(H-308) Optimization and Validation of Utilizing Alginate-Based Casts to Model and Characterize the Physical Properties associated with Bioprinted Tissue Constructs

Introduction:
The field of bioprinting has recently gained heightened attention as providing a promising technology that gives rise to achieving successful therapeutic outcomes where other methods have been met with limitations. Coaxial bioprinting in particular has been a source promise in its capacity to encapsulate larger cell types and tissues such as human islets to treat Type 1 Diabetes. However, bioprinting 3-Dimensional (3D), layered-tissue constructs requires optimization of several aspects. These include the appropriate size/shape of the desired construct, pressures to avoid shear stress, cell type, nozzle size, bioink composition, presence of extracellular matrix (ECM), and the volume of bioink needed to sustain the printing process. To measure the behavior and rheology of bioprinted tissue constructs in response to terminal mechanical assays independent of the aforementioned factors, other models may be more suitable. It is also necessary to identify the effect of using different alginates (LVM or MVG), crosslinkers (CaCl2 or SrCl2) as well as the inclusion or lack thereof of ECM on bioprinted constructs without necessitating the use of bioprinting itself. Therefore, I propose that an alginate cast/hydrogel, with the same composition and crosslinkers of traditional bioink, may be used as a suitable alternative to bioprinting in order to extrapolate the results of assays aimed at modeling the physical properties of 3D bioprinted tissue constructs in vitro.
Materials and Methods:

Alginate hydrogels are typically used in tissue engineering and regenerative medicine applications due to their physical characteristics such as biocompatibility, biodegradability, and encapsulation of certain cell types or cellular clusters such as human islets. These proposed alginate casts have been demonstrative in their capacity to withstand the effects of both physiological and mechanical pressure and respond to assays designed to measure physical characterization and cytocompatibility. The casts are cylindrical and composed of primarily 1.5% UltraPure Low Viscosity Monolaurate (UP LVM) or UltraPure Medium Viscosity guluronate (UP MVG) and 2.5% Gelatin. The casts are crosslinked with either 100 mM calcium or 25 mM strontium, respectively. The cylinders occupy a volume of ~40 µL with a diameter of 5 mm and height of 2 mm. There are several aims and assays designed to assess the rheology of the alginate and physically characterize the mechanical properties of an alginate analog similar to the bioprinted constructs. The mechanical components of hydrogels such as their stiffness and elasticity are significant factors that may be influential on cellular behavior, tissue formation, and vascularization. 3D characterization of these properties can inform the design of biomaterials for therapeutic applications.
Results, Conclusions, and Discussions:
In order to determine the cytocompatibility of human islets and Min6 spheroids encapsulated within these hydrogels, a number of different functional as well as viability assays were conducted and assessed. To test cytocompatibility of spheroids encapsulated in a cast, the MultiTox-Fluor Multiplex cytotoxicity assay was used. The Multitox assay exploits the biological difference between live/dead cells in the capacity of live cells to integrate the dye GF-AFC and cleave AFC, whereas the dye bis-AAF-R110 can only be cleaved by the dead-cells to release R110. Live/Dead imaging using Calcein AM (live) and Ethidium Homodimer-1 (dead) were used to qualitatively evaluate viability. Alamar Blue and Cell-Titer Glo were utilized to determine metabolic activity of these cell and tissue types through ATP quantification, cellular proliferation, and metabolism.