Cell Flocculation and Phase-Separation Support Macro-Scale Tissue Slab Construction in a Scaffold-Free Manner

Abstract

Creating a macro-scale tissue without a scaffold is challenging, as it requires cells to bind cohesively throughout the tissue. When adherent cells are seeded in an attachment-deprived state, they typically form micro-sized spheroids rather than a single piece of cohesive tissue. In this study, cells flocculated with 0.1% hyaluronic acid and subjected to phase-separation by adding 5% polyethylene glycol (PEG) adhered to each other to form a single tissue mass. Further incubation in PEG-containing media induced a macromolecular crowding effect, creating stable cell-ECM interactions and a homogeneous texture. This process produced a macro-scale, slab-like tissue structure without scaffolds. The resulting slab tissues, constructed with bone marrow-derived multipotent stem cells (BM-MSCs) or chondrocytes, demonstrated high cellularity and a stabilized microstructure. Upon differentiation, these tissues showed excellent functionality, with superior chondrogenic potential in vitro. This study is the first report to demonstrate that cellular-level flocculation and phase-separation can control cell aggregation behavior to create macro-scale slab-like tissues without scaffolds while maintaining excellent cell functionality.

Keywords: Flocculation, Phase-separation, Macromolecular crowding, Scaffold-free Tissue Engineering, Chondrocytes, BM-MSCs