细胞生物学

Neurons are highly polarized cells, with axons that may innervate distant target regions. In the brain, basal forebrain cholinergic neurons (BFCNs) possess extensive axons that project to several target regions such as the cortex, hippocampus, and amygdala, and may be exposed to a specific microenvironment in their axon targets that may have retrograde effects on neuronal health. Interestingly, BFCNs express the pan-neurotrophin receptor p75NTR throughout life while also concomitantly co-expressing all Trk receptors, making them capable of responding to both mature and precursor neurotrophins to promote survival or apoptosis, respectively. Levels of these trophic factors may be modulated in the BFCN axon or soma microenvironment under neurodegenerative conditions such as seizure and brain injury. In this protocol, BFCNs are established in microfluidic devices for compartmental culture, with the aim of studying the effects of axon- or soma-specific stimulation of BFCNs for an in vitro representation of distal axon vs. soma environments as seen in vivo. This study further establishes a novel method of tracing and imaging live BFCNs exposed to stimuli in their distal axons with the aim of assessing retrograde cell death. The in vitro compartmental culture system of BFCNs that allows live imaging may be applied to investigate various effects of axon- or soma-specific stimuli that affect BFCN health, maintenance, and death, to model events that occur in the context of brain injury and neurodegenerative disorders.

Neurons are complex cells with two distinct compartments: the somatodendritic and the axonal domains. Because of their polarized morphology, it is challenging to study the differential cellular and molecular mechanisms that occur in axons and impact the soma and dendrites using conventional in vitro culture systems. Compartmentalized cultures offer a solution by physically and chemically separating the axonal from the somatodendritic domain of neurons. The microfluidic chamber model presented in this work is valuable for studying these mechanisms in primary cortical cultures derived from rat and mouse. In addition, this chamber model is compatible with various microscopy methods, such as phase contrast, and fluorescence imaging of living and fixed cells.

Key features

• Preparation and attachment of PDMS microfluidic chambers to glass coverslips.

• Primary culture of cortical neurons and plating cortical neurons in microfluidic chamber.

• Confirmation of compartmentalization using the retrograde transport of the fluorescently labeled form of cholera toxin subunit B (f-Ctb).

• Immunofluorescence and multilabeling of compartmentalized cortical neurons.

• Retrograde transport of fluorescently labeled BDNF.

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.