Organoids are simple tissue-engineered cell-based in vitro models that recapitulate many aspects of the complex structure and function of the corresponding in vivo tissue. They can be dissected and interrogated for fundamental mechanistic studies on development, regeneration and repair in human tissues, and can also be used in diagnostics, disease modelling, drug discovery and personalized medicine. Organoids are derived from either pluripotent or tissue-resident stem (embryonic or adult) or progenitor or differentiated cells from healthy or diseased tissues, such as tumours. To date, numerous organoid engineering strategies that support organoid culture and growth, proliferation, differentiation and maturation have been reported. This Primer highlights the rationale underlying the selection and development of these materials and methods to control the cellular/tissue niche; and therefore, the structure and function of the engineered organoid. We also discuss key considerations for generating robust organoids, such as those related to cell isolation and seeding, matrix and soluble factor selection, physical cues and integration. The general standards for data quality, reproducibility and deposition within the organoid community are also outlined. Lastly, we conclude by elaborating on the limitations of organoids in different applications, and the key priorities in organoid engineering for the coming years.

Culturing cells at microscales allows control over microenvironmental cues, such as cell–cell and cell–matrix interactions; the potential to scale experiments; the use of small culture volumes; and the ability to integrate with microsystem technologies for on-chip experimentation. Microfluidic perfusion culture in particular allows controlled delivery and removal of soluble biochemical molecules in the extracellular microenvironment, and controlled application of mechanical forces exerted via fluid flow. There are many challenges to designing and operating a robust microfluidic perfusion culture system for routine culture of adherent mammalian cells. The current literature on microfluidic perfusion culture treats microfluidic design, device fabrication, cell culture, and micro-assays independently. Here we systematically present and discuss important design considerations in the context of the entire microfluidic perfusion culture system. These design considerations include the choice of materials, culture configurations, microfluidic network fabrication and micro-assays. We also present technical issues such as sterilization; seeding cells in both 2D and 3D configurations; and operating the system under optimized mass transport and shear stress conditions, free of air-bubbles. The integrative and systematic treatment of the microfluidic system design and fabrication, cell culture, and micro-assays provides novices with an effective starting point to build and operate a robust microfludic perfusion culture system for various applications.

We have developed a microfluidic 3D hepatocyte chip (3D HepaTox Chip) for in vitro drug toxicity testing to predict in vivo drug hepatotoxicity. The 3D HepaTox Chip is based on multiplexed microfluidic channels where a 3D microenvironment is engineered in each channel to maintain the hepatocytes' synthetic and metabolic functions. The multiplexed channels allow for simultaneous administration of multiple drug doses to functional primary hepatocytes while an incorporated concentration gradient generator enables the in vitro dose-dependent drug responses to predict in vivo hepatotoxicity. The IC₅₀ values of 5 model drugs derived from the dose-dependent on-chip testing correlate well with the reported in vivo LD₅₀ values. The 3D HepaTox Chip can be integrated with on-chip sensors and actuators as the next generation cell-based on-chip drug testing platform.

Mammalian cells cultured on 2D surfaces in microfluidic channels are increasingly used in drug development and biological research applications. These systems would have more biological or clinical relevance if the cells exhibit 3D phenotypes similar to the cells in vivo. We have developed a microfluidic channel based system that allows cells to be perfusion-cultured in 3D by supporting them with adequate 3D cell–cell and cell–matrix interactions. The maximal cell–cell interaction was achieved by perfusion-seeding cells through an array of micropillars; and 3D cell–matrix interactions were achieved by a polyelectrolyte complex coacervation process to form a thin layer of matrix conforming to the 3D cell shapes. Carcinoma cell lines (HepG2, MCF7), primary differentiated (hepatocytes) and primary progenitor cells (bone marrow mesenchymal stem cells) were perfusion-cultured for 72 hours to 1 week in the microfluidic channel, which preserved their 3D cyto-architecture and cell-specific functions or differentiation competence. This transparent 3D microfluidic channel-based cell culture system also allows direct optical monitoring of cellular events for a wide range of applications.

We have developed a multi-channel 3D microfluidic cell culture system (multi-channel 3D-µFCCS) with compartmentalized microenvironments for potential application in human drug screening. To this end, the multi-channel 3D-µFCCS was designed for culturing different 3D cellular aggregates simultaneously to mimic multiple organs in the body. Four human cell types (C3A, A549, HK-2 and HPA) were chosen to represent the liver, lung, kidney and the adipose tissue, respectively. Cellular functions were optimized by supplementing the common medium with growth factors. However, TGF-β1 was found to enhance A549 functions but inhibit C3A functions. Therefore, TGF-β1 was specifically controlled-released inside the A549 compartment by means of gelatin microspheres mixed with cells, thus creating a cell-specific microenvironment. The function of A549 cells was enhanced while the functions of C3A, HK-2 and HPA cells were uncompromised, demonstrating the limited cross-talk between cell culture compartments similar to the in vivo situation. Such a multi-channel 3D-µFCCS could be potentially used to supplement or even replace animal models in drug screening.

Microtubules facilitate the maturation of phagosomes by favoring their interactions with endocytic compartments. Here, we show that phagosomes move within cells along tracks of several microns centrifugally and centripetally in a pH- and microtubuledependent manner. Phagosome movement was reconstituted in vitro and required energy, cytosol and membrane proteins of this organelle. The activity or presence of these phagosome proteins was regulated as the organelle matured, with “late” phagosomes moving threefold more frequently than “early” ones. The majority of moving phagosomes were minus-end directed; the remainder moved towards microtubule plus-ends and a small subset moved bi-directionally. Minus-end movement showed pharmacological characteristics expected for dyneins, was inhibited by immunodepletion of cytoplasmic dynein and could be restored by addition of cytoplasmic dynein. Plus-end movement displayed pharmacological properties of kinesin, was inhibited partially by immunodepletion of kinesin and fully by addition of an anti-kinesin IgG. Immunodepletion of dynactin, a dynein-activating complex, inhibited only minus-end directed motility. Evidence is provided for a dynactin-associated kinase required for dyneinmediated vesicle transport. Movement in both directions was inhibited by peptide fragments from kinectin (a putative kinesin membrane receptor), derived from the region to which a motility-blocking antibody binds. Polypeptide subunits from these microtubule-based motility factors were detected on phagosomes by immunoblotting or immunoelectron microscopy. This is the first study using a single in vitro system that describes the roles played by kinesin, kinectin, cytoplasmic dynein, and dynactin in the microtubule-mediated movement of a purified membrane organelle.