Spatial distribution of encapsulated cells dramatically alters biomechanical properties and microstructure of 3D printed cellular structures

Additive manufacturing to fabricate tissue/organ models finds various applications in tissue engineering and regenerative medicine. The bioink containing extracellular matrix and living cells is layer-by-layer printed into 3D structures with cells encapsulated. During bioprinting, cells in the bioink sediment and aggregate with each other, resulting in non-uniform spatial distribution of cells encapsulated in the printed structure. In the following post-printing incubation, the encapsulated cells interact with the extracellular matrix resulting in eventual functionality of the printed tissues/organs. This is the first paper to elaborate how the spatial distribution of encapsulated cells dramatically alters biomechanical properties and microstructure of 3D printed cellular structures. Specifically, the effects of the cell aggregates on post-printing properties and microstructure have been investigated in terms of cell viability, growth & proliferation, mechanical properties, and pore size & microchannels. Main conclusions include: (1) Nonuniform distribution results in a reduced spreading area and increased aspect ratio due to limited space for cell expansion/spreading in compacted aggregates; (2) Cell viability notably decreases from 96 % to 60 % under nonuniform distribution, due to insufficient oxygen/nutrient exchange in the core of large aggregates; (3) The measured (kPa)/normalized Young's Modulus at local aggregates under nonuniform distribution over control notably decreases from 8.6/0.96 to 2.1/0.35, compared to that decreasing from 8.9/1 to 4.0/0.65 under uniform distribution. Nonuniform larger cavities (10–50 μm) were exclusively observed in SEM, confirming the local microstructure degradation by aggregates. Microchannels in width of 5–20 μm and length of >100 μm were also observed as cell migration traits.