A comprehensive comparison of cell seeding methods using highly porous melt electrowriting scaffolds

Cell seeding is challenging in the case of additively manufactured 3-dimensional scaffolds, as the open macroscopic pore network impedes the retention of the seeding solution. The present study aimed at comparing several seeding conditions (no fetal bovine serum, 10% or 100% serum) and methods (Static seeding in Tissue Culture Treated plate (CT), Static seeding of the MES in non-Culture Treated plate (nCT), Seeding in nCT plate placed on an orbital shaker at 20 rpm (nCTR), Static seeding of the MES previously incubated with 100% FBS for 1 h to allow for protein adsorption (FBS)) commonly utilised in tissue engineering using highly porous melt electrowritten scaffolds, assessing their seeding efficacy, cell distribution homogeneity and reproducibility. Firstly, we demonstrated that the incubation in 100% serum was superior to the 10% serum pre-incubation and that 1 h only was sufficient to obtain enhanced cell attachment. We further compared this technique to the other methods and demonstrated significant and beneficial impact of the 100% serum pre-incubation, which resulted in enhanced efficacy, homogeneous cell distribution and high reproducibility, leading to accelerated colonisation/maturation of the tissue engineered constructs. We further showed the superior performance of this method using 3D-printed scaffolds also made of different polymers, demonstrating its capacity for up-scaling. Therefore, the pre-incubation of the scaffold in 100% serum is a simple yet highly effective method for enhancing cell adhesion and ensuring seeding reproducibility. This is crucial for tissue engineering applications, especially when cell availability is scarce, and for product standardisation from a translational perspective.