Brain Aggregate Cell Cultures as an In Vitro Model for Neurotoxicity Testing
Published: February 2, 2009
Paul Honegger, Ph.D., Associate Professor
Florianne Monnet-Tschudi, Ph.D., Master of Education and Research
Marie-Gabrielle Zurich, Ph.D., Chief of Projects
Department of Physiology
Faculty of Biology and Medicine
University of Lausanne
Rue du Bugnon 7
Of particular urgency is the availability of cell-based testing systems for organ-specific toxicity, since it emerged clearly that simple cell culture systems, used successfully for basal cytotoxicity tests, often fail to detect organ-specific toxicity. Target organs of special interest include liver, kidney, immune system, and nervous system. The nervous system, comprising peripheral and central nervous systems (PNS and CNS), was found to be particularly vulnerable to diverse xenobiotics such as plant and animal poisons, heavy metals, industrial chemicals, agrochemicals, and pharmaceuticals.
Organ-specific toxicity testing in vitro calls for culture systems capable of representing the structural and functional particularities of a given target organ. With respect to the nervous system, typical structural specificities comprise the cellular heterogeneity (i.e., the presence of numerous neuronal subtypes, astrocytes, oligodendrocytes or Schwann cells, and microglia); elaborate neuronal networks with different types of chemical synapses; and myelinated axons. Typical functional particularities of the nervous system include the neuronal electrical activity and synaptic neurotransmission; neuron-glia interactions such as the metabolic coupling between neurons and astrocytes, the participation of astrocytes in synaptic integration, and the myelination of axons by oligodendrocytes or Schwann cells; and glia-glia interactions as exemplified by the reactivity of microglial cells and astrocytes in response to diverse brain insults, including neurotoxicant-induced perturbations.
In the CNS, neuronal energy metabolism, neurotransmitter synthesis, electrical activity, and glutathione homeostasis rely on the metabolic coupling between neurons and astrocytes. Neurons utilize glucose as the almost exclusive substrate for energy metabolism and neurotransmitter synthesis, but lack the anaplerotic enzymes necessary to replenish the tricarboxylic acid (TCA) cycle. Therefore, they depend on neighboring astrocytes for the supply of glutamine and glutamate. Astrocytes not only express the anaplerotic pathways lacking in neurons, they also are able to store glycogen, and to use it as a substrate for glutamate and glutamine synthesis. In contrast to neurons, astrocytes also metabolize acetate and ammonia. In the CNS, astrocytes are the only cell type able to detoxify ammonia, a regular byproduct of the neuronal metabolism of glutamate and other amino acids. In addition, astrocytes participate significantly in the metabolism of xenobiotics, predominantly via phase I enzymes, and they play a crucial role in the protection of neurons from oxidative stress through the homeostasis of glutathione and several additional antioxidants.
The metabolic dependency of neurons from astrocytes greatly contributes to the particular vulnerability of neurons to oxidative stress, vitamin deficiencies, and perturbations of metabolic pathways including glycolysis, TCA cycle, and the respiratory chain. Taken together, the multiple cell-cell interactions involved in critical physiological and pathogenic pathways imply that the presence of functionally interacting neurons and glial cells has to be an integrant part of any in vitro model to be used for neurotoxicity testing.
Brain aggregate cell cultures are 3-dimensional (3-D) cell cultures, most easily prepared from mechanically dissociated embryonic brain tissue (1). Under gyratory agitation, the dissociated immature cells re-aggregate spontaneously into even-sized spheroids, which are kept in suspension culture by continuous agitation at 80 revolutions per minute (rpm). These cultures contain all the different brain cell types, including the major neuronal subpopulations, astrocytes, oligodendrocytes, and microglial cells. The different cell types are present in proportions similar to the brain in vivo, and they are able to undergo histotypic maturation and extensive cell-to-cell interactions.
Initially, the re-aggregated brain cells contain considerable proportions of neuroblasts, glioblasts, and stem cells, the latter giving rise to progenitor cells that further differentiate into astrocytes and oligodendrocytes. Within 3 to 4 weeks in vitro, all cell types express their differentiated phenotype, and the cultures exhibit elaborate neuronal connectivity, myelinated axons, and numerous structurally and functionally mature synapses. Spontaneous and evoked electrical activity measured in the aggregates demonstrated the presence of functional neuronal networks. From a practical point of view, brain aggregate cultures were the first neural cell cultures to be grown in a chemically defined medium (2). This enabled the study of epigenetic factors, showing for the first time a central action of nerve growth factor (3) and of insulin-like growth factor I (4), against the then held view that these factors occur and act exclusively outside the CNS.
Aggregate cultures have been used successfully for neurotoxicological investigations, e.g., to study the neurotoxicity of heavy metals (5-10), neuron-specific toxicants (11-13), demyelinating agents (14), organophosphorous compounds (15), mycotoxins (16-17), and ammonia (18-20) as well as to investigate developmental neurotoxicity (21-27). Many observations confirm the importance of cell-cell interactions in xenobiotic neurotoxicity pathways, e.g., the decreased gliotoxicity of lead in 3-D neuron-glia co-cultures as compared to glia-enriched cultures (8); the high neurotoxicity of the gliotoxin glufosinate, an irreversible inhibitor of glutamine synthetase, in aggregate cultures but not in isolated neuronal cultures, suggesting physiological metabolic coupling between neurons and astrocytes in 3-D cultures; the extreme neurotoxicity of organophosphorous compounds such as parathion and chlorpyrifos in aggregate cultures indicating metabolic activation through glial phase I enzymes (16,26); the reactivity of microglial cells and astrocytes to various neuronal insults indicating inflammatory responsiveness (28).
In a recent project sponsored by the European Commission and aimed at the development of testing strategies for acute systemic toxicity (ACuteTox), some 60 reference compounds were tested in various in vitro systems. Using aggregate cell cultures prepared from embryonic rat brain and a multiparametric endpoint scheme, all chemicals known to be highly toxic in humans also showed high toxicity (significant effects in the lower micromolar range) to extreme toxicity (significant effects at nanomolar concentrations) in aggregate cultures. In comparison with the results obtained by other groups using cell lines for basal cytotoxicity testing, aggregate cultures showed at least 10-fold higher sensitivity (tentatively taken as a criterion for outliers) for 46% of the compounds when compared with a 3T3 cell line, and for 37% of the compounds when compared with a NHK cell line. Aggregate cultures also showed more accurate prediction with respect to human neurotoxicity when compared with a human neuroblastoma cell line (SH-SY5Y) and with monolayer cultures of normal rat brain neurons.
These studies also confirmed the high reproducibility of aggregate replicate cultures, and the importance of multiparametric endpoints to detect adverse effects (altogether, 14 individual endpoints were measured). Furthermore, they showed the possibility to automate the most time-consuming parts of culture maintenance and endpoint analyses. The fact that the aggregates are kept as free-floating cultures and that their diameter is only 200 –300 microns allowed simple pipetting for the preparation of culture replicates. Some 1200-1500 aggregates were derived from one rat embryo. In the ACuteTox study, each replicate culture contained 200-250 aggregates to ensure high robustness and high reproducibility of the method. In total, 450 individual tests were performed in some 100 experiments, using about 4000 replicate cultures of 22 different culture batches. Less than 1% of all tests had to be rejected because they did not meet the acceptance criteria, illustrating the robustness of this model.
The available evidence clearly shows the suitability of brain aggregate cultures as a model system for neurotoxicity testing, due to their organotypic structural and functional features. Additional advantages of these cultures include their high yield enabling the parallel testing of a series of xenobiotics and multiparametric endpoint determinations; their growth and maturation in a chemically defined culture medium; the easy handling of the free-floating aggregates; the possibility of repetitive sampling; and the high reproducibility of the resulting data. Beside the neurotoxicity testing with mature cultures at acute or prolonged exposure conditions, aggregate cultures offer also a model for developmental neurotoxicity studies, since they were shown to reproduce a series of crucial maturational events to attain their final level of differentiation.
For the ultimate validation of this culture system, it will be necessary to increase the throughput for routine testing, and to select a set of endpoints that are sufficiently sensitive and predictive for neurotoxicity detection. An increased throughput could be achieved by a reduction of the size of the replicate cultures, and by the use of automated techniques for culture maintenance, replicate preparation, and endpoint measurements. With respect to the choice of suitable enpoints, it has to be taken into account that the molecular and cellular targets for xenobiotics are numerous and in most cases unknown. Furthermore, most of the toxic reactions induced by xenobiotics seem to converge towards a limited number of identified toxicity pathways. It therefore appears most advantageous to probe for “downstream” indicators of xenobiotic adverse effects, referring either to the perturbation of cell type-specific structural or functional traits (e.g., changes in synaptic, cytosketal and myelin components; changes in energy metabolism; changes in cellular transduction pathways) or to the activation of homeostatic defense mechanisms (e.g., changes in the expression of heat shock proteins, oxidative stress responses, inflammatory responses, apoptosis).
Our previous work showed that multiparametric (high content) analyses were most efficient and most reliable in the detection of adverse effects. A routine test protocol was used successfully for the simultaneous measurement of LDH release (indicating cell membrane leakage); glucose consumption (reflecting either changes in neuronal electrical activity or perturbations in cellular pathways of energy metabolism); the rate of total RNA synthesis; and changes in the expression of neurofilament protein, glial fibrillary acidic protein, myelin basic protein, and HSP-32 by real-time PCR using an automated workstation. Depending on the aim of the study, an extended genomic assay, and additional indicators of functional perturbations were used including changes in the activity of cell type-specific enzymes and apoptosis executors. For developmental studies, changes in mitotic activity and growth factor responsiveness as well as changes in the expression of cell type-specific traits were found to be reliable endpoints.
Until now, brain aggregate cell cultures were prepared mainly from brain tissue of rat or mouse embryos. Most recently, a new procedure was published for the preparation of 3-D neural cell cultures from human stem cells (29). It remains to be explored whether this method can provide organotypic cultures in sufficient quantities for routine testing, and whether these cultures can be enriched with microglial cells or macrophages. If so, this approach would reach the ultimate level of aggregate cell culture elaboration, since it would allow the complete replacement of animals in neurotoxicity testing, and avoid species-specific confounding factors. However, the importance of species-specific differences in xenobiotic neurotoxicity is likely to be overestimated, since in most cases it may concern differences in ADME and PBPK, for which complementary models will be available, and clearly will be required for whatever in vitro system of neurotoxicity testing will finally reach validation.
At any rate, the susceptibility to (neuro)toxic chemicals varies also among human beings, depending on the relative activity of individual phase I and phase II enzymes, which may differ considerably among individuals due to the genetic polymorphism of some of these metabolic enzymes, and even in the same subject because of many additional physiological and chemical modifying factors. There will always remain some uncertainty with respect to the distribution, metabolism and action of chemicals in the human organism, which ultimately may be resolved by direct human exposure data and eventual in-life testing.
©2009 Paul Honegger, Florianne Monnet-Tschudi & Marie-Gabrielle Zurich
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