Long-term viability and differentiation of bovine oviductal monolayers: Bidimensional versus three-dimensional culture

Different in vitro models have been developed to study the interaction of gametes and embryos with the maternal tract. In cattle, the interaction of the oviduct with gametes and embryos have been classically studied using oviductal explants or monolayers (OMs). Explants are well differentiated but have to be used within 24 h after collection, whereas OMs can be used for a longer time after cell confluence but dedifferentiate during culture, losing cell polarity and ciliation. Herein, OMs were cultured either in M199 plus 10% fetal calf serum or in a semidefined culture medium (Gray's medium), in an immersed condition on collagen-coated coated microporous polyester or polycarbonate inserts under air-liquid interface conditions. The influence of culture conditions on long-term viability and differentiation of OMs was evaluated through scanning electron microscopy, localization of centrin and tubulin at the confocal laser scanning microscope, and assessment of maintenance of viability of sperm bound to OMs. Findings demonstrated that OMs cultured in an immersed condition with Gray's medium retain a better morphology, do not exhibit signs of crisis at least until 3 wks postconfluence, and maintain the viability of bound sperm significantly better than parallel OMs cultured in M199 plus 10% fetal calf serum. OM culture with Gray's medium in air-liquid interface conditions on porous inserts promotes cell polarity, ciliation, and maintenance of bound sperm viability at least until 3 wks postconfluence. In conclusion, oviduct culture in Gray's medium in an immersed or air-liquid condition allows long-term culture and, in the latter case, also ciliation of bovine OMs, and may represent in vitro systems that mimick more closely the biological processes modulated by the oviduct in vivo.     

Co-culture of rabbit 2-cell embryos with rabbit oviduct epithelial cells and other somatic cells

Rabbit 2-cell embryos were co-cultured in Basel Synthetic Medium II + 10% fetal bovine serum with one of the following: primary cultures of rabbit oviduct epithelial cells (ROEC), a rabbit kidney epithelioid cell line (RK13), a rabbit epidermal epithelioid cell line (Sf1), or a rabbit skin fibroblast-like cell line (RAB9). Embryos cultured in medium alone served as controls. After 4 d of culture at 39 degrees C in 5% CO2 in air, 77-93% of the rabbit embryos which were co-cultured with somatic cells had reached the blastocyst stage, and 60-76% were hatching through their zonae pellucidae. These percentages, however, were not significantly different (P greater than .05) from those of embryos in medium alone, of which 90% had reached the blastocyst stage and 83% were hatching. Mean intrazonal embryo diameters also did not differ significantly among treatments (239-302 microns). Bovine 1-8-cell embryos were also co-cultured with ROEC. This stimulated 60% of these embryos to develop beyond the so-called "16-cell block" in vitro, whereas 0% of the embryos cultured in medium alone developed past this block. Evaluation of the ROEC cultures by light microscopy, immunocytochemistry, and gel electrophoretic analysis of conditioned medium, together with the positive results with bovine embryos, indicate that the ROEC culture partially simulates oviductal conditions in vivo. Therefore, our results suggest that oviduct epithelial cells may play a less pivotal role in regulating early development in the rabbit than in the cow.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bovine oviductal epithelial cells: their cell culture and applications in studies for reproductive biology

Epithelial cells of the mammalian oviduct play an important role in reproductive and developmental events that occur there. Oviductal epithelial cells from several mammalian species can be isolated and cultured in serum or serum-free medium in vitro and cell culture of bovine oviductal epithelial cells (BOEC) has been described by many investigators. Cultured BOEC show a wide variety of secretory activities and these secretory factors may influence early embryonic development or sperm function. Monolayer cultures of BOEC have been widely used for in vitro co-culture of bovine preimplantation embryos. The use of BOEC co-culture systems has improved embryonic development in nearly all the studies conducted. In addition, interaction of bovine spermatozoa with BOEC, in a similar manner to that observed for spermatozoa in vivo, induced specific changes in sperm capacitation and consequently improved the fertilizing capacity of bovine spermatozoa in vitro. Thus co-culture systems with BOEC may not only offer an excellent model for studying the mechanisms of capacitation and acrosome reaction of bovine spermatozoa but also provide a useful tool for the improvement of embryo development in vitro.     

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.     

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.     

Bovine trophoblastic cell vesicle attachment to polarized endometrial epithelial cells in vitro

Summary Interactions between bovine trophoblastic cell vesicles and bovine endometrial epithelial cells were investigated by light and electron microscopy and lectin histochemistry in a cell culture model of early blastocyst attachment. Primary lines of bovine endometrial epithelial cells were polarized by subculturing on substrata and maintaining cultures at the air-medium interface. Trophoblastic cell vesicles were obtained from elongated Day 14 blastocysts. In co-cultures, trophoblastic cell vesicles adhered to endometrial epithelial cells through microvillus interdigitation and formation of primitive membrane junctional complexes. After 3 d in co-culture, a multilayered cellular plaque formed at the trophoblastic cell-endometrial epithelial cell interface. The type of cells contributing to this local proliferative response could not be identified specifically as trophoblastic or endometrial cells, and areas of membrane fusion between cells were noted. Ultrastructural features of vesicle adhesion in cultures were similar to features of conceptus attachment in vivo. Lectins bound to apical membranes of trophoblastic cells and endometrial epithelial cells in all locations except contact sites between vesicles and endometrial cells. These findings suggest that local cellular proliferation and membrane fusion between trophoblastic and endometrial epithelial cells may be early events in conceptus implantation in the cow and these events can be reproduced in culture. This work was supported by a grant from U.S. Department of Agriculture Animal Health and Disease Program, Washington, DC.     

Effect of human oviductal epithelial cell cultural medium on cryopreserved human sperm survival

The human oviduct is known as a functional site for gamete transportation, retention, fertilization and zygote development. Previous studies have shown that human oviductal epithelial cell cultural medium (OECCM) has a positive effect on prolongation of sperm motility for some cryopreserved human sperm without cryodamage. However, for most cryopreserved sperm, OECCM could not improve their survival prolongation. In this study, we assessed the influence of human OECCM on the motility longevity of cryopreserved human sperm with an in vitro incubation method.     

豚鼠肾小管上皮原代细胞的培养

目的建立一种简便易行的豚鼠原代肾小管上皮细胞培养方法。方法运用筛网分离法和多种酶消化法获取高纯度的肾小管上皮细胞。利用免疫组化法和形态学观察法鉴定培养的肾小管上皮细胞性质及纯度。结果通过肾小管节段贴壁,胶原酶消化组织节段和细胞等方法,有效地促进肾小管原代细胞增殖;胰酶节段消化法的细胞贴壁效果稍差,细胞传代状态不理想;胰酶消化法则细胞贴壁较少,细胞生长状态较差。结论培养豚鼠原代。肾小管上皮细胞是可行的。     

Albumin and mammalian cell culture: implications for biotechnology applications

Albumin has a long historical involvement in design of media for the successful culture of mammalian cells, in both the research and commercial fields. The potential application of albumins, bovine or human serum albumin, for cell culture is a by-product of the physico-chemical, biochemical and cell-specific properties of the molecule. In this review an analysis of these features of albumin leads to a consideration of the extracellular and intracellular actions of the molecule, and importantly the role of its interactions with numerous ligands or bioactive factors that influence the growth of cells in culture: these include hormones, growth factors, lipids, amino acids, metal ions, reactive oxygen and nitrogen species to name a few. The interaction of albumin with the cell in relation to these co-factors has a potential impact on metabolic and biosynthetic activity, cell proliferation and survival. Application of this knowledge to improve the performance in manufacturing biotechnology and in the emerging uses of cell culture for tissue engineering and stem cell derived therapies is an important prospect.     

Effects of species and cellular activity of oviductal epithelial cells on their dialogue with co-cultured mouse embryos

An efficient co-culture system, especially with oviductal or uterine epithelial cells, is important not only for the production of high quality embryos, but also for the study of the molecular dialogue between embryos and their maternal environment. Although mouse embryos have been co-cultured successfully with oviductal epithelial cells (OECs) from several species, studies on the effects of species and functionality of OECs are few. Reports concerning the necessity of direct contact between the embryo and OECs and about the culture of mouse embryos in medium conditioned with heterologous OECs have been controversial. In this study, pronuclear embryos from Kunming mice, characterized by an obvious two-cell block in vitro, were co-cultured with mouse, goat, and chick OECs. The functionality of OECs was determined by analyzing the cell cycle, apoptosis, the numbers of mitochondria and cilia, and the ability both to support embryonic development and to remove hypoxanthine from the culture medium. The necessity of direct contact between OECs and embryos was studied by repeated renewal of culture medium with fresh conditioned medium, the culture of embryos in plastic wells connected by tunnels to wells with OEC monolayers, and the co-culture of embryos separated from OECs by a filter. Both goat and chick OECs supported mouse embryonic development, but their embryotrophic lifespan was shorter than that of the mouse OECs. Whereas media conditioned with mouse OECs supported mouse embryonic development satisfactorily, medium conditioned with goat OECs supported little development. Immediate dialogue between heterologous OECs and embryos was essential for efficient co-culture, whereas direct contact between the two cell types was not; neither dialogue nor contact was needed between isologous OECs and embryos. Embryotrophic activity and the ability to remove hypoxanthine from conditioned medium declined with time after confluence and number of passages of OECs, mainly because of apoptosis and dedifferentiation. Thus, the species and functionality of OECs have profound effects on their molecular dialogue with co-cultured embryos, and efficient co-culture depends upon both positive and negative conditioning.This study was supported by grants from the China National Natural Science Foundation (nos. 30430530 and 30571337) and the “973” Project of the Chinese Science and Technology Ministry (no. G200016108).     

Feeding lactate for CHO cell culture processes: impact on culture metabolism and performance

Lactate has long been regarded as one of the key metabolites of mammalian cell cultures. High levels of lactate have clear negative impacts on cell culture processes, and therefore, a great amount of efforts have been made to reduce lactate accumulation and/or to induce lactate consumption in the later stage of cultures. However, there is virtually no report on the impact of lactate depletion after initial accumulation. In this work, we observed that glucose uptake rate dropped over 50% at the onset of lactate consumption, and that catabolism of alanine due to lactate depletion led to ammonium accumulation. We explored the impact of feeding lactate as well as pyruvate to the cultures. In particular, a strategy was employed where CO(2) was replaced by lactic acid for culture pH control, which enabled automatic lactate feeding. The results demonstrated that lactate or pyruvate can serve as an alternative or even preferred carbon source during certain stage of the culture in the presence of glucose, and that by feeding lactate or pyruvate, very low levels of ammonia can be achieved throughout the culture. In addition, low levels of pCO(2) were also maintained in these cultures. This was in strong contrast to the control cultures where lactate was depleted during the culture, and ammonia and pCO(2) build-up were significant. Culture growth and productivity were similar between the control and lactate-fed cultures, as well as various product quality attributes. To our knowledge, this work represents the first comprehensive study on lactate depletion and offers a simple yet effective strategy to overcome ammonia and pCO(2) accumulation that could arise in certain cultures due to early depletion of lactate.     

Phenotypic characterization of telomerase-immortalized primary non-malignant and malignant tumor-derived human prostate epithelial cell lines

In vitro human prostate cell culture models are critical for clarifying the mechanism of prostate cancer progression and for testing preventive and therapeutic agents. Cell lines ideal for the study of human primary prostate tumors would be those derived from spontaneously immortalized tumor cells; unfortunately, explanted primary prostate cells survive only short-term in culture, and rarely immortalize spontaneously. Therefore, we recently have generated five immortal human prostate epithelial cell cultures derived from both the benign and malignant tissues of prostate cancer patients with telomerase, a gene that prevents cellular senescence. Examination of these cell lines for their morphologies and proliferative capacities, their abilities to grow in low serum, to respond to androgen stimulation, to grow above the agar layer, to form tumors in SCID mice, suggests that they may serve as valid, useful tools for the elucidation of early events in prostate tumorigenesis. Furthermore, the chromosome alterations observed in these immortalized cell lines expressing aspects of the malignant phenotypes imply that these cell lines accurately recapitulate the genetic composition of primary tumors. These novel in vitro models may offer unique models for the study of prostate carcinogenesis and also provide the means for testing both chemopreventive and chemotherapeutic agents.     

Isolation and long-term culture of rat,rabbit, and human nasal turbinate epithelial cells

Summary Nasal turbinate epithelial cells were isolated from rats, rabbits, and humans using either a surgical or an in situ enzyme incubation technique. The culture conditions that permit optimal cell attachment and selective growth of the nasal epithelial cells were determined. These conditions will permit the long-term culture of these cells where typically 20 to 30 population doublings were observed. Differences between rat and human nasal epithelial cells were seen in substrate requirements, colony-forming efficiency, and response to fetal bovine serum and bovine serum albumin. These methodology and results will permit mechanistic studies of normal and abnormal cellular function and comparative response studies between nasal epithelial cells from rats and humans. This work was supported under U.S. Environmental Protection Agency contract 68-02-4032.     

Isolation and characterization of a novel human prostatic stromal cell culture: DuK50

Summary A novel human prostatic stromal cell culture, designated DuK50, has been passedin vitro >12 mo. Tissue cultures were obtained from material harvested within a normal region of a radical prostatectomy specimen. These monolayers exhibited normal fibroblastic characteristics with each cell having a flattened, elongated appearance. Karyotypic analysis revealed a normal, male 46, XY chromosomal content with no numerical or structural abnormalities. DNA analysis using a Cell Analysis Systems Image Analyzer confirmed a euploid DNA content (7.9 pg DNA). Cellular markers for verification of stromal cell type were performed by immunohistochemical techniques. DuK50 stained positive for vimentin and fibronectin. Immunostains for epithelial cytokeratins and prostate-specific antigen were negative, which ruled out contamination with prostatic epithelial cells. Negative immunostaining with desmin monoclonal antibody and light staining with smooth muscle actin alpha is consistent with the staining pattern of myofibroblasts. Response to various androgens, measured by a microculture tetrazolium assay technique, revealed a significant growth stimulation of DuK50. Soft agar invasiveness assays and tumorigenicity studies in nude mice were negative. DuK50 exhibits a rapid doubling time with excellent plating efficiency, thrives in a readily available media supplemented with fetal bovine serum, and passes with routine trypsin protocols. The availability of this prostatic stromal cell culture may facilitate studies on this cell type’s role in growth factor modulation, drug and steroid metabolism, and stromal-epithelial interactions in the prostate. Presented at the Congress on Cell and Tissue Culture, Research Triangle Park, NC, June 1994.     

Long term endocrine regulation of nucleoside transporters in rat intestinal epithelial cells

We studied the regulation of nucleoside transporters in intestinal epithelial cells upon exposure to either differentiating or proliferative agents. Rat intestinal epithelial cells (line IEC-6) were incubated in the presence of differentiating (glucocorticoids) or proliferative (EGF and TGF-alpha) agents. Nucleoside uptake rates and nucleoside transporter protein and mRNA levels were assessed. The signal transduction pathways used by the proliferative stimuli were analyzed. We found that glucocorticoids induce an increase in sodium-dependent, concentrative nucleoside transport rates and in protein and mRNA levels of both rCNT2 and rCNT1, with negligible effects on the equilibrative transporters. EGF and TGF-alpha induce an increase in the equilibrative transport rate, mostly accounted for by an increase in rENT1 activity and mRNA levels, rENT2 mRNA levels remaining unaltered. This effect is mimicked by another proliferative stimulus that functions as an in vitro model of epithelial wounding. Here, rENT1 activity and mRNA levels are also increased, although the signal transduction pathways used by the two stimuli are different. We concluded that differentiation of rat intestinal epithelial cells is accompanied by increased mature enterocyte features, such as concentrative nucleoside transport (located at the brush border membrane of the enterocyte), thus preparing the cell for its ultimate absorptive function. A proliferative stimulus induces the equilibrative nucleoside activities (mostly through ENT1) known to be located at the basolateral membrane, allowing the uptake of nucleosides from the bloodstream for the increased demands of the proliferating cell.     

Isolation and characterization of epithelial cells from bovine pancreatic duct

Summary Epithelial cells derived from bovine pancreatic duct have been grown continuously in culture for 30 weeks (approximately 90 doublings of the cell population). The cells were grown in Eagle's minimal essential medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 0.1 mM nonessential amino acids, and antibiotics. In confluent cultures, the cells are multilayered and form circular structures. When tested at various passages, the cells neither formed colonies in soft agar nor produced tumors after inoculation into athymic, nude mice. Hydrocortisone (1 and 5 μg per ml) and insulin (1,5 and 10 μg per ml) had no effect on the growth of the cells. β-Retinyl acetate inhibited growth rate and cell yield at a concentration of 5 μg per ml but was not growth-inhibitory at lower concentrations. By electron microscopy the cells have numerous mitochondria, Golgi and microvilli. Mucous droplets were observed in a small proportion of the cells. Desmosome-like structures and occluding junctions were observed more frequently between cells that had been transferred as aggregates than between cells transferred as single cells. Cytochemical studies indicated that some cells produce PAS positive granules that were not removed after treatment of the cultures with diastase. Eleven cell clones were isolated from the mass culture. The growth rates of the clones are different as well as the period of time in which the clones can be propagated in vitro. This work was supported in part by Y01 CP 60204 and N01 CP 43237.     

Establishment of primary cell culture from the temperate symbiotic cnidarian, Anemonia viridis

The temperate symbiotic sea anemone Anemonia viridis, a member of the Cnidaria phylum, is a relevant experimental model to investigate the molecular and cellular events involved in the preservation or in the rupture of the symbiosis between the animal cells and their symbiotic microalgae, commonly named zooxanthellae. In order to increase research tools for this model, we developed a primary culture from A. viridis animal cells. By adapting enzymatic dissociation protocols, we isolated animal host cells from a whole tentacle in regeneration state. Each plating resulted in a heterogeneous primary culture consisted of free zooxanthellae and many regular, small rounded and adherent cells (of 3–5 μm diameter). Molecular analyses conducted on primary cultures, maintained for 2 weeks, confirmed a specific signature of A. viridis cells. Further serial dilutions and micromanipulation allowed us to obtain homogenous primary cultures of the small rounded cells, corresponding to A. viridis “epithelial-like cells”. The maintenance and the propagation over a 4 weeks period of primary cells provide, for in vitro cnidarian studies, a preliminary step for further investigations on cnidarian cellular pathways notably in regard to symbiosis interactions.