Growth and characterization of isolated bovine tracheal gland cells in culture. Influence of a reconstituted basement membrane matrix

We describe a method for establishing the culture of bovine tracheal submucosal gland (BTG) cells, in which we have also examined the influence of a reconstituted basement membrane matrix derived from the Engelbreth-Holm-Swarm tumor (EHS) on the growth and morphological differentiation of these cells. BTG cells have been isolated by tissue enzymatic digestion using trypsin, deoxyribonuclease I, elastase, hyaluronidase and EGTA for 1 hr at 37 degrees C. Afterwards, cells and tissue were collected by centrifugation and were incubated for 15 min with 15% newborn calf serum to inactivate the proteolytic enzymes. Enzymatic digestion using only trypsin, centrifugation and inactivation steps were repeated three times. Using this protocol, we obtained 15 +/- 4 (X 10(6] cells per g of tracheal submucosa with 72 +/- 2% (n = 5) cell viability. On microscopic observation, isolated cells were mainly composed of serous type glandular cells. Cells were cultured in a 1:1 medium of Dulbecco's Modified Eagle's/Ham's F12 supplemented with 10% fetal calf serum and subcultured in either plastic flasks or flasks coated with EHS matrix. On the plastic, the BTG cells exhibited at confluency an epithelioid appearance. They stained positively with the immunofluorescent anticytokeratin antibody and contained PAS-staining granules. By electron microscopy, lactoferrin, a protein marker specific to the serous cells, was demonstrated immunocytochemically in small secretory vesicles. BTG cells cultured on EHS matrix revealed a significantly increased growth in comparison to those cultured on plastic. In post-confluent culture of BTG cells on EHS matrix, we observed numerous dome-like structures formed by differentiated cells which were joined together around luminal spaces.     

Regulation of cytoskeletal structure in human mammary carcinoma cells MCF-7 by culture substrata

Three-dimensional cellular structures formed by MCF-7 human mammary carcinoma cells within collagen gels were isolated with collagenase and cultivated on plastic substratum to examine whether the cytoskeleton specific for cells forming cellular structures (S-type) changes to that specific for cells grown as monolayers (M-type). The cytoskeleton isolated as 0.05% Triton-insoluble fraction from the cellular structures after culture for 1 day on plastic was exclusively S-type. However, both types of cytoskeletons were observed in the cellular structures cultivated for 7 days on plastic as well as in the cells grown as monolayers for 2 days after dissociation of the cellular structures with trypsin. By use of an antibody raised against a 65-kD polypeptide that was specific for the M-type cytoskeleton, the presence of the polypeptide was found to be restricted to the cells grown out as monolayers from the edge of the cellular structures. In the cells grown for 2 days as monolayers, a mixture of cells both having and lacking the polypeptide was observed. After a 7-day culture of the dissociated cells as monolayers on plastic, however, most of the cells had M-type cytoskeletons. The present results show that the apparent change in the cytoskeleton of MCF-7 cells from S-type to M-type does not occur in cells involved in the three-dimensional cellular structures even in the absence of collagen gels, but that it occurs in cells which are grown as monolayers for at least 7 days on plastic substratum.     

Effects of substrata on the polarization of bovine endometrial epithelial cells in vitro

Summary Epithelial-cell function requires cellular polarity in which apical membrane surfaces have unique characteristics and cellular organelles are stratified. Physiological investigations of endometrial, epithelial cells would be enhanced greatly by the ability of a method to polarize cells in culture. This study investigates the effects of different substrata on polarization of cultured bovine endometrial epithelial cells. Fetal bovine endometrial epithelial-cell lines were developed from explant outgrowth. Epithelial monolayers were subcultured onto amniotic membranes, Millicell-HA membranes, or Millicell-CM membranes coated with rat-tail collagen, Matrigel, laminin, Vitrogen,or fibronectin. Cultures on these substrata were maintained at the air/liquid interface. Cells grown on either collagen-coated or uncoated Milli-cell membranes also were maintained submerged in medium. Excellent polarized morphology was attained in cultures grown at the air/liquid interface on amniotic membranes and rat-tail collagen-coated membranes. Lectin-binding patterns, to apical membranes of polarized epithelial cell cultures paralleled patterns of binding to bovine endometrial surfaces in vivo. Cultures on rat-tail collagen were maintained for several weeks. These methods provide a valuable system for studying the endometrium in vitro.     

Evidence that hepatocyte growth factor abrogates contact inhibition of mitosis in Madin-Darby canine kidney cell monolayers.

It is becoming increasingly apparent that hepatocyte growth factor (HGF) plays an important role in kidney development, regeneration, and transformation to carcinoma. Previous in vitro studies have shown that HGF stimulates cell scattering, but not proliferation, in the renal epithelial cell line Madin-Darby canine kidney (MDCK) when grown on plastic at low density. This communication demonstrates that HGF treatment of confluent monolayers of MDCK also stimulates DNA synthesis and cell division. HGF stimulated thymidine incorporation in confluent MDCK cell monolayers grown on plastic in a dose dependent fashion, but did not stimulate thymidine incorporation in MDCK cells at 10-20% confluency on plastic. Additionally, basolaterally, but not apically, applied HGF stimulated thymidine incorporation in confluent MDCK cell monolayers grown on filters. Immunofluorescent labeling of nuclei in control and HGF treated MDCK cell monolayers grown on filters demonstrated an increase in mitotic figures. Confocal X-Z section views and direct cell counts of MDCK cell monolayers grown on filters demonstrated an increase in cell number after HGF treatment compared to controls. This is the first report of HGF stimulating cell proliferation in previously quiescent renal epithelial cell monolayers. This model will be useful for studying the mechanisms controlling cell proliferation rates in epithelial tissue.     

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

Incubation of cow oviducts flushed with 0.1 mg collagenase/ml, for 90 min helped to dislodge large numbers of ciliated and secretory cells. About 90-95% of the isolated epithelial cells were viable. The epithelial cells suspended in DMEM:F-12 + 10% serum attached to the plastic culture dish in 18-20 h after seeding. The ciliated cells which attached to the plastic dish lost their cilia after 4-5 days in culture. The attached cells, which proliferated to form a confluent monolayer 8-10 days after seeding in a 35-mm dish, could be subcultured at least 3 successive times. Some cell aggregates which did not attach to the culture dish proliferated into floating balls of cells. The ciliated cells in the unattached floating colonies maintained the ciliary movement for 9-10 days in the same culture medium. The primary cultures of the ciliated and the secretory cells maintained most of the histoarchitecture observed in intact epithelium. The secretory cells maintained their secretory activity of specific proteins in culture as indicated by immunocytology. The cultured cells contained keratin, a specific cytoskeletal component of epithelial cells.     

Cell-specific localization of prostaglandin E2-sensitive adenylate cyclase in rabbit endometrium

Epithelial and stromal cells were isolated from endometrium of Day 1 pseudopregnant rabbits by enzymatic digestion with trypsin or trypsin:collagenase:deoxyribonuclease. Dispersed cells were grown in RPMI 1640 supplemented with 10% whole or steroid-depleted fetal bovine serum (FBS). Epithelial and stromal cells reached confluency after 6 to 7 days in culture and showed specific characteristics. Cells could be differentiated according to morphology, growth patterns, electrophoretic patterns, and response to estrogen or progesterone. Hormonal stimulation of adenylate cyclase activity was measured in broken cell preparations by catalytic transformation of alpha-32P-adenosine triphosphate into 32P-adenosine 3'-5' cyclic monophosphate (cAMP). Adenylate cyclase activity was present in fresh endometrial tissue and in dispersed cells after 7 days in culture. The enzyme activity was significantly higher in stromal than in epithelial cells at all stimulation levels: basal (9.2 +/- 1.0 vs. 2.3 +/- 0.6, p less than 0.001) and guanosine triphosphate (GTP, 300 microM) (25.4 +/- 2.9 vs. 7.0 +/- 1.6, p less than 0.001). Net response to prostaglandin E2 (PGE2, 10 microM) was three times higher (p less than 0.001) in stromal (17 +/- 2) than in epithelial (5.0 +/- 1) cells. These results suggest that PGE2 can stimulate adenylate cyclase in rabbit endometrium and that the enzyme is preferentially localized in the stroma. Our results are in agreement with the hypothesis that cAMP formed in endometrium in response to PGE2 might be involved in the decidual reaction.     

CA 125 is an excretory product of human endometrial glands

The present investigation was undertaken to study the cellular localization and kinetics of synthesis of CA 125 in the endometrium. CA 125 was localized by immunohistochemistry to the infranuclear region of epithelial cells during the proliferative phase and to the apical luminal border during the secretory phase. In gestational endometrium, both the cytoplasm and the apical luminal border of epithelial cells were intensely positive. No staining was seen in endometrial stromal cells during the normal cycle or in decidualized endometria. Results obtained from in vitro cultures of separated glandular and stromal cells were similar to those obtained by immunohistochemistry. That is, epithelial cells released between 5 and 25 times more CA 125 into the culture medium than did stromal cells. The release of CA 125 was highest in epithelial and stromal cells obtained during the early secretory phase. CA 125 concentrations were markedly elevated in endometrial aspirations obtained during the secretory phase or in endometria with crumbling stroma compared to plasma levels. Plasma levels of CA 125 were slightly elevated during menses. These results suggest that CA 125 is an exocrine product of endometrial epithelial cells. Plasma levels of CA 125 may be of endometrial origin only when the membrane barriers, which normally prevent its entry into the circulation, are damaged.     

PC12 cells grown on cellulosic filters differentiate in response to NGF and exhibit a polarity not seen when they are grown on solid substrata

Summary Studies were performed with cellulosic filters and standard culture plates to compare methods of cell culture and differentiation of the cell line PC12, a clone originating from a rat pheochromocytoma. PC12 cells respond to nerve growth factor (NGF) by flattening of the cell body and subsequent extension of neurite-like processes. When PC12 cells are cultured in dishes without NGF, they have a diameter of approximately 3 to 7 μm and exhibit short processes of no longer than 3 to 5 μm. If PC12 cells are grown on a cellulosic filter they have the same average soma diameter and similar short processes extending laterally, but in addition have branching processes which extend as far as 10 to 15 μm into the filter substrate. When dish-cultured and filter-cultured cells are incubated with 50 ng/ml NGF they both exhibit differentiation-specific ultrastructural changes by 3 d of treatment. In the case of dish-cultured cells, large cytoplasmic processes exhibit an increase in the number of chromaffin cell-like secretory granules by 3 d of treatment. This characteristic is also demonstrated by filter-cultured cells, but the processes containing these granules are found concentrated within the cellulosic meshwork. Thus the timing of the NGF-elicited differentiation program is similar to both filter-cultured and dish-cultured cells, but the ultrastructural consequences are different. The filter-cultured PC12 cells exhibit a polarity not demonstrated by dish-cultured cells. Growing PC12 cells on cellulosic filters is a technique useful for “anchoring” neurons without the complication of the addition of extracellular matrix components. Filter-culture may represent a more in vivo-like method for studying neuronal growth and differentiation. This work was supported by grants S07RR07149-13 to R.V.B. and CA40929 to J.A.W. from the National Institutes of Health, Bethesda, MD.     

Normal human endometrial cells in culture: Characterization and immortalization of epithelial and stromal cells by SV 40 large T antigen

Summary— Thirty endometrial biopsies were cultured in order to separate stromal and epithelial cells. Using epidermal growth factor (EGF), cortisol, cholera toxin, insulin with 5% horse serum for epithelial cells or a medium with 20% fetal calf serum for stromal cells, we could specifically enrich endometrial culture in epithelial or stromal cells and culture them for 1 or 2 months. These cultures retained the phenotypic characteristics of epithelial (cytokeratins, mucin HMFG 1) and stromal (vimentin, smooth muscle actin, desmin) lineage by immunostaining analysis. Epithelial and stromal cultures from one individual were respectively immortalized by the SV 40 large T antigen. The immortalized cell lines kept the phenotype of the normal cells from which they derived.     

Expression of interferon receptor subunits,IFNAR1 and IFNAR2, in the ovine uterus

Interferon-tau (IFN-tau) is the antiluteolytic factor released by concepti of ruminant ungulate species prior to implantation. All type I interferons, including IFN-tau, exert their action through a common receptor, which consists of two subunits, IFNAR1 and IFNAR2c, but the distribution of the two polypeptides in uterine endometrium has not been examined. In situ hybridization and immunohistochemistry on sections from pregnant and nonpregnant ovine uteri at Days 14 and 15 after estrus and mating showed that both IFNAR1 and IFNAR2 mRNA and protein were strongly expressed in endometrial luminal epithelium (LE), superficial glandular epithelium (GE), and stromal cells, within but not outside caruncles. Similar staining patterns were noted in pregnant and nonpregnant uteri for both subunits. Western blot analysis of membrane fractions from cell lines derived from endometrial LE, GE, and stromal cells, and affinity cross-linking experiments with radioactively labeled IFN-tau performed on crude endometrial membranes indicated the presence of both high ( approximately 110 kDa) and low (75-80 kDa) molecular mass forms of the two receptor subunits. To localize where IFN-tau binds when it is introduced into the uterine lumen, immunohistochemistry with an antiserum against IFN-tau was performed on sections of uteri from Day 14 nonpregnant ewes whose uteri had previously been infused with IFN-tau. Staining was concentrated on the LE and superficial GE cells, and was absent from the deeper regions of the glands and from the stromal tissues. These studies demonstrate the heavy concentration of IFNAR1 and IFNAR2 in cells of the LE and superficial GE, which appear to be the main targets for IFN-tau.     

The release of PGF2 alpha and PGE2 from separated cells of human endometrium and decidua

The capacity of separated glandular and stromal cells from endometrium and first trimester decidua to release prostaglandins (PGs) was studied over 48 hours in culture. Glandular preparations released more PGs than stromal preparations in all tissues. Stromal release of PGs did not alter throughout the cycle or in early pregnancy but the capacity of glandular preparations to release PGs varied considerably. Proliferative glands released most PGF2 alpha and PGE2 followed by secretory glands and decidua. Histamine (10(-5)) stimulated PG release from endometrial and decidual glands but the response of proliferative glands was greatest. Actinomycin D stimulated release of PGF2 alpha and PGE2 from glandular cells of secretory endometrium and decidua. These results suggest that in vitro release of PGs is suppressed after ovulation and is in part due to inhibition of PG release by a protein or proteins synthesized in the glandular fraction of secretory endometrium or decidua.     

Growth and hormonal responsiveness of human endometrial stromal cells in culture

The present review describes and discusses published results on growth and hormonal responsiveness of human endometrial stromal cells in culture. The proliferative potential of serially subcultured cells, that is, the number of cell doublings before cells enter mitotic senescence and cease to divide, was unusually high in stromal cells from several endometrial specimens, a property that may reflect the unique proliferative capacity of human endometrium when compared to other adult tissues. Fluorescent visualization of microfilaments revealed distinct age-related changes in the distribution of cytoskeletal fibers. Addition of ovarian steroids to the culture medium of stromal cells resulted in significant morphologic changes. From comparative studies using different culture media it became evident that medium components remarkably influenced cell morphology during early culture periods in an irreversible manner. Cultured stromal cells yielded interesting results in experiments designed to define the role of polyamines in growth regulation. Proliferation was greatly inhibited when polyamine levels were reduced by specific inhibition of ornithine decarboxylase, the first and rate limiting enzyme in polyamine synthesis which produces putrescine by catalytic conversion from ornithine. The antiproliferative effects were reversed by addition of putrescine to the culture medium. These results clearly establish a causal link between polyamine depletion and growth deficiencies and reveal an essential function of polyamines in stromal cell proliferation. Hormonally regulated parameters in cultured stromal cells include aromatase activity, pregnancy-associated plasma protein-A, 51K secreted protein, prolactin and laminin. The hormonally regulated production of prolactin and laminin, both considered markers of decidualization, together with morphologic changes of stromal cells to decidual-like cells, strongly suggest that human endometrial stromal cells, when subjected to appropriate hormonal stimulation, are capable of differentiating into decidual cells in culture. Cultured stromal cells therefore offer a unique opportunity to examine the complex changes in gene expression associated with decidualization. In addition, in vitro decidualization may prove to be an effective diagnostic tool in certain cases of infertility. Finally, decidualization of cultured stromal cells represents a relevant end point for testing compounds of potential clinical importance, such as synthetic progestins or antifertility drugs.     

Androgen metabolism and actions in rat ventral prostate epithelial and stromal cell cultures

The rat ventral prostate requires androgens for normal development, growth, and function. To investigate the relationship between androgen metabolism and its effects in the prostate and to examine differences between the epithelial and stromal cells, we have established a system of primary cell cultures of immature rat ventral prostate cells. Cultures of both cell types after reaching confluency (6-7 days) actively metabolized 3H-labelled testosterone (T), 5 alpha-dihydrotestosterone (5 alpha-DHT), 5 alpha-androstane-3 alpha,17 beta-diol, and 5 alpha-androstane-3 beta,17 beta-diol. The epithelial cells actively reduced T to 5 alpha-DHT and formed significant amounts of 5 alpha-androstane-3,17-dione from T, 5 alpha-DHT, and 5 alpha-androstane-3 alpha,17 beta-diol. All substrates were converted to significant amounts of C19O3 metabolites. The stromal cells also metabolized all substrates, but very little 5 alpha-androstane-3,17-dione was formed. The metabolism studies indicate that both cell types have delta 4-5 alpha-reductase, 3 alpha- and 3 beta-hydroxysteroid oxidoreductase and hydroxylase activities. The epithelial cells have significant 17 beta-hydroxysteroid oxidoreductase activity. The epithelial cells cultures grown in the presence of T have higher acid phosphatase (AP) contents (demonstrated histochemically and by biochemical assay). Tartrate inhibition studies indicate that the epithelial cells grown in the presence of T are making secretory AP. Stromal cell AP is not influenced by T. The results indicate that the cultured cells maintain differentiated prostatic functions: ability to metabolize androgens and, in the case of the epithelial cells, synthesize secretory AP.     

Establishment and characterization of a cell line (OMC-9) originating from a human endometrial stromal sarcoma

Cell lines are very useful for clinical and basic research. The establishment of uterine malignant tumor cell lines with unusual histology is especially important. We describe the establishment and characterization of a new human endometrial stromal sarcoma cell line of the uterus. The cell line OMC-9 was established from a tumor mass in the uterine body of a 55-year-old woman. Characteristics of the cell line studied include morphology, chromosome analysis, heterotransplantation, tumor markers and chemosensitivity. This cell line has grown well for 196 months and has been subcultured more than 50 times. Monolayer cultured cells are polygonal in shape, appear to be spindle-shaped or multipolar and have a tendency to pile up without contact inhibition. The cells exhibit a human karyotype with a modal chromosomal number in the diploid range. The cells were able to be transplanted into the subcutis of nude mice and produced tumors resembling the original tumor. OMC-9 cells produced tissue polypeptide antigen. Both CD10, a sensitive and diagnostically useful marker of endometrial stromal neoplasms, and vimentin were identified immunohistochemically in the original tumor and the heterotransplanted tumor. The cells were sensitive to actinomycin D, doxorubicin, carboplatin, cisplatin and etoposide, drugs used commonly in the treatment of gynecologic cancer. Only three reports of uterine endometrial stromal sarcoma cell lines have thus far been reported in the literature. OMC-9 is the first endometrial stromal sarcoma cell line in which CD10 expression and chemosensitivity have been identified.     

Change of insulin-like growth factor gene expression in Chinese hamster ovary cells cultured in serum-free media

Although the sera used in animal cell culture media provide the macromolecules, nutrients, hormones, and growth factors necessary to support cell growth, it could also be an obstacle to the production of recombinant proteins in animal cell culture systems used in many sectors of the biotechnology industry. For this reason, many research groups, including our laboratory, have been trying to develop serum-free media (SFM) or serum-supplemented media (SSM) for special or multi-purpose cell lines. The Chinese hamster ovary (CHO) cell, for example, is frequently used to produce proteins and is especially valuable in the large-scale production of pharmaceutically important proteins, yet information about its genome is lacking. Also, SFMs have only been evaluated by comparing growth patterns for cells grown in SFMs with those grown in SSM or by measuring the titer of the target protein obtained from cells grown in each type of medium. These are not reliable methods of obtaining the type of information needed to determine whether an SFM should be replaced with an SSM. We carried out a cDNA microarray analysis to evaluate MED-3, an SFM developed in our laboratory, as a CHO culture medium. When CHO cells were cultured in MED-3 instead of an SSM, several genes associated with cell growth were down-regulated, although this change diminished over time. We found that the insulin-like growth factor (IGF) gene was representative of the proteins that were down-regulated in cells cultured in MED-3. When several key supplements-including insulin, transferrin, ethanolamine, and selenium-were removed from MED-3, theIGF expression was consistently down-regulated and cell growth decreased proportionately. Based on these results, we concluded that when an SFM is used as a culture medium, it is important to supplement it with substances that can help the cells maintain a high level ofIGF expression. The data presented in this study, therefore, might provide useful information for the design and development of SFM or SSM, as well as for the design of genome-based studies of CHO cells to determine how they can be used optimally for protein production in pharmaceutical and biomedical research.     

Isolation and culture of glandular epithelial and stromal cells from the endometrium of mares.

Glandular epithelial and stromal cells were isolated from the endometrium of mares by collagenase digestion and were incubated on plastic for 7-9 days until the cells formed confluent monolayers. The cells differed in morphology: epithelial cells appeared polyhedral and stromal cells were spindle like. The monolayers were incubated in the presence and absence of oxytocin. Medium was removed from wells after 2, 8 and 24 h of incubation. Concentrations of prostaglandin F (PGF) in the medium increased significantly during this time. Glandular epithelial cells produced significantly more PGF than did stromal cells. Both types of cell responded significantly to oxytocin stimulation by increased secretion of PGF; the response of glandular epithelial cells tended to be greater than that of stromal cells. Secretion of PGF by cultured cells was not affected by cycle stage or pregnancy.     

IL-1-induced procoagulant and fibrinolytic activities of human endothelium grown on carbodiimide crosslinked proteins

We examined the regulation of procoagulant activity and the production of fibrinolytic components by human vascular endothelium grown on coating membranes of gelatin, pure or mixed with albumin, crosslinked by carbodiimide ((G)C, (AG)C) in comparison with plastic culture dishes. Confluent monolayers were stimulated by human recombinant interleukin (IL-1) and responses in terms of tissue factors like procoagulant activity, tissue plasminogen activator (tPA) and plasminogen activator inhibitor (PAI-1) were followed for up to 72 h. Procoagulant activity of cell extracts displayed similar patterns whatever the substratum tested. Quantitative immunological assays revealed a 2-fold increase in tPA antigen released from monolayers grown on (G)C and on (AG)C compared to cells grown on plastic. Exposure of monolayers to IL-1 reduced the secretion of tPA antigen which still reached higher values on coated than on uncoated substratrum. We found that the quasi-totality of tPA formed stable complexes with PAI-1, thereby suppressing measurable fibrinolytic activity. IL-1 stimulated the release of PAI-1 antigen quantified by immunoassay and the kinetics of secretion were comparable on both coated and uncoated substratum.     

The kinetics of granulopoiesis in long-term mouse bone marrow culture. Part I

The spontaneous stratification in long-term bone marrow cultures was illustrated and quantified. The cultures were separated into three hematopoietic layers: nonadherent cells in the supernatant medium, lightly adherent cells on top of the stromal layer, and remaining cells buried within the stromal layer. The cells of each layer were subcultured for 10 days in plastic tubes that inhibit the formation of a stromal layer. Daily samplings with absolute and differential cell counts were obtained. We identified three families of cell disappearance curves and cell types: CFU-s, hemocytoblasts, myeloblasts, and promyelocytes (G1, 2); myelocytes (G3); and postmitotic granulocytes (G4). Also, the numbers of mitotic and necrotic cells were determined. The longest half-time of CFU-s was 2.5 days. Lacking stromal support, CFU-s disappeared faster than other differentiated cells. Generally, these cells maintained their numbers for the first week of subcultures, which was attributable to a temporarily maintained balance of cell death and fresh cell production. After more than 7 days, there was a rapid decline of all differentiated cell types.