Chronic Lymphocytic Leukemia (CLL) represents the most common leukemia in the western world and remains incurable. Leukemic cells organize and interact in the lymphoid tissues, however what actually occurs in these sites has not been fully elucidated yet. Studying primary CLL cells in vitro is very challenging due to their short survival in culture and also to the fact that traditional two-dimensional in vitro models lack cellular and spatial complexity present in vivo. Based on these considerations, we exploited for the first time three-dimensional (3D) bioprinting to advance in vitro models for CLL. This technology allowed us to print CLL cells (both primary cells and cell lines) mixed with the appropriate, deeply characterized, hydrogel to generate a scaffold containing the cells, thus avoiding the direct cell seeding onto a precast 3D scaffold and paving the way to more complex models. Using this system, we were able to efficiently 3D bioprint leukemic cells and improve their viability in vitro that could be maintained up to 28 days. We monitored over time CLL cells viability, phenotype and gene expression, thus establishing a reproducible long-term 3D culture model for leukemia. Through RNA sequencing (RNAseq) analysis, we observed a consistent difference in gene expression profile between 2D and 3D samples, indicating a different behavior of the cells in the two different culture settings. In particular, we identified pathways upregulated in 3D, at both day 7 and 14, associated with immunoglobulins production, pro-inflammatory molecules expression, activation of cytokines/chemokines and cell-cell adhesion pathways, paralleled by a decreased production of proteins involved in DNA replication and cell division, suggesting a strong adaptation of the cells in the 3D culture. Thanks to this innovative approach, we developed a new tool that may help to better mimic the physiological 3D in vivo settings of leukemic cells as well as of immune cells in broader terms. This will allow for a more reliable study of the molecular and cellular interactions occurring in normal and neoplastic conditions in vivo, and could also be exploited for clinical purposes to test individual responses to different drugs.

Introduction: For decades, in vitro 2D cell culture techniques have been employed in research, but they fail to recapitulate the complexity of natural tissues. 3D bioprinting could potentially overcome this drawback due to the possibility to control the spatial disposition of living cells and the geometry of the 3D scaffold.Materials and methods: This study reports the design and characterization of a novel bioink for extrusion bioprinting, analyzing different blend formulations composed of alginate, gelatin, and methylcellulose, suitable as cell-laden bioink for lymphoid cells, in particular those isolated from patients with Chronic Lymphocytic Leukemia (CLL). The rheological properties as a function of temperature and the printability of the formulations were investigated to define the optimal printing parameters. In vitro stability of the printed scaffolds was investigated under culture conditions and compression tests were performed on printed and bioprinted scaffolds to compare their mechanical properties with those of fresh lymphoid tissue. Finally, MEC1, a CLL cell line, was bioprinted to investigate cell viability, cell density, and cell capability to be released from the scaffold over time.Results and discussion: Results showed that, for the selected blends, good shape fidelity and printing accuracy were achieved with a limitation on the number of printed layers. Scaffolds withstood culture conditions showing stability for up to 3 weeks and their mechanical properties were similar to those of lymphoid tissues already reported in the literature. High cell viability after 21 days was observed for both MEC1 and primary peripheral mononuclear cells, confirming the possibility to use the selected formulation to successfully bioprint lymphoid cells by possibly mimicking their native lymphoid microenvironment.

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