Millicell Inserts Research Articles

Development of anex vivo patient-derived tumor model (PDTM) to assess the tumor microenvironment in renal cell carcinoma (RCC)

Abstract Background With the rise of immune checkpoint inhibitors (ICIs) as the primary treatment option for metastatic RCC, investigating the role of T cells within the tumor microenvironment (TME) is a critical component of understanding both treatment response and resistance. Prior efforts, including single-cell transcriptomic approaches, have provided an important landscape of T cell transcriptional phenotypes. However, these immuno-profiling efforts require validation through functional interrogation of the TME to facilitate the development of novel immunomodulatory therapies. Thus, we established a patient derived tumor model (PDTM) system to directly assess the effect of inhibitory immune interactions on T cell function and anti-tumor activity in the RCC TME. In this initial proof-of-concept study, we evaluated T cell activation in the RCC TME using the PDTM system. Methods Fresh tumor samples were obtained from surgical resections of RCC at Yale-New Haven Hospital. The tumor was minced to ~1-3 mm³ pieces and suspended in an air-liquid interface system, consisting of tumor fragments embedded in a collagen matrix on an insert with a semi-permeable membrane, exposed to culture media. The tumor fragment and matrix suspension were carefully pipetted onto the Millicell insert, which served as the top layer. The PDTM setup includes an inner dish containing the bottom gel layer and the tissue-containing top layer. To complete the assembly, 1.5 ml of DMEM media with or without an anti-CD3 monoclonal antibody (aCD3mAb) and 500 nM of anti-PD-1 monoclonal antibody (aPD1mAb) was added to the outer dish surrounding the insert. Results We successfully optimized PDTM experimental workflows for culture, dissociation, and analysis using immunohistochemistry (IHC), flow cytometry (FCM), and enzyme-linked immunosorbent assays (ELISA). Hematoxylin and eosin (H&E) staining and IHC showed that the TME cellular architecture and immune cell composition was broadly preserved during the three day experimental period. Using FCM to analyze the dissociated tumor samples, we identified well-preserved CA9+ tumor cells, CD4+ and CD8+ T cell populations, CD4+CD25+ regulatory T cells, CD56+ natural killer cells, CD20+ B cells, and CD14+CD11b+ myeloid subsets including monocytes and CD163-/+macrophages. Among the T cells, we detected PD1+, LAG3+, TIM3+, and TIGIT+ cells. For dose-response analysis of aCD3mAb, we observed that stimulation with 0.025 ng/mL aCD3mAb for T cells from healthy donor peripheral blood mononuclear cells (PBMCs) yielded significant but non-saturating levels of interferon gamma (IFN-γ) production as quantified by ELISA. We tested the effect of the anti-PD-1 antibody on PDTM under the low-dose of aCD3mAb, and importantly, found that treatment of the PDTM with aPD1mAb resulted in more activated CD8 T cells and higher IFN-γ production than the control samples. Conclusions Through optimization of assays evaluating T cell cytokine production, we were able to assess multiple axes of T-cell function in the RCC TME. This study revealed that our PDTM system preserves the RCC TME for functional interrogation. Furthermore, our system for assessing T cell phenotype and cytokine production successfully demonstrated the activity of PD-1 blockade ex vivo. Taken together, this novel ex vivo PDTM system has extensive applications in the study of RCC, including assessing the impact of ICIs on T cell function. DOD CDMRP Funding: yes

10-7M genistein partially alleviates 10-7M MEHP unfavorable effects in a new modified fetal rat testis culture system.

Background: Recent studies revealed that some common endocrine-disrupting chemicals (EDCs) including phthalates and phytoestrogens may exhibit low-dose effects properties. However, how low dose of these EDCs and their mixture would affect fetal rat testis development still needs further investigation. Moreover, testis organ culture system also needs further modification to provide an effective tool for ex vivo EDCs study. Methods: We firstly modified the agarose organ culture system, in which fetal rat testes were cultured for 4 days (d1 to d4) on agarose gels held by Millicell inserts. Then we used the modified agarose culture system to study the combined effects of multiple EDCs exposure. 15.5 dpc fetal rat testes were isolated and treated with vehicle, MEHP (0.1 μmol/L), GEN (0.1 μmol/L) or MEHP (0.1 μmol/L) + GEN (0.1 μmol/L). Parameters concerning testicular cell development and function were evaluated, trying to gain insight into the early molecular events after multiple EDCs exposure. Results: The development of somatic, germ cells and seminiferous tubule in 15.5 dpc fetal rat testis was better sustained in the modified agarose culture system. Based on the modified system, we found that MEHP at 0.1 μmol/L induced alterations in gonocyte markers, antioxidative enzyme activity as well as transient reduction of testosterone production, accompanied by mitochondria swelling in gonocytes and Sertoli cells. No obvious morphological and histological alterations were observed in all treated groups. However, coadministration of genistein at 0.1 μmol/L partially alleviated MEHP-induced fetal testis damage ex vivo through enhancement of antioxidative action. MEHP at low dose still showed weak endocrine disrupting properties but did not exhibit typical low-dose effects. Conclusion: Our findings indicated that the modified agarose culture system could better mimic testicular microenvironment without obvious hypoxic cell damage. Furthermore, low dose of MEHP induced mild disruption to fetal testis development, cotreatment of genistein at low dose attenuated MEHP induced fetal testis injuries in part by balancing redox state, indicating that low dose of genistein may partially protect fetal testis from phthalates induced injury.

In-vitro studies of the effects of interferons on endometrial metabolism in sheep

Primary cultures of ovine epithelial and stromal cells have been used to examine paracrine interactions between the endometrium and the preimplantation sheep blastocyst, and in particular the actions of the blastocyst alpha-interferon, ovine trophoblast protein-1 (oTP-1), on endometrial cell metabolism. The synthesis and secretion of several 'pregnancy-related' acidic proteins with molecular weights in the range 70,000-120,000 can be induced by addition of oTP-1 or human recombinant interferon (IFN) to cultured enriched epithelial endometrial cells. Consistent with the antiluteolytic role of oTP-1, dose-dependent attenuation of both PGE and PGF-2 alpha release has been demonstrated. Arachidonic acid added to the cells increased overall PG release but the inhibitory effects of interferons were still apparent. Immunocytochemical analysis of PG synthase demonstrated marked cyclic variation of its localization within the endometrium, but no differences in distribution or intensity of staining were apparent in endometrium of early pregnancy (Day 15) compared with that of the cycle (Day 15). It appears that conceptus-induced changes in PG release do not occur via changes in concentration or localization of PG synthase but rather by modifying its activity. Highly purified epithelial cells cultured on matrigel-coated millicell inserts retain important morphological features seen in vivo. Under such conditions, PGF-2 alpha and PGE release into the basal compartment was greater than that into the apical compartment. Stromal fibroblasts cultured under similar conditions secreted less PGF-2 alpha but more PGE than epithelial cells. Whilst the limitations of in-vitro studies are acknowledged, these findings are compatible with and markedly extend what is known of the action of embryonic interferons in vivo in the establishment of pregnancy in sheep.

Transmigration of Neural Stem Cells across the Blood Brain Barrier Induced by Glioma Cells

Transit of human neural stem cells, ReNcell CX, through the blood brain barrier (BBB) was evaluated in an in vitro model of BBB and in nude mice. The BBB model was based on rat brain microvascular endothelial cells (RBMECs) cultured on Millicell inserts bathed from the basolateral side with conditioned media (CM) from astrocytes or glioma C6 cells. Glioma C6 CM induced a significant transendothelial migration of ReNcells CX in comparison to astrocyte CM. The presence in glioma C6 CM of high amounts of HGF, VEGF, zonulin and PGE2, together with the low abundance of EGF, promoted ReNcells CX transmigration. In contrast cytokines IFN-α, TNF-α, IL-12p70, IL-1β, IL-6, IL-8 and IL-10, as well as metalloproteinases -2 and -9 were present in equal amounts in glioma C6 and astrocyte CMs. ReNcells expressed the tight junction proteins occludin and claudins 1, 3 and 4, and the cell adhesion molecule CRTAM, while RBMECs expressed occludin, claudins 1 and 5 and CRTAM. Competing CRTAM mediated adhesion with soluble CRTAM, inhibited ReNcells CX transmigration, and at the sites of transmigration, the expression of occludin and claudin-5 diminished in RBMECs. In nude mice we found that ReNcells CX injected into systemic circulation passed the BBB and reached intracranial gliomas, which overexpressed HGF, VEGF and zonulin/prehaptoglobin 2.

Development of a Model for Functional Studies of ABCG2 (Breast Cancer Resistance Protein) Efflux Employing a Standard BeWo Clone (B24)

Human choriocarcinoma-derived BeWo cells express high levels of breast cancer resistance protein (BCRP/ABCG2) with no functional P-glycoprotein (P-gp) (ABCB1) activity, making them a potential model to study bidirectional ABCG2-mediated drug transport. However, the original BeWo clone (B24) available to researchers does not form confluent monolayers with tight junctions required by the model. Our aim was to adapt culture conditions to attempt to generate confluent BeWo monolayers for drug transport studies using the standard B24 clone. BeWo cells (B24; American Type Culture collection [ATCC]) were cultured in six-well plates or polycarbonate millicell inserts in a number of media formulations, growth supplements, and basement membrane substitutes. Cells were examined for confluence by microscopy, and transepithelial electrical resistance (TEER) was measured daily; monolayer permeability was assessed when TEER had stabilized. Optimal growth rates were achieved in culture conditions consisting of Medium 199 (M199) supplemented with epidermal growth factor (EGF; 20 ng/mL), vitamin supplements, and 10% fetal calf serum (FCS) with collagen coating. A TEER of 170 Ω in 0.6 cm(2) inserts was achieved 2 weeks after seeding under optimal conditions. The cell-impermeable diffusion marker 5(6) carboxy-2,7dichlorodihydrofluorescein (C-DCDHF) had a permeability coefficient of 3.5×10(-6) cm/s, indicative of minimal paracellular permeability. ABCG2 expression, as determined by immunoblotting, remained unaffected by confluency. In conclusion, we describe culture conditions for the B24 BeWo clone that facilitate the formation of monolayers with tighter junctions and reduced paracellular transport compared to previously published models. These growth conditions provide a good model of ABCG2-mediated drug transport in a human placental cell line.

311. Oncolytic Adenovirus CG5757 Preferentially Infects Primary Human Tumor Tissues and Shows Strong Anti-Tumor Activity in Tumor Models Following Systemic Administration

Top of pageAbstract A human adenovirus serotype 5 based oncolytic virus CG5757 was designed to preferentially replicate in and kill cancer cells that have a defective retinoblastoma (Rb) pathway and overexpress telomerase. Tumor selectivity of CG5757 was examined using a newly developed ex vivo primary tumor tissue culture model. Primary human colorectal tumors were obtained after surgical resection and placed in Millicell insert with media containing hormones, which can keep tissue viable for about 5 days. The paired tumor or normal tissues from same tissue sample were used in a viral infection experiment to determine the tumor selectivity of CG5757. Cultured tissues were infected with CG5757 or wild type (wt) Ad5 for five days, and progeny viral production was determined by TCID50 assay. Results of the paired tissue cultures from five colon cancer patients indicate that wt Ad5 has similar viral productivity in tumor and normal tissues, whereas CG5757 has higher productivity in tumor tissues. In general, CG5757 produces 100- to 1,000-fold more virus in tumor tissue compared to normal tissue. Immunohistochemical staining for adenoviral E1A shows that CG5757 has positive infection only in the cancer tissue (colon), whereas no signal is detected in normal tissues of colon, pancreas or spleen. Similarly in vitro viral specificity characterization using a panel of tumor cell lines also suggest a strong tumor selectivity of CG5757. Anti-tumor efficacy of CG5757 following intravenous injection was examined in a subcutaneous tumor model. In this study, pre-established prostate cancer LNCaP xenografts in nude mice were treated with two intravenous injections of 4|[times]|1010 viral particles of CG5757 given at a 3-day interval. Significant anti-tumor efficacy was observed in CG5757-treated animals. Eight weeks after treatment, CG5757-treated animals exhibited approximately 72% inhibition of tumor growth. These studies demonstrate the potential therapeutic efficacy of such dual promoter-controlled oncolytic adenoviruses in cancers that are Rb-defective and telomerase-positive.

Growth of differentiated ovine tracheal epithelial cells in vitro.

Culture of ovine tracheal epithelial cells is a useful tool for conducting various in vitro studies. We describe herein an in vitro technique and the conditions for culturing primary epithelial cells derived from tracheas of adult sheep. Ovine tracheas were surgically removed from 2- to 3-month-old healthy sheep and tracheal epithelial cells were isolated by 0.15% pronase digestion. After epithelial cells isolation, a Millicell insert with porous membrane was coated with 0.05% human placental collagen and the epithelial cells were added to the membrane. To create an air-liquid interface environment for the cells, the apical compartment of the membrane containing the tracheal epithelial cells was left exposed to 5% CO(2) at 37 degrees C for 2 days then increased to 9% CO(2) while cells in the basolateral compartment underneath the membrane contained the growth medium necessary for cells nourishment. Pepsin digestion was more effective in reducing the number of fibroblasts than other procedures. Cells were allowed to grow for 6-7 days to form a confluent monolayer and nearly 21 days for cilia formation on the apical surface as determined by light microscopy of haematoxylin and eosin-stained sections of membranes. In order to further confirm the epithelial origin of cells, cells were stained for cytokeratin antigen by immunohistochemistry. Most ciliated epithelial cells were immunoreactive for cytokeratin. This is the first report of differentiated ovine tracheal epithelial cells growth and isolation. This technique can be used in numerous in vitro investigative studies in ovine species as an animal model for human disease.

New model for studying the migration of immune cells into intestinal epithelial cell monolayers.

A novel cell culture system was constructed to analyze the direct interaction between intestinal epithelial cells and immune cells. Human intestinal epithelial Caco-2 cells were monolayer-cultured on the under side of a permeable membrane (12 mum pore size) in a Millicell insert. Integrated monolayers of Caco-2 cells had formed after 12 days of culture. Human monocyte/macrophage-like THP-1 cells were then added to the upper chamber of the insert, and their migration into the Caco-2 cell monolayers was observed by confocal laser scanning microscopy, after staining the cells with specific antibodies. When MCP-1, a beta-chemokine, was added to the apical side of the monolayer, a greater number of THP-1 cells migrated into the Caco-2 cell monolayers. This cell culture system will be useful for studying the behavior of macrophages in the intestinal epithelial cell monolayers at the initial stage of an intestinal immune reaction.

Regeneration of fertile plants from isolated zygotes of rice (Oryza sativa).

A simple mechanical method has been developed which allows the routine isolation of unfertilized and fertilized egg cells from ovules of Japonica and Indica rice varieties. In the experiments described, the majority of the egg cells and zygotes survived the isolation procedure when the donor plants were in a vigorous state. About 40% of the surviving zygotes underwent sustained development when cultured in Millicell inserts with a non-morphogenic rice feeder-cell culture. Nearly all zygote-derived callus cultures regenerated multiple shoots, which could be subsequently rooted with high efficiency. Zygote-derived plantlets matured to fertile plants when transplanted to soil. So far, about 80 independent plants each from the Japonica variety 'Taipei309' and the Indica variety 'IR58' have been regenerated. The potential of this single-cell regeneration system for marker gene-free transformation is discussed.

Prolonged survival of human spermatozoa when co-incubated with epididymal cell cultures.

Human epididymal tissue was recovered from 11 patients undergoing orchidectomy without anti-androgen treatment. Everted epithelial fragments from the caput and corpus epididymis of six patients were successfully cultured in a modified RPMI 1640 medium supplemented with HEPES and androgens for up to 110 days (mean 56 +/- 28) in 5% CO(2) in air at 37 degrees C. Epithelial cells from human oviduct and non-reproductive tract cells (breast epithelial cells, fibroblasts) were also cultured for comparison. The proportion of epididymal epithelial cells in primary cultures assessed by immunofluorescent localization using a cytokeratin monoclonal antibody was shown to be >70% for the first 6-8 weeks of culture. Light and electron microscopy indicated that epithelial cells maintained polarity and some normal morphology during the culture period. Washed epididymal or ejaculated spermatozoa prepared by a 'swim-up' procedure were co-incubated (i) directly with epididymal cells in culture wells, (ii) in 12 mm Millicell inserts within culture wells, thereby preventing contact of spermatozoa with culture cells; and (iii) in culture medium alone. A significant proportion of spermatozoa in direct contact with culture cells or in Millicell inserts were viable after 6 days of co-incubation (30-45%) and exhibited progressive motility, while all spermatozoa in medium alone were non-motile by 3 days. Using computer-assisted sperm analysis it was shown that the progressive motility of viable spermatozoa decreased gradually for the first 5 days in culture and then remained constant (approximately 30 microm/s, average path velocity). After 12 days of co-incubation, 15 +/- 4% of spermatozoa in direct contact with epithelial cells remained motile; in one experiment, a few spermatozoa (<1%) were motile at 17 days. Light and electron microscope observations indicated that prolonged sperm survival was associated with close apposition of spermatozoa (by equatorial segment) to the apical membrane of epithelial cells. Oviductal epithelial cells were also beneficial for sperm survival, but other cell types had no effect.

Immunohistochemical and ultrastructural study of adult porcine endocrine pancreas during the different steps of islet isolation.

Treatment of diabetes mellitus by transplantation of isolated pancreatic islets could constitute an alternative to human pancreas allograft. Before transplantation, porcine islets are submitted to a procedure of isolation and purification. The quality of islets through these different steps may be assessed by morphological and functional studies. The aim of this work was the histological characterization of the four main cell types of porcine adult endocrine islets during the different steps of the isolation procedure using immunohistochemistry (IHC) applied in light (LM) and electron microscopy (EM). In fresh pancreas, islets were various sizes and shapes in LM. The number was not found different between the different portions of the pancreas. In IHC, insulin (Ins)-secreting cells accounted for the majority of the islet cells, while glucagon(Glu)-somatostatin (Som)- and polypeptide(PP)-immunoreactive cells, in decreasing number, were found in the mantle around the core of Ins-cells. In EM, B-cells contained poly-hedric granules with a dense central core and clear halo. Glu granules were spherical and very dense. D-cells and PP-cells were characterized by numerous granules, rather spherical and of inequal density for Som and more ellipsoidal for PP granules. After purification in Euroficoll, in EM, the four cellular types remained recognizable, but underwent vacuolization, mitochondrial swelling, and enlargement of intercellular spaces. After 3 days of culture on plastic dishes, as on Biopore membranes in a Millicell insert, microvilli appeared and vacuolization increased in EM. At the seventh day of culture, in EM, most of the cells were lysed in contrast to LM where at the same time, the four cell types were clearly identified by IHC but only in collagen matrix. Important discrepancies were noticed between LM and EM. This fact emphasizes the complementarity of morphological and functional studies in assessment of the quality of an islet isolation.

Aged median eminence glial cell cultures promote survival and neurite outgrowth of cocultured neurons.

We have recently shown that tanycytes, a particular type of glial cell that has morphological and biochemical similarities with radial glial cells, constitute a preferential support for the regeneration of lesioned neurohypophysial axons. The present study was designed to explore the possible neurotrophic role of tanycytes in vitro. Glial cells derived from the median eminence or from the cerebral cortex of 10-day-old rats were cultured for 4-7 weeks. At these times the majority of the cells identified in the median eminence cultures exhibited immunostaining patterns of tanycytes, as detected in the mediobasal hypothalamus of 10-day-old and adult rats, i.e., they were immunoreactive to vimentin (VIM), to DARPP-32 (a dopamine- and adenosine 3':5'-monophosphate-regulated phosphoprotein), and to a lesser extent to glial fibrillary acidic protein (GFAP) antibodies. On the other hand, the majority of cells in cortex cultures showed immunostaining patterns of astrocytes, i.e., they were intensely immunoreactive to GFAP and VIM antibodies but negative to DARPP-32. Cells obtained from the dissociation of 3-day-old rat mesencephalon, cortex, and hypothalamus were cocultured on these glial monolayers, and the number of surviving neurons and their neurite length were quantified after 8 days. Our data showed that, when compared with astrocytes, tanycytes greatly improved both survival (six-to ten-fold higher) and neurite outgrowth (two- to five-fold longer) of cocultured neurons whatever their origin. Experiments performed by coculturing neurons on millicell inserts placed above the glial monolayers showed that diffusible factors from median eminence glial cells slightly increased survival (1.7-fold higher) of cocultured neurons but had no significant effect on neurite outgrowth. These observations indicate: 1) that aged tanycytes have a capacity to support survival and neurite outgrowth for a variety of postnatal neurons; and 2) that this neurotrophic effect is exerted mainly by means of specific molecules bound to the tanycytic plasmalemma limiting membrane and/or to the extracellular matrix.

Production of Somatic Hybrid Calli between Hordeum vulgare L. and Hordeum bulbosum L.

Summary As a step towards the development of a somatic hybridization system, protoplast fusion between Hordeum vulgare L. and Hordeum bulbosum L. was performed. In H. vulgare an embryogenic cell suspension was chosen as a protoplast donor. In H. bulbosum the npt II gene was introduced into protoplasts and then one of the resulting lines was used for hybridization experiments. Protoplasts were fused by a newly developed method using polyethyleneglycol (PEG) and Millicell inserts. Selection of hybrid cells was based on iodoacetoamide (IOA) inactivation and antibiotic resistance. RFLP analysis using rDNA probes confirmed the production of somatic hybrid cell lines between H. vulgare and H. bulbosum .

Dermal fibroblasts activate keratinocyte outgrowth on collagen gels

The effects of dermal fibroblasts on keratinocyte outgrowth on collagen substrata was studied using an in vitro keratinocyte-collagen gel composite model. Skin fibroblasts were seeded inside collagen gels, which remained attached to the cell culture plastic substratum. Fibroblasts incorporated in collagen gels were either kept viable throughout the study, or were lysed hypotonically with water at different time intervals (2 hours and 5 days). Results show that very little keratinocyte outgrowth occurred on either plain collagen gels or gels that had previously contained viable fibroblasts for 2 hours. A 3- to 4-fold increase in keratinocyte outgrowth occurred on collagen gels that had previously contained viable fibroblasts for 5 days. A striking increase (20-fold) in keratinocyte outgrowth was observed on collagen gels that contain viable fibroblasts. The effect of fibroblast diffusible factors on keratinocyte outgrowth was further studied with a co-culture system using Millicell inserts. It was found that the co-culture of fibroblasts with the composite enhanced keratinocyte outgrowth on collagen gels that had previously contained viable fibroblasts for 5 days. Among all, however, the keratinocyte outgrowth was far better on gels containing viable fibroblasts. Addition of keratinocyte growth factor or its neutralizing antibody did not affect keratinocyte outgrowth. These results suggest that dermal fibroblasts can activate keratinocyte outgrowth on collagen matrices through some diffusible factors other than keratinocyte growth factor, and epithelial-mesenchymal interactions exert some special effects on keratinocyte outgrowth on collagen gels.

Co-culture of sheep zygotes on long-term cultures of estrus or luteal-phase oviductal cells on millicell inserts

Epithelium of oviductal ampulla was studied in normal and in superovulated sheep using morphologic analysis and lectin glycohistochemistry. The lining epithelium consisted of two types of cells, ciliated and nonciliated cells. Unlike superovulated samples, the nonciliated cells from control ewes showed apical protrusions indicating an apocrine secretory activity. The ciliated cells showed lectin-binding sites mainly at the level of the cilia which bound all the used lectins except Peanut agglutinin, suggesting the lack of glycans terminating with Galβ1,3GalNAc. In superovulated specimens, the ciliated cells with high mannosylated glycans Concanavalin A (Con A) and GlcNAc and GalNac termini Griffonia simplicifolia agglutinin II (GSA II) and Dolicurus biflorus agglutinin (DBA) decreased. The luminal surface of nonciliated cells showed all investigated sugar residues in controls, whereas it was lacking in high mannosylated (Con A) and terminal GalNAcα1,3(LFucα1,2)Galβ1,3/4GlcNAcβ1 sequence (DBA) in superovulated ewes. Apical protrusions from control ampullae nonciliated cells showed glycans containing mannose, GlcNac, GalNAc, galactose, and α2,3-linked sialic acid (Con A, KOH-sialidase- Wheat germ agglutnin [WGA], GSA II, SBA, Griffonia simplicifolia agglutinin-isolectin B4 [GSA I-B4], Maackia amurensis agglutinin II [MAL II]). The supranuclear cytoplasm of nonciliated cells expressed terminal GlcNAc (GSA II) in all specimens, also O-linked glycans (mucin-type glycans) with GalNAc and sialic acid termini (Helix pomatia agglutinin [HPA] and MAL II) in control animals, and also N-linked glycans with fucose, galactose, lactosamine, and α2,3-linked sialic acid termini (Ulex europaeus agglutinin I [UEA I], GSA I-B4, Ricinus communis agglutinin120 [RCA120], and Sambucus nigra agglutinin [SNA] ) in superovulated ewes. These results report for the first time that the superovulation treatment affects the secretory activity and the glycan pattern of the epithelium lining the sheep oviductal ampulla.

Porcine sperm binding to oviductal explants in culture

Explants taken from the mucosa of the oviductal isthmus and ampulla of gilts 15 days after mating were cultured on Matrigel-coated Millicell inserts in modified minimal essential medium (MEM) containing 70 pg/ml estradiol and 0.5 ng/ml progesterone. After two weeks, ejaculated boar spermatozoa washed in MEM were added to the explants within the inserts to a final concentration of 2×10 6sperm/ml. Then the medium in the outer well surrounding the inserts was replaced with MEM containing 70 pg/ml estradiol and 0.5 ng/ml progesterone (peak systemic levels during estrus) or 2 pg/ml estradiol and 0.5 ng/ml progesterone (systemic levels during diestrus) or 0.01% dimethylsulfoxide (DMSO) carrier alone. Spermatozoa attached to the epithelial surface of the explants within minutes, and more than 70% remained motile for 42 to 44 hours. Greater numbers of spermatozoa (P< 0.05) bound to explants from the isthmus than from the ampulla. Both in the isthmus and on the ampulla, binding of spermatozoa was greater (P < 0.05) in the presence of estrus levels of steroids than in medium without steroids. These results indicate that the binding of spermatozoa to oviductal epithelium is influenced by region and hormonal status. In addition to binding to the epithelial surface, some spermatozoa were trapped in a mucous secretion produced by isthmic explants. Pronase but not trypsin, heparin or hyaluronidase was found to release spermatozoa from the mucus. Binding to epithelial surfaces and entrapment in mucus may serve to create an oviductal reservoir of spermatozoa prior to fertilization.

Anin vitro model system to evaluate pulmonary macrophage, endothelial cell, and neutrophil interactions

Pasteurella haemolytica, the cause of fibrinous pleuropneumonia in cattle, produces extensive microvascular endothelial cell (EC) damage. We have developed an in vitro model system to study the inflammatory process of this disease involving the interaction of pulmonary alveolar macrophages (AM), neutrophils (PMN), and EC. Bovine EC are grown to confluency in 24 well tissue culture plates. To mimic the vascular component, 10(6) PMN are later added to the EC monolayers. Bovine AM are plated onto millicell inserts and placed into the wells containing EC and PMN. The millicell insert serves as a semi-permeable barrier between AM and EC, allowing the exchange of only diffusible material. Preliminary work demonstrates that P. haemolytica stimulated AM resulted in EC damage presumably due to both soluble lipopolysaccharide and AM secreted products.

In vitro Development and Functions of Human Mast Cells

A long-term co-culture of mononuclear cells of human umbilical cord blood with mouse embryo-derived 3T3 fibroblasts resulted in the development of human mast cells. These mast cells are morphologically and functionally mature cells, containing 1.4-2.8 micrograms histamine per 10(6) cells and bear approximately 10(5) Fc epsilon RI per cell. The mast cells sensitized with human IgE released histamine upon challenge with anti-IgE. Electron-microscopic analysis of the cells showed that these cells were mature human mast cells, and clearly different from basophilic granulocytes. Most of the mast cells contained some granules with regular crystalline arrays and both tryptase and chymase, resembling human skin mast cells. When mononuclear cells of cord blood were seeded in a millicell insert which was placed on 3T3 fibroblasts monolayer, the number of mast cells developed in the millicell inserts was comparable to those developed in the co-culture of the same cord blood cells with 3T3 fibroblasts. Recent observations that mast cells developed in the presence of concentrated culture supernatants of 3T3 fibroblasts without fibroblasts feeder layers, confirmed that soluble factors released from 3T3 fibroblasts are essential and sufficient for the differentiation of human mast cell progenitors in vitro. Analysis of functional characteristics of cultured mast cells revealed that they respond to anti-IgE, Ca2+ ionophore A23187 and substance P for histamine release, but failed to respond to compound 48/80 and FMLP. Upon anti-IgE challenge, sensitized mast cells generated approximately 80 ng PGD2 per 10(6) cells, and approximately 50 ng of LTC4 per 10(6) cells but no detectable generation of LTB4.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclosporin A and vehicle toxicity in primary cultures of rabbit renal proximal tubule cells

The capability of cyclosporin to produce direct injury to primary proximal tubular renal cells was studied. These cells, when grown on Millicell inserts, retain the functional polarity of the proximal tubule, i.e., generate a transepithelial pH gradient (apical compartment acidic) that is reversibly blocked by amiloride addition only if it is added to the apical compartment. Administration of ouabain to the basal compartment also blocks the generation of the transepithelial pH gradient. Additionally, the cells were more responsive to parathyroid hormone (PTH), a proximal tubule characteristic, than to arginine vasopressin (AVP), a distal tubule characteristic. The following substances were tested for their effect on the capacity of these cells to generate a pH gradient: Sandimmune, the commercial form of cyclosporin A; the free form of the drug; Cremophor EL, the vehicle used in the commercial preparation; and ethanol, the vehicle used to dissolve the free form. Sandimmune, at 25-50 microM, inhibited the generation of the pH gradient within 24 h. Surprisingly, Cremophor also blocked the development of a pH gradient, although somewhat less effectively. In contrast, 10 microM cyclosporin, regardless of the form tested, had no effect for up to 96 h. These findings show that cyclosporin, in the form of Sandimmune, has a direct toxic effect on these cells; they also suggest that the vehicle, Cremophor, may contribute to the well-established nephrotoxicity of cyclosporin A.

Separation and Culture of Ovine Endometrial Epithelial and Stromal Cells: Evidence of Morphological and Functional Polarity1

Epithelial and stromal cells from endometria of ovariectomized estradiol-treated Corriedale ewes were separated and purified after collagenase digestion. The separation method utilized differences in the speed and ease of detachment of cultured epithelial and stromal cells attached to plastic in response to brief trypsin exposure. Cells were characterized according to morphological, growth, and histochemical criteria. Contamination of each cell type with the other was less than 1%. Separated cells were grown on plastic or on Matrigel-coated Millicell inserts with nitrocellulose membranes. Transmission and scanning electron microscope analyses demonstrated the existence of tight junctions and prominent microvilli in the epithelial cultures on inserts but not on plastic. Asymmetrical secretion of prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E (PGE) by epithelial cells provided further evidence of polarization. Epithelial cell secretion of PGF2 alpha was greater than that by stromal cells whereas PGE secretion by stromal cells was greater than that by epithelial cells. Epithelial secretion in the basal direction was approximately 4 and 3 times that of apical secretion for PGF2 alpha and PGE, respectively. The separation protocol provides pure populations of ovine endometrial epithelial and stromal cells and the cultured epithelial cells exhibit characteristics of in vivo morphology and polarized function.