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.
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.
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.
The aim of this feasibility study was to test the ability of fluorescent nanodiamond particles (F-NDP) covalently conjugated with bitistatin (F-NDP-Bit) to detect vascular blood clots in vivo using ...extracorporeal near-infrared (NIR) imaging. Specifically, we compared NIR fluorescence properties of F-NDP with N-V (F-NDP
) and N-V-N color centers and sizes (100-10,000 nm). Optimal NIR fluorescence and tissue penetration across biological tissues (rat skin, porcine axillary veins, and skin) was obtained for F-NDP
with a mean diameter of 700 nm. Intravital imaging (using in vivo imaging system IVIS) in vitro revealed that F-NDP
-loaded glass capillaries could be detected across 6 mm of rat red-muscle barrier and 12 mm of porcine skin, which equals the average vertical distance of a human carotid artery bifurcation from the surface of the adjacent skin (14 mm). In vivo, feasibility was demonstrated in a rat model of occlusive blood clots generated using FeCl
in the carotid artery bifurcation. Following systemic infusions of F-NDP
-Bit (3 or 15 mg/kg) via the external carotid artery or femoral vein (N=3), presence of the particles in the thrombi was confirmed both in situ via IVIS, and ex vivo via confocal imaging. The presence of F-NDP
in the vascular clots was further confirmed by direct counting of fluorescent particles extracted from clots following tissue solubilization. Our data suggest that F-NDP
-Bit associate with vascular blood clots, presumably by binding of F-NDP
-Bit to activated platelets within the blood clot. We posit that F-NDP
-Bit could serve as a noninvasive platform for identification of vascular thrombi using NIR energy monitored by an extracorporeal device.
Custom prostheses could be used to recreate the ossicular chain and improve hearing.
Ossicular discontinuity or fixation occurs in 55% of cases of conductive hearing loss, with most cases involving ...the incus. Reconstruction has been achieved by a variety of methods; however, there has been little improvement in hearing outcomes in decades.
Precise measurements of anatomic dimensions, weight, and center of gravity were taken from 19 cadaveric incudes. These measurements were combined with measurements from the medical literature and micro-computed tomography (micro-CT) of cadaveric temporal bones to generate a rasterizable incus model. As a proof of concept, incudal replacements including possible anatomic variations were then three-dimensionally (3-D) printed and inserted into a cadaveric temporal bone.
Our measurements of cadaveric incudes corresponded well with those from the medical literature. These measurements were combined with anatomical information from micro-CT allowing identification of critical features of the incus, which remained constant. Other model features were modified to increase stability and facilitate synthesis, including broadening and thickening of the lenticular process and the incudomalleolar articulation. 3-D printed incudal replacements based on this model readily fit into a cadaveric temporal bone and successfully bridged the gap between malleus and incus.
We have generated a model for custom 3-D synthesis of incudal prostheses. While current 3-D printing in biocompatible materials at the size required is limited, the technology is rapidly advancing, and 3-D printing of incudal replacements with polylactic acid (PLA) is of the correct size and shape.
Electrospinning is an increasingly popular technique to generate 3D fibrous tissue scaffolds that mimic the submicron sized fibers of extracellular matrices. A major drawback of electrospun scaffolds ...is the small interfibrillar pore size, which normally prevents cellular penetration in between fibers. In this study, we introduced a novel process, based on electrospinning, to manufacture a unique gradient porous fibrous (GPF) scaffold from soy protein isolate (SPI). The pore sizes in the GPF scaffolds gradually increase from one side of the scaffold to the other, ranging from 7.8 ± 2.5 μm in the small pore side, 21.4 ± 10.3 μm in the mid layer to 58.0 ± 23.6 μm in the large pore side. The smallest pores of the GPF scaffolds appeared to be somewhat larger than those in conventionally electrospun SPI scaffolds (4.2 ± 1.3 μm). Hydrated GPF scaffolds exhibited J-shaped stress-strain curves, reminiscent of those for soft biological scaffolds. Attachment, spreading, and proliferation of human dermal fibroblasts (HDFB) were supported on both the small and the large pore sides of the GPF scaffolds. Cultured HDFB and murine RAW 264.7 macrophages penetrated significantly deeper (98.7 ± 24.2 μm and 53.3 ± 9.6 μm, respectively) into the larger pores than when seeded onto the small pore side of GPF scaffolds (22.8 ± 6.2 μm and 25.7 ± 7.3 μm) and control SPI scaffolds. (11.3 ± 3.8 μm and 15.3 ± 3.1 μm). This study introduces a novel fabrication technique, which, by convergence of several biofabrication technologies, produces scaffolds with enhanced cellular penetration.
Infection and inflammation are common complications that seriously affect the functionality and longevity of implanted medical implants. Systemic administration of antibiotics and anti-inflammatory ...drugs often cannot achieve sufficient local concentration to be effective, and elicits serious side effects. Local delivery of therapeutics from drug-eluting coatings presents a promising solution. However, hydrophobic and thick coatings are commonly used to ensure sufficient drug loading and sustained release, which may limit tissue integration and tissue device communications. A calcium-mediated drug delivery mechanism was developed and characterized in this study. This novel mechanism allows controlled, sustained release of minocycline, an effective antibiotic and anti-inflammatory drug, from nanoscale thin hydrophilic polyelectrolyte multilayers for over 35 days at physiologically relevant concentrations. pH-responsive minocycline release was observed as the chelation between minocycline and Ca(2+) is less stable at acidic pH, enabling 'smart' drug delivery in response to infection and/or inflammation-induced tissue acidosis. The release kinetics of minocycline can be controlled by varying initial loading, Ca(2+) concentration, and Ca(2+) incorporation into different layers, enabling facile development of implant coatings with versatile release kinetics. This drug delivery platform can potentially be used for releasing any drug that has high Ca(2+) binding affinity, enabling its use in a variety of biomedical applications.
We hereby report on studies aimed to characterize safety, pharmacokinetics, and bio-distribution of fluorescent nanodiamond particles (NV)-Z~800 (FNDP-(NV)) administered to rats by intravenous ...infusion in a single high dose.
Broad scale biological variables were monitored following acute (90 minutes) and subacute (5 or 14 days) exposure to FNDP-(NV). Primary endpoints included morbidity and mortality, while secondary endpoints focused on hematology and clinical biochemistry biomarkers. Particle distribution (liver, spleen, lung, heart, and kidney) was assessed by whole organ near infrared imaging using an in vivo imaging system. This was validated by the quantification of particles extracted from the same organs and visualized by fluorescent and scanning electron microscopy. FNDP-(NV)-treated rats showed no change in morbidity or mortality and preserved normal motor and sensory function, as assessed by six different tests.
Blood cell counts and plasma biochemistry remained normal. The particles were principally distributed in the liver and spleen. The liver particle load accounted for 51%, 24%, and 18% at 90 minutes, 5 days, and 14 days, respectively. A pilot study of particle clearance from blood indicated 50% clearance 33 minutes following the end of particle infusion.
We concluded that systemic exposure of rats to a single high dose of FDNP-(NV)-Z~800 (60 mg/kg) appeared to be safe and well tolerated over at least 2 weeks. These data suggest that FNDP-(NV) should proceed to preclinical development in the near future.
Thromboembolic events are a major cause of heart attacks and strokes. However, diagnosis of the location of high risk vascular clots is hampered by lack of proper technologies for their detection. We ...recently reported on bio-engineered fluorescent diamond-(NV)-Z~800nm (FNDP-(NV)) conjugated with bitistatin (Bit) and proven its ability to identify iatrogenic blood clots in the rat carotid artery in vivo by Near Infra-Red (NIR) monitored by In Vivo Imaging System (IVIS).
The objective of the present research was to assess the in vivo biocompatibility of FNDP-(NV)-Z~800nm infused intravenously to rats. Multiple biological variables were assessed along this 12 week study commissioned in anticipation of regulatory requirements for a long-term safety assessment.
Rats were infused under anesthesia with aforementioned dose of the FNDP-(NV), while equal number of animals served as control (vehicle treated). Over the 12 week observation period rats were tested for thriving, motor, sensory and cognitive functions. At the termination of study, blood samples were obtained under anesthesia for comprehensive hematology and biochemical assays. Furthermore, 6 whole organs (liver, spleen, brain, heart, lung and kidney) were collected and examined ex vivo for FNDP-NV) via NIR monitored by IVIS and histochemical inspection.
All animals survived, thrived (no change in body and organ growth). Neuro-behavioral functions remain intact. Hematology and biochemistry (including liver and kidney functions) were normal. Preferential FNDP-(NV) distribution identified the liver as the main long-term repository. Certified pathology reports indicated no outstanding of finding in all organs.
The present study suggests outstanding biocompatibility of FNDP-(NV)-Z~800nm after long-term exposure in the rat.
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