3D spheroid culture is a bridge between standard 2D cell culture and in vivo research which mimics the physiological microenvironment in scaffold-free conditions. Here, this 3D technique is being investigated as a potential method for engineering bone tissue in vitro. However, spheroid culture can exhibit limitations, such as necrotic core formation due to the restricted access of oxygen and nutrients. It is therefore important to determine if spheroids without a sizeable necrotic core can be produced. This study aims to understand necrotic core formation and cell viability in 3D bone cell spheroids using different seeding densities and media formulations.

Differentiated rat osteoblasts (dRObs) were seeded in three different seeding densities (1×10⁴, 5×10⁴, 1×10 cells) in 96 well U-bottom cell-repellent plates and in three different media i.e., Growth medium (GM), Mineralisation medium 1 (MM1) and MM2. Spheroids were analysed from day 1 to 28 (N=3, n=2). Cell count and viability was assessed by trypan blue method. One way ANOVA and post-hoc Tukey test was performed to compare cell viability among different media and seeding densities. Histological spheroid sections were stained with hematoxylin and eosin (H&E) to identify any visible necrotic core.

Cell number increased from day 1 to 28 in all three seeding densities with a notable decrease in cell viability. 1×10⁴ cells proliferated faster than 5×10⁴ and 1×10⁵ cells and had proportionately similar cell death. The necrotic core area was relatively equivalent between all cell seeding densities. The larger the spheroid size, the larger is the size of the necrotic core.

This study has demonstrated that 3D spheroids can be formed from dRobs at a variety of seeding densities with no marked difference in necrotic core formation. Future studies will focus on utilising the bone cell spheroids for engineering scalable scaffold-free bone tissue constructs.