulated a 3D self-rolled biosensor array (3D-SR-BA), allowing electrical measurements in 3D. Bii, A confocal microscopy 3D image of an encapsulated cardiac spheroid labeled with Ca²⁺ indicator dye (green fluorescence) in a 3D-SR-BA. Scale bar = 50 μm. Bii_i, 2D map of the microelectrodes in the biosensor array. Biv, Representative field potential (FP) traces measured from the channels of the array. Reproduced from [252] with permission from the American Association for the Advancement of Science 2019.