Failure of complex supplementation of minimal cultures to elicit a shift-up response in Pseudomonas putida

Primary cell cultures are established from 8-day quail embryo livers. During the first three days the culture is made up of areas of epithelial-like cells and scattered fibroblasts. The cytoplasm of the epithelial cells shows a high glycogen content as detected by the PAS reaction controlled with salivary amylase digestion. During the following days an important increase in the number of fibroblastic cells is observed. After 6-7 days of cultivation, the epithelial cells have disappeared and the culture is entirely fibroblastic. PAS technique does not show any trace of glycogen in these cultures which have been prolonged up to 45 days. Six-to 45-day primary cultures entirely made up of fibroblasts were associated with hepatic or pulmonary mesenchyme in organotypic culture for 3-4 days. In some cases the explant was first cultivated in vitro for 2 days and then grafted into a 5-day-old chick embryo on the chorioallantoic membrane for 6 days. In the secondary cultures hepatocytes showing an epithelial arrangement and a high glycogen content were observed. It appears from this observation that some of the primary culture fibroblasts are in fact dedifferentiated parenchymal cells. Such a dedifferentiation is a reversible phenomenon since the cells retain the ability to express their initial determination if they are placed in convenient environmental conditions. The role of the specific tissular arrangement in the stability of the differentiated state is discussed.     

Transformation of tracheal epithelium exposed in vitro to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

Using an organ culture/cell culture system, we transformed rat tracheal epithelial cells in vitro by exposure to MNNG. Ten tracheal organ cultures per group were exposed twice (at days 3 and 6) to 0,0.001, 1.0 or 10.0 microgram MNNG/ml of medium. Following this exposure, the explants were placed on the bottom of culture dishes to initiate epithelial cell outgrowths and establish primary cultures. Each explant was replanted 8-10 times to produce multiple outgrowths. The number of primary cultures and the number of subsequently established cell lines obtained was carcinogen-dose-dependent. The average number of primary epithelial cell cultures per explant after exposure to 0, 0.001 1.0 and 10.0 microgram MNNG/ml was 1.3, 1.5, 3.3, and 4.6, respectively. The average yield of cell lines per explant in these groups was 0, 0.8, 1.3, and 2.0, respectively. It is apparent that cell lines could only be established from carcinogen-exposed epithelium. These cell lines are currently being tested for tumorigenicity in vivo. To date, of 35 cell lines tested between the 5th and 15th passages, 5 cell lines from the 10 microgram MNNG/ml group and 2 from the 1.0 microgram MNNG/ml group have produced palpable tumors upon injection into immunosuppressed recipients.     

Substrate induction and glucose repression of maltose utilization by Streptomyces coelicolor A3(2) is controlled by malR, a member of the lacl-galR family of regulatory genes

The rat treated with bile duct ligation (BDL) and furan is a unique animal model of massive bile ductular hyperplasia in which normal liver parenchyma is largely replaced with well-differentiated proliferated bile ductules. We have now developed a simple cell isolation procedure to obtain and culture viable bile ductular epithelial cells in high numbers and with a high degree of purity from the livers of BDL/furan-treated rats. Primary monolayer cell cultures were readily established when the isolated bile ductular epithelial cells were cultured in plastic tissue culture wells coated with rat tail tendon type I collagen plus bovine plasma fibronectin. Under these conditions, epidermal growth factor (EGF) was mitogenic for the cultured cells, and they retained phenotypic features typical of hyperplastic bile ductular epithelium but did not show evidence of ductal morphogenesis in vitro. In contrast, when the isolated bile ductular cells were cultured for 7 to 16 days in the presence of 25 ng EGF/mL and 10% fetal bovine serum on type I collagen gels, they formed into branching ductal structures whose ultrastructural features very closely resembled those of polarized hyperplastic bile ductules/ducts in vivo. Histological preparations of these gel cultures further showed that numerous ductal structures with defined lumens were present. Phenotypically, these ductal structures were completely surrounded by a thickened basement membrane that was strongly immunoreactive for laminin. Like their in vivo biliary cell counterparts, the epithelial cells comprising these ductal structures in culture also exhibited strong immunocytochemical staining reactions for cytokeratins 8 and 19, for glutathione S-transferase pi 7, and for luminal gamma-glutamyl transpeptidase, but they did not express immunoreactive albumin or alpha-fetoprotein. Occasional epithelial cells of the ductal structures, when examined in 10-day-old primary gel culture, showed strong nuclear staining for incorporated 5-bromo-2'deoxyuridine, indicating active cell proliferation. Our results support the development of a novel biliary epithelial cell culture model that has the potential of serving as a powerful tool for investigation of factors that regulate hyperplastic bile ductular morphogenesis, cell proliferation, and polarized cell functions in a structural form in vitro that mimics that of hyperplastic bile ductules induced in vivo.     

A simple technique for the long-term non-polarised and polarised culture of human fallopian tube epithelial cells

Fallopian tubes were obtained from 25 women undergoing abdominal hysterectomy. Pieces of fallopian tube mucosa were placed in culture flasks containing minimum essential medium in Earle's salts supplemented with fetal bovine serum. First passage was carried out after 7-10 days and subcultures in 4-5 days. For polarised cell culture, epithelial cells were seeded onto an extracellular matrix system. New epithelial cells were seen on day 2-3 of the primary culture and epithelial patches on day 7-10. Cells reached confluence in 4-5 days in subcultures. The cells could be subcultured for 7-11 passages with a life span of 42-60 days. Epithelial origins of the cells were confirmed by immunofluorescence staining with anti-cytokeratin antibody. Polarised cells showed a columnar pattern, microvilli on their apical surface and basally located nucleus whereas non-polarised cells were flat. It was concluded that the human fallopian tube epithelial cells can be cultured in vitro to create non-polarised and polarised cell layers by using a simple and reproducible technique and this system can be a potential model to study function of the fallopian tube.     

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.     

Establishment of primary cell culture from stria vascularis explants. Morphological and functional characterization

To provide the prerequisite for long-term study of the inner ear related to structural and functional integrity, tissue of stria vascularis with spiral ligament was isolated from Wistar rat cochleas and cultured using the explant-culture technique. The following culture media were used: EMEM with Hepes buffer, hydrocortisone (400 ng/ml), transferrin (5 micrograms/ml). triiodothyronine (10(-9) M), cholera toxin (10(-10) M), insulin (5 micrograms/ml), and epidermal growth factor (10 ng/ml). To characterize the cells growing out from the explant, immunofluorescence with cytokeratin (cytokeratin 18) and ultrastructural examination with SEM and TEM were performed. The marginal cell function was investigated by expression of Na+, K(+)-ATPase antisera against beta 2 subunit of rat Na+, K(+)-ATPase and P-NPPase. We were able to maintain the cultured cells for 3 weeks or more. Monolayered marginal cells were observed beyond 14 days in vitro and the expression of cytokeratin 18 was especially enhanced. The cultured marginal cells were almost identical to in vivo cells both as regards ultrastructural features and Na+, K(+)-ATPase activity. The present results suggest that the primary explant culture technique is a reliable in vitro model of strial marginal cells. However, establishment of the cell line is needed for long-term study.     

Clonal characterization of mouse mammary luminal epithelial and myoepithelial cells separated by fluorescence-activated cell sorting

Lineage analysis in vitro of heterogeneous tissues such as mammary epithelium requires the separation of constituent cell types and their growth as clones. The separation of virgin mouse mammary luminal epithelial and myoepithelial cells by fluorescence-activated cell-sorting, their growth at clonal density, and the phenotyping of the clones obtained with cell-type specific markers are described in this paper. Epithelial cells were isolated by collagenase digestion followed by trypsinization, and the luminal and myoepithelial cells were flow-sorted with the rat monoclonal antibodies 33A10 and JB6, respectively. Sorted cells were cloned under, using low oxygen conditions (<5% vol/vol), in medium containing cholera toxin and insulin, with an irradiated feeder layer of 3T3-L1 cells. Clones were characterized morphologically, and antigenically by multiple immunofluorescence with a panel of antibodies to cytoskeletal antigens specific to either luminal epithelial or myoepithelial cells in situ. Whereas sorted myoepithelial cells gave a single clone type, sorted luminal cells gave three morphological clone types, two of which grew rapidly. All myoepithelially derived clones showed a limited proliferative capacity in vitro, in contrast to their rat and human counterparts, as shown in previous studies. The present results with sorted mouse cells have also allowed the stability of the differentiated phenotype in mouse, rat, and human mammary luminal epithelial and myoepithelial cells in primary clonal culture to be compared. They show that the mouse mammary cells are the least stable in terms of expression of differentiation-specific cytoskeletal markers in vitro.     

Evaluation of congenital vena cava anomalies and acquired vena cava obstructions after atrial switch operation using spiral computerized tomography and 3-dimensional reconstruction

The aim of this study was to establish an in vitro model of ovine luteinizing hormone-releasing hormone (LHRH) neurones. Olfactory placodes from 26 day-old sheep embryos (E26) were used for explant culture. Cultures were maintained successfully up to 35 days, but were usually used at 17 days for immunocytochemistry. LHRH and neuronal markers such as neurofilament (NF) were detected by immunocytochemistry within and/or outside the explant. Three main types of LHRH positive cells are described: (1) neuroblastic LHRH and NF immunoreactive cells with round cell body and very short neurites found mainly within the explant, (2) migrating LHRH bipolar neurones with an fusiform cell body, found outside the explant, (3) network LHRH neuron, bipolar or multipolar with long neurites connecting other LHRH neurons. Cell morphology was very similar to that which has been described in the adult sheep brain. These results strongly suggest that LHRH neurones in the sheep originate from the olfactory placode. This mode may represent a useful tool to study LHRH neurones directly in the sheep.     

Culture of human retinal pigment epithelial cells from peripheral scleral flap biopsies

PURPOSE: We studied various methods for harvesting retinal pigment epithelium (RPE) biopsies from cadaver human eyes of donors over age 60 years. Our goal was to harvest cells for possible autologous RPE cell transplantation in patients with age-related macular degeneration and to test the viability of the RPE after isolation by evaluating explant growth in culture. METHODS: Choroid-RPE biopsies were excised from enucleated human eyes. The RPE was separated from the choroid by treatment with type IV collagenase. RPE patches were cultured. After 100-500 cells had grown out from the explant, the primary cultures were passaged. RESULTS: There was no clear effect of donor age on the ability to establish primary RPE cultures with good morphology from biopsies 2 x 2-10 x 10 mm2 in size. Biopsies 6 x 6 mm2 or larger produced satisfactory primary cultures more than 70% of the time. The number of viable RPE cells (defined as the number of cells adherent to the culture dish 24 h after plating) obtained after enzymatic separation of the RPE and choroid was an important determinant of our ability to establish primary cultures and passage the cells. Primary cultures with good cellular morphology were obtained 100% of the time when RPE explants > 4 mm2 in size were obtained from the biopsy specimen. Seventy-three percent of the biopsies yielding explants > 4 mm2 in size were successfully passaged. CONCLUSIONS: These results suggest that peripheral scleral flap biopsies in aging donors can be used to establish RPE explant primary cultures. These cultures may be suitable as a source for autologous RPE transplantation in patients.     

Tonsil stromal-cell lines expressing FDC-like properties: isolation, characterization, and interaction with B lymphocytes

The microenvironment of secondary lymphoid organs consists of two major populations of cells, the lymphoid cells and a population of stromal cells that contribute to both tissue architecture and function. Interactions of both populations are essential for the development and control of humoral immune responses. In this study, stromal-cell preparations were obtained by a multistage process. This involved culturing 300-400-microm slices of human tonsil for 6-8 days at 25 degrees C, trypsin digestion of the residual explant, followed by CD45-positive-cell depletion using magnetic beads, and a final period of culture for 4 days to remove remaining nonadherent cells. Phenotyping with a panel of monoclonal antibodies revealed that the cells express HLA-DR, CD54 (ICAM-1), CD44, but no CD45 nor a range of other markers for epithelial and endothelial cells. Immunoassays of supernatants from stromal cells revealed that IL-6 was produced constitutively, and its production was increased by treatment with TNF-alpha and IFN-gamma. In contrast IL-1, IL-2, IL-4, IL-7, IL-8, IL-10, IL-12, TNF-alpha, and IFNgamma were not produced. Functional tests showed that these cells express follicular dendritic cell-like properties. Coculturing of tonsilar B cells with stromal cells resulted in enhanced proliferation and also led to increased production of immunoglobulins and IL-6, suggesting crucial signaling between these populations.     

Mechanism for inhibition of deoxyribonuclease activity by antisera

The cultivation of cells from primary breast cancers is very unpredictable. The majority of breast-cancer-derived cell lines are of metastatic origin. To define the characteristics of tumor cells which govern their ability to grow in vitro as primary cultures as well as continuous or established culture cell lineages, human mammary epithelial cancer (HMEC) cells from 18 cases of unselected primary breast cancer were propagated in culture. Propagation of HMEC cells in vitro as monolayers in primary culture was successful in 10 out of 18 (55.5%) cases, which showed continous proliferation of tumor cells only up to 6-8 passages before they reached senescence. An investigation of the effects of phenotypic expression of estrogen receptors (ER), the progesterone receptors, c-erbB-2 oncoprotein and epidermal growth factor receptors (EGFR) on the capacity of HMEC cells to grow in vitro as monolayers showed that expression of ER and EGFR is required for controlling tumor proliferative activity in vitro. Expression of ER protein made the growth of HMEC cells more difficult, while expression of EGFR protein made their growth in vitro easier. Phenotypic characteristics of floating HMEC cells were found to be different from those grown on cover slips as adherent cultures, suggesting a selective growth of HMEC cells of a specific phenotype in culture. Cultured HMEC cells in subsequent passages showed a decrease in their proliferative capacity, alterations in phenotypic characteristics and development of morphologic features of terminal differentiation, resulting in senescence.     

Gene transfer by guanidinium-cholesterol cationic lipids into airway epithelial cells in vitro and in vivo

Synthetic vectors represent an attractive alternative approach to viral vectors for gene transfer, in particular into airway epithelial cells for lung-directed gene therapy for cystic fibrosis. Having recently found that guanidinium-cholesterol cationic lipids are efficient reagents for gene transfer into mammalian cell lines in vitro, we have investigated their use for gene delivery into primary airway epithelial cells in vitro and in vivo. The results obtained indicate that the lipid bis(guanidinium)-tren-cholesterol (BGTC) can be used to transfer a reporter gene into primary human airway epithelial cells in culture. Furthermore, liposomes composed of BGTC and dioleoyl phosphatidylethanolamine (DOPE) are efficient for gene delivery to the mouse airway epithelium in vivo. Transfected cells were detected both in the surface epithelium and in submucosal glands. In addition, the transfection efficiency of BGTC/DOPE liposomes in vivo was quantitatively assessed by using the luciferase reporter gene system.     

Phenotypic mapping of the chicken embryonic thymic microenvironment developing within an organ culture system

The chicken thymic microenvironment, as it developed in an embryonic thymus organ culture system, was phenotypically mapped using a panel of mAb defining both epithelial and nonepithelial stromal cell antigens. We have previously reported that thymocyte proliferation and differentiation with proceed for up to 6-8 days in thymus organ culture, hence demonstrating the functional integrity of the thymic microenvironment in vitro. During this time, the stromal component reflected that of the normal embryo with cortical and medullary epithelial areas readily identifiable by both morphology and surface-antigen expression. An abundance of subcapsular and cortical epithelial antigens was detected in the cultured thymus, particularly those normally expressed by the epithelium lining the capsule, trabeculae, and vascular regions (type I epithelium) in the adult and embryonic thymus. Medullary epithelial antigens developed in organ culture, although were present in lower frequency than observed in the age-matched embryonic thymus. MHC class II expression by both epithelial and nonepithelial cells was maintained at high levels throughout the culture period. With increasing time in culture, the ratio of epithelial to nonepithelial cells decreased, concurrent with a decrease in thymocyte frequency and suggestive of a bidirectional interaction between these two cell types. Thus, a functionally intact thymic microenvironment appears to be maintained in embryonic thymus organ culture, a model that is currently being exploited to assess the role of stromal antigens, as defined by our mAb, in the process of thymopoiesis.     

Cell, tissue and organ culture as in vitro models to study the biology of squamous cell carcinomas of the head and neck

In vitro models are currently being used to study head and neck squamous cell carcinoma (HNSCC). Several hundred HNSCC cell lines have been established by various investigators and used to study a broad spectrum of questions related to head and neck cancer. The head and neck model with respect to multistage carcinogenesis is now complete. Several techniques exist for the culture of normal epithelial cells from the upper aerodigestive tract (UADT). The biology of these UADT cells (oral cavity, oropharynx, hypopharynx and larynx) is being studied. Successful culture of premalignant lesions (dysplastic mucosa, leukoplakia, erythroplakia) has resulted in establishment of a limited number of premalignant cell lines and cell cultures. HPV infection of normal oral epithelial cells for immortalization (approximately premalignant cells) coupled with transformation with carcinogens (malignant cells) has established an experimental model for progression. Two in vivo models for oral carcinogenesis, the 7,12 dimethylbenz(a)anthracene-induced hamster cheek pouch model and the 4-nitroquinoline-N-oxide rat oral model, have been established in culture. Thus, multistage carcinogenesis models have been established from both human tissues and animal models and include cultures of normal, premalignant and malignant cells. Culture techniques for growing dissociated primary tumor cells for short term experimental analysis are being used. The culture of normal or tumor tissue as organ/explant cultures allows for the maintenance of normal cell-cell and cell-matrix interaction, but limits experimentation since these cultures cannot be propagated. Several three dimensional model systems are being used to obtain this histological complexity but allow for experimentation. The ability to culture normal, premalignant and malignant cells coupled with the use of a variety of culture techniques, should allow for the continued growth and experimentation in head and neck cancer research.     

Proliferation and agglutinability of primary and transformed human epithelial cells in culture

Primary epithelial populations (HAM) were obtained by dissociation of the amniotic membrane stripped from human placentae. Agglutinability of cells from such normal populations and of cells from the transformed epithelial line WISH was then compared using concavanalin A as mediator. Extensive similar studies have previously been reported with cell strains isolated from other species. Freshly dissociated HAM cells from primary cultures agglutinated much less readily than did cells from WISH populations. Furthermore, the former exhibited a drastic decline in agglutinability as a function of time in suspension culture after trypsinization. Short-term exposure (60 h) of HAM cells in monolayer culture to 5-bromodeoxyuridine (BrdU) elicited heightened agglutinability detectable through 22 days in vitro. Addition of the protease inhibitors n-tosyl-L-lysyl-chloromethyl ketone (TLCK) or p-tosyl-L-arginine-methyl ester (TAME) to the culture medium inhibited proliferation of the WISH line by 40--50% while effecting only a 10-15% inhibition of HAM cells. These results also confirm data with other cell species indicating that high proteolytic activity at the surface of transformed cells may be related to the rapid proliferation rate.     

Increased 3-nitrotyrosine and oxidative damage in mice with a human copper/zinc superoxide dismutase mutation

The structural and functional barrier preventing the free advancement of microbial plaque subgingivally along the tooth surface is formed by the junctional epithelial (JE) cells directly attached to the tooth (DAT cells). The mechanism leading to degeneration of the DAT cells is not known. In the present study we examined the possible role of short chain fatty acids (SCFAs) on epithelial cells by making use of 2 epithelial cell cultures (HaCaT and ERM) and an explant culture model of human JE. The SCFAs butyrate and propionate were used in concentrations found in human plaque and gingival crevicular fluid (0.25-16.0 mM). The SCFAs had no effect on primary cell adhesion nor on the epithelial attachment apparatus (EAA). By contrast, even 0.25 mM of butyrate significantly retarded epithelial cell growth. Similar effects with propionate were first observed at concentrations higher than 1.0 mM. The retardation of epithelial cell growth was found to be due to inhibition of cell division. Furthermore, after butyrate treatment dense accumulations of intermediate filaments and cytoplasmic vacuolization were characteristically seen in cells adjacent to cells of normal appearance. This suggests that some cells of the growing epithelial cell population are more sensitive to the SCFAs than others, and agrees with previous reports on the DAT cells of periodontally-involved teeth in vivo. The results suggest that SCFAs are microbial factors that play a role in the initiation and progression of periodontal pocket formation by impairing epithelial cell function rather than having a direct effect on the EAA.     

An evaluation of the effectiveness of osteopathic treatment on symptoms associated with myalgic encephalomyelitis. A preliminary report

To improve the study of epithelial function in rat ductuli efferentes (efferent ductules) and initial segment epididymis, we developed a primary cell culture system with modification of the Klinefelter method (1992). The cultured efferent ductal epithelium was grown to confluence and the cells maintained many of the organelles characteristic of these cells in vivo, including dense-staining granules, indented nuclei and apical cilia. Ciliary beat was observed for up to 10 days in culture, Cultured initial segment epithelial cells were elongated and characterized by apical branched microvilli. Electron microscopy revealed intact cell junctions, and endocytotic apparatus and lysosomal granules. Ultrastructurally, the initial segment epithelium contained a well developed Golgi apparatus. For both epithelia, cell characteristics were also confirmed by indirect immunofluorescent staining for cytokeratins 8, 18. Endocytotic activity was detected by the uptake of cationic ferritin at the apical surface and within vesicles. Estrogen receptor and clusterin mRNAs were expressed in the cultured epithelia and no difference was found in their expressions when cultured with or without 10(-9)M 17-beta estradiol. Indirect immunofluorescent staining for clusterin further indicated that this protein was present in the cultures. In conclusion, these in vitro methods will be useful for the investigation of epithelial function in the head of the epididymis.     

A family of gram-negative bacterial outer membrane factors that function in the export of proteins, carbohydrates, drugs and heavy metals from gram-negative bacteria

Differentiation of biliary epithelial cells from hepatic endodermal cells of the mouse embryo was examined with a special attention to the role of the connective tissue. When the whole liver primordium of the 9.5-day mouse embryo was cultured in vitro for 5 days, the endodermal cells differentiated into mature hepatocytes expressing carbamoylphosphate synthetase I (CPSI) and accumulating glycogen. Intrahepatic bile duct cells and connective tissue were poorly developed in this culture. However, when the hepatic endoderm was recombined with the 4-day embryonic chick lung mesenchyme and cultured in vitro, the endodermal cells differentiated into many ductal epithelial cells as well as mature hepatocytes with abundant connective tissue development. These results suggest that the ducts might be bile ducts, and that connective tissue is very important for bile duct development. In addition, this in vitro culture system might be useful for the study of mechanisms of bile duct differentiation and congenital biliary atresia.     

Properties of cochlear nucleus neurons in primary culture

Dissociated primary cell cultures were derived from the cochlear nuclei (CN) of postnatal rats using standard techniques. Cultured cells differentiated morphologically, but their dendritic profiles were generally less specialized than those of CN cells in vivo. Physiologically, cultured cells could be divided into three classes: tonic, phasic and non-spiking cells, which differed in many of their fundamental biophysical properties. The percentage of cultured cells that spiked repetitively increased over time to a maximum of 85% at 6 days. However, the percentage of cells that produced action potentials decreased with time in culture, from 91% during the first 8 days to less than 40% after 9 days. CN cells were successfully cultured in both serum-supplemented and serum-free (Neurobasal) media. More neurons survived at low plating densities in Neurobasal than in medium containing serum, although neuronal survival was similar at higher densities. Few neurons raised in the serum-free medium were spontaneously active; other response properties were similar to those of cells grown in the presence of serum. Although differentiation of CN cells in culture did not completely mirror the in vivo developmental pattern, these experiments demonstrate that primary culture represents a viable method for the in vitro study of CN neurons.     

Isolation, cell culture, and characterization of oviduct epithelial cells of the cow

This report describes an easy method of isolation and cell culture of the epithelial cells of cow oviduct. Incubation of cow oviduct with 0.1 mg/ml collagenase in the lumen for 90 minutes helped to dislodge large numbers of ciliated and secretory epithelial cells. The isolated cells, when seeded on plastic, proliferated very quickly and became confluent in 8-10 days in 35 mm Petri dishes. The isolated ciliated cells which attached to the plastic dish lost their cilia after 4-5 days in culture. The cultured epithelial cells were keretin positive. The isolated bovine oviduct epithelial cells, when cultured on plastic precoated with 10 mg/ml matrigel, organized themselves into hollow tubes or spheres with microvilli directed towards the lumen. The epithelial cells seeded on 2 mg/ml matrigel became subconfluent in 15-20 days after seeding. The histoarchitecture of the secretory cells growing in vitro on matrigel resembled that of intact oviduct secretory epithelial cells. Occasional ciliated cells containing large number of mitochondria were observed in the monolayer cultured on 2 mg/ml matrigel substratum but possessed few cilia. The oviduct epithelial cells cultured on 2 mg/ml matrigel incorporated 35S-methionine linearly into protein up to 8 hours in the presence of estradiol or progesterone. The fluorograph of the newly synthesized proteins indicated the presence of an additional 60 kd protein in the cell extract of epithelial cells incubated with estradiol.