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ICE Krakow

ICE Krakow

ul. Marii Konopnickiej 17 30-302 Kraków


Banicevic, Ivana (University of Belgrade, Faculty of Technology and Metallurgy)


Cancers are one of the leading causes of death worldwide. Failure to obtain effective treatment could be attributed to the irrelevancy of commonly utilized two-dimensional (2D) in vitro and animal models in drug evaluation. Three-dimensional (3D) in vitro models aim to provide more resemblance to in vivo cancer cell microenvironment and therefore represent promising tools for reliable anticancer drug screening. Here, we present the development of a 3D in vitro model mimicking in vivo osteosarcoma cell microenvironment regarding extracellular matrix and hydrodynamic conditions by utilising bone-mimicking macroporous composite alginate scaffolds in conjunction with perfusion biomimetic bioreactor.
Macroporous composite alginate scaffolds with incorporated mineral powder (2 wt% alginate, 2 wt% hydroxyapatite) were produced as previously described1. Scaffolds in dry and rehydrated forms (~10 mm in diameter, ~4.5 mm thick) were analyzed regarding porosity and the average pore size. The optimal cell seeding method was based on partial rehydration of the scaffolds in the cell culture medium, followed by manual seeding with K7M2-wt osteosarcoma cells (ATCC® CRL-2836™) at the cell density of 15x106 cells cm-3 of the scaffold volume. The cell-seeded scaffolds were then cultivated for 24 h under static conditions followed by cultivation in biomimetic perfusion bioreactors (“3D Perfuse”, Innovation Center of the Faculty of Technology and Metallurgy, Belgrade, Serbia) under continuous medium flow (0.27 cm3 min-1; superficial medium velocity of 40 µm s-1) for the next 7 days. Static 3D cultures served as controls. Cell viability was investigated by the MTT test, while the cell morphology and distribution within the scaffolds were analyzed by field-emission scanning electron microscopy (FE-SEM, MIRA 3 XMU instrument, TESCAN, Brno, Czech Republic) and histological staining (H&E) of scaffold cross-sections.
Biocompatible macroporous alginate scaffolds had an average porosity of 60%, which falls within the porosity range of trabecular bone2. Efficiency of the cell seeding method was above 80% while the cultivation under medium flow had negligible effects on the cell loss. MTT test confirmed that the cells remained viable and even more metabolically active in perfusion cultures compared to static conditions (control). Moreover, the cells retrieved from the scaffolds were able to attach to the cell culture plastic and proliferate over 48 h in 2D in vitro model. The FE-SEM analysis revealed the presence of cell aggregates attached to the pores. In addition, osteosarcoma cells spontaneously formed spheroids within scaffold pores confirmed by histological examination.
In this study, a 3D model for osteosarcoma cell culture was developed by using murine cells, macroporous composite alginate-based scaffolds and perfusion bioreactors. The cells attached to the scaffold interiors and spontaneously formed spheroids during a 7-day culture. Perfusion had positive effects on the cells indicated by the increased metabolic activity as compared to static controls. It can be suggested that this developed 3D model has the potential for utilisation in osteosarcoma drug testing, which is the next phase of model evaluation.

  1. Stojkovska, J., J. Biomedic. Mater. Res. Part B Appl. Biomater., 109(12),1-12 (2021).
  2. Monteiro, C.F., Adv. Therap.,2: 1800108 (2019)

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