Bioreactor Technologies for Enhanced Organoid Culture.

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.

Gradient porous fibrous scaffolds: a novel approach to improving cell penetration in electrospun scaffolds.

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.

Preliminary Model for the Design of a Custom Middle Ear Prosthesis.

HYPOTHESIS: Custom prostheses could be used to recreate the ossicular chain and improve hearing. BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Alimentary 'green' proteins as electrospun scaffolds for skin regenerative engineering.

As a potential alternative to currently available skin substitutes and wound dressings, we explored the use of bioactive scaffolds made of plant-derived proteins. We hypothesized that 'green' materials, derived from renewable and biodegradable natural sources, may confer bioactive properties to enhance wound healing and tissue regeneration. We optimized and characterized fibrous scaffolds electrospun from soy protein isolate (SPI) with addition of 0.05% poly(ethylene oxide) (PEO) dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol, and from corn zein dissolved in glacial acetic acid. Fibrous mats electrospun from either of these plant proteins remained intact without further cross-linking, possessing a skin-like pliability. Soy-derived scaffolds supported the adhesion and proliferation of cultured primary human dermal fibroblasts. Using targeted PCR arrays and qPCR validation, we found similar gene expression profiles of fibroblasts cultured for 2 and 24 h on SPI substrates and on collagen type I at both time points. On both substrates there was a pronounced time-dependent upregulation of several genes related to ECM deposition remodelling, including MMP-10, MMP-1, collagen VII, integrin-α2 and laminin-ß3, indicating that both plant- and animal-derived materials induce similar responses from the cells after initial adhesion, degrading substrate proteins and depositing extracellular matrix in a 'normal' remodelling process. These results suggest that 'green' proteins, such as soy and zein, are promising as a platform for organotypic skin equivalent culture, as well as implantable scaffolds for skin regeneration. Future studies will determine specific mechanisms of their interaction with skin cells and their efficacy in wound-healing applications.

In vivo multicolor molecular MR imaging using diamagnetic chemical exchange saturation transfer liposomes.

A variety of (super)paramagnetic contrast agents are available for enhanced MR visualization of specific tissues, cells, or molecules. To develop alternative contrast agents without the presence of metal ions, liposomes were developed containing simple bioorganic and biodegradable compounds that produce diamagnetic chemical exchange saturation transfer MR contrast. This diamagnetic chemical exchange saturation transfer contrast is frequency-dependent, allowing the unique generation of "multicolor" images. The contrast can be turned on and off at will, and standard images do not show the presence of these agents. As an example, glycogen, L-arginine, and poly-L-lysine were encapsulated inside liposomes and injected intradermally into mice to image the lymphatic uptake of these liposomes. Using a frequency-dependent acquisition scheme, it is demonstrated that multicolor MRI can differentiate between different contrast particles in vivo following their homing to draining lymph nodes. Being nonmetallic and bioorganic, these diamagnetic chemical exchange saturation transfer liposomes form an attractive novel platform for multicolor imaging in vivo.

Size-induced enhancement of chemical exchange saturation transfer (CEST) contrast in liposomes.

Liposome-based chemical exchange saturation transfer (lipoCEST) agents have shown great sensitivity and potential for molecular magnetic resonance imaging (MRI). Here we demonstrate that the size of liposomes can be exploited to enhance the lipoCEST contrast. A concise analytical model is developed to describe the contrast dependence on size for an ensemble of liposomes. The model attributes the increased lipoCEST contrast in smaller liposomes to their larger surface-to-volume ratio, causing an increased membrane water exchange rate. Experimentally measured rates correlate with size, in agreement with the model. The water permeability of liposomal membrane is found to be 1.11 +/- 0.14 microm/s for the specific lipid composition at 22 degrees C. Availability of the model allows rational design of the size of liposomes and quantification of their properties. These new theoretical and experimental tools are expected to benefit applications of liposomes to sensing the cellular environment, targeting and imaging biological processes, and optimizing drug delivery properties.