Organotypic brain slice cultures: an efficient and reliable method for neurotoxicological screening and mechanistic studies

This paper reviews the current state of the use of organotypic brain slice cultures for neurotoxicological and neuropharmacological screening and mechanistic studies, as exemplified by excitotoxin application. At present, no in vitro systems have been approved by the regulatory authorities for neurotoxicity testing. For the evaluation of the slice culture method, organotypic hippocampal slice cultures were exposed to toxic doses of the excitotoxins, glutamate, N-methyl-D-aspartate (NMDA), kainic acid and 2-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and the glial toxin, DL-alpha-aminoadipic acid (DLAAA). Neuronal cell death was quantified by propidium iodide (PI) uptake, and visualised by Fluoro-Jade (FJ) staining. General cell death was monitored by lactate dehydrogenase (LDH) release into the culture medium. EC50 values for the different compounds, based on PI uptake after exposure for 48 hours in entire cultures, were: glutamate, 3.5 mM; DL-AAA, 2.3 mM; kainic acid, 13 microM; NMDA, 11 microM; and AMPA, 3.7 microM. In the slice cultures, the hippocampal subfields displayed the same differences in vulnerability as those observed in vivo. When subfield analysis was performed on the cultures, the CA1 subfield was most susceptible to glutamate, NMDA and AMPA, while CA3 was most susceptible to kainic acid. The amount of LDH release for DL-AAA was about four times that of L-glutamate, in accordance with the additional toxic effect on glial cells, which was also found by confocal microscopy to stain for FJ. In conclusion, it was found that organotypic brain slice culture, combined with standardised protocols and quantifiable markers, such as PI and FJ staining, is a relevant and feasible in vitro system for neurotoxicity testing. Considering the amount and quality of the available published data, it is recommended that the brain slice culture method could be subjected to pre-validation and formal validation for inclusion in a tiered in vitro neurotoxicity testing scheme to supplement and replace conventional animal tests.     

Cytosine arabinofuranoside-induced activation of astrocytes increases the susceptibility of neurons to glutamate due to the release of soluble factors

Activation of astrocytes occurs during many forms of CNS injury, but its importance for neuronal survival is poorly understood. When hippocampal cultures of neurons and astrocytes were treated from day 2–4 in vitro (DIV 2–4) with 1 μM cytosine arabinofuranoside (AraC), we observed a stellation of astrocytes, an increase in glial fibrillary acidic protein (GFAP) level as well as a higher susceptibility of the neurons to glutamate compared with cultures treated from DIV 2–4 with vehicle. To find out whether factors released into the culture medium were responsible for the observed differences in glutamate neurotoxicity, conditioned medium of AraC-treated cultures (MCMAraC) was added to vehicle-treated cultures and conditioned medium of vehicle-treated cultures (MCMvh) was added to AraC-treated cultures 2 h before and up to 18 h after the exposure to 1 mM glutamate for 1 h. MCMAraC increased glutamate neurotoxicity in vehicle-treated cultures and MCMvh reduced glutamate neurotoxicity in AraC-treated cultures. Heat-inactivation of MCMvh increased, whereas heat-inactivation of MCMAraC did not affect glutamate toxicity suggesting that heat-inactivation changed the proportion of factors in MCMvh inhibiting and exacerbating the excitotoxic injury. Similar findings were obtained using conditioned medium of pure astrocyte cultures of DIV 12 treated from DIV 2–4 with vehicle or 1 μM AraC suggesting that heat-sensitive factors in MCMvh were mainly derived from astrocytes. Treatment of hippocampal cultures with 1 mM dibutyryl-cAMP for 3 days induced an activation of the astrocytes similar to AraC and increased neuronal susceptibility to glutamate. Our findings provide evidence that activation of astrocytes impairs their ability to protect neurons after excitotoxic injury due to changes in the release of soluble and heat-sensitive factors.     

Validation of organotypical hippocampal slice cultures as an ex vivo model of brain ischemia: different roles of NMDA receptors in cell death signalling after exposure to NMDA or oxygen and glucose deprivation

N-Methyl-D-aspartate receptors (NMDARs) are essential mediators of synaptic plasticity under normal physiological conditions. During brain ischemia, these receptors are excessively activated due to glutamate overflow and mediate excitotoxic cell death. Although organotypical hippocampal slice cultures are widely used to study brain ischemia in vitro by induction of oxygen and glucose deprivation (OGD), there is scant data regarding expression and functionality of NMDARs in such slice cultures. Here, we have evaluated the contribution of NMDARs in mediating excitotoxic cell death after exposure to NMDA or OGD in organotypical hippocampal slice cultures after 14 days in vitro (DIV14). We found that all NMDAR subunits were expressed at DIV14. The NMDARs were functional and contributed to cell death, as evidenced by use of the NMDAR antagonist MK-801 (dizocilpine). Excitotoxic cell death induced by NMDA could be fully antagonized by 10 μM MK-801, a dose that offered only partial protection against OGD-induced cell death. Very high concentrations of MK-801 (50–100 μM) were required to counteract cell death at long delays (48–72 h) after OGD. The relative high dose of MK-801 needed for long-term protection after OGD could not be attributed to down-regulation of NMDARs at the gene expression level. Our data indicate that NMDAR signaling is just one of several mechanisms underlying ischemic cell death and that prospective cytoprotective therapies must be directed to multiple targets.     

Adult neural stem/progenitor cells reduce NMDA-induced excitotoxicity via the novel neuroprotective peptide pentinin

Although the potential of adult neural stem cells to repair damage via cell replacement has been widely reported, the ability of endogenous stem cells to positively modulate damage is less well studied. We investigated whether medium conditioned by adult hippocampal stem/progenitor cells altered the extent of excitotoxic cell death in hippocampal slice cultures. Conditioned medium significantly reduced cell death following 24 h of exposure to 10 μM NMDA. Neuroprotection was greater in the dentate gyrus, a region neighboring the subgranular zone where stem/progenitor cells reside compared with pyramidal cells of the cornis ammonis. Using mass spectrometric analysis of the conditioned medium, we identified a pentameric peptide fragment that corresponded to residues 26–30 of the insulin B chain which we termed 'pentinin'. The peptide is a putative breakdown product of insulin, a constituent of the culture medium, and may be produced by insulin-degrading enzyme, an enzyme expressed by the stem/progenitor cells. In the presence of 100 pM of synthetic pentinin, the number of mature and immature neurons killed by NMDA-induced toxicity was significantly reduced in the dentate gyrus. These data suggest that progenitors in the subgranular zone may convert exogenous insulin into a peptide capable of protecting neighboring neurons from excitotoxic injury.     

Transient increase in the high affinity [3H]-L-glutamate uptake activity during in vitro development of hippocampal neurons in culture

The glial GLAST and GLT-1 glutamate transporters are transiently expressed in hippocampal neurons as shown by immunocytochemistry (Plachez et al., 2000. J. Neurosci. Res., 59, 587-593). In order to test if this transient expression is associated to a transient glutamate uptake activity, [3H]-glutamate uptake was studied during the in vitro development of embryonic hippocampal neurons cultured in a defined (serum free) medium. In these cultures, the ratio of the number of glial cells to the number of neurons increased from 1.7 to 11.3% during the first 10 days of culture, while 77% of the neurons died. The number of neurons then remains stable up to 23 days of culture. The initial glutamate uptake velocity at 20 and 200 microM [3H]-glutamate usually increased about five times between 1 and 10 days in vitro (DIV). Interestingly, at 2 microM [3H]-glutamate, the uptake initial velocity showed a biphasic pattern, with a transient peak between 1 and 6 DIV, the maximum being reached at 2 DIV and a delayed regular increase from 8 to 23 DIV. The concentration-dependent curves were best fitted with two saturable sites high and low affinities, at both 2 and 10 DIV. To pharmacologically characterize the transient increased glutamate uptake activity, four uptake inhibitors, L-threo-3-hydroxy-aspartic acid (THA), L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-2,4-PDC), dihydrokainate (DHK), and DL-threo-beta-benzyloxyaspartate (TBOA) were tested. THA, L-trans-2,4-PDC and DL-TBOA inhibited glutamate uptake both at 2 and 10 DIV, while the GLT-1 selective uptake inhibitor DHK neither strongly affected the uptake at 2, nor at 10 DIV. These data indicated that, besides the regular increase in the glial-dependent glutamate uptake activity, a transient high-affinity, DHK insensitive, glutamate transport activity in hippocampal neurons in culture is present. This latter activity could potentially be related to the transient expression of the glial GLAST transporter in neurons.     

Cytoprotection by pyruvate through an anti-oxidative mechanism in cultured rat calvarial osteoblasts

Although we have previously shown drastic cell death by pyruvate deficiency in osteoblasts at the proliferative stage, the exact mechanism remains unclear so far. Cell survivability was significantly decreased in rat calvarial osteoblasts cultured for 0 to 3 days in vitro (DIV) following replacement of the eutrophic alpha-modified minimum essential medium (alpha-MEM) with Dulbecco's modified eagle medium (DMEM) for cultivation. The addition of pyruvate enriched in alpha-MEM, but not in MEM, entirely prevented cell death induced by the medium replacement throughout a culture period from 0 to 3 DIV. Both cysteine and reduced glutathione protected cell death in cells cultured for 3 DIV without significantly affecting that in cells cultured for 1 DIV, however, while none of lactate, acetate and insulin significantly prevented the cell death irrespective of the culture period up to 3 DIV. A marked increase was detected in intracellular reactive oxygen species (ROS) levels 4 h after the medium replacement. In osteoblasts cultured in alpha-MEM for 3 DIV, but not in those for 7 DIV, hydrogen peroxide (H2O2) markedly decreased cell survivability when exposed for 2 to 24 h. Furthermore, H2O2 was effective in significantly decreasing cell survivability in osteoblasts cultured in DMEM for 7 DIV. Pyruvate at 1 mM not only prevented cell death by H2O2, but also suppressed the generation of intracellular ROS in osteoblasts exposed to H2O2. These results suggest that pyruvate could be cytoprotective through a mechanism associated with the anti-oxidative property rather than an energy fuel in cultured rat calvarial osteoblasts.     

Cell Suspension and Callus Culture from Somatic Tissue of Maize

Cell suspension and callus cultures from somatic tissue of inbred lines of maize (Zea mays L.) were cultured on media that were defined via modification of a Linsmaier and Skoog preparation. Germlings incubated on the primary medium originally employed required long-term incubation for callus induction. Modification of the primary medium with high levels of iron and (ethylene dinitrillo)tetraacetic acid (EDTA), B vitamin amendments and vitamin E, shortened incubation by 75% and nearly doubled the percentage of germlings which produced callus. Callus did not remain viable in subcultures to the secondary medium originally employed, whereas a preparation, developed via modification of the original secondary medium, enabled perpetuation of callus through repeated subculture. Modification with high levels of iron and EDTA, plug B vitamins and vitamin E, with decreased concentrations of five inorganic salts, suppressed aberrant organogenesis and stabilized culture growth as viable callus. Similar modification, with the exception that EDTA was omitted, was employed for the development of a liquid medium. Tonicity of the medium was adjusted with a lowered level of sucrose, with the liquid further modified by addition of acetate. Upon development of this liquid, maize became the sixth monocot species for which somatic cells remain viable through repeated subculture in liquid suspensions.     

Ultrastructural analysis of chemical synapses and gap junctions between Drosophila brain neurons in culture

Dissociated cultures from many species have been important tools for exploring factors that regulate structure and function of central neuronal synapses. We have previously shown that cells harvested from brains of late stage Drosophila pupae can regenerate their processes in vitro. Electrophysiological recordings demonstrate the formation of functional synaptic connections as early as 3 days in vitro (DIV), but no information about synapse structure is available. Here, we report that antibodies against pre-synaptic proteins Synapsin and Bruchpilot result in punctate staining of regenerating neurites. Puncta density increases as neuritic plexuses develop over the first 4 DIV. Electron microscopy reveals that closely apposed neurites can form chemical synapses with both pre- and postsynaptic specializations characteristic of many inter-neuronal synapses in the adult brain. Chemical synapses in culture are restricted to neuritic processes and some neurite pairs form reciprocal synapses. GABAergic synapses have a significantly higher percentage of clear core versus granular vesicles than non-GABA synapses. Gap junction profiles, some adjacent to chemical synapses, suggest that neurons in culture can form purely electrical as well as mixed synapses, as they do in the brain. However, unlike adult brain, gap junctions in culture form between neuronal somata as well as neurites, suggesting soma ensheathing glia, largely absent in culture, regulate gap junction location in vivo. Thus pupal brain cultures, which support formation of interneuronal synapses with structural features similar to synapses in adult brain, are a useful model system for identifying intrinsic and extrinsic regulators of central synapse structure as well as function.     

Characterization of cortical neuronal and glial alterations during culture of organotypic whole brain slices from neonatal and mature mice

Background

Organotypic brain slice culturing techniques are extensively used in a wide range of experimental procedures and are particularly useful in providing mechanistic insights into neurological disorders or injury. The cellular and morphological alterations associated with hippocampal brain slice cultures has been well established, however, the neuronal response of mouse cortical neurons to culture is not well documented.

Methods

In the current study, we compared the cell viability, as well as phenotypic and protein expression changes in cortical neurons, in whole brain slice cultures from mouse neonates (P4–6), adolescent animals (P25–28) and mature adults (P50+). Cultures were prepared using the membrane interface method.

Results

Propidium iodide labeling of nuclei (due to compromised cell membrane) and AlamarBlue™ (cell respiration) analysis demonstrated that neonatal tissue was significantly less vulnerable to long-term culture in comparison to the more mature brain tissues. Cultures from P6 animals showed a significant increase in the expression of synaptic markers and a decrease in growth-associated proteins over the entire culture period. However, morphological analysis of organotypic brain slices cultured from neonatal tissue demonstrated that there were substantial changes to neuronal and glial organization within the neocortex, with a distinct loss of cytoarchitectural stratification and increased GFAP expression (p<0.05). Additionally, cultures from neonatal tissue had no glial limitans and, after 14 DIV, displayed substantial cellular protrusions from slice edges, including cells that expressed both glial and neuronal markers.

Conclusion

In summary, we present a substantial evaluation of the viability and morphological changes that occur in the neocortex of whole brain tissue cultures, from different ages, over an extended period of culture.     

Astroglial-Conditioned Media and Growth Factors Modulate Proliferation and Differentiation of Astrocytes in Primary Culture

Astroglial conditioned media (ACM) influence the development and maturation of cultured nerve cells and modulate neuron-glia interaction. To clarify mechanisms of astroglial cell proliferation/differentiation in culture, incorporation of [methyl-³H]-thymidine or [5,6-³H]-uridine in cultured astrocytes was assessed. Cultures were pre-treated with epidermal growth factor (EGF), insulin (INS), insulin-like growth factor-I (IGF-I), and basic fibroblast growth factor (bFGF) and subsequently with ACM. DNA labeling revealed a marked stimulatory effect of ACM from 15 days in vitro (DIV) cultures in 30 DIV astrocytes after12 h pre-treatment with growth factors. The main effects were found after INS or EGF pre-treatment in 30 DIV cultures. ACM collected from 15 or 60 or 90 DIV increased RNA labeling of 15 and 30 DIV astrocyte cultures, being the highest value that of 30 DIV cultures added with ACM from 90 DIV. The findings of increased DNA labeling after EGF or INS pre-treatment in 30 DIV cultures, followed by addition of ACM from 15 DIV cultures, suggest that these phenomena may depend by extra cellular signal-regulated kinase 1 (ERK1) activation.     

A profile of the insulin receptor in cultured hippocampal slices

OBJECTIVES: Insulin receptors (IRs) are distributed in a region-specific fashion throughout the brain, and may play a role in processes related to learning and memory. The hippocampus, which participates in spatial memory formation, is one region in which the IR is abundantly expressed. Organotypic hippocampal slice cultures (OHSCs) are an in vitro model that permits the easy manipulation of growth conditions, yet retains much of the source structure's cytoarchitecture. To assess OHSCs as a model for the study of hippocampal IRs, ligand-binding and the expression and cellular distribution of the β-subunit (which transduces the insulin signal) were examined over time in culture. Design & Results: Fluorescently conjugated insulin was used to assess neural insulin receptor binding, and revealed that labelling remained similar over three weeks in culture (a typical length of OHSC maintenance). Cross-linking of surface proteins helped to show that approximately half of β-subunits were found at the cell surface, and that this relative proportion remained stable over several weeks. In contrast, expression of the β-subunit protein progressively declined to a plateau approximately 60% less than that seen when the cultures were prepared. Conclusions: Our results provide a foundation for subsequent studies to employ OHSCs to explore neural IRs; for instance, the dissonance between the progressive decline in expression of the IR β-subunit and the relative stability of receptor-mediated binding suggests the presence of an active process to hold steady the ability of cells to respond to insulin stimulation.     

Primary culture of striatal neurons: a model of choice for pharmacological and biochemical studies of neurotransmitter receptors

Striatal neurons were cultured from fetal mouse brain and maintained in serum-free medium for 14-21 days in vitro (DIV). A double coating of culture dishes with polyornithine and fetal calf serum was needed in order to obtain synaptic differentiation. Synaptic vesicles were present in axon terminals as well as in varicosities along extended axons. The presence of differentiated synapses was confirmed by the immunostaining of the preparation with synapsin I antibody. After 13 days in vitro synapsin I was present in axonal varicosities and particularly concentrated at contact points between axonal terminals and postsynaptic sites on adjacent axons or perikarya. On a surface of 429 mm2 on which 2211 cells were observed under phase contrast microscopy only 7% were stained with an antibody against GFAP (glial fibrillary acidic protein). One or two days after the formation of differentiated synapses (11 DIV), a Ca2+-dependent liberation of GABA was observed. These cultures are an excellent model for studying the coupling of some neurotransmitter receptors with an adenylate cyclase. In particular using this preparation we were able to demonstrate that dopamine (D2) and serotonin-(5-HT1) receptors are negatively coupled with an adenylate cyclase. These cultures are also an excellent model to study the coupling of some neurotransmitter receptors with inositol phosphate producing enzymes. We demonstrated for the first time that the quisqualate subtype of glutamate receptors is able to increase inositol phosphate production in striatal neurons.     

Nuclear and mitochondrial DNA synthesis and energy metabolism in primary rat glial cell cultures

DNA synthesis in nuclei and mitochondria purified from serum-supplemented rat glial cell cultures at different days after plating was studied. Furthermore in mitochondria, some enzymatic activities related to energy transduction (citrate synthase, malate dehydrogenase, total NADH-cytochromec reductase, cytochrome oxidase and glutamate dehydrogenase) were measured. For DNA labeling [methyl-³H]thymidine was added to the culture medium at different days after plating. During the culture times studied the specific activity of total, nuclear, and mitochondrial DNA decreased from 8 days in vitro (DIV) to 21 DIV and increased at 30 DIV. The specific activity of nuclear DNA was always higher than that of mitochondrial DNA. The specific activity of the above mentioned mitochondrial enzymes increased from 8 DIV up to 21 DIV and decreased at 30 DIV, suggesting a relationship between the energy metabolism and the differentiation of glial cells in culture.The AA. would like to dedicate this paper to the memory of Dr. Ida Serra, Associate Professor of Biochemistry at the Medical Faculty, University of Catania, who prematurely died, after this paper was submitted for publication.     

Isolation and culture of rat embryonic neural cells: a quick protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.     

Mixed primary culture and clonal analysis provide evidence that NG2 proteoglycan-expressing cells after spinal cord injury are glial progenitors

NG2(+) cells in the adult rat spinal cord proliferate after spinal cord injury (SCI) and are postulated to differentiate into mature glia to replace some of those lost to injury. To further study these putative endogenous precursors, tissue at 3 days after SCI or from uninjured adults was dissociated, myelin partially removed and replicate cultures grown in serum-containing or serum-free medium with or without growth factors for up to 7 days in vitro (DIV). Cell yield after SCI was 5-6 times higher than from the normal adult. Most cells were OX42(+) microglia/macrophages but there were also more than twice the normal number of NG2(+) cells. Most of these coexpressed A2B5 or nestin, as would be expected for glial progenitors. Few cells initially expressed mature astrocyte (GFAP) or oligodendrocyte (CC1) markers, but more did at 7 DIV, suggesting differentiation of glial precursors in vitro. To test the hypothesis that NG2(+) cells after SCI express progenitor-like properties, we prepared free-floating sphere and single cell cultures from purified suspension of NG2(+) cells from injured spinal cord. We found that sphere cultures could be passaged in free-floating subcultures, and upon attachment the spheres clonally derived from an acutely purified single cell differentiated into oligodendrocytes and rarely astrocytes. Taken together, these data support the hypothesis that SCI stimulates proliferation of NG2(+) cells that are glial progenitor cells. Better understanding the intrinsic properties of the NG2(+) cells stimulated by SCI may permit future therapeutic manipulations to improve recovery after SCI.     

Coupling of organotypic brain slice cultures to silicon-based arrays of electrodes.

Fetal or early postnatal brain tissue can be cultured in viable and healthy condition for several weeks with development and preservation of the basic cellular and connective organization as so-called organotypic brain slice cultures. Here we demonstrate and describe how it is possible to establish such hippocampal rat brain slice cultures on biocompatible silicon-based chips with arrays of electrodes with a histological organization comparable to that of conventional brain slice cultures grown by the roller drum technique and on semiporous membranes. Intracellular and extracellular recordings from neurons in the slice cultures show that the electroresponsive properties of the neurons and synaptic circuitry are in accordance with those described for cells in acutely prepared slices of the adult rat hippocampus. Based on the recordings and the possibilities of stimulating the cultured cells through the electrode arrays it is anticipated that the setup eventually will allow long-term studies of defined neuronal networks and provide valuable information on both normal and neurotoxicological and neuropathological conditions.     

Comparison of different culture modes for long-term expansion of neural stem cells

Neural stem cells (NSCs) can be cultured in two modes of suspension and monolayer in vitro. The cultured cells are different in both the ability to proliferate and heterogeneity. In order to find the appropriate methods for large-scale expansion of NSCs, we systematically compared the NSCs cultured in suspension with those cultured in monolayer. The forebrain tissue was removed from embryonic day 14 (E14) mice, then the tissue was dissociated into single-cell suspension by Accutase and mechanical trituration. The cells were cultured in both suspension and monolayer. The NSCs cultured in suspension and in monolayer were compared on viability, ability to proliferate and heterogeneity by fluorescent dyes, immunofluorescence and flow cytometry on DIV21 (21 days in vitro), DIV56 and DIV112, respectively. The results indicated that the NSCs cultured in both suspension and monolayer represented good viability in long-term cultures. But they displayed a distinct ability to proliferate in long-term cultures. The NSCs cultured in monolayer preceded those cultured in suspension on the ability to proliferate on DIV21 and DIV56, but no obvious difference on DIV112. The NSCs population cultured in suspension displayed more nestin-positive cells than those in monolayer during the whole process of culture. The NSCs population cultured in monolayer, however, displayed more βIII tubulin-positive cells than those in suspension in the same period. The suspension culture mode excels the monolayer culture mode for large-scale expansion of NSCs.     

Conditions affecting primary cell cultures of functional adult rat hepatocytes. II. Dexamethasone enhanced longevity and maintenance of morphology.

Primary monolayer cell cultures of adult rat hepatocytes underwent change in morphology and substantial cell loss between 1 and 3 days postinoculation. Dexamethasone-supplementation (1 micronM) of the culture medium maintained the polygonal epithelial morphology of the hepatocytes and increased longevity such that over 80% of the cells survived for 3 days and at least 30% for 8 or 9 days. This enhancement of survival was obtained up to 48 hr postinoculation, but the earlier the time of dexamethason supplementation the greater the effect. Removal of dexamethasone resulted in a decrease in longevity. The positive effect of dexamethasone on longevity was observed following dexamethasone replacement of insulin in supplemented cultures, but the combination of insulin and dexamethasone resulted in poorer survival than with dexamethasone alone. The results are interpreted to indicate that dexamethasone provided a requirement of the in vitro environment for survival and suggest that elaboration of a complex medium is required to maintain hepatocytes in culture.     

Adult human brain cell culture for neuroscience research

Studies of the brain have progressed enormously through the use of in vivo and in vitro non-human models. However, it is unlikely such studies alone will unravel the complexities of the human brain and so far no neuroprotective treatment developed in animals has worked in humans. In this review we discuss the use of adult human brain cell culture methods in brain research to unravel the biology of the normal and diseased human brain. The advantages of using adult human brain cells as tools to study human brain function from both historical and future perspectives are discussed. In particular, studies using dissociated cultures of adult human microglia, astrocytes, oligodendrocytes and neurons are described and the applications of these types of study are evaluated. Alternative sources of human brain cells such as adult neural stem cells, induced pluripotent stem cells and slice cultures of adult human brain tissue are also reviewed. These adult human brain cell culture methods could benefit basic research and more importantly, facilitate the translation of basic neuroscience research to the clinic for the treatment of brain disorders.