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Introduction

Tissue Engineering (TE) is an interdisciplinary field combining cells, biomaterials and

biochemical cues, which aims to recreate human tissues in vitro with authentic structure and
function. These structures can then be used for in vitro diagnostic applications or to restore the

functionality of damaged tissue in vivo 1,2 . In the past, two-dimensional (2D) cell culture

systems, which poorly represent the in vivo niche that is a highly complex microenvironment
including three-dimensional (3D) biochemical and biomechanical cues, were predominant.

This ongoing over-simplification of the intricate tissue microenvironment leads to the lack of
biophysical traits, that in turn impact the performance of TE models. Notably, gene expression

and oxygen gradient differences between 2D and 3D models critically influence the cell
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response and of typical cell behavior . As a response to these shortcomings, modern 3D cell
culture constructs aim to mimic the in vivo environment more accurately, providing cell-cell

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interaction and the physiological cues of the emulated microenvironment .
The formation of engineered tissue constructs is to date realized by either scaffold-based or the

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scaffold-free approaches . The scaffold-based approach uses natural or synthetic materials to
provide cells with a biodegradable structural support that promotes cellular adhesion and

proliferation. It also defines the mechanical properties of the engineered construct and can even

shield cells from external mechanical damage.

Scaffolding materials offer excellent opportunities for TE. However, conventional 3D porous

scaffolds, a crucial type of engineered biomaterials, pose challenges due to large seeding
variability, especially deep inside, and relatively low initial cell densities. Hydrogels and

hydrogel-based microparticles, another commonly used group, can directly embed cells,

ensuring their even distribution. Yet, they often have limited mechanical properties, similar to
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soft tissues, which can be problematic for applications like cartilage or bone TE . Notably,
scaffold-based approaches limit the overall architecture of the engineered construct mostly to
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the initial shape and size of the biomaterial scaffold prior cell seeding .
While advanced technologies, such as 3D bioprinting can fabricate complex tissue
architectures, they still face limitations in achieving high cell density and rapid tissue

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maturation due to bioink constraints and mechanical stresses during printing .
Typical scaffold-free approaches involve the use of cell aggregates (also referred to as
spheroids or pellets) and even cell-sheets, which can be stacked to produce building blocks for

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