Effects of initial cell number, maturation stage, and compressive strain on the viscoelastic response of normal human dermal fibroblast spheroids.

Mono- and co-culture spheroids have emerged as important building blocks for tissue engineering and advanced three-dimensional in vitro models. However, the time-dependent mechanical behavior of juvenile normal human dermal fibroblast (NHDF) spheroids during early maturation remains insufficiently understood, limiting the optimization of tissue-engineering strategies and the development of predictive mechanical models. This study investigated the viscoelastic response of NHDF spheroids as a function of initial cell number, maturation state, compression strain, and donor identity. Spheroids generated by the liquid overlay technique (LOT) from 5,000, 10,000, and 50,000 cells each were characterized on days 2, 4, and 7 after formation. Spheroid diameter increased with initial cell number and decreased during maturation, indicating progressive structural remodeling. Uniaxial compression-relaxation experiments were performed using a nanoindenter at compression strains of 15-45%, followed by a 180 s hold phase. Force-displacement and force-time data were analyzed using a generalized Maxwell model comprising one fast and one slow relaxation element combined with a Hertzian contact-area approximation. Linear mixed-effects models were used to evaluate the effects of cell number, maturation state, compression strain, and donor-specific variability. The generalized Maxwell model consistently provided good fits and resolved distinct fast and slow relaxation timescales. The equilibrium effective modulus decreased markedly during maturation in 50,000-cell spheroids, from 2.57 kPa on day 2 to 1.13 kPa on day 7. In contrast, the relaxation amplitudes varied depending on the combination of cell number and maturation state. The slow relaxation time increased with initial cell number, whereas the fast relaxation time exhibited only minor overall changes. The loading condition, reflecting coupled changes in compressive strain and nominal strain rate, affected several fitted parameters, indicating that the apparent viscoelastic properties depend on the applied deformation protocol. Overall, the viscoelastic behavior of juvenile NHDF spheroids was determined by the combined effects of cell number, maturation, loading condition, and biological variability. These findings provide quantitative insights into the evolution of spheroid viscoelasticity during early maturation and support the development of predictive mechanical models for tissue-engineering applications.