## 1 Introduction  
Risk assessment is of paramount importance during the development of novel biomaterials, ensuring human health and their safest clinical use [1]. In this context, the first screening to assess the toxicological risk is performed using in vitro models with cell culture, as an important step prior to in vivo preclinical and clinical assessments. Bidimensional (2D) monolaminar models represent the main cell culture methodology and have provided, for decades, important data on the cytotoxicity of several compounds and materials, being the scope of international standards for material testing (ISO 10993-5:2018). However, several inherent limitations of in vitro assays have reduced their predictability and applicability. Regarding tests employing monolaminar culture, cells are devoid of a consistent extracellular matrix (ECM), and the reduced cell-cell interactions and mechanical stimuli interfere with cell behavior, morphology, and phenotype [2], increasing the distance with expected clinical outcomes. The high rate of failure of materials during clinical test phases demonstrates the need for more predictive models.  
Technologies involving cell culture have continually improved throughout the 20 th century [3]. Concerning Toxicity Testing for the 21 st Century, organotypic culture techniques such as 3D models have considered as promising models to fill the gap between the in vitro and in vivo or clinical results [4]. This model allows cells to further interact with each other and with ECM, mimicking some of the physiological responses that occur in a tissue, permitting cells to make complex interactions with adjacent cells, receiving and transmitting signals [5]. Furthermore, 3D models provide gradients of oxygen, nutrients, metabolites, soluble signals, with increased heterogeneity of cells, and the expression of cell-signaling factors and responses to drugs and materials more similar to in vivo conditions [6]. Several studies have investigated 3D models, which includes spheroidal organoids or spheroids, which may be produced by the liquid overlay technique [7].  
Osteoblast and osteoblast-like cells lines are important for toxicology evaluation and risk assessment of drugs related to bone diseases such as osteoporosis, osteosarcoma, and nanoparticles [8] and implantable biomaterials [9], including the use of international standards for materials testing (ISO 10993-5:2009). Different osteoblast and osteoblast-like-based 3D models are being developed nowadays [10; 11; 12; 13; 14]. However, there is a lack of further characterization of these 3D models. Moreover, there is a gap in the evidence of the suitability of spheres of osteoblastic lines to standardized cytotoxicity assessments. As long as these factors are not fully approached, the confidence in these tests will remain limited. In this context, this work aimed to (i) characterize the stability, integrity, and uniformity of a 3D model, composed of aggregates of preosteoblastic cells, intended for the in vitro evaluation of biocompatibility of bone-substitute biomaterials, and (ii) to investigate the suitability of the 3D model as a substitute of monolaminar culture in the performance of some of the most widely used in vitro cytotoxicity tests for the evaluation of biomaterials.