Poster N2 - Characterization of Extrusion Based Bioprinted Tissue Constructs

Introduction: The aim of this research is to develop a bioprinted 3D tissue model system for disease modeling and treatment. 3D bioprinting has become an important technology for studying tissue regeneration, modeling various diseases, and developing pharmaceuticals to prevent these diseases. 3D cell tissue has significant advantages over its 2D counterparts, including more physiologically relevant representations of cell morphology, proliferation gradients, response to drugs, gene expression, and cell health. Manually producing 3D culture models has proven to be a difficult and expensive task for many researchers, often resulting in high sample-to-sample variability. This research proposes a novel approach to quantifying and assessing the capabilities of modern bioprinting. A GelMA-based bioink is a standard hydrogel material used for encapsulating cells to provide a supportive extracellular matrix (ECM) environment and safeguard cells during printing. GelMA provides the ideal base bioink for mimicking the ECM environment and should be capable of bioprinting structures with high resolution, mechanical properties, excellent biodegradability, biocompatibility, and cellular viability. An extrusion-based six-head bioprinter is used for bioprinting constructs. These bioprinted tissue constructs are then imaged using fluorescence microscopy to assess cell attachment and cell distribution. The cellular viability of the bioprinted tissue constructs is assessed using a live-dead assay to determine viability and effectiveness. Thus, a 3D tissue model generated by the bioprinting process will help understand the role that ECM plays as a key regulator in tissue architecture, proliferation, differentiation, migration, apoptosis, metastasis, and others.

Materials and

Methods: PhotoGel® 50% degree of methacrylation (DOM) was prepared with a bioink concentration of 10% (w/v). The photoinitiator solution consisted of a 10% lithium phenyl phosphate (LAP) photoinitiator, which was solubilized in Reconstitution Agent P. This ink was seeded with cells to have a cell density of about 9 million cells per 1 mL of GelMA. The CELLINK BioX6 3D bioprinter was utilized for bioprinting printing of the tissue constructs. Bioprinting occurred in a sterile environment with a printhead temperature of 23.5° and a pressure range of 45-55kPa. The printed constructs have eight-layers of rectangular grids having 40% rectilinear infill.
The viability of the cells within the 3D bioprinted constructs was assessed using the LIVE/DEAD® Viability/Cytotoxicity Kit *for mammalian cells* protocol (Molecular Probes™). From the prepared stock, 200 µL of the live/dead staining solution was extruded onto the construct and then incubated in the staining solution for 30 minutes at 37°C, protected from light. The stained constructs were then imaged using confocal microscopy to visualize the spatial distribution of live (green) and dead (red) cells throughout the 3D bioprinted structure. Z-stack images were captured at multiple depths to assess cell viability within the full bioprinted construct volume. This live/dead assay provided qualitative and quantitative data on the biocompatibility of the 3D bioprinting process. It enabled the evaluation of cell survival and distribution within the bioprinted tissue constructs over a period of 14 days.

Results, Conclusions, and Discussions: The bioprinted tissue constructs exhibited cellular adhesion and distribution, confirmed by fluorescence microscopy and live-dead assays, demonstrating cellular viability and adhesion at various time points. This indicates the constructs' potential for functional applications. Figure 1 shows the architecture of the bioprinted construct. This design enables the creation of tissue structures with precise control over cellular organization and spatial distribution.
Figure 2 depicts the volume view of the construct, highlighting the successful integration of living cells into the printed framework. The 4X magnification view offers a perspective on the construct's cellular organization and spatial distribution (Figure 3).
Confocal microscopy imaging of the samples provided a detailed understanding of cellular integrity and structure. Notably, cellular viability remained exceptionally high, with 96.21% viability on Day 7, gradually decreasing to 82.56% on Day 10 and 77.19% on Day 14. These findings demonstrate the efficacy of the bioprinting approach in creating functional tissue constructs with excellent cellular viability and structural integrity. The results suggest that the bioprinted constructs can potentially serve as viable alternatives for animal testing, offering new avenues for research and clinical applications. Overall, this study highlights the potential of bio-printing technology to create complex, functional tissue constructs that mimic native tissue architecture that mimic native tissue architecture.
While these viability levels are promising, strategies to further improve and maintain high cellular viability over extended periods could involve optimizing the bioink composition, incorporating growth factors, and refining the bioprinting parameters and handling methods to minimize cellular stress.

Acknowledgements (Optional): The authors would like to acknowledge funding support from National Science Foundation – Grant Number CBET 2317757.