Cell Biology

Due to cell heterogeneity, the differences among individual cells are averaged out in bulk analysis methods, especially in the analysis of primary tumor biopsy samples from patients. To deeply understand the cell-to-cell variation in a primary tumor, single-cell culture and analysis with limited amount of cells are in high demand. Microfluidics has been an optimum platform to address the issue given its small reaction volume requirements. Digital microfluidics, which utilizes an electric signal to manipulate individual droplets has shown promise in cell-culture with easy controls. In this work, we realize single cell trapping on digital microfluidic platform by fabricating 3D microstructures on-chip to form semi-closed micro-wells. With this design, 20% of 30 x 30 array can be occupied by isolated single cells. We also use a low evaporation silicon oil and a fluorinated surfactant to lower the droplet actuation voltage and prevent the drop from evaporation, while allowing cell respiration during the long term of culture (24 h). The main steps for single cell trapping on digital microfluidics, as illustrated in this protocol, include 3D microstructures design, 3D microstructures construction on chip and oil film with surfactant for single cell trapping on chip.

The deposition of misfolded, aggregated tau protein is a hallmark of several neurodegenerative diseases, collectively termed “tauopathies”. Tau pathology spreads throughout the brain along connected pathways in a prion-like manner. The process of tau pathology propagation across circuits is a focus of intense research and has been investigated in vivo in human post-mortem brain and in mouse models of the diseases, in vitro in diverse cellular systems including primary neurons, and in cell free assays using purified recombinant tau protein. Here we describe a protocol that takes advantage of a minimalistic neuronal circuit arrayed within a microfluidic device to follow the propagation of tau misfolding from a presynaptic to a postsynaptic neuron. This assay allows high-resolution imaging as well as individual manipulation of the releasing and receiving neuron, and is therefore beneficial for investigating the propagation of tau and other misfolded proteins in vitro.

In the study of neurodegenerative diseases, it is imperative to study the cellular and molecular changes associated with pathogenesis in the relevant cell type, central nervous system neurons. The unique compartmentalized morphology and bioenergetic needs of primary neurons present complications for their study in culture. Recent microculture techniques utilizing microfluidic culture devices allows for environmental separation and analysis of neuronal cell bodies and neurites in culture. Here, we present our protocol for culture of primary neurons in microfluidic devices and their chronic treatment with the Parkinson’s disease (PD) relevant toxicant rotenone. In addition, we present a method for reuse of devices for culture. This culture methodology presents advantages for evaluating early pathogenic cellular and molecular changes in neurons in a compartment-specific manner.