Development and Optimization of a Preservable Three-Dimensional Bio-Construct

Abstract

Many cellular culture and assay models are still considered in “two dimensions (2D)”, despite the obvious need for biologically relevant, three dimensional (3D) systems. Forcing cells to adapt to a pseudo 2D environment causes phenotypic changes in growth, metabolism, and functionality, leading to poor clinical translation. In recent years, the need for more biologically relevant 3D systems have become a major focus of research (i.e. lab on chip, organoids) to attempt to resolve these geometric issues. With the creation of these increasingly complex cell and tissue systems requires a parallel need for transportation and storage solutions via preservation techniques. The preservation of these culture and assay products must allow ample viability and functionality afterwards. The material of this dissertation can be divided into two major components. Early focus was towards cryoprotective solution (CPA) formulation and characterization of suspension freezing to improve upon post-thaw outcome. As the shift in industrial focus was gleaned towards more complex, 3D cellular systems, focus shifted towards the translation of knowledge in suspension cryobiology to creating a cryopreservation methodology for simple 3D constructs. Thermodynamic studies (using differential scanning calorimetry (DSC)) and a Raman spectroscopic based analysis of hydrogen bonding network were used to study mechanisms of non-penetrating cryoprotectants. Alginate hydrogel suspension is one method of creating a 3D pseudo extra cellular matrix for cells to be housed in. Alginate’s biocompatibility and easily tunable properties make it an ideal candidate for biological mimicking tissue systems which have the potential to be cryopreserved. Optimization studies on liver carcinoma cells were performed to develop a comprehensive system for cryopreserving cell-laden alginate hydrogels. Optimization studies were separated into 3 major components: alginate precursor formulation, protocol modulation, and CPA formulation; however, the final two received bulk focus due to previous expertise. Studies found that liver carcinoma (HepG2) cells could be cryopreserved in alginate hydrogels with substantial post-thaw cell viability and metabolic recovery. Subtle changes were made to slow suspension freezing protocol to adapt to 3D preservation. In addition, osmotic pressure was found to be an important factor in post-thaw outcome. Cells of differing origins (neurological and immunological) were also evaluated using the same cryopreservation and encapsulation techniques. It was found that although the HepG2 optimized technique was viable for other cell types, an optimization study is recommended for all cell types to enhance post thaw outcome. This research provides a framework for the development of 3D culture systems and their cryopreservation outcome characterization. This methodology can be applied for future work to create more complex and biologically relevant constructs with long-term storage implications.