Determining the mechanical properties of living cells accurately and repeatably is critical to understanding developmental, disease, and repair biology. The cellular environment is composed of fibrous proteins of a mix of diameters organized in random and aligned configurations. In the past two decades, several methods, including modified atomic force microscopy (AFM) and micro-pipette aspiration have been developed to measure cellular viscoelastic properties at single-cell resolution. We inquired if the fibrous environment affected cellular mechanobiology. Using our non-electrospinning Spinneret based Tunable Engineered Parameters (STEP) fiber manufacturing platform, we developed fused nanonets to measure single-cell forces and viscoelasticity. Using computer-controlled probes, we stretched single cells attached to two-fiber and three-fiber systems precisely and recorded the relaxation response of cells. The viscoelastic properties were determined by fitting the data to the standard linear viscoelastic solid model (SLS), which includes a spring (k0) in parallel with a spring (km)-damper (cm) series. In cases in which cells are seeded on two fibers, we tested hMSCs and BJ-5TA cells, and the viscoelastic components measurements k0, km, and cm are 26.16 ± 3.38 nN/µm, 5.81 ± 0.81 nN/µm, and 41.15 ± 5.97 nN-s/µm, respectively for hMSCs, while the k0, km, and cm, measurements of BJ-5TA cells are 20.02 ± 2.89 nN/µm, 4.62 ± 0.75 nN/µm, and 45.46 ± 6.00 nN-s/µm respectively. Transitioning to the three-fiber system resulted in an overall increase in native contractility of the cells while allowing us to understand how the viscoelastic response was distributed with an increasing number of fibers. Viscoelastic experiments were done twice. First, we pulled on the outermost fiber similar to the two-fiber case. The cell was then allowed to rest for two hours, sufficient time to regain its pre-stretching contractility. The cell was then excited by pulling on the middle fiber. The experimental results of cell seeding on three fibers proved that the viscoelastic property measurements depend on the excitation position. Overall, we present new knowledge on the cellular viscoelasticity of cells attached to ECM-mimicking fibers. / Master of Science / Investigating living cell mechanical properties including the viscoelastic properties of single living cell is critical to understanding developmental, disease, and repair biology. With the advancement of micrometer scale technologies, researchers are able to excite individual living cells. Current methods are mostly based on perturbing cells attached to flat 2D surfaces with limited physiological relevance. Since the native environment of cells is fibrous in nature, we inquired if cellular viscoelasticity could be measured of cells attached to suspended fibers. Using our non-electrospinning Spinneret based Tunable Engineered Parameters (STEP) fiber manufacturing platform, we developed fused nanonets to measure single-cell forces and viscoelasticity. Our suspended, aligned nanonet provides a unique way for us to pull on individual living cells using computercontrolled probes. By controlling the aligned fiber spacing, we are able to determine how many fibers the cells were seeded on. We first measured the viscoelastic properties of human mesenchymal stem cells(hMSCs) and human fibroblast BJ-5TA cells seeded on two fibers. The standard linear solid (SLS) model, which includes a spring in parallel with a spring-damper series, was used to quantitatively analyze the viscoelastic properties of cells. By giving the excitation on one fiber and measuring the cell forces on the other fiber, we calculated the corresponding spring constants and damping coefficients of the model. Then we investigated the viscoelastic properties of hMSCs seeded on three fibers by giving the excitation on the outermost fiber and then the middle fiber. Between the two excitations, the cell was allowed to relax for two hours and regain contractility. Our results confirm that the viscoelastic properties measurements depend on the excitation position. Overall, we present a new fiber-based force measurement system capable of determining the viscoelastic response of cells repeatably.