Long-Term Culture of Rat Hippocampal Neurons at Low Density in Serum-Free Medium: Combination of the Sandwich Culture Technique with the Three-Dimensional Nanofibrous Hydrogel PuraMatrix

The primary culture of neuronal cells plays an important role in neuroscience. There has long been a need for methods enabling the long-term culture of primary neurons at low density, in defined serum-free medium. However, the lower the cell density, the more difficult it is to maintain the cells in culture. Therefore, we aimed to develop a method for long-term culture of neurons at low density, in serum-free medium, without the need for a glial feeder layer. Here, we describe the work leading to our determination of a protocol for long-term (>2 months) primary culture of rat hippocampal neurons in serum-free medium at the low density of 3×10⁴ cells/mL (8.9×10³ cells/cm²) without a glial feeder layer. Neurons were cultured on a three-dimensional nanofibrous hydrogel, PuraMatrix, and sandwiched under a coverslip to reproduce the in vivo environment, including the three-dimensional extracellular matrix, low-oxygen conditions, and exposure to concentrated paracrine factors. We examined the effects of varying PuraMatrix concentrations, the timing and presence or absence of a coverslip, the timing of neuronal isolation from embryos, cell density at plating, medium components, and changing the medium or not on parameters such as developmental pattern, cell viability, neuronal ratio, and neurite length. Using our method of combining the sandwich culture technique with PuraMatrix in Neurobasal medium/B27/L-glutamine for primary neuron culture, we achieved longer neurites (≥3,000 µm), greater cell viability (≥30%) for 2 months, and uniform culture across the wells. We also achieved an average neuronal ratio of 97%, showing a nearly pure culture of neurons without astrocytes. Our method is considerably better than techniques for the primary culture of neurons, and eliminates the need for a glial feeder layer. It also exhibits continued support for axonal elongation and synaptic activity for long periods (>6 weeks).     

Serum-free medium for murine and human lymphoid and hybridoma cell lines

We established a serum-free medium of low protein content(125g/ml) TYI 100, consisting of three hormones and five growth factors for the growth of lymphoid and hybridoma cell lines. In TYI 100 medium, mouse and human hybridomas grew equally well as in RPMI 1640 supplemented with 10% fetal bovine serum (10% FBS) without adaptation to the serum-free medium. TYI 100 medium allowed several passages of mouse hybridoma lines and the total cell number was more than in 10% FBS. TYI 100 medium also supported growth of myelomas and anchorage dependent cell lines, Bowes and CHO, well. TYI 100 medium is composed of inexpensive supplements and is therefor applicable to large scale culture.Abbreviations FBS Fetal Bovine Serum - IMDM Iscove's Modification of Dulbecco's Medium - PBS Phosphate-Buffered Saline - TPA Tissue Plasminogen Activator     

Serum depletion induces changes in protein expression in the trophoblast-derived cell line HTR-8/SVneo

Background

How nutrition and growth factor restriction due to serum depletion affect trophoblast function remains poorly understood. We performed a proteomic differential study of the effects of serum depletion on a first trimester human immortalized trophoblast cell line.

Methods

The viability of HTR-8/SVneo trophoblast cells in culture with 0, 0.5 and 10 % fetal bovine serum (FBS) were assayed via MTT at 24, 48 and 64 h. A comparative proteomic analysis of the cells grown with those FBS levels for 24 h was performed using two-dimensional electrophoresis (2DE), followed by mass spectrometry for protein spot identification, and a database search and bioinformatics analysis of the expressed proteins. Differential spots were identified using the Kolmogorov-Smirnov test (n?=?3, significance level 0.10, D?>?0.642) and/or ANOVA (n?=?3, p?<?0.05).

Results

The results showed that low serum doses or serum depletion differentially affect cell growth and protein expression. Differential expression was seen in 25 % of the protein spots grown with 0.5 % FBS and in 84 % of those grown with 0 % FBS, using 10 % serum as the physiological control. In 0.5 % FBS, this difference was related with biological processes typically affected by the serum, such as cell cycle, regulation of apoptosis and proliferation. In addition to these changes, in the serum-depleted proteome we observed downregulation of keratin 8, and upregulation of vimentin, the glycolytic enzymes enolase and pyruvate kinase (PKM2) and tumor progression-related inosine-5’-monophosphate dehydrogenase 2 (IMPDH2) enzyme. The proteins regulated by total serum depletion, but not affected by growth in 0.5 % serum, are members of the glycolytic and nucleotide metabolic pathways and the epithelial-to-mesenchymal transition (EMT), suggesting an adaptive switch characteristic of malignant cells.

Conclusions

This comparative proteomic analysis and the identified proteins are the first evidence of a protein expression response to serum depletion in a trophoblast cell model. Our results show that serum depletion induces specific changes in protein expression concordant with main cell metabolic adaptations and EMT, resembling the progression to a malignant phenotype.

     

Effect of lipid supplements on the production and glycosylation of recombinant interferon-γ expressed in CHO cells

The effects of lipids on the glycosylation of recombinant human interferon- expressed in a Chinese Hamster Ovary cell line were investigated in batch culture. Lipids form an essential part of the N-glycosylation pathway, and have been shown to improve cell viability. In control (serum-free) medium the proportion of fully-glycosylated interferon- deteriorated reproducibly with time in batch culture, but the lipoprotein supplement ExCyte was shown to minimise this trend. Partially substituting the bovine serum albumin content of the medium with a fatty-acid free preparation also improved interferon- glycosylation, possibly indicating that oxidised lipids carried on Cohn fraction V albumin may damage the glycosylation process.Abbreviations BSA bovine serum albumin - CHO chinese hamster ovary - DHFR dihydrofolate reductase - FCS foetal calf serum - IFN- human interferon-gamma - q _IFN specific interferon production rate - specific growth rate - 2N doubly-gycosylated - 1N singly-glycosylated - ON non-glycosylated     

无血清培养上调N2a细胞钙反应性反式激活因子的表达

目的探讨钙反应性反式激活因子(calcium-responsive transactivator,CREST)是否与神经细胞的分化有关。方法选用在促分化因素作用下具有分化为神经元能力的小鼠神经母细胞瘤(N2a)细胞,用无血清培养(血清撤除)诱导N2a细胞分化。接种和培养N2a细胞12～24h后,将其分为对照组和无血清诱导组,对照组继续在含5%胎牛血清(FBS)的培养基中培养,无血清诱导组在不含FBS的培养基中培养。采用免疫印迹技术、免疫荧光技术和RT-PCR检测无血清诱导分化对N2a细胞CREST蛋白和mRNA表达的影响。结果在含5%胎牛血清(FBS)培养基培养的对照N2a细胞中,CREST蛋白水平在各时间点均无明显变化,CREST mRNA水平仅在培养48h后略有升高。而在无血清诱导分化的N2a细胞中,CREST蛋白表达水平在无血清诱导12h时无明显变化,但在诱导24h后明显升高,诱导48h后进一步升高;RT-PCR检测显示,CREST mRNA在无血清诱导12h后即开始升高,诱导24h和48h则升高更为明显。结论无血清诱导分化的N2a细胞中CREST的表达上调,提示CREST可能参与神经细胞的分化。     

Cell growth stimulating effect of <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> spores and their potential application for Chinese hamster ovary K1 cell cultivation

In this work, water-soluble extracts of Ganoderma lucidum spores (Gls), a Chinese medicinal herb that possesses cell growth stimulating function, were found to be an effective growth factor for Chinese hamster ovary (CHO) cell cultivation. The Gls extract was prepared and supplemented to CHO K1 cell culture media with various serum levels. Our results obtained from both the static culture and the spinner-flask suspension culture showed that use of small-amount Gls extract effectively promoted cell growth and suppressed cell apoptosis induced by serum deprivation with normal cell cycle maintained in a low-serum medium. The low-serum medium containing 1 % (v/v) fetal bovine serum (FBS) and 0.01 % (w/v) Gls extract showed a comparable performance on both cell growth and fusion protein productivity with the conventional CHO culture medium containing 10 % (v/v) FBS and a commercial serum-free medium. This is the first study of the potential of Gls extracts for use as an alternative cell growth factor and nutrient for CHO cells. The findings have presented a new approach to economic cultivation of CHO cells for therapeutic protein production.     

Protective effect of salidroside against H2O2-induced cell apoptosis in primary culture of rat hippocampal neurons

Salidroside, a phenylpropanoid glycoside separated from a medicinal plant Rhodiola rosea, has been documented to have protective effects on neuronal cells in vitro. This study investigated whether salidroside was able to extend its unique neuroprotection to primary cultured rat hippocampal neurons against hydrogen peroxide (H₂O₂)-induced cell damage. Cell viability tests and cell apoptosis assays confirmed that salidroside pretreatment attenuated H₂O₂-stimulated apoptotic cell death in primary culture of hippocampal neurons in a concentration-dependent manner. The measurements of caspase-3 activity, nitric oxide (NO) production, and NO synthase (NOS) activity suggest that the protection of salidroside, shown in this study, might be mediated by inhibiting caspase-3 activity, and antagonizing NO production and NOS activity during H₂O₂ stimulation. Perhaps, this study might contribute to the development of salidroside as a broad-spectrum agent for preventing and/or treating neuronal damage in neurodegenerative disorders.     

Intramitochondrial pyruvate attenuates hydrogen peroxide-induced apoptosis in bovine pulmonary artery endothelium

In the hydrogen peroxide (H₂O₂) apoptosis model of the murine thymocyte, redox reactant and antioxidant pyruvate prevents programmed cell death. We tested the hypothesis that such protection was mediated, at least in part, via pyruvate handling by mitochondrial metabolism. Cultured bovine pulmonary artery endothelial cells were incubated for 30 min with 0.5 mM H₂O₂ in the absence and presence of 0.5 mM -cyano-3-hydroxycinnamate, as a selective inhibitor of the mitochondrial pyruvate transporter. In controls H₂O₂ decreased cell viability by 30% within 24 h; this was associated with apoptosis-like bodies, nuclear condensation, and biochemical DNA damage consistent with programmed cell death. Pyruvate (0.1–20 mM) enhanced cell viability in a dose-dependent manner, with 85% viable cells at 3 mM and no DNA laddering, no positive nick-end labeling (TUNEL), and no detectable Annexin V or propidium iodide staining. In contrast, using 5 mM L-lactate as a cytosolic reductant or acetate as a redox-neutral substrate, cell death increased to 40%, which was associated with intense DNA laddering, positive TUNEL and Hoechst 33258 assays. -Cyano-3-hydroxycinnamate alone did not significantly decrease endothelial viability but reduced viability from 85 ± 3 to 71 ± 4% (p = 0.023) in presence of 3 mM pyruvate plus H₂O₂; pathological cell morphology and DNA laddering under the same conditions suggested loss of pyruvate protection against apoptosis. Since -cyano-3-hydroxycinnamate re-distributed medium pyruvate and L-lactate consistent with selective blockade of pyruvate uptake into the mitochondria, the findings support the hypothesis that pyruvate protection against H₂O₂ apoptosis is mediated in part via the mitochondrial matrix compartment. Possible mediators include anti-apoptotic bcl-2 and/or products of mitochondrial pyruvate metabolism such as citrate that affect metabolic regulation and anti-oxidant status in the cytoplasm.     

Methods for increasing monoclonal antibody production in suspension and entrapped cell cultures: biochemical and flow cytometric analysis as a function of medium serum content.

The growth and antibody production of the SP2/0-derived hybridoma HB124 (ATCC) grown in media containing varying amounts of fetal bovine serum (FBS) were monitored using biochemical and flow cytometric methods. Hybridomas grown in 100 ml spinner flasks with RPMI-1640 containing varying amounts of serum demonstrated that cell growth, viability and IgG production show significant changes when serum content is decreased from 10.0 to 5.5 to 1.0 and 0.5%. A longer lag phase resulted when the lower serum content media were used. Cellular rates of glucose uptake showed a significant increase as serum levels were lowered. Similarly, exponential phase IgG production rates increased as the amount of serum was decreased, probably as a result of the decreased rate of exponential growth. Flow cytometric analysis showed a similar increase in cellular IgG content as medium serum levels declined. In contrast, the maximum IgG concentrations were found in flasks containing 1% FBS or above with the lowest concentration in the 0.5% FBS flask being due to the lower numbers of viable cells. Cells grown in microporous hollow fiber reactors were fed with medium containing serum which was decreased stepwise with time. Decreasing medium serum content stepwise from 10 to 2.5% resulted in increased antibody production. However, complete removal of serum from the medium resulted in a significant drop in antibody productivity. Cumulative antibody production was equivalent for cells grown entirely in medium containing 10% FBS and for those which experienced a drop to 2.5% FBS. To compare a defined serum-free medium preparation with medium containing 10% FBS, cells were again grown in batch suspension culture and analyzed. The growth rates were similar but there was a significant difference in IgG production rates. The serum-free culture exhibited both higher cellular production rates and higher IgG concentrations. These results indicate that decreasing medium serum content can adversely affect antibody yield because of lower cell viabilities, not because of lower production rates. Use of a defined serum-free medium, as done in this study, results in higher yields because of a higher IgG production rate as well as good cell growth and viability.     

Insulin binding in cultured Chinese hamster kidney epithelial cells: The effect of serum in the medium

Summary [¹²⁵I]Insulin (porcine) binding to an epithelial cell line established from a Chinese hamster kidney, CHK-AC_E−100, showed an optimum at pH 8.0 and reached a maximum after 2.5 h incubation at 25°C. Dissociation of bound [¹²⁵I]insulin was facilitated by the addition of unlabeled insulin in the dilution buffer. Porcine insulin effectively competed for [¹²⁵I]insulin binding to the cultured cells and was 30 and 90 times as potent as guinea pig insulin and porcine proinsulin in causing 50% inhibition of [¹²⁵I]insulin binding; glucagon was completely ineffective. Scatchard analysis of the binding data yielded a curvilinear plot and a capacity of 0.6 ng/10⁶ cells; the average affinity of the empty receptor, , was calculated to be 1.78×10⁸ M ⁻¹ and that of the filled receptor, , 0.57×10⁸ M ⁻¹. Substitution of fetal bovine serum (FBS) in the culture medium with bovine calf, bovine newborn, or bobby calf serum altered insulin binding characteristics in the cells and reduced cell growth. Insulin binding characteristics of cells grown in hormone-supplemented medium containing 0 to 0.1% FBS were similar to those of cells grown in minimum essential medium (MEM) containing 2 to 5% FBS. The data indicated that the established Chinese hamster kidney epithelial cell line CHK-AC_E−100 possessed specific insulin receptors and the characteristics of the receptors could be manipulated by changing the serum in culture medium.     

Yeast hydrolysate as a low-cost additive to serum-free medium for the production of human thrombopoietin in suspension cultures of Chinese hamster ovary cells

To enhance the performance of a serum-free medium (SFM) for human thrombopoietin (hTPO) production in suspension cultures of recombinant Chinese hamster ovary (rCHO) cells, several low-cost hydrolysates such as yeast hydrolysate (YH), soy hydrolysate, wheat gluten hydrolysate and rice hydrolysate were tested as medium additives. Among various hydrolysates tested, the positive effect of YH on hTPO production was most significant. When 5 g l^–1 YH was added to SFM, the maximum hTPO concentration in batch culture was 40.41 g ml^–1, which is 11.5 times higher than that in SFM without YH supplementation. This enhanced hTPO production in YH-supplemented SFM was obtained by the combined effect of enhanced q_hTPO (the specific rate of hTPO production). The supplementation of YH in SFM increased q_hTPO by 294% and extended culture longevity by >2 days if the culture was terminated at a cell viability of 50%. Furthermore, cell viability throughout the culture using YH-supplemented SFM was higher than that using any other hydrolysate-supplemented SFM tested, thereby minimizing degradation of hTPO susceptible to proteolytic degradation. In addition, YH supplementation did not affect in vivo biological activity of hTPO. Taken together, the results obtained demonstrate the potential of YH as a medium additive for hTPO production in serum-free suspension cultures of rCHO cells.     

Bifemelane Hydrochloride Protects Against Cytotoxicity of Hydrogen Peroxide on Cultured Rat Neuroblastoma Cell Line

Free radicals are involved in neuronal damage. Bifemelane hydrochloride has been reported to protect neural tissues against ischemic damage and age-related neurodegeneration. We examined the protective effects of bifemelane HCl and the relation between its effectiveness and free radical formation in hydrogen peroxide (H₂O₂)-induced cytotoxicity using cultured rat neuroblastoma cell line (B50). Cytotoxicity was examined by using the lactate dehydrogenase (LDH) assay and cell viability by the WST-1 assay. H₂O₂ reduced the survival of B50 cells in a dose-dependent manner, and treatment of these cells with 75 M or 100 M H₂O₂ reduced their viability by 50% relative to the control group. B50 cells were treated with 5 or 10 M bifemelane for 2 days followed by treatment with 75 M or 100 M H₂O₂. H₂O₂ cytotoxicity was reduced by pretreatment with bifemelane. We also examined the effect of bifemelane on lipid peroxide formation in B50 cells using thiobarbituric acid reactive substances assay. Pretreatment of B50 cells with 10 M bifemelane for 2 days reduced lipid peroxide formation to approximately 54% of the control group. Our results suggest that bifemelane hydrochloride provides a protective effect against H₂O₂ cytotoxicity partly due to its anti-oxidative properties.     

Characterization of Hydrogen Peroxide Toxicity in Cultured Rat Forebrain Neurons

We investigated the ability of hydrogen peroxide (H₂O₂) to cause apoptotic cell death in cultured rat forebrain neurons and the potential mechanisms by which oxidative stress triggers delayed neuronal death. H₂O₂ (25 M for 5 min) reduced cell viability to 34.5 ± 8.3% of untreated controls 20 h after exposure, and resulted in a significant proportion of neurons which exhibited apoptotic nuclear morphology. Using single cell fluorescence assays, we measured H₂O₂-induced changes in DNA strand breaks, 27 dichlorofluorescin fluorescence, reduced glutathione, intracellular free Ca²⁺, and mitochondrial membrane potential. DNA strand breaks in response to H₂O₂ were not evident immediately following exposure, but were increased 12h and 20h after exposure. Millimolar concentrations of H₂O₂ caused increases in the fluorescence of the oxidant-sensitive fluorescent dye, 27-dichlorofluorescin. H₂O₂ treatment decreased reduced glutathione following 30 minutes of exposure using the fluorescent indicator, 5-chloromethylfluorescein diacetate, and increased intra-neuronal free Ca²⁺ levels in a subpopulation of neurons. Mitochondrial membrane potential, measured by rhodamine 123 localization was unaffected by 25 H₂O₂, while higher concentrations of H₂O₂ (10 or 30 mM) depolarized mitochondria. These studies demonstrate that H₂O₂ is a potent and effective neurotoxin that produces oxidative stress, as well as apoptotic neuronal death     

Role of Reactive Oxygen Species and Glutathione in Inorganic Mercury-Induced Injury in Human Glioma Cells

The present study was undertaken to examine the role of reactive oxygen species (ROS) and glutathione (GSH) in glia cells using human glioma cell line A172 cells. HgCl₂ caused the loss of cell viability in a dose-dependent manner. HgCl₂-induced loss of cell viability was not affected by H₂O₂ scavengers catalase and pyruvate, a superoxide scavenger superoxide dismutase, a peroxynitrite scavenger uric acid, and an inhibitor of nitric oxide N^G-nitro-arginine Methyl ester. HgCl₂ did not cause changes in DCF fluorescence, an H₂O₂-sensitive fluorescent dye. The loss of cell viability was significantly prevented by the hydroxyl radical scavengers dimethylthiourea and thiourea, but it was not affected by antioxidants DPPD and Trlox. HgCl₂-induced loss of cell viability was accompanied by a significant reduction in GSH content. The GSH depletion was almost completely prevented by thiols dithiothreitol and GSH, whereas the loss of viability was partially prevented by these agents. Incubation of cells with 0.2 mM buthionine sulfoximine for 24 hr, a selective inhibitor of -glutamylcysteine synthetase, resulted in 56% reduction in GSH content without any change in cell viability. HgCl² resulted in 34% reduction in GSH content, which was accompanied by 59% loss of cell viability. These results suggest that HgCl₂-induced cell death is not associated with generation of H₂O₂ and ROS-induced lipid peroxidation. In addition, these data suggest that the depletion of endogenous GSH itself may not play a critical role in the HgCl₂-induced cytotoxicity in human glioma cells.     

Application of percent labeled mitoses (PLM) analysis to the investigation of melanoma cell responsiveness to MSH stimulation throughout the cell cycle

Dissociated embryonic chick dorsal root ganglionic cells were plated on collagen-coated tissue culture dishes in Eagle's basal medium containing 10% fetal calf serum (FCS). After 48 h, which allowed adequate cell attachment, the cultures were washed with serum-free medium and then received fresh medium supplemented with 10% FCS or serum-free defined medium (N1), which was supplemented with insulin, transferrin, progesterone, putrescine and selenium. In addition, both media required the addition of Nerve Growth Factor (NGF). N1 medium selectively maintained the neurons and did not support proliferation or even survival of almost all non-neuronal elements (fibroblasts and Schwann cells). Survival of neurons in N1 was initially as good and eventually better than in serum-containing medium. After 6 days in N1 the cultures consisted almost entirely of neurons (>95%), which had smaller cell bodies but more extensive process formation than in serum-supplemented medium. The omission of any one of the supplements resulted in a reduction of neuron survival. The ability to generate cultures of pure neurons in a serum-free defined medium may be useful for studying (i) the role of specific hormones and growth factors normally supplied by serum in the maintenance of neurons and (ii) biochemical parameters of neurons in the absence of the substantial background due to non-neuronal elements.     

Optimizing the transport and storage conditions of current Good Manufacturing Practice –grade human umbilical cord mesenchymal stromal cells for transplantation (HUC-HEART Trial)

Background

The HUC-HEART Trial is a clinical study of intramyocardial delivery of current Good Manufacturing Practice (cGMP)-grade human umbilical cord multipotent stromal cells (HUC-MSCs) in ischemic cardiomyopathy where 2?×?10⁷ cells are administered to peri-infarcted myocardium. Prior to the onset of the trial, we aimed to optimize the transport/storage conditions for obtaining the highest cell viability and proliferation rate of cells to be transplanted.

Cultures of cells from fetal rat brain: Methods to control composition,morphology, and biochemical activity

Fetal tissue transplantation is a promising new approach for the treatment of neurodegenerative diseases, but the optimal conditions for preparing cells for transplantation have not been defined. The growth of a population of septal brain cells, primarily containing cholinergic neurons and glia, was characterized after seeding at densities from 5 × 10⁴ to 6 × 10⁵ cells/cm², on polystyrene‐, collagen‐, laminin‐, and fibronectin‐coated surfaces, in the presence of serum and/or serum‐free medium. Differentiated glial cells were selected by culture on fibronectin or laminin surfaces, in the presence of low amounts of serum (2.5% FBS) and G5, a soluble factor containing EGF and insulin. Differentiated neuronal cells were selected by culture on laminin, in the presence of low amounts of serum (2.5% FBS) and N2, a soluble factor containing supplemental hormones. In each case, a minimum seeding density of 1 × 10⁵ cells/cm² was required. Neuronal growth could be maintained long term (21 days) with high levels of neuronal activity (ChAT activity). © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 461–467, 1999.     

Expansion of human bone marrow-derived mesenchymal stromal cells: serum-reduced medium is better than conventional medium

Background aimsHuman mesenchymal stromal cells (hMSC) are of enormous interest for various clinical applications. For the expansion of isolated hMSC to relevant numbers for clinical applications, 10% fetal bovine serum (FBS)-supplemented medium is commonly used. The main critical disadvantage of FBS is the possibility of transmission of infectious agents as well as the possibility of immune rejection of the transplanted cells in response to the bovine serum. Therefore, we tested a commercially available medium, Panserin 401, that was specifically developed for serum-free cell cultivation.MethodshMSC were isolated from bone marrow (BM) and expanded in either Dulbecco's modified Eagle medium (DMEM) or Panserin 401 alone, or combined with FBS (2% or 10%), with or without supplementary growth factors. Cell proliferation and cytotoxicity were monitored twice a week for 3 weeks.Results and ConclusionsNo proliferation was observed in any of the serum-free media. However, DMEM/10% FBS (the conventional culture medium for hMSC) and DMEM/2% FBS with growth factors revealed moderate proliferation. Interestingly, the best proliferation was obtained using Panserin 401 supplemented with 2% FBS and growth factors (as well as with 10% FBS). Analysis of cell growth in Panserin 401 supplemented with 2% FBS only or with growth factors only revealed no proliferation, demonstrating the necessity of the combination of 2% FBS and growth factors. Efficient isolation and expansion of hMSC from cancellous bone could also be performed using Panserin 401 with 2% FBS and growth factors. Furthermore, these isolated cultures maintained multipotency, as demonstrated by adipogenic and osteogenic differentiation.     

Neurons and Neuronal Stem Cells Survive in Glucose-Free Lactate and in High Glucose Cell Culture Medium During Normoxia and Anoxia

Several questions concerning the survival of isolated neurons and neuronal stem and progenitor cells (NPCs) have not been answered in the past: (1) If lactate is discussed as a major physiological substrate of neurons, do neurons and NPCs survive in a glucose-free lactate environment? (2) If elevated levels of glucose are detrimental to neuronal survival during ischemia, do high concentrations of glucose (up to 40 mmol/L) damage neurons and NPCs? (3) Which is the detrimental factor in oxygen glucose deprivation (OGD), lack of oxygen, lack of glucose, or the combination of both? Therefore, in the present study, we exposed rat cortical neurons and NPCs to different concentrations of d-glucose ranging from 0 to 40 mmol/L, or 10 and 20 mmol/L l-lactate under normoxic and anoxic conditions, as well as in OGD. After 24 h, we measured cellular viability by biochemical assays and automated cytochemical morphometry, pH values, bicarbonate, lactate and glucose concentrations in the cell culture media, and caspases activities. We found that (1) neurons and NPCs survived in a glucose-free lactate environment at least up to 24 h, (2) high glucose concentrations >5 mmol/L had no effect on cell viability, and (3) cell viability was reduced in normoxic glucose deprivation to 50% compared to 10 mmol/L glucose, whereas cell viability in OGD did not differ from that in anoxia with lactate which reduced cell viability to 30%. Total caspases activities were increased in the anoxic glucose groups only. Our data indicate that (1) neurons and NPCs can survive with lactate as exclusive metabolic substrate, (2) the viability of isolated neurons and NPCs is not impaired by high glucose concentrations during normoxia or anoxia, and (3) in OGD, low glucose concentrations, but not low oxygen levels are detrimental for neurons and NPCs.     

Bilobalide prevents apoptosis through activation of the PI3K/Akt pathway in SH-SY5Y cells

Bilobalide, a sesquiterpene trilactone constituent of Ginkgo biloba leaf extracts, has been proposed to exert protective and trophic effects on neurons. However, mechanisms underlying the protective effects of bilobalide remain unclear. Using human SH-SY5Y neuroblastoma cells and primary hippocampal neurons, this study investigated the neuroprotective effects of bilobalide. We mimicked aging-associated neuronal impairments by applying external factors (beta amyloid protein (Aβ) 1-42, H₂O₂ and serum deprivation) consequently inducing cell apoptosis. As markers for apoptosis, cell viability, DNA fragmentation, mitochondrial membrane potential and levels of cleaved caspase 3 were measured. We found that, bilobalide prevented Aβ 1-42-, H₂O₂- and serum deprivation-induced apoptosis. To better understand the neuroprotective effects of bilobalide, we also tested the ability of bilobalide to modulate pro-survival signaling pathways such as protein kinase C (PKC), extracellular-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. It was found that, bilobalide dose-dependently increased PI3K activity and levels of phosphorylated Akt (p-Akt Ser473 and Thr308), which could be maintained up to at least 2 h after bilobalide withdrawal in cells treated with or without Aβ 1-42, H₂O₂ or serum-free medium. In addition, application of PI3K/Akt inhibitor LY294002 could abrogate both the protective effects of bilobalide against Aβ 1-42-, H₂O₂- and serum deprivation-induced apoptotic cell damage and bilobalide-induced increase in PI3K activity and levels of p-Akt (Ser473 and Thr308). In contrast, application of PKC inhibitor staurosporine (STS) did not affect the protective effects of bilobalide. Moreover, no change in levels of phosphorylated ERK1/2 (p-ERK1/2) was observed in bilobalide-treated cells. These results further suggested that the PI3K/Akt pathway might be involved in the protective effects of bilobalide. Since modern technology allows production of purified bilobalide with high bioavailability, bilobalide may be useful in developing therapy for diseases involving age-associated neurodegeneration.