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Description
Introduction
Chronic Lymphocytic Leukemia (CLL) is the most common hematological malignancy in the Western World [1], caused by the expansion and accumulation of B lymphocytes in peripheral blood, bone marrow, lymph nodes and spleen. To date, the comprehension of the interactions between CLL cells and the tumor microenvironment (TME) is challenging to implement novel therapiesIn this study, we designed and prepared 3D bioprinted in vitro models to investigate CLL cells behavior in TME with different stiffness.
Materials and Methods
Low, medium, and high viscosity alginate was dissolved in DPBS (2, 4% w/v). Calcium carbonate (CaCO3) and D-glucono-lactone (GDL) were mixed with deionized water and added dropwise (alginate:CaCO3:GDL=2:1:1) [2]. Rheological analyses of pre-crosslinked alginate inks were performed to investigate the optimal pre-crosslinking condition to ensure adequate printability. Thixotropy tests were performed to evaluate recovery ability of the inks after the printing. Methacrylate gelatin (GelMA) was synthetized as reported elsewhere [3]. Biomaterial inks were prepared using pre-crosslinked alginate at high viscosity, obtaining ALG_1 and ALG_2. Additionally, a blended bioink (ALG_2_GELMA_0.4RF) was obtained by mixing ALG_2 with GelMA, using a visible light specific photoinitiator (RF/SPS). Cylindrical samples were 3D printed by optimizing the printing parameters. ALG_1 and ALG_2 hydrogels were ionically crosslinked, whereas ALG_2_GELMA_0.4RF samples were photocrosslinked during the printing process. Stability in vitro tests and compressive mechanical tests were performed to evaluate stability in RPMI medium and elastic modulus. CLL cell line MEC1 were collected and suspended in ALG_1, ALG_2, ALG_2_GELMA_0.4RF. 3D bioprinted samples were cultured up to 14 days. AlamarBlue assay and Live/Dead staining were performed to investigate cells’ behavior in the hydrogels.
Results and Discussion
Alginate gelation occurred in 48 h at 4°C and at room temperature. Herschel-Bulkley model evidenced that printable inks can be obtained from high viscosity alginate and CaCO3/GDL gelation at 4°C. Thixotropy tests evidenced the highest recovery rate at 4°C (Figure 1a). The biomaterial inks composition affected stability and mechanical properties. Alginate low concentration (ALG_1) resulted in weaker hydrogels, whereas ALG_2 and ALG_2_GELMA_0.4RF were stable up to 10 days. Mechanical tests on ALG_1, ALG_2 and ALG_2_GELMA_0.4RF exhibited an elastic modulus of 7.51, 13.67 and 30.68 kPa, respectively (Figure 1b). MEC1 cells proliferated in cell-laden bioprinted scaffolds. Cell metabolism progressively increased in time for ALG_2 and ALG_2_GELMA_0.4RF, whereas decreased for ALG_1, due to a faster degradation. Live/Dead staining evidenced how cells differently behaved in microenvironments with different stiffness. By increasing hydrogel stiffness, cells formed tumor-like clusters, especially after 7 days of culture. Moreover, in ALG_1 and ALG_2, MEC1 cells assumed a rounded shape, whereas the presence of RGD motifs in the blended bioink allowed cells to adhere onto the surrounding microenvironment.
Conclusion
Varying the composition of the bioink it was possible to obtain in vitro models that allow a different CLL cell behavior. In particular, ALG-GelMA bioprinted models resulted promising 3D in vitro models for CLL study.
References
1. Scielzo C. et al., 2020, 103389/fonc.2020.607608.
2. Hazur J. et al., 2020, 101088/1758-5090/ab98e5.
3. Pitton M. et al., 2024, 101016/j.jmbbm.2024.106675.
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