Biofabrication of 3D cell-encapsulated tubular constructs using dynamic optical projection stereolithography.

It has been widely recognized that one of the critical limitations in biofabrication of functional tissues/organs is lack of vascular networks which provide tissues and organs with oxygen and nutrients. Biofabrication of 3D vascular-like constructs is a reasonable first step towards successful printing of functional tissues and organs. In this paper, a dynamic optical projection stereolithography system has been implemented to successfully fabricate 3D Y-shaped tubular constructs with living cells encapsulated. The effects of operating conditions on the cure depth of a single layer have been investigated, such as UV intensity, exposure time, and cell density. A phase diagram has been constructed to identify optimal operating conditions. Cell viability immediately after printing has been measured to be around 75%. Post-printing mechanical properties, swelling properties, and microstructures of the gelatin methacrylate hydrogels have been characterized. The resulting fabrication knowledge helps to effectively and efficiently print tissue-engineered vascular networks with complex geometries.

Effects of Encapsulated Cells on the Physical-Mechanical Properties and Microstructure of Gelatin Methacrylate Hydrogels.

Gelatin methacrylate (GelMA) has been gaining popularity in recent years as a photo-crosslinkable biomaterial widely used in a variety of bioprinting and tissue engineering applications. Several studies have established the effects of process-based and material-based parameters on the physical-mechanical properties and microstructure of GelMA hydrogels. However, the effect of encapsulated cells on the physical-mechanical properties and microstructure of GelMA hydrogels has not been fully understood. In this study, 3T3 fibroblasts were encapsulated at different cell densities within the GelMA hydrogels and incubated over 96 h. The effects of encapsulated cells were investigated in terms of mechanical properties (tensile modulus and strength), physical properties (swelling and degradation), and microstructure (pore size). Cell viability was also evaluated to confirm that most cells were alive during the incubation. It was found that with an increase in cell density, the mechanical properties decreased, while the degradation and the pore size increased.

Establishing a reproducible approach to study cellular functions of plant cells with 3D bioprinting.

Capturing cell-to-cell signals in a three-dimensional (3D) environment is key to studying cellular functions. A major challenge in the current culturing methods is the lack of accurately capturing multicellular 3D environments. In this study, we established a framework for 3D bioprinting plant cells to study cell viability, cell division, and cell identity. We established long-term cell viability for bioprinted Arabidopsis and soybean cells. To analyze the generated large image datasets, we developed a high-throughput image analysis pipeline. Furthermore, we showed the cell cycle reentry of bioprinted cells for which the timing coincides with the induction of core cell cycle genes and regeneration-related genes, ultimately leading to microcallus formation. Last, the identity of bioprinted Arabidopsis root cells expressing endodermal markers was maintained for longer periods. The framework established here paves the way for a general use of 3D bioprinting for studying cellular reprogramming and cell cycle reentry toward tissue regeneration.

Effect of topography parameters on cellular morphology during guided cell migration on a graded micropillar surface.

PURPOSE: Guided cell migration refers to the engineering of local cell environment to specifically direct cell migration and has important applications such as utilization in cell sorting and wound healing assays. Graded micropillar surfaces have been utilized for achieving guided cell migration. Topographic parameters such as micropillar diameter and spacing gradient may have effects on the morphology of attached cells. It is critical to understand this interaction between the cells and the underlying microscale structures. METHODS: In this study, a graded micropillar substrate has been fabricated to investigate the effects of the microtopography on the cell morphology in terms of the cell aspect ratio and cell circularity. RESULTS: It is found that 1) with the increase of the micropillar diameter, the cell aspect ratio has no significance change. At the small spacing gradients, the aspect ratio is smaller than that at the large spacing gradients; 2) statistical analysis shows both the micropillar diameter and spacing gradient have no significant effect on the cell aspect ratio compared to the flat surface; 3) the cell circularity at the small micropillar diameters is higher than that at the large micropillar diameters. The cell circularity at the micropillar gradient of 0.1 µm is higher than those at the other micropillar gradients; 4) three microtopographic conditions are considered to have statistically significant effect on the cell circularity compared to the flat surface, including the micropillar diameters of 5 µm and 10 µm and the spacing gradient of 0.1 µm.

Tissue Regeneration with Hydrogel Encapsulation: A Review of Developments in Plants and Animals.

Hydrogel encapsulation has been widely utilized in the study of fundamental cellular mechanisms and has been shown to provide a better representation of the complex in vivo microenvironment in natural biological conditions of mammalian cells. In this review, we provide a background into the adoption of hydrogel encapsulation methods in the study of mammalian cells, highlight some key findings that may aid with the adoption of similar methods for the study of plant cells, including the potential challenges and considerations, and discuss key findings of studies that have utilized these methods in plant sciences.

Effects of Irgacure 2959 and lithium phenyl-2,4,6-trimethylbenzoylphosphinate on cell viability, physical properties, and microstructure in 3D bioprinting of vascular-like constructs.

Photocrosslinkable polymers such as gelatin methacrylate (GelMA) have various 3D bioprinting applications. These polymers crosslink upon exposure to UV irradiation with the existence of an appropriate photoinitiator. Two photoinitiators, Irgacure 2959 and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) are commonly used. This study systematically investigates the effects of photoinitiator types on the cell viability, physical properties, and microstructure in 3D bioprinting of GelMA-based cellular constructs. The main conclusions are: (1) during the 3D bioprinting, the cell viability generally decreases as the photoinitiator concentration and printing time increase using both Irgacure 2959 and LAP. At the low photoinitiator concentrations (such as 0.3% and 0.5% (w/v)), the overall cell viability is good within the printing time of 60 min using both Irgacure 2959 and LAP. However, at the high photoinitiator concentrations (such as 0.7% and 0.9% (w/v)), the overall cell viability using LAP is much higher than that using Irgacure 2959 within the printing time of 60 min; (2) after the 3D bioprinting, the photoinitiator types, either Irgacure 2959 or LAP, have negligible effects on the post-printing cell viability after crosslinking; (3) after the 3D bioprinting, GelMA samples cured with Irgacure 2959 have slightly larger pore size, faster degradation rate, and greater swelling ratio compared to those cured with LAP; (4) 3D GelMA-based vascular-like constructs have been fabricated using dynamic optical projection stereolithography, and the measured dimensions have been compared with the designed dimensions showing good shape fidelity.

Biofabrication of three-dimensional cellular structures based on gelatin methacrylate-alginate interpenetrating network hydrogel.

Hydrogels have been widely used as extracellular matrix materials in various three-dimensional bioprinting applications. However, they possess limitations such as insufficient mechanical integrity and strength, especially in the vascular applications requiring suture retention and tolerance of systemic intraluminal pressure. Interpenetrating network hydrogels are unique mixtures of two separate hydrogels with enhanced properties. This paper has demonstrated the fabrication of three-dimensional cellular constructs based on gelatin methacrylate-alginate interpenetrating network hydrogels using a microgel-assisted bioprinting method. Filament formation was investigated in terms of the filament diameter under different nozzle speed and dispensing pressure, and a phase diagram to identify the optimal conditions for continuous and uniform filaments was prepared. Three-dimensional hollow cellular constructs were fabricated and the cell viability was 75% after 24-hour incubation. The post-printing properties were characterized including mechanical properties, degradation and swelling properties, and pore size. The interpenetrating network hydrogels with different concentrations were compared with their individual components. It is found that the interpenetrating network hydrogels exhibit stronger mechanical properties, faster degradation and larger pore sizes than their individual components.