Page 101 - Live-cellanalysis handbook

Kinetic Assays for Studying Neuronal Cell Models

Assays for studying neuronal cell health, function and morphology long-term

Effective modeling for neurological disease, injury, and attributes are (1) relevant, informative analyses based on the
development is critical for identifying novel and effective minimization of artifacts arising from cell perturbation, (2) long-
treatments. However, due to the complexity and plasticity of term monitoring of biological events that unfold over days, weeks
the nervous system, inaccessibility of human diseased tissue, or even months, and (3) sufficient miniaturization, throughput and
sensitivity of neurons to perturbation, and lack oftranslational ease of use to enable replication, controls and overall experimental
value of animal models, elucidating the function of the nervous productivity. The attributes of live-cell analysis offer simple and
system and identifying novel treatments is challenging. With reliable assays which offer new biological insights into basic and
advances in stem cell technologies, the promise to create advanced neuronal cell models.In the subsequent chapters, the
differentiated neurons and support cells (e.g. microglia, astrocytes) assays are described in detail, demonstrating their utility for
that accurately represent human phenotypes in order to build characterizing and validating translational cell models for the
translational and patient-specific human models now exists. To discovery of novel neurotherapeutics.
fulfill this promise, considerable work is required to optimize the
reprogramming and differentiation methods, and to build and Live-cell imaging and analysis approaches for studying
validate cellular bioassays that are representative of the native neuronal cell dynamics
human pathophysiology.
Building on existing methodologies employed to study cell health,
To facilitate the evaluation and characterization of human induced morphology and function, novel strategies were developed in
pluripotent stem cells (hiPSCs), many technologies are employed order to quantify long-term changes in neurite dynamics, neuronal
to measure various aspects of cell health, morphology and activity, as well as readouts for neuroimmune functions, such as
function, such as immunocytochemistry methods, microelectrode phagocytosis and chemotaxis of microglia. The IncuCyte Live-
electrophysiology techniques, and flow cytometry. Unfortunately, Cell Analysis methodology consists of instrumentation, software
they are not amenable to monitoring long-term changes (days modules, lab-tested protocols and reagents that enable continuous
and weeks) and sample preparation can perturb fragile neuronal interrogation of sensitive neuronal cell models. In order to
cells, thus compromising accurate analysis of dynamic biological accomplish this, instrumentation was designed to quantify cell
changes and responses to treatment. The ability to evaluate long- behavior with uninterrupted incubation provided by a cell culture
term changes and characterize complex biological models in a incubator and a mobile optical train the enables sample to stay
non-invasive manner from a physiologically relevant environment stationary and reduce physical disturbance. Purpose-built software
offers considerable advantages in characterizing the function of analysis modules automatically segment and analyze images to
the nervous system. create full time course plots for each well in 96- and 384-well
plates. Finally, combining novel, non-perturbing reagents that
Live-cell analysis with the IncuCyte addresses the inherent deploy longer wavelength fluorophores designed for neuronal-
shortcomings of traditional assays through non-invasive, specific measurements along with lab-tested protocols ensures
automated time-lapse imaging and analysis from within a cell reproducible and unprecedented access to phenotypic information.
incubator. Phase-contrast, brightfield and fluorescence images The flexibility of this instrument, novel reagents, and standardized
are acquired under uninterrupted environmental control, and protocols, combined with higher throughput capabilities and
then analyzed and quantified in real time to report changes in capacity for long-term, unperturbed culture enables this system
morphology, movement, activity and function. Time-lapse videos to overcome many of the challenges associated with traditional
can be created to verify the experimental outcomes. The critical endpoint workflows.

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