Paul Honegger

Optimization of chemically defined cell culture media - Replacing fetal bovine serum in mammalian in vitro methods

Quality assurance is becoming increasingly important. Good laboratory practice (GLP) and good manufacturing practice (GMP) are now established standards. The biomedical field aims at an increasing reliance on the use of in vitro methods. Cell and tissue culture methods are generally fast, cheap, reproducible and reduce the use of experimental animals. Good cell culture practice (GCCP) is an attempt to develop a common standard for in vitro methods. The implementation of the use of chemically defined media is part of the GCCP. This will decrease the dependence on animal serum, a supplement with an undefined and variable composition. Defined media supplements are commercially available for some cell types. However, information on the formulation by the companies is often limited and such supplements can therefore not be regarded as completely defined. The development of defined media is difficult and often takes place in isolation. A workshop was organised in 2009 in Copenhagen to discuss strategies to improve the development and use of serum-free defined media. In this report, the results from the meeting are discussed and the formulation of a basic serum-free medium is suggested. Furthermore, recommendations are provided to improve information exchange on newly developed serum-free media.

Insulin-like growth factor I (IGF I) stimulates DNA synthesis in fetal rat brain cell cultures

Addition of insulin, IGF I or IGF II to serum-free cultures of fetal rat brain cells (gestation day 15/16) significantly stimulates DNA synthesis. The dose-response curves show that IGF I is more potent than insulin; half maximal stimulation of [3H]thymidine incorporation is obtained at about 0.4 nM IGF I and 14 nM insulin, respectively. Cultures initiated 2 days later (gestation day 17/18) showed a decreased responsiveness to both peptides. No additive effect was observed after combined addition of both peptides at near-maximal doses. Both peptides show a latency of action of about 12-18 h. In the presence of either IGF or insulin, neuronal as well as glial enzymes are increased, suggesting that neuronal and glial precursor cell division is influenced. IGF I and IGF II interact with a specific binding site for which insulin competes very weakly; however IGF I and IGF II bind with relatively high affinity to the insulin specific binding site. The present results support the hypothesis that both insulin and IGF stimulate mitotic activity by interacting with specific somatomedin receptors and suggest a physiological role of IGF in the developing brain.

Demyelination Induced in Aggregating Brain Cell Cultures by a Monoclonal Antibody Against Myelin/Oligodendrocyte Glycoprotein

A monoclonal antibody (8–18C5) directed against myelin/oligodendrocyte glycoprotein (MOG) induced demyelination in aggregating brain cell cultures. With increasing doses of anti‐MOG antibody in the presence of complement, myelin basic protein (MBP) concentrations decreased in a dose‐related manner. A similar, albeit less pronounced, effect was observed on specific activity of 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase. In the absence of complement, anti‐MOG antibody did not induce detectable demyelination. In contrast to the effect of anti‐MOG antibody and as expected, anti‐MBP antibody did not demyelinate aggregating brain cell cultures in the presence of complement. These results provide additional support to the suggestion that MOG, a quantitatively minor myelin component located on the external side of the myelin membrane, is a good target antigen for antibody‐induced demyelination. Indeed, they show that a purified anti‐MOG antibody directed against a single epitope on the glycoprotein can produce demyelination, not only in vivo as previously shown, but also in cultures. Such an observation has not been made with polyclonal antisera raised against purified myelin proteins like MBP and proteolipid protein, the major protein components of the myelin membrane, or myelin‐associated glycoprotein. These observations may have important implications regarding the possible role of anti‐MOG antibodies in demyelinating diseases.

Workgroup Report: Incorporating In Vitro Alternative Methods for Developmental Neurotoxicity into International Hazard and Risk Assessment Strategies

This is the report of the first workshop on Incorporating In Vitro Alternative Methods for Developmental Neurotoxicity (DNT) Testing into International Hazard and Risk Assessment Strategies, held in Ispra, Italy, on 19–21 April 2005. The workshop was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) and jointly organized by ECVAM, the European Chemical Industry Council, and the Johns Hopkins University Center for Alternatives to Animal Testing. The primary aim of the workshop was to identify and catalog potential methods that could be used to assess how data from in vitro alternative methods could help to predict and identify DNT hazards. Working groups focused on two different aspects: a) details on the science available in the field of DNT, including discussions on the models available to capture the critical DNT mechanisms and processes, and b) policy and strategy aspects to assess the integration of alternative methods in a regulatory framework. This report summarizes these discussions and details the recommendations and priorities for future work.

Peroxisome proliferator-activated receptor beta regulates acyl-CoA synthetase 2 in reaggregated rat brain cell cultures.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that regulate the expression of many genes involved in lipid metabolism. The biological roles of PPARα and PPARγ are relatively well understood, but little is known about the function of PPARβ. To address this question, and because PPARβ is expressed to a high level in the developing brain, we used reaggregated brain cell cultures prepared from dissociated fetal rat telencephalon as experimental model. In these primary cultures, the fetal cells initially form random aggregates, which progressively acquire a tissue-specific pattern resembling that of the brain. PPARs are differentially expressed in these aggregates, with PPARβ being the prevalent isotype. PPARα is present at a very low level, and PPARγ is absent. Cell type-specific expression analyses revealed that PPARβ is ubiquitous and most abundant in some neurons, whereas PPARα is predominantly astrocytic. We chose acyl-CoA synthetases (ACSs) 1, 2, and 3 as potential target genes of PPARβ and first analyzed their temporal and cell type-specific pattern. This analysis indicated that ACS2 and PPARβ mRNAs have overlapping expression patterns, thus designating the ACS2 gene as a putative target of PPARβ. Using a selective PPARβ activator, we found that the ACS2 gene is transcriptionally regulated by PPARβ, demonstrating a role for PPARβ in brain lipid metabolism.

Triiodothyronine enhancement of neuronal differentiation in aggregating fetal rat brain cells cultured in a chemically defined medium

Summary Triiodothyronine (30 nM) added to serum-free cultures of mechanically dissociated re-aggregating fetal (15–16 days gestation) rat brain cells greatly increased the enzymatic activity of choline acetyltransferase and acetylcholinesterase throughout the entire culture period (33 days), and markedly accelerated the developmental rise of glutamic acid decar☐ylase specific activity. The enhancement of choline acetyltransferase and acetylcholinesterase specific activities in the presence of triiodothyronine was even more pronounced in cultures of telecephalic cells. If triododthyronine treatment was restricted to the first 17 culture days, the level of choline acetyltransferase specific activity at day 33 was 84% of that in chronically treated cultures and 270% of that in cultures receiving triiodothyronine between days 17 and 33, indicating that relatively undifferentiated cells were more responsive to the hormone. Triiodothyronine had no apparent effect on the incorporation of [3H]thymidine at day 5 or on the total DNA content of cultures, suggesting that cellular differentiation, rather than proliferation was affected by the hormone. Our findings in vitro are in good agreement with many observations in vivo, suggesting that rotation-mediated aggregating cell cultures of fetal rat brain provide a useful model to study thyroid hormone action in the developing brain.

Involvement of environmental mercury and lead in the etiology of neurodegenerative diseases.

The incidence of neurodegenerative disease like Parkinsons disease and Alzheimers disease (AD) increases dramatically with age; only a small percentage is directly related to familial forms. The etiology of the most abundant, sporadic forms is complex and multifactorial, involving both genetic and environmental factors. Several environmental pollutants have been associated with neurodegenerative disorders. The present article focuses on results obtained in experimental neurotoxicology studies that indicate a potential pathogenic role of lead and mercury in the development of neurodegenerative diseases. Both heavy metals have been shown to interfere with a multitude of intracellular targets, thereby contributing to several pathogenic processes typical of neurodegenerative disorders, including mitochondrial dysfunction, oxidative stress, deregulation of protein turnover, and brain inflammation. Exposure to heavy metals early in development can precondition the brain for developing a neurodegenerative disease later in life. Alternatively, heavy metals can exert their adverse effects through acute neurotoxicity or through slow accumulation during prolonged periods of life. The pro-oxidant effects of heavy metals can exacerbate the age-related increase in oxidative stress that is related to the decline of the antioxidant defense systems. Brain inflammatory reactions also generate oxidative stress. Chronic inflammation can contribute to the formation of the senile plaques that are typical for AD. In accord with this view, nonsteroidal anti-inflammatory drugs and antioxidants suppress early pathogenic processes leading to Alzheimers disease, thus decreasing the risk of developing the disease. The effects of lead and mercury were also tested in aggregating brain-cell cultures of fetal rat telencephalon, a three-dimensional brain-cell culture system. The continuous application for 10 to 50 days of non-cytotoxic concentrations of heavy metals resulted in their accumulation in brain cells and the occurrence of delayed toxic effects. When applied at non-toxic concentrations, methylmercury, the most common environmental form of mercury, becomes neurotoxic under pro-oxidant conditions. Furthermore, lead and mercury induce glial cell reactivity, a hallmark of brain inflammation. Both mercury and lead increase the expression of the amyloid precursor protein; mercury also stimulates the formation of insoluble beta-amyloid, which plays a crucial role in the pathogenesis of AD and causes oxidative stress and neurotoxicity in vitro. Taken together, a considerable body of evidence suggests that the heavy metals lead and mercury contribute to the etiology of neurodegenerative diseases and emphasizes the importance of taking preventive measures in this regard.

Microglial reaction induced by noncytotoxic methylmercury treatment leads to neuroprotection via interactions with astrocytes and IL‐6 release

Microglial cells react early to a neurotoxic insult. However, the bioactive factors and the cell‐cell interactions leading to microglial activation and finally to a neuroprotective or neurodegenerative outcome remain to be elucidated. Therefore, we analyzed the microglial reaction induced by methylmercury (MeHgCl) using cell cultures of different complexity. Isolated miroglia were found to be directly activated by MeHgCl (10−10 to 10−6 M), as indicated by process retraction, enhanced lectin staining, and cluster formation. An association of MeHgCl–induced microglial clusters with astrocytes and neurons was observed in three‐dimensional cultures. Close proximity was found between the clusters of lectin‐stained microglia and astrocytes immunostained for glial fibrillary acidic protein (GFAP), which may facilitate interactions between astrocytes and reactive microglia. In contrast, immunoreactivity for microtubule‐associated protein (MAP‐2), a neuronal marker, was absent in the vicinity of the microglial clusters. Interactions between astrocytes and microglia were studied in cocultures treated for 10 days with MeHgCl. Interleukin‐6 release was increased at 10−7 M of MeHgCl, whereas it was decreased when each of these two cell types was cultured separately. Moreover, addition of IL‐6 to three‐dimensional brain cell cultures treated with 3 × 10−7 M of MeHgCl prevented the decrease in immunostaining of the neuronal markers MAP‐2 and neurofilament‐M. IL‐6 administered to three‐dimensional cultures in the absence of MeHgCl caused astrogliosis, as indicated by increased GFAP immunoreactivity. Altogether, these results show that microglial cells are directly activated by MeHgCl and that the interaction between activated microglia and astrocytes can increase local IL‐6 release, which may cause astrocyte reactivity and neuroprotection. GLIA 37:43–52, 2002.

Ammonium-Induced Impairment of Axonal Growth Is Prevented through Glial Creatine

Hyperammonemia in neonates and infants affects brain development and causes mental retardation. We report that ammonium impaired cholinergic axonal growth and altered localization and phosphorylation of intermediate neurofilament protein in rat reaggregated brain cell primary cultures. This effect was restricted to the phase of early maturation but did not occur after synaptogenesis. Exposure to NH4Cl decreased intracellular creatine, phosphocreatine, and ADP. We demonstrate that creatine cotreatment protected axons from ammonium toxic effects, although this did not restore high-energy phosphates. The protection by creatine was glial cell-dependent. Our findings suggest that the means to efficiently sustain CNS creatine concentration in hyperammonemic neonates and infants should be assessed to prevent impairment of axonogenesis and irreversible brain damage.

Biochemical Differentiation in Serum-Free Aggregating Brain Cell Cultures

The basic methodology of rotation-mediated aggregating cell culture was introduced by Moscona (1961), who took advantage of the finding that dissociated immature cells in vitro reassemble spontaneously to form histotypic, three-dimensional structures (Moscona, 1960). Subsequent morphological investigations have revealed that such cell aggregates prepared from fetal brain form patterns of cell alignment similar to those in vivo (Moscona, 1965; DeLong, 1970; DeLong and Sidman, 1970; Garber and Moscona, 1972; Levitt et al., 1976; Garber 1977), and undergo extensive morphological differentiation, including synaptogenesis and myelination (Seeds and Vatter, 1971; Kozak et al.,1977; Matthieu et al., 1978,1981; Seeds and Haffke, 1978; Honegger et al.,1979; Matthieu and Honegger, 1979; Trapp et al., 1979, 1982; Lu et al., 1980).