Development and validation of a multi-modal customized device to stimulate in vitro cell culture systems

This work focuses on the development and validation of a multi-modal stimulation device for in vitro cell culture systems. The device was designed to stimulate cells or tissues placed on 12-well culture plates. It is connected to customized software that controls the parameters of photobiomodulation (PBM) and ultrasound stimulation (US) through light-emitting diodes and piezoelectric disks, respectively. A wide range of stimulation protocols can be explored by modulating central frequency or wavelength, power density, and duration. Four different cell lines were used to validate the safety and functionality of the device. Human osteoblasts, chondrocytes, periodontal ligament fibroblasts, and mouse-derived neuronal cells were cultured and stimulated daily with ultrasound (1.0 MHz, 100 mW/cm², 5 min), light (810 nm, 7.5 mW/cm², 5 min) and combined stimuli. After three days, metabolic activity and proliferation were assessed. Different cell types demonstrated distinct biological responses to the stimuli, as osteoblasts and chondrocytes showed increased metabolic activity after combined stimulation or PBM, while the metabolic activity of human fibroblasts or neuronal-like cells was unchanged after three days. This highlights the importance of a rigorous optimization of stimulation protocols according to the target tissue. The safety of the device and its sterilization conditions were demonstrated as there was no cell death or contamination during in vitro stimulation. This work features a feasible, safe, and effective multi-modal stimulation device that can provide a wide range of stimulation protocols to better understand their effect on cells or tissues.