Establishment and evaluation of the primary cultured tibial osteoblast model of broiler chicks

Osteoblasts are considered as a major factor contributing to bone development and mineralization,however,few studies have been done to establish and evaluate the primary cultured tibial osteoblast model of broiler chicks.Therefore,in the present study,two experiments were conducted to establish and evaluate the primary cultured tibial osteoblast model of broiler chicks.In experiment 1,osteoblasts were isolated from the tibia of one-day-old Arbor Acre male broiler chicks using the explant method and identified through the cell morphology,alkaline phosphatase(ALP)and alizarin red staining.Experiment 2 was carried out to evaluate the vitality and mineralization of primary cultured tibial osteoblasts of broilers on days 4,8,12,16,20,24,28 and 32 after incubation,respectively.The results from experiment 1 demonstrated that primary cultured tibial osteoblasts of broilers showed a spindle-shaped,triangular or polygonal morphology.More than 95%of the cells were stained blue-black after ALP staining,and mineralized nodules were formed after 4 days of continuous incubation.In experiment 2,lactate dehydrogenase(LDH)activity stayed at a relatively stabilized level although incubation time affected(P=0.0012)it during the whole culture period.Additionally,incubation time affected(P≤0.0001)the number and proportion of the area of mineralized nodules.They increased linearly and quadratically(P<0.04)with the increase of incubation time,and remained at a stabilized level from 24 to 32 days of incubation.The estimates of the optimal incubation time were 17 and 26 days based on the best fitted broken-line or quadratic models(P<0.0001)of the number and proportion of the area of mineralized nodules,respectively.These results indicate that the primary cultured tibial osteoblast model of broilers has been established successfully by the explant method,and it showed typical osteoblast morphology and characteristics of ALP activity and mineralization,and could maintain a relatively stabilized vitality from 4 to 32 days of incubation;and the optimal incubation time of primary tibial osteoblasts was 17 to 26 days.Therefore,it could be used to further study the underlying mechanisms of bone development and mineralization of broiler chicks.

Primary Culture of Bovine Mammary Epithelial Cells

[ Objective] To investigate the feasibility of the primary culture of bovine mammary epithelial cells in biochemical incubator. [ Method] In vitro, bovine mammary epithelial cells were isolated and cultured by the tissue explant method in order to investigate the optimal culture conditions. The morphology observation and identification of the cultured cells were performed by inverted microscope observation, Giemsa staining and cytokeratin immunohistochemistry. [ Result] Observed with inverted microscope, most of the bovine mammary epithelial cells were polygonal and displayed typical slabstone-like appearance. As it can be seen from cell staining results, the cell body was big and the nucleus was stained dark blue and was round or oval in shape, with clearly visible nucleoli, generally 2 -4 nucleoli. The tissue-specific expression of cytokeratin 14 and cytokeratin 18 genes in mammary epithelial cells was identified by cytokeratin immunohistochemistry. [ Conclusion] Primary bovine mammary epithelial cells were successfully cultured in biochemical incubator.

Oxidative damage of primary cultured hippocampal neurons Does androgen have an antagonistic effect?

BACKGROUND： Evidence illustrates that androgen has a neuroprotective role. However, whether androgen also has the protective effect on hippocampal neurons during free radical mediated injury remains unclear. OBJECTIVE： To investigate the neuroprotective effect of androgen on hippocampal neurons during free radical damage. DESIGN, TIME AND SETTING： A controlled in vitro experiment was performed at the Department of Human Anatomy, Cell Culture Lab, and Neuroendocrinology Lab, Basic Medical School, Hebei Medical University from February to June 2009. MATERIALS： Testosterone was provided by Tianjin Jinyao Amino Acid Company, China. METHODS： Primary cultured neurons from 24 Sprague Dawley rats were randomly assigned into four groups： control, H202, testosterone, and testosterone （pre-added） plus H2O2 groups. MAIN OUTCOME MEASURES： The positive cell ratio of microtubule associated protein-Ⅱ and neuron specific enolase was determined by immunocytochemistry. Neuronal morphology was observed by hematoxylin-eosin staining and Nissl staining. Cell vitality and viability were determined using an inverted phase contrast microscope. The content of nitric oxide synthase, malondialdehyde, and superoxide dismutase were measured with a spectrophotometer. RESULTS： As compared with the control group, cell vitality and viability, and superoxide dismutase level were significantly decreased in the H202 group （P 〈 0.05）, while nitric oxide synthase and malondialdehyde levels were significantly increased （P 〈 0.05）. Neuronal vitality and viability as well as superoxide dismutase level in the testosterone plus H2O2 group were significantly greater than in the H2O2 group （P 〈 0.05）, and nitric oxide synthase and malondialdehyde levels were significantly less than in the H2O2 group （P〈 0.05）. CONCLUSION： Androgen partially reversed H2O2-induced neuronal damage and protected neurons.

Transplantation of primary cultured embryonic mesencephalic neural precursor cells for treating Parkinsonian rats

BACKGROUND： Choosing proper donor cells is one of keys in experimental and clinical studies on cell replacement therapy （CRT） for treating Parkinson disease （PD）. Embryonic mesencephalic precursor cells （MPCs） can stably differentiate into dopaminergic neuron after in vitro proliferated culture. As compared with embryonic stem cell and neural stem cell strains, cell composition of embryonic MPCs after primary culture is also the most close to that of embryonic mesencephalic ventral cell suspension without proliferated culture. Successful experience accumulated in the latter suggests that primary cultured embryonic MPCs might be the most potential donor cells in clinical application with CRT for treating PD so far. OBJECTIVE： To investigate the feasibility of primary cultured embryonic precursor cells cultured primarily as donor cells in CRT for treating PD in rats. DESIGN ： A randomized and controlled trial taking SD rats as experimental animals.SETTING： Department of Neurosurgery, Huashan Hospital Affiliated to Fudan University.MATERIALS： This experiment was carried out at the Institute of Neuroscience, Shanghai Institute for Biological Science, Chinese Academy of Sciences from July 2003 to June 2004. Totally 26 female SD rats, with body mass of 200 to 220 g, were provided by Shanghai Experimental Animal Center of Chinese Academy of Sciences. METHODS ： Stereotaxic injection of 6-hydroxydopamine into the medial forebrain bundle were perfored to develop PD model rat. Among 26 SD rats, 20 rats achieved a more than 5 turns/min in apomorphine induced rotation test, reaching the standard of PD model rats. Immunohistochemical detection was performed on 1 out of 20 model rats after execution, and the other 19 rats were randomly divided into control group （n=5）, sham transplantation group （n=5）and cell grafted group （n=9）. Primary cultured E12 MPC cell suspension （1.2×10^11 L^-1）were used as donor cells. 4μL primary cultured E12 MPC cell suspension prepared freshly was injected into the lesioned corpus striatum of rats in cell grafted group, and 4μL D-Hank＇s solution was injected in sham transplantation group in the same way. There was no injection in control group. Apomorphine-induced rotation rate of PD rats were recorded respectively in cell grafted group and sham transplantation group pre-operation （initial value） and at postoperative 2, 4, 6 and 16 weeks. Apomorphine-induced rotation rate of PD rats was recorded in control group at postoperative 2 months （initial value） and following 2,4,6 and 16 weeks. To determine TH antigen with immunohistological ABC method （DAB developing） at 6 months post-transplantation to investigate the differentiation and survival of donor cells in the host body.MAIN OUTCOME MEASURES： Apomorphine-induced rotation behavior before and after transplantation and the survival and differentiation of implanted cells in the host body at 6 months post-transplantation. RESULTS： Among 19 model rats, one rat died after transplantation respectively in the cell grafted group and sham transplantation group; finally 17 model rats entered the stage of result analysis. Relative apomorphine-induced rotation rate was significantly decreased in the cell grafted group as compared with that before transplantation , with significant difference （P 〈 0.01 .P 〈 0.05）;the mean value of relative apomorphine-induced rotation rate was significantly decreased at postoperative 16 weeks in cell grafted group as compared with that of corresponding relative rotation rate in control group , also with significant difference （P 〈 0.05）.Immunohistological results showed that donor cells could differentiate into large and multi-polar dopaminergic neurons in the host body. CONCLUSION ： Primary cultured embryonic MPCs can be used as the donor cells in CRT for treating PD.

Establishment of primary cultures of craniopharyngioma cells

Craniopharynigoma samples were collected from 36 patients. Out of the 36 samples, 29 achieved successful sub-culturing, with a success rate of 80.6%. Immunohistochemistry staining showed that cytokeratin-7 was positively expressed in the cytomembrane and cytoplasm of craniopharyngioma cells at 6-8 passages, confirming that all cultured cells were squamous epithelial cells. The doubling time of craniopharyngioma cells was 3 days, as confirmed by the 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide assay. In this study, craniopharyngioma cells cultured in vitro were established; however, establishment of immortalized craniopharyngioma cell lines requires further research.

Primary Culture of Alveolar Epithelial Type Ⅱ Cells and Its Bionomic Study

To establish a better method of primary culture for alveolar epithelial type Ⅱ cells （AEC Ⅱ ） and to study its bionomics, alveolar epithelial type Ⅱ cells were isolated by digestion with trypsin and collagenase, which were then purified by plated into culture flask coated with rat immunoglobulin （i The purified AEC Ⅱ were identified by alkaline phosphatase staining, electron mi- croscopy, immunocytochemical staining of pulmonary surfactant protein A （SPA）. The SPA expression and transfection characteristics were compared with those of A549 cell line. The results showed that AEC Ⅱ could be isolated by digestion with trysin and collagenase and purified by adhesive purification by using IgG, with a yield of about 2-3 × 10^7, and a purity of about 75%-84 %. Cells could be quickly identified with AKP staining. AEC Ⅱ were different from A549 cell line in terms of SPA expression and transfection characteristics. It is concluded that adhesive purification with IgG can improve the purity of AEC Ⅱ, and AKP staining is simple in cell identification. AEC Ⅱ can not be completely replaced by A549 cells in some studies because the differences between them, such as SPA expression.

The study of selective primary culture and determination of a breast cancer cell line in vitro

Objective The successful establishment of a tumor cell bank is based on the premise that the target cells can be cultured by a legitimate approach.In this experiment,we used primary culture to select and detect breast cancer cells in vitro,which can provide experimental ideas and methods for the establishment of a living tumor tissue cell bank.Methods Fifty-two specimens were collected over a two-year period from people with breast cancer who needed surgical treatment in our hospital.Cells were isolated and used to establish successful cell culture.Cell activity and cell purity were measured before liquid nitrogen cryopreservation.Results(1)At the initial culture stage,cells grew with adherence.Cell multiplication could be seen after the cell medium was exchanged three times.Cell viability was above 86%,while the viability of the target cells was above 75%,as detected by hematoxylin and eosin(HE)staining.(2)The number of breast cancer cells decreased,while the number of fibroblasts increased after five rounds of passage.(3)The success rate was 73.08%,which did not include polluted cells and those that were not successfully cryopreserved.Conclusion(1)breast cancer cells could be selected from primary culture in vitro through an appropriate method.(2)Exchange of the cell medium and further cell passage improved cell multiplication.(3)The experimental results could be monitored using trypan blue and HE staining.(4)The success of breast cancer cell culture in vitro could be used as a reference for other cell culture,so as to establish a tumor tissue cell bank.

Cytotoxicity of Modified Nonequilibrium Plasma with Chlorhexidine Digluconate on Primary Cultured Human Gingival Fibroblasts

The aim of this study was to investigate the cytotoxicity of modified nonequilibrium plasma with chlorhexidine digluconate（CHX） on human gingival fibroblasts（HGFs）, and to evaluate the biosecurity of modified nonequilibrium plasma with 2% CHX as a new method of root canal treatment. Tissue samples taken from human gingiva were primarily cultured and passaged. Cells from passages 3–7 were used. HGFs were treated by modified nonequilibrium plasma with 2% CHX for 0 min（control group）, 30 s, 1 min, 1.5 min, 3 min, 5 min, and 10 min, respectively, and then they were incubated for 0, 24, and 48 h. After that, cell counting kit-8（CCK-8） assay was applied to analyze the cytotoxicity of modified nonequilibrium plasma with 2% CHX on HGFs. There was no significant difference between the 0 h group treated with the modified nonequilibrium plasma for 1 min and the control group（P〉0.05）. However, there were significant differences between all the other treated groups and the control group（P〈0.05）. When treated for 1.5 min or shorter, the cell viability was obviously increased; while treated for 3 min or longer, it was obviously reduced. Moreover, when successively cultured for 0, 24, and 48 h, cell viability was decreased at first and then increased in the 3-min-treated and 5-min-treated groups. The modified nonequilibrium plasma with 2% CHX was of no influence on cell viability in 1.5 min treatment, and it could be safely used on root canal treatment.

Effect of the gene silencing of phosphorus transporters on phosphorus absorption across primary cultured duodenal epithelial cell monolayers of chick embryos

The aim of the study was to investigate whether phosphorus(P) transporters, type IIb sodium-dependent phosphate cotransporter(NaP-IIb) and inorganic phosphate transporter 2(PiT2), were directly involved in P absorption across primary cultured duodenal epithelial cell monolayers of chick embryos. The siRNAs against NaP-IIb or PiT2 were designed, synthesized and transfected into primary cultured duodenal epithelial cells of chick embryos. Then, the inhibitory efficiency of siRNAs against NaP-IIb or PiT2 was analyzed, and the most efficacious siRNAs were selected to be used for subsequent P absorption experiments. Briefly, primary cultured duodenal epithelial cells of chick embryos were transfected with either NaP-IIb or PiT2 siRNAs and grown in confluent monolayers on transwell plates. The untransfected or transfected cell monolayers were then incubated in an uptake medium containing 0 or 0.25 mmol L^(–1) of P as KH_(2) PO_(4) to measure the P absorption across duodenal epithelial cell monolayers. The results showed that among the siRNAs designed, si-1372 and si-890 were demonstrated to be the most effective in inhibiting the NaPIIb and PiT2 expressions, respectively. Supplemental P increased(P=0.065) the protein abundance of PiT2 and enhanced(P<0.0001) P absorption in primary cultured duodenal epithelial cell of chick embryos. Furthermore, NaPIIb silencing decreased(P=0.07) P absorption across duodenal epithelial cell monolayers, while PiT2 silencing had no effect(P=0.345). It is concluded that the NaP-IIb, but not PiT2, might be directly involved in the P absorption of chick duodenal epithelial cells.

Primary culture and identify of cerebral cortical astrocytes from Sprague-Dawley rats

OBJECTIVE To establish a method for primary cultured and iden-tified Sprague-Dawley(SD) rats cerebral cortical astrocytes. METHODS Cerebral co-rtex of SD neonatal rats within 24 h was taken with stereo microscope and was cut topieces(1 mm^3),digested by Accutase and 0.1% DNAase(37℃,15 min),anddispersed cell suspension was made by mechanical method and filtered. The fibroblast cells and microglia were removed through differential adhesion and sha-ke. Passaged cells were identified by immunofluorescent with anti-Glial fibrillaryacidic protein(GFAP) antibody. RESULTS The astrocytes of rats cerebral cortex were cultured in this method,which had a large number of cells,good activity,high purity,abundant and elongated cell processes,and were interwoven into a network,showing a typical and good growth state. The third generation of the cel s comprised >95% astrocytes. CONCLUSION This simple and reliable cultivation method of astrocyte from rat cerebral tissue is established with high purity,and in a good growth condition.

Primary culture and identification about brain microvascular endothelial cells of rabbits

Objective:To establish a simple and efficient culture method of primary rabbit brain microvascular endothelial cells,provide important carriers and tool cells for the research of related cerebrovascular diseases.Methods:The cerebral cortexes of rabbits were collected aseptic and inoculated after cutting,passing through cell sieve,bovine serum albumin density gradient centrifugation,typeⅡcollagenase digestion,finally inoculated and cultured.The cultured cells were identified by cell morphological observation and angiogenesis experiment.Results:Under the inverted microscope,the cells were short fusiform or polygonal,and grew in clusters and adhere to the wall.After the cells were densely fused,they would be in a typical monolayer flat,“pebbled"mosaic arrangement.Tube formation test had the ability to form tubes structure.Conclusion:This method can successfully separate and cultivate primary rabbit brain microvascular endothelial cells.

Nutrients Modulate T1r2 Transcript Levels in MIN 6 and Primary Cultured Taste Buds Cells under High Glucose Condition

The sweet taste receptors comprised of T1r2 and T1r3, sense glucose concentrations in the gastrointestine. While hyperglycemia was reported to decrease the T1R2 and T1R3 tanscript levels in healthy subjects, no change was observed in type 2 diabetes patients. We investigated which glucose level and nutrients affect those transcript levels in MIN 6 and primary cultured taste buds cells using quantitative Reverse Trancription Polymerase Chain Reaction. High glucose diminished T1r2 transcript levels in MIN 6 and primary cultured taste buds cells. Resveratrol and its analogue augmented transcript levels of T1r1 and T1r2 above normal levels in MIN 6 cells in the medium with 25 mM glucose. Adenine, but not guanine, augmented T1r2 transcript levels of MIN 6 cells in the medium with 25 mM glucose. These results imply that nutrients in meals could affect sweet taste sensitivity by modulating T1r2 transcript levels in response to blood glucose levels.

An improved primary culture method for hippocampal neurons in fetal rats and MAP2 identification

Objective： To establish a simple, effective and high-purity primary culture method for fetal rat hippocampalneurons. Methods： Wistar rats of gestational age 18 days were taken and the brain tissue was separated under themicroscope. Single neuronal cells were obtained by digestion with Brain Dissociation Kit, and then were seeded incell plates to observe the basic morphologic structure after 24h, 3d, and 5d. Immunofluorescence of microtubuleassociated protein 2 was applied to assess cell purity of the culture. Results： The hippocampal neurons obtained inthis culture method are in good condition and grow vigorously. On the 7th day after culture, the purity of neuronswas up to 99.62%. Conclusion： The method is simple and effective for obtaining the high-purity and stableneurons.

Protective Effects of Quercetin on Cadmium-induced Cytotoxicity in Primary Cultures of Rat Proximal Tubular Cells

Objective To investigate the protective effects of quercetin on cadmium-induced cytotoxicity in primary cultures of rat proximal tubular （rPT） cells. Methods Primary cultures of rPT cells undergoing exponential growth were incubated with 1.0 ug/mL quercetin and/or cadmium （2.5, 5.0 umol/L）, in a serum-free medium at 37℃ at different time intervals. Commercial kits were used and flow cytometric analyses were performed on rPT cell cultures to assay apoptosis and oxidative stress. Results Exposure of rPT cells to cadmium acetate （2.5, 5.0 umol/L） induced a decrease in cell viability, caused an increase in apoptotic rate and apoptotic morphological changes. Simultaneously, elevation of intracellular reactive oxygen species, malondialdehyde and calcium levels, depletion of mitochondrial membrane potential and intracellular glutathione, and inhibition of Na＋, K＋ -ATPase, Ca2＋ -ATPase, glutathione peroxidase （GSH-Px）, catalase （CAT）, and superoxide dismutase （SOD） activities were revealed during the cadmium exposure of rPT cells. However, simultaneous supplementation with 1 ug/mL quercetin protected rPT cells against cadmium-induced cytotoxicity through inhibiting apoptosis, attenuating lipid peroxidation, renewing mitochondrial function and elevating the intracellular antioxidants （non-enzymatic and enzymic） levels. Conclusion The present study has suggested that quercetin, as a widely distributed dietary antioxidant, contributes potentially to prevent cadmium-induced cytotoxicity in rPT cells.

Bis(7)-Tacrine, a Promising Anti-Alzheimer's Agent,Attenuates Glutamate-Induced Cell Injury in Primary Cultured Cerebrocortical Neurons of Rats

The effects of bis(7) tacrine, a novel dimeric acetylcholinesterase (AChE) inhibitor, on glutamate induced cell injury were investigated in primary cerebral cortical neurons of rats. Exposure of cultured neurons (12 days after plating) to 0.5 mmol/L glutamate for 30 min resulted in significant cell damage. Pretreatment with bis(7) tacrine (0.03 1.0 μmol/L) reduced the glutamate induced neurotoxicity in a concentration dependent manner and the maximal response was seen at 1 μmol/L with approximately 30% protection. A receptor binding assay showed that bis(7) tacrine can completely displace MK 801 binding to rat cortical membrane with an IC 50 of 0.57 μmol/L. These findings suggest that bis(7) tacrine can directly interact with N methyl D aspartate receptor channel complex, which may contribute to the inhibitor's protective effects against glutamate induced excitotoxicity. Thus, it is possible that anti glutamate/anti AChE synergism is responsible for potentially better Alzheimer's therapy of bis(7) tacrine relative to tacrine.

Effect of neuronal excitotoxicity on Munc18-1 distribution in nuclei of rat hippocampal neuron and primary cultured neuron

Objective Muncl8-1 has an important role in neurotransmitter release, and controls every step in the exocy- totic pathway in the central nervous system. In the present study, whether epileptic seizure causes a change of Muncl8 localization in neuronal nuclei was analyzed. Methods Epilepsy models were established by injection of kainic acid （KA） solution into hippocampus of Sprague-Dawley （SD） rats or intraperitoneal injection of KA in Kunming mice. The hippocampal neurons were prepared from embryonic day 18 SD rats, and cultured in neurobasal medium, followed by treatment with glutamate for 3 h. Neuronal and glial nuclei of hippocampus were separated by sucrose density gradient centrifugation. The nucleus-enriched fractions were stained with 0.1% Cresyl Violet for morphological assay. Immuno- chemistry and immunoelectron microscopy with anti-Muncl 8-1 antibody were used to determine the nuclear locatization of Munc 18-1. Immunoblotting was used to detect the protein level of Munc 18-1. Results The localization of Munc 18-1 in nucleus of rat hippocampal neuron was confirmed by immunochemistry, immunoelectron microscopy, and immunob- lotting detection of neuronal nucleus fraction. In animals receiving intrahippocampal or intraperitoneal injection of KA, immunostaining revealed that the expression of Muncl 8-1 decreased in pyramidal cell layer of CA regions, as well as in hilus and granular cell layer of dentate gyrus in hippocampus. Moreover, immunoblotting analysis showed that the expres- sion level of Muncl 8-1 in nucleus fraction of hippocampus significantly decreased in KA-treated animals. The relation- ship between the change of Muncl8-1 expression in neuronal nuclei and neuronal over-activation was also tested in pri- mary cultured neurons. After treatment with 50 ~tmol/L glutamate acid for 3 h, Muncl8-1 level was decreased in nucleus fraction and increased in cytoplasmic fraction of primary cultured neurons. Conclusion These results suggest that excit- atory stimulation can induce the distribution change of Munc 18-1 in neuron, which may subsequently modulate neuronal functions in brain.

Reversing effect of exogenous WWOX gene expression on malignant phenotype of primary cultured lung carcinoma cells

Background Whether WW domain containing oxidoreductase （WWOX） gene is a tumor-suppressor is still controversial. Some researchers found that the transcription of the WWOX gene was lacking not only in tumor tissues but also in non-tumorous tissues and sometimes in normal tissues. Hence it is important to explore the role of the expression of the exogenous WWOXgene in the proliferation and apoptosis of primary cultured lung carcinoma cells. Methods Lipofection technique was used to determine primary cultured lung carcinoma cells containing the highly expressed exogenous WWOX gene and primary cultured cells with vectors as controls. An animal model of lung cancer was made by subcutaneous implantation of tumor cells into nude mice. RT-PCR, Western blotting, flow cytometry, and TUNEL were used to detect the transcription, expression of the exogenous gene and the effect of the expression of targeted genes on the proliferation and apoptosis of the primary cultured lung carcinoma cells. Results The growth, clone formation rate （CFR） （（5.33±1.53）%） of the primary lung cancer cells transfected with the WWOX gene, tumor size and weight were significantly lower than those of the non-transfected lung cancer cells （CFR： （14.33±1.53）%） and the primary tung cancer cells transfected with blank plasmids （CFR： （11.00±1.73）%, P 〈0.05）. The apoptosis level of primary lung cancer cells transfected with the WWOX gene （（40.72±5.20）%） was significantly higher than that of the non-transfected lung cancer cells （（2.76±0.02）%） and the primary lung cancer cells transfected with blank plasmids （（2.72±0.15）%, P 〈0.05）. Conclusion The expression of the exogenous WWOXgene can significantly inhibit the proliferation of lung cancer cells and induce their apoptosis, suggesting that the WWOX gene possesses tumor-suppressing effect.

Effects of Single and Repeated Exposure to a 50-Hz 2-mT Electromagnetic Field on Primary Cultured Hippocampal Neurons

The prevalence of domestic and industrial electrical appliances has raised concerns about the health risk of extremely low-frequency magnetic fields（ELF-MFs）. At present, the effects of ELF-MFs on the central nervous system are still highly controversial, and few studies have investigated its effects on cultured neurons. Here, we evaluated the biological effects of different patterns of ELF-MF exposure on primary cultured hippocampal neurons in terms of viability, apoptosis, genomic instability,and oxidative stress. The results showed that repeated exposure to 50-Hz 2-mT ELF-MF for 8 h per day after different times in culture decreased the viability and increased the production of intracellular reactive oxidative species in hippocampal neurons. The mechanism was potentially related to the up-regulation of Nox2 expression.Moreover, none of the repeated exposure patterns had significant effects on DNA damage, apoptosis, or autophagy, which suggested that ELF-MF exposure has no severe biological consequences in cultured hippocampal neurons.

Differences in nutrient uptake between the fat body and embryonic primary cultures of silkworm （Bombyx mori）

Nutrition utilization and by-product formation in cultured insect cells has been investigated in several insect cells and has been of great interest to cell culturists and physiologists. In this research the biochemical changes in embryonic and fat body primary cultures of silkworm, Bombyx mori, have been compared. TC-100 medium supplemented with 10% and 20% FBS was used in embryonic and fat body primary cultures, respectively. Medium was renewed every week and the amount of glucose, uric acid, urea, total protein and alkaline phosphatase were measured in the samples from medium of primary cultures using spectrophotometeric methods. All biochemical macromolecules except uric acid showed significant changes. Glucose decreased in embryonic tissues, while in fat body culture its amount increased. Urea accumulation in embryonic culture was higher than in the fat body cultures. Since urea is a by-product, this accumulation could be due to higher utilization of amino acids. Total protein showed considerable changes and was consumed by embryonic culture more than the fat body＇ s. Alkaline phosphatase showed stronger activity in embryonic cells.

Establishment of a novel method for primary culture of normal human cervical keratinocytes

Background Cervical keratinocytes are recovered at a low numbers and frequently associated with contaminating human fibroblasts which rapidly overgrow the epithelial cells in culture with medium supplemented with 10% fetal bovine serum （FBS）. However, it is difficult to initiate keratinocyte cultures with serum-free keratinocyte growth medium alone because cell attachment can be poor. Therefore, the culture of these cells is extremely difficult. In this study, we described a modified culture medium and coated culture plastics for growing normal human cervical epithelial cells in vitro. Methods Normal cervical epithelial tissue pieces were obtained and digested with type I collagenase to dissociate the cells and a single cell suspension produced. The cells were cultured on plastic tissue culture substrate alone or substrate coated with collagen type I from rat tail, with modified keratinocyte serum-free medium （K-SFM） supplemented with 5% FBS. After attachment, the medium were replaced with K-SFM without FBS. The expression of basal keratins of the ectocervical epithelium, K5, K14 and K19 were assayed by immunofluorescence with monoclonal antibodies to identify the cell purity. Results Our results indicate that cells attached to the culture plastic more quickly in K-SFM supplemented with 5% FBS than in K-SFM alone, as well as to tissue culture plastic coated with collagen type I than plastic alone. The modified medium composed of K-SFM and 5% FBS combined with a specific tissue culture plastic coated with collagen type I from rat tail was the best method for culture of normal cervical epithelial cells. K5, K14 and K19 were assayed and keratinocyte purity was nearly 100%. Conclusion A novel, simple and effective method can be used to rapidly obtain highly purified keratinocytes from normal human cervical epithelium.