A Novel Microfluidic Platform for Biomechano-Stimulations on a Chip.

Mechanical stress has been proven to be an important factor interfering with many biological functions through mechano-sensitive elements within the cells. Despite the current interest in mechano-transduction, the development of suitable experimental tools is still characterized by the strife to design a compact device that allows high-magnification real-time imaging of the stretched cells, thus enabling to follow the dynamics of cellular response to mechanical stimulations. Here we present a microfluidic multi-layered chip that allows mechanical deformation of adherent cells maintaining a fixed focal plane, while allowing independent control of the soluble microenvironment. The device was optimized with the aid of FEM simulation and fully characterized in terms of mechanical deformation. Different cell lines were exposed to tunable mechanical strain, which results in continuous area deformation up to 20%. Thanks to the coupling of chemical glass etching, 2-dimensional deformation of a thin elastomeric membrane and microfluidic cell culture, the developed device allows a unique combination of cell mechanical stimulation, in line imaging and accurate control of cell culture microenvironment.

Optimal periodic perfusion strategy for robust long-term microfluidic cell culture.

Long-term cell culture in microfluidic devices is an essential prerequisite for "on a chip" biological and physiological based studies. We investigated how medium delivery, from continuous to periodic perfusion, affects long-term cell cultures in a microfluidic platform. Computational simulations suggested that different delivery strategies result in different temporal profiles of accumulation and washing out of endogenous (EnF) and exogenous (ExF) factors, respectively. Thus, cultures exposed to the same overall amount of medium with different temporal profiles were analysed in terms of homogeneity, cell morphology and phenotype. Murine and human cell lines (C2C12 and HFF) and mouse embryonic stem cells (mESC) were cultured in microfluidic channels. An ad hoc experimental setup was developed to perform continuous and periodic medium delivery into the chip, tuning the flow rate, the perfusion time, and the interval of perfusion while using the same amount of medium volume. Periodic medium delivery with a short perfusion pulse ensured cell homogeneity compared to standard cell culture. Conversely, a continuous flow resulted in cell heterogeneity, with abnormal morphology and vesiculation. Only dramatic and unfeasible increasing of perfused medium volume in the continuous configuration could rescue normal cell behaviour. Consistent results were obtained for C2C12 and HFF. In order to extend these results to highly sensitive cells, mESC were cultured for 6 days in the microfluidic channels. Our analysis demonstrates that a periodic medium delivery with fast pulses (with a frequency of 4 times per day) resulted in a homogeneous cell culture in terms of cell viability, colony morphology and maintenance of pluripotency markers. According to experimental observations, the computational model provided a rational description of the perfusion strategies and of how they deeply shape the cell microenvironment in microfluidic cell cultures. These results provide new insight to define optimal strategies for homogeneous and robust long-term cell culture in microfluidic systems, an essential prerequisite for lab on chip cell-based applications.