Cell Biology

Pathological conditions of the cervix ranging from cervical cancer to structural dysfunction associated with preterm labor all have limited treatment options. Thus, there is a need for physiologically relevant preclinical models that recapitulate the structure and function of this human organ. Here, we describe a protocol for engineering and studying a highly functional in vitro model of the human cervix that is composed of a commercially available, dual-channel, microfluidic, organ-on-a-chip (Organ Chip) device lined by primary cervical epithelial (CE) cells interfaced across a porous membrane with cervical stromal cells. The provision of dynamic and customized media flow through both the epithelial and stromal compartments results in cell growth and differentiation, including the accumulation of a thick mucus layer overlying the epithelium. The resulting model closely mimics the structure, epithelial barrier, mucus composition and structure, and biochemical properties of the in vivo human cervix, as well as its responsiveness to female hormones, pH, and microbiome. This Cervix Chip protocol also includes noninvasive techniques for longitudinal monitoring of the live 3D tissue model. The Cervix Chip offers a powerful preclinical platform for replicating in vivo cervical physiology, studying disease mechanisms, and facilitating the development of new therapeutics and diagnostics.

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.