21 3.10 – in vitro Neuronal Models
A variety of in vitro models can be used to examine circuits as well as to test many of the techniques mentioned in this unit and to help understand development of the nervous system. Often these in vitro models have been developed to provide a simplified understanding of the much more complex in vivo condition. These reduced complexity models allow for a simplified approach to studying key neuronal processes on both the cellular and molecular level. At the same time, some tissue preparations suffer several limitations due to their simplicity, reducing direct in vivo comparisons.
Expression systems and immortalized cell lines
The simplest in vitro electrophysiological models include heterologous and recombinant expression systems which are cells/cell lines that can be maintained in culture for an extended period of time. The cells/cell lines typically used as heterologous (e.g., Xenopus oocytes; or recombinant expression systems (e.g., human embryonic kidney 293 (HEK-293) cells, Chinese hamster ovary (CHO) cells)) that are easily maintained, allow for manual and automated electrophysiological techniques and express high levels of desired protein within a short period of time that can be consistently observed. As such, these systems have been used extensively to evaluate the pharmacological properties and structure-function relationships of multiple neuron ion-channels. However, despite their simplicity and ubiquitous use, these cells lack many of the complexities associated with neuronal function within the intact brain (e.g. network associations, glial interactions, and developmental regulation) — a disadvantage when modeling the brain. Furthermore, these cells are typically of a non-neuronal origin and thus lack the same sophisticated level of cellular architecture, sub-cellular organization or biochemistry associated with native neuronal preparations.
Early efforts to address these non-neuronal concerns focused on neuronal cells derived from mouse neuroblastoma C-1300 tumor (e.g. N1E-115)) or the human SH-SY5Y neuroblastoma cell line. However, subsequent advances in molecular biology enable the use of neural stem cells (NSCs). NSCs are uncommitted cells with self-renewal potential and the ability to differentiate into cells of all neural lineages. These cells can be derived from several sources such as pluripotent embryonic stem cells isolated from the blastocyst, human umbilical cord blood, induced pluripotent stem cells and multipotent somatic progenitors derived from several tissues including the CNS. Electrophysiologically, these cells possess Na+, K+ and Ca2+ currents that resemble the known patterns described for their in vivo neuronal counterparts, even at early stages of differentiation. Furthermore, these cells are also capable of forming rudimentary, yet functional, glutamatergic and GABAergic synapses in culture. Limitations in the use of cells obtained from adults offer limited neural lineage potential and senesce after only a few passages (Jakel, Schneider, & Svendsen, 2004). Moreover, NSC cultures may possess mixtures of both undifferentiated and differentiated neurons, for which some neurons are developmentally immature, and thus hinder extrapolation of data to the adult in vivo condition.
Dissociated neuronal primary cultures
Increasing in complexity, dissociated neuronal primary cultures represent another common tissue preparation. These cultures are mechanically and enzymatically dissociated from various brain regions (e.g., hippocampus, cortex, cerebellum, striatum, midbrain, superior cervical ganglion, etc.) and consist of either one predominant neuronal cell type, a co-mixture of different neuronal populations or mixed neuronal–glial cultures. Dissociated neurons and astrocytes retain much of their functional capacity in vitro enabling these preparations to address many important processes observed in the in vivo condition such as network dynamics and neuronal-glial interactions but dissociated neurons cannot be maintained in culture for extended periods of time and thus are required to be freshly isolated and grown on a regular basis.
Three-dimensional (3D) neuronal organoid models
The 3D neuronal model represents the next level of complexity for CNS in vitro models. Like the two-dimensional (2D) preparations discussed above, 3D brain cell cultures can consist of a co-mixture of different neuronal and non-neuronal populations. Interestingly, instead of being cultured in a traditional planar monolayer, 3D brain cultures are created up to 10 cell diameters thick within reaggregate or spherical cultures (i.e. spheroids), hydrogel/scaffold cultures or rotary bioreactor cultures with cell aggregates or microcarriers. When grown in a 3D environment, neural cells demonstrate better survivability and behave differently when compared to traditional 2D-models. As such, these models promote better development of native voltage-gated ion-channel functionality, resting membrane potentials, intracellular Ca2 + dynamics, Na+/H+ exchange, enhanced neurogenesis and differentiation, synapse formation, neuronal mobility and axon myelination (Lancaster & Knoblich, 2014; Lancaster et al., 2013; LaPlaca et al., 2010; van Vliet et al., 2007).