The use of Alamar Blue assay for quantitative analysis of viability, migration and invasion of choriocarcinoma cells

BACKGROUND: The current techniques for quantifying trophoblast viability, migration and invasion are mainly limited by the need to sacrifice the cells during the test procedure. In this study, the vital dye AB (AB) was used to quantify cell number and viability of BeWo and JEG-3 choriocarcinoma cells, as well as their migration and invasion through fibronectin-coated filters. METHODS :AB was directly added to culture medium of incubated test and control cells. At various time intervals, the redox reaction, in which AB is reduced by the cells, was measured by absorbance readings at 540 and 630 nm. For cell migration and invasion, cells were cultured onto uncoated or fibronectin-coated inserts, respectively. AB reduction of migrated cells was normalized to that of control cells to calculate percentages of migration. This model was also tested in the presence of a reported inhibitor, transforming growth factor (TGF) beta. RESULTS: The curve of %AB reduction versus cell number was linear, with intra- and inter-assay Coefficient of Variations of 1.88%and 2.94%, respectively. AB reduction increased with both seeding concentrations and incubation time with AB. TGFbeta treatment caused a modest decrease in AB reduction in both JEG-3 and BeWo cells. TGFbeta treatment also decreased migration in BeWo, but not in JEG-3, cells. CONCLUSIONS: AB assay is a simple and reliable method for quantifying trophoblast viability, migration and invasion.     

Relevance of syndecan-1 in the trophoblastic BeWo cell syncytialization

Citation Prakash GJ, Suman P, Gupta SK. Relevance of syndecan‐1 in the trophoblastic BeWo cell syncytialization. Am J Reprod Immunol 2011; 66: 385–393 Problem To investigate the role of syndecan‐1 in the differentiation of the BeWo cells into syncytiotrophoblast. Method of study BeWo cells were stimulated with forskolin to form syncytia, and the expression of syndecan‐1, desmoplakin I+II, human chorionic gonadotrophin (hCG) and angiogenesis‐associated factors was analyzed. Syndecan‐1 was silenced by siRNA to evaluate its involvement in the forskolin‐mediated syncytia formation. Results Treatment of the BeWo cells with forskolin led to a significant increase in the syncytia formation. It was associated with an increase in the expression of syndecan‐1 with a concomitant decrease in the expression of desmoplakin I+II. Forskolin treatment of the BeWo cells also led to an increase in the secretion of soluble endoglin, whereas no change was observed in the soluble fms‐like tyrosine kinase‐1. Silencing of the syndecan‐1 expression in BeWo cells led to a significant decrease in cell fusion both in the presence and in the absence of forskolin. It was associated with a significant decrease in hCG level in the conditioned medium. Conclusion Syndecan‐1 is up‐regulated in BeWo cells during differentiation and its silencing inhibits syncytialization and thus could be a useful biomarker for syncytiotrophoblast formation.     

Behaviour of normal and neoplastic cultured mouse cells on the chorioallantoic membrane of the chicken.

Cultivated normal and transformed fibroblasts of the mouse (short-term cultures of lung fibroblasts and L-cells) have been implanted onto the chorioallantoic membrane of the chick embryo (CAM). Both cell types formed macroscopically visible nodules on the CAM where they induced a weak angiogenic reaction. Labelling of the cells with activated charcoal or 3H-thymidine gave evidence of their invasion into the CAM mesoderm, where they induced the formation of new capillaries. The successive multiplication of the cells led to the formation of tumours, resp. tumour-like cellular accumulations in the hypertrophied mesoderm of the CAM. Treatment of L-cells with the protease-inhibitor Contrykal reduced the invasive properties of the cells. The results presented clearly demonstrate invasive and angiogenic properties of the normal and malignantly transformed cell cultures of the mouse used in our experiments.     

Manipulation of CD98 Expression Affects both Trophoblast Cell Fusion and Amino Acid Transport Activity during Syncytialization of Human Placental BeWo Cells

The physiological importance of CD98 surface antigen in regulating placental trophoblast cell fusion and amino acid transport activity has been studied in parallel in a cell model of syncytialization (the cytotrophoblast cell line BeWo following increased intracellular cAMP by forskolin treatment) using antisense oligonucleotides. CD98 protein abundance (determined by Western blot) was decreased by 50 % following antisense oligonucleotide transfection. Transfection with antisense oligonucleotide altered the responses of BeWo to forskolin. Cell fusion (determined by a quantitative flow cytometry assay) was inhibited by 57 %, and both human chorionic gonadotropin secretion and L-leucine influx through system L were suppressed. These findings show that CD98 is involved in the process of cell fusion necessary for syncytiotrophoblast formation and that during this physiologically important event, amino acid transport activity is also regulated through expression of this membrane protein.     

Differential effects of inducers of syncytialization and apoptosis on BeWo and JEG-3 choriocarcinoma cells

BACKGROUND: The interactions of trophoblasts with the cytokine network at the fetomaternal interface determine the pathway the cell undertakes, e.g. proliferation, differentiation and apoptosis. METHODS: We used cultures of fusigenic BeWo and non-fusigenic JEG-3 choriocarcinoma cells to study the effects of inducers of syncytialisation (forskolin) and apoptosis [tumour necrosis factor-alpha (TNFalpha)] on differentiation, viability, proliferation and apoptosis. RESULTS: E-cadherin immunostaining showed that syncytium formation was confined to BeWo and not JEG-3 cells, while secretion of hCG was promoted by forskolin in both cell types implying a 'dissociation' between morphological and biochemical differentiation. Forskolin also had differential effects on cell viability (MTT reduction test) and proliferation (Ki67 immunostaining with MIB-1 monoclonal antibody), both decreasing in BeWo and increasing in JEG-3 cells. TNFalpha increased apoptosis (cytokeratin neo-epitope immunostaining with M30 monoclonal antibody) in both cell types, an effect which was blocked by epidermal growth factor selectively in JEG-3 cells. CONCLUSION: Our results suggest that the differential responses of BeWo and JEG-3 cells to inducers of syncytialization and apoptosis might be related to their fusigenic capacity. Caution is needed when extrapolating results obtained by these models to normal trophoblast populations. However, we speculate that these models can help identify key factors involved in trophoblast differentiation at the placental bed.     

Bone sialoprotein promotes tumor cell migration in both in vitro and in vivo models

The present study was conducted to determine the effects of bone sialoprotein (BSP) in promoting vascular invasion of tumor cells in metastasis. We used a Matrigel system and the MDA-231 human breast cancer cells transfected with human BSP cDNA (MDA-231/BSP). Quantative analysis indicated an average of 1.7-fold increase in cell numbers that migrated through the endothelial cells in MDA-231/BSP cells compared with empty vector-transfected MDA-231 cells (MDA-231/EV). In an in vivo assay, the MDA-231 cells were incubated with or without BSP antibodies and were then inoculated onto the upper chorioallantoic membrane (CAM) of chicken embryos, in which the only route for the tumor cells to reach the lower CAM was to migrate through the embryonic vasculature. PCR amplification using human Alu primers and genomic DNA from harvested lower CAM showed an average reduction of 67% in the samples treated with BSP antibodies. These preliminary data suggest that, in metastasis, BSP may enhance the penetrating ability of tumor cells through endothelial cells and basement membrane into blood vessels. BSP antibodies can specifically hinder this effect in an in vivo system.     

Bone Sialoprotein Promotes Tumor Cell Migration in Both In Vitro and In Vivo Models

The present study was conducted to determine the effects of bone sialoprotein (BSP) in promoting vascular invasion of tumor cells in metastasis. We used a Matrigel system and the MDA-231 human breast cancer cells transfected with human BSP cDNA (MDA-231/BSP). Quantative analysis indicated an average of 1.7-fold increase in cell numbers that migrated through the endothelial cells in MDA-231/BSP cells compared with empty vector-transfected MDA-231 cells (MDA-231/EV). In an in vivo assay, the MDA-231 cells were incubated with or without BSP antibodies and were then inoculated onto the upper chorioallantoic membrane (CAM) of chicken embryos, in which the only route for the tumor cells to reach the lower CAM was to migrate through the embryonic vasculature. PCR amplification using human Alu primers and genomic DNA from harvested lower CAM showed an average reduction of 67% in the samples treated with BSP antibodies. These preliminary data suggest that, in metastasis, BSP may enhance the penetrating ability of tumor cells through endothelial cells and basement membrane into blood vessels. BSP antibodies can specifically hinder this effect in an in vivo system.     

Generation and Characterization of Monoclonal Antibodies Specific to Surface Antigens of Human Trophoblast Cells

PROBLEM : To generate and utilize specific monoclonal antibodies for routine fetal cell isolation from the maternal circulation. METHODS : Monoclonal antibodies specific to human trophoblast cell surface antigens were generated and characterized. After cell fusion, antibodies secreted by hybridomas were screened by enzyme-linked immunosorbent assay and immunohistochemical assays. RESULTS : By using cultured BeWo choriocarcinoma cells or the membrane fraction of human placenta as the immunogen, seven (BW-108, 110, 123, 124, HP-15, 16 and 17) antibodies specific to the surface antigens of trophoblast were produced. They were shown to have little cross-reactivity to other human tissues. Among the antibodies raised against human sperm, HSA-10 was also found to cross-react with human trophoblast, but not detected in other tissues. When immobilized to magnetic beads, these antibodies were shown to react only with BeWo cells in suspension, but not blood cells and ovarian carcinoma cell line, OC-3-VGH. CONCLUSION : Therefore, these antibodies may have potential application in fetal trophoblast cell isolation from the maternal circulation for prenatal genetic diagnosis.     

Ultrastructural evidence for loss of the trophoblastic layer in the chorioallantoic placenta of Australian Bandicoots (Marsupialia: Peramelidae)

In most marsupials, placentation involves only the yolk sac; however, in the bandicoot family, Peramelidae, a functional chorioallantoic placentation develops in addition (Hill, 1895, 1897, 1900; Flynn, ′22, ′23). This duality is viewed as having evolutionary significance because most eutheria have both placentae. Furthermore, the bandicoot trophoblast was reported to vanish from the chorioallantoic site in late gestation (Hill, 1897; Flynn, ′23); whereas, the eutherian trophoblast is identifiable throughout later pregnancy and may act as an immunological barrier between maternal and fetal genotypes (Kirby, ′68). Thus we have re-examined this singular chorioallantoic placenta of the bandicoot in plastic sections with light and electron microscopy. A distinctive feature of bandicoot placentation is the transformation of the uterine simple columnar luminal epithelium into a highly vascular lining composed almost entirely of discrete syncytial masses (homokaryons). Endometrial blood vessels penetrate among the homokaryons to create a rich network of large diameter capillaries at extremely superficial locations near the maternal surface. In the chorioallantoic placenta (7 mm to 10–11 mm crown-rump embryos) the microvillous surface of the maternal homokaryons interdigitates with the microvillous border of the fetal trophoblast with desmosomal interaction. This trophoblast consists of a single layer of tall columnar undifferentiated cells rich in ribosomes-polysomes, poor in cytoplasmic membranes, and with large nuclei that have distinct clumps of heterochromatin and conspicuous nucleoli. It is thus remarkable that these undifferentiated cells disappear as a recognizable layer later in gestation (12 mm crown-rump embryos). Flynn's hypothesis that the trophoblastic cells disapppear by fusing with maternal syncytia gains support from the existence of two populations of nuclei in the syncytial masses only at the chorioallantoic site. One population is comparable to that occurring in the homokaryons of the yolk sac placenta, i.e., pale staining nuclei with little heterochromatin and small peripheral nucleoli. However, the other nuclei resemble those of the trophoblast cells. Since the trophoblastic cells before their disappearance as a layer possess properties associated with potential for further differentiation, the possibility of fusion between the maternal homokaryons and the fetal trophoblastic cells to form heterokaryons composed of two genotypes merits further consideration. The disappearance of the trophoblastic layer and the superficial positioning of the maternal capillaries bring the maternal and fetal bloodstreams into closest proximity near term (12 mm crown-rump embryo). The thinnest parts of the barrier consist of delicate cytoplasmic extensions from the syncytial masses (that may be maternal in origin or jointly maternal and fetal) and a layer of maternal stroma intervening between the maternal and fetal endothelia. Thus the chorioallantoic placental barrier of the marsupial bandicoot is unlike any thus far described for eutherian mammals.     

Mechanism of the induction of angiogenesis by human neoplastic lymphoid tissue: studies on the chorioallantoic membrane (CAM) of the chick embryo

The angiogenic activity of various neoplastic and control tissues, cells and extracts has been tested on the chorioallantoic membrane of the chick (CAM). The vascular response was assessed macroscopically and also by histological examination. Angiogenesis was induced by a number of neoplastic implants the most potent being derived from Hodgkin's disease, histiocytic lymphoma or glioma tissue. Boiled tumour tissue was ineffective. Lymphocytes extracted from human lymphomas, activated normal peripheral blood lymphocytes and established lymphoid cell lines of neoplastic origin were generally effective in inducing neovascularisation through millipore membranes as were 90,000-100,000 MW fractions of human tumour tissue. In all cases examined histologically a mononuclear cell infiltrate in the CAM mesoderm accompanied a positive vascular response. These results implicate host monocytes in the generation of neovascularisation by neoplastic tissue.     

Markers to sensibility and relapse on IMR-32 neuroblastoma cell line cultured in monolayer (2D) and neurosphere (3D) models cisplatin-treated

The complexity of different components of tumor stroma poses huge challenges for therapies targeting the neuroblastoma (NB) microenvironment. The present study aimed to evaluate platinum-based response in IMR-32 neuroblastoma cell line cultured in monolayer (2D) and neurosphere (3D) models. For this, we evaluated mRNA expression of heat shock proteins HSPA1A, HSPB1, TRAP1, HSPA1AL, HSPD1, and DNA damage repair gene ERCC1. After treatment, residual cells were grafted on CAM (chicken chorioallantoic membrane) to evaluate the growth capability and histological paraffin sections were made to assess Ki-67 and HER-2 proteins by immunofluorescence. Our results showed that cisplatin induces mRNA downregulation of Heat Shock Proteins and ERCC1 in IMR-32 cells cultured in 2D or 3D models. In addition, the cisplatin-treatment approach increased HER-2 expression in residual IMR-32 cells grafted on the CAM. Therefore, these insights provide many advances in neuroendocrine tumor biology and knowledge about cisplatin-response in neuroblastoma.     

不同鼻咽癌细胞株对鸡胚尿囊膜模型血管生成影响的比较研究

目的比较不同鼻咽癌细胞株在鸡胚尿囊膜模型上促进血管生成能力的差异。方法选用6日龄鸡胚,开窗后分别种植5-8F、6-10B及CNE-2三种鼻咽癌细胞株,三种细胞株细胞数为2×106/鸡胚者各一组,细胞数为5×105/鸡胚者各一组,另一组为对照组（PBS）,每组10个鸡胚。孵育6日后,统计分析新生血管数及血管面积/鸡胚面积比。结果种植细胞数为5×105/鸡胚时,5-8F、6-10B细胞部分鸡胚有移植瘤形成,CNE-2细胞鸡胚均未见成瘤。种植细胞数为2×106/鸡胚时,三种鼻咽癌细胞株均可100%成瘤,其新生血管数依次递减,分别为（38.7±2.50）、（33.5±4.43）、（29.7±2.71）,差异有统计学意义（P〈0.05）;血管面积/鸡胚面积比分别为（22.2±2.18）%、（18.7±2.45）%、（16.9±2.62）%,均高于对照组的（9.5±1.86）%,差异有统计学意义（P〈0.05）,且5-8F面积比高于其他两种细胞株（P〈0.05）。结论鼻咽癌5-8F、6-10B及CNE-2三种细胞株在鸡胚尿囊膜模型上血管生成能力依次减弱,实验研究可根据实际情况选用。     

Heritability and number of genes controlling susceptibility of chick embryo chorioallantoic membranes (CAM) to RSV

Two lines of commercial chickens differing significantly in pock responses on chorioallantoic membrane (CAM) of embryonating eggs were studied to estimate heritability and the number of pairs of genes which control this response. By using Wright's formula it was found that one pair of genes controls pock response of the CAM, suggesting a monogenic Mendelian inheritance of the trait. The heritability estimate of the trait was 0.71.     

BeWo cells stimulate smooth muscle cell apoptosis and elastin breakdown in a model of spiral artery transformation

BACKGROUND: During pregnancy, extravillous trophoblast invades the uterine wall and enters the spiral arteries. Remodelling ensues, with loss of vascular smooth muscle cells (SMCs) to create high flow, low resistance vessels. Pregnancies complicated by pre-eclampsia are characterized by incomplete arterial remodelling. Endovascular trophoblast is not easily accessible for studies to establish the pathogenesis of pre-eclampsia, so we have developed a model appropriate to carry out mechanistic studies of vessel wall transformation. METHODS AND RESULTS: Segments of human spiral artery were perfused with the choriocarcinoma cell line, BeWo; cells invaded the vessel wall and induced apoptosis of vascular SMC. Perfusion of vessels with BeWo-conditioned medium also induced SMC apoptosis, indicating the presence of a soluble apoptotic factor. BeWo express Fas ligand (FasL) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Treatment of BeWo-conditioned medium with antibodies against FasL inhibited vascular SMC apoptosis in vitro. Antibodies that blocked TRAIL receptor function had no effect. Extracellular matrix degradation is also a prerequisite for vascular remodelling; BeWo express matrix metalloproteinase-12 (MMP-12) and BeWo-conditioned medium increased MMP-12 expression in spiral artery SMC. CONCLUSIONS: BeWo induce arterial remodelling via FasL- and MMP-12-dependent mechanisms. BeWo-derived factors up-regulate protease expression in spiral artery SMC to facilitate matrix breakdown.     

乙型肝炎病毒阳性血清体外感染Hep G2细胞的实验研究

目的　建立HBV阳性血清体外感染HepG2 细胞的实验方法。方法　培养HepG2 细胞传至6孔板中,2 4h后进行HBV阳性血清体外感染HepG2 细胞的实验。感染组用HBV阳性血清,阴性对照组用HBV阴性血清,空白对照组用DMEM培养基。实验开始后HepG2 细胞继续孵育2 4h ,而后用0 0 1mol LPBS清洗8次后加入2 %DMEM培养液。收集PBS第8次洗液,收集PBS洗后每隔12h各孔细胞培养上清。ELISA检测细胞培养上清中的HBsAg。PCR检测细胞培养上清和HepG2 细胞中的HBVDNA。结果　感染组在PBS洗后12h的细胞培养上清中ELISA检测HBsAg呈阳性。PCR检测显示感染组细胞培养上清和HepG2 细胞中HBVDNA呈阳性,阴性对照组和空白对照组HBVDNA呈阴性。结论　HBV阳性血清进行HBV感染体外培养HepG2 细胞是可行的。     

Expression of membrane and nuclear progesterone receptors in two human placental choriocarcinoma cell lines (JEG-3 and BeWo): Effects of syncytialization

A vital function of the human placenta is to produce steroid hormones such as progesterone, which are essential for the maintenance of pregnancy and the onset of parturition. Although choriocarcinoma cell lines are valuable placental models for investigations of steroid hormone actions, little is known about the expression of progesterone receptors (PRs) in these cell lines. Therefore, in this study, the expression of membrane and nuclear PRs was investigated in cultures of fusigenic (BeWo) and non-fusigenic (JEG-3) human choriocarcinoma cell lines. In addition, the effects of an inducer of syncytialization (forskolin) on the PR expression in BeWo cells were assessed. Quantitative RT-PCR revealed that in fully syncytialized BeWo cells (treated with 50 μM forskolin for 72 h) there was a significant down-regulation of mPRα and up-regulation of mPRβ and of the progesterone membrane component-1 (PGRMC1) when compared with non-syncytialized BeWo cells. Expression of all the mPR and PGRMC1 mRNAs was significantly lower in JEG-3 cells compared to non-syncytialized BeWo cells. Interestingly, expression of PR-B was unaltered between the two BeWo states but was significantly higher in JEG-3 cells. Immunofluorescence analysis revealed that mPR proteins are differentially expressed in these choriocarcinoma cell lines as well as in the human placenta. The data demonstrate that human choriocarcinoma cell lines have a complex system of progesterone signalling involving multiple classes of PRs. The finding that syncytialization is accompanied by changes in the expression of these receptors may suggest that this process influences progesterone signalling.     

The chick chorioallantoic membrane as a novel in vivo model for the testing of biomaterials

Current in vivo models for testing biomaterials are time and labor intensive as well as expensive. This article describes a new approach for testing biomaterials in vivo using the chorioallantoic membrane (CAM) of the developing chicken embryo, as an alternative to the traditional mammalian models. Fertilized chicken eggs were incubated for 4 days, at which time a small window was cut in the shell of the egg. After 1 week of incubation, the CAM received several test materials, including the endotoxin LPS, a cotton thread and a Silastic tubing. One day and 1 week later, the tissue response to the test materials was assessed using gross, histological, and scanning electron microscope evaluations. The inflammatory response of the chorioallantoic membrane to biomaterials was fully characterized and found to be similar to that of the mammalian response and was also seen to vary according to test materials. We also found that the structure and geometry of the test materials greatly influenced the incorporation of the samples in the CAM. The similarity of the tissue response of the CAM with the mammalian models, plus the low cost, simplicity, and possibility to continuously visualize the test site through the shell window make this animal model particularly attractive for the rapid in vivo screening of biomaterials.     

Chorioallantoic membrane angiogenesis model for tissue engineering: a new twist on a classic model

Tissue-engineering (TE) applications include the isolation, culture, and seeding of cells into a suitable matrix or scaffold before in vivo transplantation. After transplantation, vascularization of the scaffold is a principal limiting factor for cell viability for the first 6-8 days posttransplantation. A model for systematic analysis of this process has been developed. Fertilized White Leghorn eggs were incubated (at 37.8 degrees C in 60% relative humidity) and opened on day 3 of incubation. Preadipocyte-seeded fibrin constructs were implanted in a specially designed plastic cylinder and placed through the opening on the surface of the chorioallantoic membrane (CAM) on day 8 of incubation. Vascularization of the constructs by chorioallantoic blood vessels was assessed for up to 8 days posttransplantation. The survival rate for embryos receiving transplanted constructs was about 90%. Histology confirmed transplant cell viability at day 4 posttransplantation and vascularization of the constructs by avian endothelial cells began at this time. A new in vivo model to study the effect of angiogenesis in TE constructs, including assessments of viability, proliferation, and differentiation of transplanted cells and biomaterial properties, is presented. Advantages include easy access to the vascular network of the CAM, lack of immunocompetence, low costs, and avoidance of animal experiments.     

Liver regeneration on chicken chorioallantoic membrane

To explore how the liver regenerates, liver pieces from 15-day-old chicken embryos were grafted onto the chorioallantoic membrane (CAM) of 9-day-old chicken embryos and cultured for 11 days at the longest. The cultured liver pieces were examined histologically. The liver implants were gradually engulfed into the CAM and underwent necrosis of hepatocytes, except in their peripheral areas, during the first 1-4 days after grafting. Surviving cells in the peripheral areas began to proliferate 4 days after grafting. Thereafter, the cells were assembled into normal liver tissues and represented almost all the areas of the implants 9 days after grafting. Only after penetration of blood vessels from CAM did the liver implants enter a phase of rapid regeneration to form well-organized liver tissues. At the early stage of regeneration, the cells at the peripheral areas did not produce albumin, but reproduced it in the regenerated liver tissues, implying that hepatocytes restored their functions that were temporarily lost in the process of regeneration. Thus, we concluded that the liver pieces from 15-day-old chicken embryos had the ability to form normal liver tissues on CAM and that the blood supply played an important role in liver regeneration.