An organoid is a 3D organization of cells that can recapitulate some of the structure and function of native tissue. Recent work has seen organoids gain prominence as a valuable model for studying tissue development, drug discovery, and potential clinical applications. The requirements for the successful culture of organoids in vitro differ significantly from those of traditional monolayer cell cultures. The generation and maturation of high-fidelity organoids entails developing and optimizing environmental conditions to provide the optimal cues for growth and 3D maturation, such as oxygenation, mechanical and fluidic activation, nutrition gradients, etc. To this end, we discuss the four main categories of bioreactors used for organoid culture: stirred bioreactors (SBR), microfluidic bioreactors (MFB), rotating wall vessels (RWV), and electrically stimulating (ES) bioreactors. We aim to lay out the state-of-the-art of both commercial and in-house developed bioreactor systems, their benefits to the culture of organoids derived from various cells and tissues, and the limitations of bioreactor technology, including sterilization, accessibility, and suitability and ease of use for long-term culture. Finally, we discuss future directions for improvements to existing bioreactor technology and how they may be used to enhance organoid culture for specific applications.

Therapeutic irradiation for head and neck cancer, and the autoimmune disease Sjogren's syndrome, lead to loss of salivary parenchyma. They are the two main causes of irreversible salivary gland hypofunction. Such patients cannot produce adequate levels of saliva, leading to considerable morbidity. We are working to develop an artificial salivary gland for such patients. A major problem in this endeavor has been the difficulty in obtaining a suitable autologous cellular component. This article describes a method of culturing and expanding primary salivary cells obtained from human submandibular glands (huSMGs) that is serum free and yields cells that are epithelial in nature. These include morphological (light and transmission electron microscopy [TEM]), protein expression (immunologically positive for ZO-1, claudin-1, and E-cadherin), and functional evidence. Under confocal microscopy, huSMG cells show polarization and appropriately localize tight junction proteins. TEM micrographs show an absence of dense core granules, but confirm the presence of tight and intermediate junctions and desmosomes between the cells. Functional assays showed that huSMG cells have high transepithelial electrical resistance and low rates of paracellular fluid movement. Additionally, huSMG cells show a normal karyotype without any morphological or numerical abnormalities, and most closely resemble striated and excretory duct cells in appearance. We conclude that this culture method for obtaining autologous human salivary cells should be useful in developing an artificial salivary gland.

In this study we describe the use of an electrospun soy protein-based tissue scaffold (SPS) as a bioactive wound dressing in a pig model of full thickness excisional wound healing. The time course of wound healing and the quality of the healing tissue were evaluated using histology (H&E and Masson's trichrome staining). While the overall rate of wound closure was similar in the SPS-treated vs. untreated control wounds covered with Tegaderm®, there were significant qualitative differences between the two groups. Two weeks after a single application of SPS at the time of wounding, the SPS treated wounds showed robust signs of reepithelialization, which was absent in the control wounds. After 4 weeks, the SPS treated wounds contained a stratified epithelial layer in the epidermis that looked essentially normal, while the connective tissue in the dermis was attaining a cellular, organized appearance. By contrast the nascent epidermis of the untreated controls appeared immature, while the dermis was still replete with numerous inflammatory/immune cells. Masson's trichrome staining confirmed the increased presence of collagen in the dermis of the SPS treated wounds at 4 weeks, while the control wounds were largely devoid of collagen. Finally, in addition to enhanced reepithelialization and dermal tissue regeneration, 4 weeks after application of the SPS dressing, we observed the presence of dermal appendages, such as sweat glands and hair follicles. No such appendage formation was observed in the untreated controls during the entire duration of our study. Taken together, the histological data clearly indicate that our soy protein based scaffolds accelerated and enhanced a more natural mode of tissue regeneration in the porcine model of full thickness excisional wound healing. Given the similarities between porcine and human wound healing, we anticipate that SPS will also be advantageous in clinical applications

Tight junctions (TJs) are an essential structure of fluid-secreting cells, such as those in salivary glands. Three major families of integral membrane proteins have been identified as components of the TJ: claudins, occludin, and junctional adhesion molecules (JAMs), plus the cytosolic protein zonula occludens (ZO). We have been working to develop an orally implantable artificial salivary gland that would be suitable for treating patients lacking salivary parenchymal tissue. To date, little is known about the distribution of TJ proteins in adult human salivary cells and thus what key molecular components might be desirable for the cellular component of an artificial salivary gland device. Therefore, the aim of this study was to determine the distribution of TJ proteins in human salivary glands. Salivary gland samples were obtained from 10 patients. Frozen and formalin-fixed paraffin-embedded sections were stained using IHC methods. Claudin-1 was expressed in ductal, endothelial, and ∼25% of serous cells. Claudins-2, −3, and −4 and JAM-A were expressed in both ductal and acinar cells, whereas claudin-5 was expressed only in endothelial cells. Occludin and ZO-1 were expressed in acinar, ductal, and endothelial cells. These results provide new information on TJ proteins in two major human salivary glands and should serve as a reference for future studies to assess the presence of appropriate TJ proteins in a tissue-engineered human salivary gland.

Rotating wall vessel (RWV) bioreactors have been used to produce cell spheroids and organoids at a faster rate than in other bioreactor devices and with higher structural and functional fidelity. One of the limitations of traditional RWV systems is the well-documented tendency for air bubble formation during operation. The presence of these bubbles negates key features of the RWV environment, such as zero headspace, low-shear, and simulated microgravity. In this article, we describe the design, construction, and testing of a novel RWV capable of constantly removing air bubbles from the system without interfering with the fluid dynamics that produce optimized cell culture conditions. We modeled this capacity using computational fluid dynamics and then validated the model with alginate beads and spheroid cultures of A549 human lung adenocarcinoma cells. The areas of spheroids assembled from A549 cells in the novel bioreactor in the presence of air bubbles were an order of magnitude larger than in conventional bioreactors when bubbles were present. Our results demonstrate the ability of the novel design to remove and isolate bubbles while avoiding damage to spheroid assembly, as observed in conventional RWV bioreactors in the presence of bubbles. We anticipate that the novel design will increase experimental reproducibility and consistency when using RWV bioreactors. Impact Statement: The rotating wall vessel (RWV) bioreactor is a powerful tool for the generation of sizeable, faster-growing organoids. However, the ideal, low-shear, modeled microgravity environment in the RWV is frequently disrupted by the formation of bubbles, a critical but understated failure mode. To address this, we have designed and fabricated a novel, modified RWV bioreactor capable of continuously removing bubbles while providing optimal fluid dynamics. We validated the capacity of this device with computational and empirical studies. We anticipate that our novel bioreactor will be more consistent and easier to use and may fill a unique and unmet niche in the burgeoning field of organoids.

Liver organoid technology holds great promises to be used in large-scale population-based drug screening and in future regenerative medicine strategies. Recently, some studies reported robust protocols for generating isogenic liver organoids using liver parenchymal and non-parenchymal cells derived from induced pluripotent stem cells (iPS) or using isogenic adult primary non-parenchymal cells. However, the use of whole iPS-derived cells could represent great challenges for a translational perspective. Here, we evaluated the influence of isogenic versus heterogenic non-parenchymal cells, using iPS-derived or adult primary cell lines, in the liver organoid development. We tested four groups comprised of all different combinations of non-parenchymal cells for the liver functionality in vitro. Gene expression and protein secretion of important hepatic function markers were evaluated. Additionally, liver development-associated signaling pathways were tested. Finally, organoid label-free proteomic analysis and non-parenchymal cell secretome were performed in all groups at day 12. We show that liver organoids generated using primary mesenchymal stromal cells and iPS-derived endothelial cells expressed and produced significantly more albumin and showed increased expression of CYP1A1, CYP1A2, and TDO2 while presented reduced TGF-β and Wnt signaling activity. Proteomics analysis revealed that major shifts in protein expression induced by this specific combination of non-parenchymal cells are related to integrin profile and TGF-β/Wnt signaling activity. Aiming the translation of this technology bench-to-bedside, this work highlights the role of important developmental pathways that are modulated by non-parenchymal cells enhancing the liver organoid maturation.