Long-term culture of primary porcine oviduct epithelial cells: Validation of a comprehensive in vitro model for reproductive science

Recently, we established a protocol for the cultivation of primary porcine oviduct epithelial cells (POEC), which promoted tissue-like morphology for a prolonged culture period. The present study focuses on developing this model into a comprehensive, standardized culture system, as a candidate tool for reproductive toxicity testing and basic research. We cultivated POEC isolated from 25 animals in our culture system for both 3 and 6 weeks and systematically analyzed effects of medium conditioning, supplementation with standardized sera, and culture duration in both freshly isolated and cryopreserved cells. The differentiation status was evaluated via histomorphometry, transepithelial electrical resistance (TEER) measurement, and expression analyses. The culture system possessed high reproducibility, more than 95% of cultures achieved a fully differentiated phenotype. Cells recapitulated in vivo–like morphology and ultrastructure from 3 to 6 weeks. Cryopreservation of the cells prior to cultivation did not affect culture quality of POEC. Employment of conditioned medium ensured optimal promotion of POEC differentiation, and different standardized sera induced fully differentiated phenotypes. Consistent TEER establishment indicated the presence and maintenance of cell type–specific intercellular junctions. The functionality of POEC was proven by consistent mucin secretion and stable expression of selected markers over the whole culture duration. We conclude that POEC are suitable for experiments from 3 weeks up to at least 6 weeks of culture. Therefore, this culture system could be used for in vitro estrous cycle simulation and long-term investigation of toxic effects on oviduct epithelium.     

Short-term and long-term effects of embryo culture in the surrogate sheep oviduct versus in vitro culture for different domestic species

The culture of early embryos in the surrogate xeno-oviduct was first developed in the early 1950s to allow transport of embryos at long distances. Later, it was applied to the study of culture requirements of the early embryo especially that of bovine origin. In this article, we review the data available on the culture of in vitro-matured and in vitro-fertilized embryos of Bos taurus, Sus scrofa, Equus caballus and Ovis aries in the surrogate sheep oviduct compared with data on in vitro culture in different media. Short-term and long-term cellular and molecular effects are described mainly for the bovine species where more extensive use of this technique has been made. A comparison with in vitro culture in various conditions and species indicate that embryos cultured in the sheep oviduct have close similarities to totally in vivo-derived embryos. The data provided demonstrate that the technique of in vivo culture in the surrogate sheep oviduct is versatile and allows a high rate of embryonic development in all species examined.     

Up-regulated mRNA expression of some anti-inflammatory mediators in bovine oviduct epithelial cells by urea in vitro: Cellular pathways by Reactome analysis

Increased urea concentration is a major cause of low fertility in dairy cows fed high-protein diets. A strong correlation exists between the urea concentration in the blood and oviduct fluid of dairy cows. In this study, bovine oviduct epithelial cells (BOECs) were incubated with varying concentrations of urea (0, 20, 40, and 80?mg/dL) in the absence of ovarian sex steroids (estradiol and progesterone) and luteinizing hormone. The 80?mg/dL urea reduced the cell viability, and thus was excluded in further analysis. Compared to the control (U0), the 20?mg/dL urea (U20) increased the mRNA expression of Toll-like receptor (TLR) 4, interleukin (IL) 10, IL4, and prostaglandin (PG) E synthase (mPGES) but decreased the mRNA expression of tumor necrosis factor α (TNFA). Compared to U0, the 40?mg/dL urea (U40) decreased the mRNA expression of TNFA and increased alpha-1-acid glycoprotein (AGP). U40 also increased TLR2, IL10, and IL4 mRNA expression compared to U0. In addition, compared to U20, the U40 decreased the mRNA expression of TLR4 and IL1B but increased that of AGP and TLR2. Subsequently, the mRNA expression data were then projected into the Reactome database. The Reactome analysis showed that pathways, including cytokine signaling in the immune system (i.e., TNFs bind their physiological receptors) and death receptor signaling (i.e., TNF signaling), were down-regulated in the presence of urea compared to the U0 group. These in vitro data implied that high urea level can alter the balance between pro- and anti-inflammatory responses in BOECs, thus providing a suboptimal environment for the early reproductive events or a weakened innate immune system, predisposing the oviduct to infections.     

Endoscopic approaches to manage in vitro and in vivo embryo development: Use of the bovine oviduct

The oviduct plays a major part in different reproductive processes providing the microenvironment for numerous steps in early embryogenesis. Consequently, there is a growing demand to perform comparative studies focusing on causal mechanisms related to embryo development within its environment including complex and holistic strategies. However, the routine flushing and transfer procedure of bovine embryos is limited to the morula and blastocyst stage. Additionally, the use of in vitro production of bovine embryos provides access to an extra amount of embryos at various stages. But the quality of these embryos does not reflect the quality of its ex vivo counterparts. For two decades our own studies have focused on use of the oviductal environment of different species to optimize early embryo development for different purposes. The current article briefly highlights some main characteristics of the fallopian tube and reviews the endoscopic approach to access the fallopian tube using the stepwise minimal invasive technique established in different species.     

Immunophenotype of porcine oviduct epithelial cells during the oestrous cycle: a double-labelling immunohistochemical study

The luminal epithelium of the porcine oviduct is composed of ciliated cells and secretory cells, but it is assumed for several species that under the control of steroid hormones secretory cells are able to be transformed into ciliated cells. In order to better understand such physiological changes during the different stages of the oestrous cycle, we evaluated epithelial cell proliferation together with oestrogen receptor (ER) expression of porcine ampullary oviducts. To identify the immunophenotype of proliferating cells, double immunohistochemistry was performed using anti-chromogranin A antibody (anti-CgA) as the second primary antibody. Anti-CgA, recently shown to be an immunocytochemical marker of ciliated cells of the cow, also labelled specifically the luminal surface of ciliated cells of the pig. Double labelling of sections with the monoclonal antibody MIB-1 against the proliferation-associated nuclear epitope Ki-67 and anti-CgA clearly demonstrates that MIB-1 was selectively localised in the nuclei of secretory cells. Proliferative activity was not observed in CgA-positive ciliated cells in all examined stages of the oestrous cycle. The percentage of Ki-67-positive epithelial cells was higher at pro-oestrus, compared with the other stages of the oestrous cycle. Furthermore, ER immunoreactivity was exclusively detected in the nuclei of the epithelial cells, which were negative for CgA. We conclude, therefore, that oestrogen may induce the initial proliferation of secretory cells and promote the differentiation into ciliated cells.     

The 96-well twin-layer system: a novel approach in the cultivation of microalgae

A novel system for the growth and maintenance of microalgae has been developed that allows the cultivation of a large number of strains with little manual effort. The system is based on a 96-well microtiter plate in which a membrane filter constitutes the bottom of each well. Algal strains are immobilised on the membranes and provided with culture medium through contact with layers of glass fibre located beneath the membranes in a special cultivation chamber. The configuration effectively separates culture medium from algal cells which allows the simultaneous exchange of the culture medium for 96 strains within a few minutes without the need to transfer the algae. If necessary, algal strains can be transferred using multi-channel pipettes. We demonstrate that a large variety of microalgal strains including delicate flagellates can be reliably grown in the system under axenic conditions and without cross-contamination. As an array system, the 96-well twin-layer system using immobilised algae is also amenable to high-throughput and massively parallel applications increasingly sought after in algal bio- and environmental technology.     

Evidence for the involvement of a SchistoFLRF-amide-like peptide in the neural control of locust oviduct

Summary The presence of a SchistoFLRFamide-like peptide associated with the oviducts of Locusta migratoria has been shown using sequential reversed-phase high performance liquid chromatography separation coupled with radioimmunoassay and bioassay. The peptide is present in areas of the oviduct which receive extensive innervation, with sixfold less peptide in areas that receive little innervation. Material with FMRFamide-like immunoreactivity (determined by radioimmunoassay) is also present in the oviducal nerve and VIIth abdominal ganglion.SchistoFLRFamide is a potent modulator of contraction of this visceral muscle, inhibiting or reducing the amplitude and frequency of spontaneous contractions, relaxing basal tonus, and reducing the amplitude of neurally-evoked, proctolin-induced, glutamate-induced and high potassium-induced contractions. The FMRFamide-like immunoreactivity within the oviducts which co-elutes with SchistoFLRFamide on two separations is also capable of reducing the amplitude of neurally-evoked and proctolin-induced contractions, and of inhibiting spontaneous contractions and relaxing basal tonus.The effects of SchistoFLRFamide upon this visceral muscle are not abolished by the -adrenergic receptor antagonist phentolamine and do not appear to be mediated by cyclic AMP. Thus the receptors for Schisto-FLRFamide are distinct from those of octopamine which mediate similar physiological effects but which are blocked by phentolamine and which are coupled to adenylate cyclase.The results indicate that SchistoFLRFamide, or a very similar peptide, which has previously been identified as a modulator of locust heart beat, is also associated with visceral muscle of the reproductive system, and may play a neural role in concert with octopamine, at modulating muscular activity.Abbreviations BPP Bovine pancreatic polypeptide - BSA Bovine serum albumin - EJP Excitatory junctional potential - FaRPs FMRFamide-related peptides - FLI FMRFamide-like immuno-reactivity - LMS Leucomyosuppressin - RIA Radioimmunoassay - RP-HPLC Reversed-phase high performance liquid chromatography - TFA Trifluoroacetic acid     

An integrated ELF magnetic-field generator and incubator for long-term in vitro studies.

Long-term studies of the effects of low-frequency magnetic fields on cells in culture require an incubator that is free of contaminating magnetic fields and that provides automatic control of exposure duration, uniform applied fields, a uniform and well-controlled environmental temperature, and high reliability of the total system. We describe a dual-incubator system in which the enclosed solenoid of either unit can provide a sham exposure while the other generates a magnetic induction of up to 500 microT. Each solenoid-incubator unit contains an enclosed rack that can hold as many as 140 petri dishes for culturing cells; each unit is heated radiantly by circulating warm water. Field-exposure conditions and temperatures are continuously monitored and controlled by a microcomputer.     

In vitro response as a reflection of genomic diversity in long-term cultures of Musa

Summary Ten commercial cultivars of Musa representing five different types of genomic constitutions were studied for in vitro multiplication through meristem culture. In addition, the effects of various genomic constitutions at different ploidy levels on growth and meristem proliferation in long-term cultures were analysed statistically. Plantlets were readily obtained by culturing the excised meristems on MS semisolid medium supplemented with IAA, IBA and BAP at various concentrations. The regenerative potential of all cultivars of Musa, irrespective of their genomes, remained unaffected in long-term culture, even after 28–30 months. The genomic influence on both the nature and rate of proliferative growth was evident. Statistical analysis revealed that the rates of meristem proliferation between different cultivars of the same passage and between different passages of the same cultivar were significantly different. Those cultivars having only an A genome showed a low rate of meristem proliferation, while under the same culture conditions, cultivars having one or two B genomes in addition to the A exhibited a very high rate.     

Human breast epithelium in vitro: The re-expression of structural and functional cellular differentiation in long-term culture

Summary Fragments of human breast epithelium, devoid of all stromal and basal lamina components, which maintain their in vivo topological organisation can be cultured for up to 28 days within a reconstituted rat-tail-derived collagen matrix. These organoids initially undergo a loss of structural and 3-dimensional organisation, typified by loss of lumina formed by epithelial cells, and myosin from myoepithelial cells. Their subsequent reorganisation is dependent on the presence of serum, insulin, hydrocortisone, and cholera toxin in tissue culture medium. After this preliminary phase, a reduction in the concentration of serum, insulin, hydrocortisone, and cholera toxin is necessary to allow the structural differentiation of epithelial and myoepithelial cells. The myoepithelial cells also regain their ability to produce the basal lamina component laminin. The use of bovine-dermal collagen as the matrix, rather than rat-tail-derived collagen is shown to result in more stable organisation and differentiation of the organoids. The successful use of single-cell pellets (derived by trypsinisation of the organoids) in place of organoids in such cultures illustrates that there is no requirement for pre-existing cell/ cell contact or topological organisation of cells prior to embedding within the collagen matrix.     

Gastrulation in the sea urchin embryo: a model system for analyzing the morphogenesis of a monolayered epithelium

Processes of gastrulation in the sea urchin embryo have been intensively studied to reveal the mechanisms involved in the invagination of a monolayered epithelium. It is widely accepted that the invagination proceeds in two steps (primary and secondary invagination) until the archenteron reaches the apical plate, and that the constituent cells of the resulting archenteron are exclusively derived from the veg2 tier of blastomeres formed at the 60-cell stage. However, recent studies have shown that the recruitment of the archenteron cells lasts as late as the late prism stage, and some descendants of veg1 blastomeres are also recruited into the archenteron. In this review, we first illustrate the current outline of sea urchin gastrulation. Second, several factors, such as cytoskeletons, cell contact and extracellular matrix, will be discussed in relation to the cellular and mechanical basis of gastrulation. Third, differences in the manner of gastrulation among sea urchin species will be described; in some species, the archenteron does not elongate stepwise but continuously. In those embryos, bottle cells are scarcely observed, and the archenteron cells are not rearranged during invagination unlike in typical sea urchins. Attention will be also paid to some other factors, such as the turgor pressure of blastocoele and the force generated by blastocoele wall. These factors, in spite of their significance, have been neglected in the analysis of sea urchin gastrulation. Lastly, we will discuss how behavior of pigment cells defines the manner of gastrulation, because pigment cells recently turned out to be the bottle cells that trigger the initial inward bending of the vegetal plate.     

An in vitro study of long-term potentiation in the carp (Cyprinus carpio L.) olfactory bulb

Long-term potentiation (LTP) of synaptic transmission is considered a cellular mechanism for neural plasticity and memory formation. Previously, we showed that in the carp olfactory bulb, LTP occurs at the dendrodendritic mitral-to-granule cell synapse following tetanic electrical stimulation applied to the olfactory tract, and suggested that it is involved in the process of olfactory memory formation. As a first step towards understanding mechanisms underlying plasticity at this synapse, we examined the effects of various drugs (glutamate and GABA receptor agonists and antagonists, noradrenaline, and drugs affecting cAMP signaling) on dendrodendritic mitral-to-granule cell synaptic transmission in an in vitro preparation. Two forms of LTP are involved: a postsynaptic form (tetanus-evoked LTP) and a presynaptic form. The postsynaptic form is evoked at the granule cell dendrite following tetanic olfactory tract stimulation and is suppressed by the NMDA receptor antagonist, D-AP5, enhanced by noradrenaline, and occluded by the metabotropic glutamate receptor agonist, trans-ACPD. The presynaptic form occurs at the mitral cell dendrite following blockade of the GABA_A receptor by picrotoxin and bicuculline, or via activation of cAMP signaling by forskolin and 8-Br-cAMP.     

猪胎儿肾脏成纤维细胞体外培养体系的建立

本研究旨在建立猪胎儿肾脏成纤维细胞体外培养体系,并探讨其作为猪体细胞克隆供体的可能性。使用组织块培养法从体长为10cm以上的猪胎儿分离得到猪胎儿肾脏成纤维细胞,绘制了生长曲线,鉴定了细胞类型并且进行了细胞周期同期化效果的研究。结果表明:该培养体系可以支持猪胎儿肾脏成纤维细胞的体外生长,单个细胞均为梭形细胞,抗波形蛋白免疫荧光染色显示为阳性,而抗角形蛋白免疫荧光染色为阴性,分离到的细胞为胎儿肾脏成纤维细胞。使用血清饥饿法和接触抑制法诱导细胞进入G0/G1期,并且分别比较两者同期化效率,结果显示:血清饥饿2d和4d的同期化效率差异不显著,但都比8d组的高(88.97%和87.69%比82.45%,P<0.05);接触抑制4d、6d组间同期化效率差异不显著,但都比0d组的高(85.56%和85.89%比81.82%,P<0.05)。本研究在国内首次分离得到猪胎儿肾脏成纤维细胞,已经在体外传代培养到32代,其同期化效果好,可以作为体细胞克隆供体。     

A novel medium for the development of in vitro cell culture system from Penaeus monodon

Lack of a valid shrimp cell line has been hampering the progress of research on shrimp viruses. One of the reasons identified was the absence of an appropriate medium which would satisfy the requirements of the cells in vitro. We report the first attempt to formulate an exclusive shrimp cell culture medium (SCCM) based on the haemolymph components of Penaeus monodon prepared in isosmotic seawater having 27 ‰ salinity. The SCCM is composed of 22 amino acids, 4 sugars, 6 vitamins, cholesterol, FBS, phenol red, three antibiotics, potassium dihydrogen phosphate and di-sodium hydrogen phosphate at pH 6.8–7.2. Osmolality was adjusted to 720 ± 10 mOsm kg⁻¹ and temperature of incubation was 25 ºC. The most appropriate composition was finally selected based on the extent of attachment of cells and their proliferation by visual observation. Metabolic activity of cultured cells was measured by MTT assay and compared with that in L-15 (2×), modified L-15 and Grace’s insect medium, and found better performance in SCCM especially for lymphoid cells with 107 % increase in activity and 85 ± 9 days of longevity. The cells from ovary and lymphoid organs were passaged twice using the newly designed shrimp cell dissociation “cocktail”.     

The oviduct is a brood chamber for facultative egg retention in the parthenogenetic oribatid mite Archegozetes longisetosus AOKI (Acari, Oribatida)

Archegozetes longisetosus is a parthenogenetic oribatid mite and a chelicerate model organism. We examined the localisation of processes between vitellogenesis and embryogenesis as well as the anatomy and histology of involved structures by means of light- and electron microscopy. The proximal oviduct is differentiated into an oviductal bulb, exhibiting a strong secretory epithelium. Here, solidification of the egg shell instantaneously occurs upon passing of the egg from the perivitelline space into the oviductal lumen. This is interpreted as an internalised oviposition with the generation boundary being effectively located at the ovary-oviduct transition, rendering the oviducts into functional brood chambers. The parity mode combines elements of oviparity and ovolarviparity with facultative egg retention.     

幽门螺杆菌感染新型原代人胃上皮细胞模型的建立

目的 培养三维(3D)人胃黏膜上皮类器官,转为二维(2D)原代胃黏膜细胞培养,并建成人2D原代胃上皮细胞的幽门螺杆菌感染模型。 方法 (1)从正常人胃上皮组织中分离胃腺,在含有多种生长调节和凋亡抑制等混合因子的培养基中,依附于基质胶而培养成3D类器官;(2)利用免疫荧光技术鉴定胃上皮类器官的相关分子标记;(3)研究正常原代胃上皮细胞被幽门螺杆菌感染后的形态学变化,利用免疫印迹技术鉴定幽门螺杆菌感染相关蛋白的表达水平。 结果 成功培养出可长期传代的人胃上皮3D类器官,具有典型的人胃黏膜上皮分子标记。而且3D类器官转为2D平面培养的原代胃上皮细胞,可作为幽门螺杆菌的体外原代细胞感染模型。 结论 3D胃上皮类器官,可作为2D原代胃上皮细胞的持久来源,为研究幽门螺杆菌感染人体胃上皮的分子机制带来个体化的新模型。     

Bio-electrospraying primary cardiac cells: in vitro tissue creation and functional study

Manifestations of myocardial infarctions have been recognized as one of the major killers in the Western world. Therefore, advancing and developing novel cardiac tissue repair and replacement therapeutics have great implications to our health sciences and well-being. There are several approaches for forming cardiac tissues, non-jet-based and jet-based methodologies. A unique advantage of jet-based approaches is the possibility to handle living cells with a matrix for cell distribution and deposition in suspension, either as single or heterogeneous cell populations. Our previous studies on bio-electrospraying of cardiac cells have shown great promise. Here, we show for the first time the ability to bio-electrospray the three major cell types of the myocardium, both independently and simultaneously, for forming a fully functional cardiac tissue. Several samples are characterized in vitro and found to be indistinguishable in comparison to controls. Thus, we are describing a swiftly emerging novel biotechnique for direct cardiac tissue generation. Moreover, the present investigations pave the way for the development and optimization of a bio-patterning approach for the fabrication of biologically viable cardiac tissue grafts for the potential treatment of severe heart failure after myocardial infarction.     

Development and utilization of crustacean long-term primary cell cultures: Ecdysteroid effects in vitro

Summary

Long-term maintenance of lobster, Homarus americanus and crayfish, Pacifasticus leniusculus primary cell cultures of testicular and hematopoietic tissues, for 11 and 3 months, respectively, succeeded in a modified Medium 199 supplemented with 10% fetal bovine serum (pH 7.5, 200°C). In addition, NaC1 was used to adjust the lobster culture medium to 1000 mOsm and the crayfish medium to 400 mOsm. Proline concentration was also elevated. Testes were dissociated with 200 U/ml type II collagenase 2–3 days prior to culture.

Lobster hemocytes reacted to 10^?7 M 20-hydroxyecdysone (20-HE) by reducing contact inhibition and increasing invasive behavior one week after hormonal exposure. The presence of 10^?7 M 20-HE caused mesodermal cell death and spermatogonial proliferation in lobster testicular cell cultures within one week. Crayfish testicular mesodermal cells formed vacuoles 5 days after exposure to 10^?8 M 20-HE. These results are discussed in relation to the cellular events that occur in vivo during premolt.