Monolayer cultures for immunofluorescent staining from tumor primary explants.

Epithelial cells growing around primary explants of carcinomas in plastic ware are well-suited for modern incident light immunofluorescence microscopy. Epithelial outgrowths in growth culture medium are flushed with phosphate-buffered saline (PBS) and absolute ethanol and snap-frozen in liquid nitrogen-isopentane. The walls of the plastic containers bearing the dried monolayer outgrowths are cut out to form microscopic slides. Immunofluorescence tests are made on circular areas demarcated on the monolayers by using small metal cylinders to mask against a water-repellent plastic spray. More than 20 immunofluorescence tests can be performed on a culture 40 X 50 mm.     

Human ovarian surface epithelium in primary culture

The ovarian surface epithelium (OSE) represents a minute fraction of the cell mass of the ovary but gives rise to over 80% of human ovarian carcinomas. No experimental models for the study of human OSE exist. To characterize OSE cells in culture, explants of ovarian surface from normal ovary of premenopausal women were grown on plastic, glass, and collagen gel in 25% fetal bovine serum/Waymouth's medium 752/1. About 25% of explants produced epithelial outgrowths. Morphologically, these outgrowths resembled OSE in vivo and endothelial and mesothelial cells in culture, but they differed from cultured ovarian stromal, granulosa, and luteal cells. Only OSE among ovarian cell types were intensely keratin positive by immunofluorescence. Keratin also distinguished OSE cells from the keratin-negative endothelial cells. Most but not all OSE colonies tested showed 17 beta-hydroxysteroid dehydrogenase (HSD) activity, which was absent in peritoneal mesothelial cells. Colonies from most patients were limited to a few millimetres and became stationary within a few weeks. Changes that accompanied cessation of growth included senescence, increased keratin content, or the formation of multicellular papillary aggregates. With time, OSE cells tended to assume a fibroblast-like morphology but remained keratin positive and continued to resemble OSE by scanning electron microscopy (SEM). Subcultured OSE cells persisted in a stationary keratin-positive form for many weeks. Throughout this study, all pavementlike epithelial outgrowths that were contiguous with an explant stained for keratin; thus, such colonies can be assumed to be OSE. Conversely, fibroblast-shaped cells may represent OSE as indicated by keratin content and SEM appearance. The methods presented here permit culture of normal human OSE under conditions in which the cells exhibit morphologic plasticity, variable 17 beta-HSD activity, and presence of keratin.     

Human ovarian surface epithelium in primary culture

Summary The ovarian surface epithelium (OSE) represents a minute fraction of the cell mass of the ovary but gives rise to over 80% of human ovarian carcinomas. No experimental models for the study of human OSE exist. To characterize OSE cells in culture, explants of ovarian surface from normal ovary of premenopausal women were grown on plastic, glass, and collagen gel in 25% fetal bovine serum/Waymouth's medium 752/1. About 25% of explants produced epithelial outgrowths. Morphologically, these outgrowths resembled OSE in vivo and endothelial and mesothelial cells in culture, but they differed from cultured ovarian stromal, granulosa, and luteal cells. Only OSE among ovarian cell types were intensely keratin positive by immunofluorescence. Keratin also distinguished OSE cells from the keratin-negative endothelial cells. Most but not all OSE colonies tested showed 17β-hydroxysteroid dehydrogenase (HSD) activity, which was absent in peritoneal mesothelial cells. Colonies from most patients were limited to a few millimetres and became stationary within a few weeks. Changes that accompanied cessation of growth included senescence, increased keratin content, or the formation of multicellular papillary aggregates. With time, OSE cells tended to assume a fibroblast-like morphology but remained keratin positive and continued to resemble OSE by scanning electron microscopy (SEM). Subcultured OSE cells persisted in a stationary keratin-positive form for many weeks. Throughout this study, all pavementlike epithelial outgrowths that were contiguous with an explant stained for keratin; thus, such colonies can be assumed to be OSE. Conversely, fibroblast-shaped cells may represent OSE as indicated by keratin content and SEM appearance. The methods presented here permit culture of normal human OSE under conditions in which the cells exhibit morphologic plasticity, variable 17β-HSD activity, and presence of keratin. Supported by a grant and a research associateship to N. A. by the National Cancer Institute of Canada.     

In vitro isolation and cultivation of rabbit tracheal epithelial cells using tissue explant technique

Epithelial cells from tracheal mucosa offer significant potential as a cell source in development of tissue-engineered trachea. The purpose of this study was to investigate and optimize a suitable culture system for tracheal epithelial cells, including the methods of primary culture, passage, identification, and cryopreservation. Epithelial cells were isolated from rabbit tracheal mucosa using tissue explant technique and were subjected to immunohistochemistry, immunofluorescence, and cryopreservation after purification. Epithelial cells reached confluency at 14–15 d. Immunohistochemical staining for cytokeratin showed brown yellow-positive cytoplasm and blue-counterstained nuclei, while immunofluorescence staining for cytokeratin showed green-positive cytoplasm and clear cell outline, indicating that the cultured cells had properties of epithelial cells. After recovery, epithelial cells exhibited high survival and viability. The results demonstrated that in vitro isolation and cultivation model was successfully established to provide high proliferative capacity, typical morphology and characteristics of tracheal epithelial cells from trachea mucosa by the use of the tissue explant technique.     

Ultrastructural and autoradiographic observations of hamster cheek pouch epithelium grown in vitro.

Epithelial outgrowths from hamster cheek pouch explants were cultured for varying periods of time up to 22 days. Growth of the epithelial sheets was monitored, employing colcemid for demonstrating mitotic activity and tritiated thymidine for DNA synthesis. Mitoses and thymidine uptake were observed among epithelial outgrowths at a considerable distance form the original explant. The epithelial nature of the growing cell sheets was confirmed, employing electron microscopic techniques. The cells exhibited the presence of tonofilaments, desmosomes, ribosomes, Golgi, mitochondria, and rough endoplasmic reticulum. The cultured explants were treated with cyclic nucleotides in order to investigate their modulatory effects on epithelial cell differentiation. Dibutyryl cAMP induced marked mitotic inhibition (46.3%) in our assay, which was increased to 57% with the addition of theophylline. Dibutyryl cGMP showed only a mild (5%) stimulatory effect on mitotic activity. Dibutyryl cAMP enhanced keratinization in the epithelial cell outgrowths with the biogenesis of keratohyalin granules, whereas dibutyryl cGMP did not produce any observable alterations.     

Phenotypic responses of mouse mammary tumours and normal mammary epithelium cultured in collagen gels: Correlation with tumour type and progression

Mammary tumours in female BR6/Icrf mice and the corresponding contralateral normal mammary glands were disaggregated with collagenase and the epithelial structures released ('organoids') separated from other cellular components by filtration. The organoids were established in primary culture in a collagen matrix and the outgrowths obtained were studied by light microscopy and time-lapse cinemicroscopy. The pattern of three-dimensional outgrowths produced was found to be specific to the original tissue. Organoids from normal tissue formed a characteristic outgrowth designated Pattern A. Normal tissue from pregnant mice formed an additional characteristic outgrowth (Pattern A') which has not been described previously. Pregnancy-dependent tumours produced a distinctive phenotypic outgrowth designated Pattern D, whereas pregnancy-independent tumours gave a different distinctive Pattern B as well as a unique specific outgrowth designated Pattern C. Outgrowths of Pattern D from a pregnancy-dependent tumour were removed from culture and implanted into a syngeneic female mouse. Tumours arising in the host were found to be pregnancy-independent and showed phenotypic outgrowths in subsequent culture of pregnancy-independent Patterns B and C. The results show that the type of outgrowths in these cultures correlates with the biology of the tissue in vivo and that changes in tumour progression in vivo are accompanied by alterations in phenotypic outgrowths in culture.     

Antigenic and ultrastructural markers associated with urothelial cytodifferentiation in primary explant outgrowths of mouse bladder.

The purpose of this study was to establish an in vitro culture model that closely resembles whole mouse urothelial tissue. Primary explant cultures of mouse bladder were established on porous membrane supports and explant outgrowths were analysed for morphology and the presence of antigenic and ultrastructural markers associated with urothelial cytodifferentiation. When examined at the ultrastructural level, the cultured urothelium was polarized and organized as a multilayered epithelium. Differentiation was found to increase from the porous membrane towards the surface and from the explant towards the periphery of the culture. Scanning and transmission electron microscopical analysis of the most superficially-located cells revealed four successive differentiation stages: cells with microvilli, cells with ropy microridges, cells with rounded microridges, and highly-differentiated cells with asymmetric unit membrane (AUM) plaques forming rigid microridges and fusiform vesicles. The more highly-differentiated cells were numerous at the periphery of the culture, but rare close to the explant. Epithelial organization was stabilized by well developed cell junctions. Immunolabeling demonstrated that superficial urothelial cells in culture: (1) develop tight junctions, E-cadherin adherens junctions and abundant desmosomes and (2) express uroplakins and cytokeratin 20 (CK 20). Using a culture model of primary explant outgrowth we have shown that non-differentiated mouse urothelial cells growing on a porous membrane show a high level of de novo differentiation.     

Characterization in primary monolayer culture of separated cell types from rabbit endometrium

Epithelial cells and stromal cells of the rabbit endometrium were separated by successive enzymic digestion of the uterine mucosa. Isolated cell types were obtained in high yield, with good viability, and were maintained in monolayer cultures for up to 2 weeks. Epithelial cells in monolayers appeared as polygonal cells, displayed contact inhibition, and showed the presence of microvilli on the cell surface, with many desmosomes. Stromal cells grew rapidly to confluence, displayed overgrowth, and had a fibroblastic appearance with an absence of junctional complexes between cells. Indirect immunofluorescence showed uteroglobin on the surface of epithelial but not of stromal cells, and only epithelial cells secreted uteroglobin into the medium. These results confirm the identity of the cells and provide biochemical evidence for the epithelial cellular origin of uteroglobin. The method allows the culture of separate endometrial cell types, which retain their morphology and differentiated function in vitro.     

Morphology and microfilament organization in human blood lymphocytes : Effects of substratum and mitogen exposure

During culture in serum-containing medium normal human blood lymphocytes, depleted of phagocytic and adherent cells, do not attach to adhesive surfaces. Concanavalin A (ConA) or phytohemagglutinin (PHA) in appropriate concentrations mediate adhesion of these lymphocytes to tissue culture plastic or glass. This process consists of two phases.

1. 1. The mitogen-mediated contact with a surface induces an almost instantaneous alteration of cell shape and a simultaneous redistribution of actin in the majority of the cells.

2. 2. The initial morphological changes are accompanied by an accumulation of actin-containing material in prominent peripheral cytoplasmic outgrowths formed by the spread cells. The contact-induced spreading and rearrangement of actin are inhibited by cytochalasin B (CB) but not by colchicine or vinblastine. The distribution of detectable actin in spread lymphocytes is similar to the distribution of footprints of actin after detachment of spread cells suggesting that actin is involved in the attachment of lymphocytes to substratum. In contrast to lymphocytes on glass or tissue culture plastic which show morphological changes and redistribution of actin cells cultured with ConA on non-adhesive surfaces of bacterial plastic or poly-2-hydroxy-methacrylate do not exhibit any morphological alterations and no rearrangement of actin.

The present approach enables visualization of cytoskeletal structures in lymphocytes to an extent which is not possible using conventional methods with the cells in suspension. The results indicate that contact is a regulator of lymphocyte shape and that actin-containing structures mediate and maintain contact-induced changes of lymphocyte morphology.     

Ultrastructural and autoradiographic observations of hamster cheek pouch epithelium grown in vitro

Summary Epithelial outgrowths from hamster cheek pouch explants were cultured for varying peroids of time up to 22 days. Growth of the epithelial sheets was monitored, employing colcemid for demonstrating mitotic activity and tritiated thymidine for DNA synthesis. Mitoses and thymidine uptake were observed among epithelial outgrowths at a considerable distance from the original explant. The epithelial nature of the growing cell sheets was confirmed, employing electron microscopic techniques. The cells exhibited the presence of tonofilaments, desmosomes, ribosomes, Golgi, mitochondria, and rough endoplasmic reticulum. The cultured explants were treated with cyclic nucleotides in order to investigate their modulatory effects on epithelial cell differentiation. Dibutyryl cAMP induced marked mitotic inhibition (46.3%) in our assay, which was increased to 57% with the addition of theophylline. Dibutyryl cGMP showed only a mild (5%) stimulatory effect on mitotic activity. Dibutyryl cAMP enhanced keratinization in the epithelial cell out-growths with the biogenesis of keratohyalin granules, whereas dibutyryl cGMP did not produce any observable alterations.     

Desmin-positive epithelial cells outgrowing from rat encapsulated glomeruli

Decapsulated glomeruli, encapsulated glomeruli and tubular fragments were each selected and cultured in order to identify the origin of polygonal epithelial cells in glomerular outgrowths and to characterize them by double-label immunofluorescence microscopy using antibodies against cytoskeletal proteins. Polygonal cells outgrew from less than 1% of decapsulated glomeruli, 34.8 to 65.1% of encapsulated glomeruli and 62.4 to 79.7% of tubular fragments in culture. These data support the idea that polygonal cells in glomerular culture are derived mainly from parietal epithelial cells of Bowman's capsule but not visceral cells, and indicate that polygonal cells of tubular epithelial origin are also present. Polygonal cells from encapsulated glomeruli consisted of intensely vimentin-positive (IV) cells and weakly vimentin-positive (WV) ones. Most of the IV cells showed various degrees of staining with anti-desmin antibody, and some of them also expressed cytokeratins and alpha-smooth muscle actin. By contrast, all of the WV cells were stained with anti-cytokeratin antibody but not with anti-desmin antibody. Polygonal cells in cultures of tubular fragments were negative for desmin. These findings suggest that parietal cells express desmin in culture, even though they show no desmin staining in kidney sections.     

Substrate properties influencing ultrastructural differentiation of mammary epithelial cells in culture

Epithelial cells dissociated from mammary glands of midpregnant mice and cultured with lactogenic hormones on plastic or collagen gel substrates have been shown to vary in their extent of differentiation, as identified by the presence of secretory organelles and accumulation and secretion of casein. Morphological and biochemical differentiation was obtained on floating collagen gels. At least four unique factors provided by the floating collagen gel substrates are not found on plastic substrates: access of nutrients to basolateral cell surfaces, close proximity of cells to the medium surface and gas phase, interaction of epithelial cells with stromal elements, and substrate flexibility permitting cell shape change. In this study, we have attempted to assess the relative contributions of these factors in the ultrastructural differentiation of mammary cells in culture. None of these factors alone is responsible for the differentiation achieved when all are present. The novel aspect of this research is the identification of the cells' apparent requirements for basolateral access to nutrients and for freedom to assume a preferred shape in order to achieve differentiation.     

Protein secretion by the mouse trophoblast during attachment and outgrowth in vitro

Protein secretion from mouse blastocysts undergoing attachment and trophoblast outgrowth in vitro was assessed. When Day 5 blastocysts were cultured in serum-containing medium, secretion of several 'attachment-associated' proteins (PAS) was initiated within 24 h, coincident with attachment and outgrowth. Those proteins characteristic of the pre-attachment blastocyst disappeared or made-up only a small portion of the secretions once attachment began. The major secreted protein from attached embryos, PA1, is a 35,000-45,000 Mr acidic glycoprotein with multiple isoelectric forms. PA2, a group of basic 40,000 Mr proteins and PA3 a group of 72,000 Mr proteins were also produced during outgrowth. PAS were secreted during outgrowth on fibronectin-coated plastic in serum-free medium, but not by blastocysts held in a non-attachment state during culture in serum-free medium on uncoated plastic. In pre-attachment blastocysts, secreted proteins were produced by trophoblast vesicles, but not by isolated inner cell masses. Both trophoblast vesicles growing out in vitro and surgically isolated trophoblast from spreading blastocysts had secreted protein patterns qualitatively similar to those of intact blastocyst outgrowths. The results indicate that development of trophoblast protein secretion continues through the period of outgrowth and giant cell transformation. These changes are apparently dependent on attachment of the blastocyst to a suitable substrate, but not dependent on any other serum influence.     

Alternative pathways of tobacco placental development: time of commitment and analysis of a mutant

Previous work from our lab identified mutants, Mgr3 and Mgr9, of tobacco (Nicotiana tabacum) that produced unusual elongated green outgrowths from placentae in vivo. Similarly appearing stigmatoid growths were described developing from some in vitro cultures of excised placentae of tobacco (Hicks and McHughen, 1974, 1977). Here we report a developmental analysis and comparison of the unusual stigmatoid outgrowths seen in in vitro cultures of wild-type and mutant placentae, as well as the green outgrowths seen in vivo in the mutants. The growths produced by wild-type and mutant placental cultures in vitro, and the growths produced by the mutants in vivo, are identified as abnormal stigmas and styles. Wild-type in vitro placental cultures also produce outgrowths identified as homologs of whole carpels. Carpel fusion is not required for differentiation of stigma, style, transmitting tract, vascular traces, ovary, and ovules in these structures. The type and extent of stigmatoid growth production depends upon the age of the explant at excision and culture initiation. Before ovule primordium initiation, few growths are seen in culture; for a short window of time afterward, the primordia are competent to give rise to stigmatoid and carpelloid growths when cultured. After commitment to ovule development occurs, the primordia produce only ovules when cultured. The behavior of the mutant placental cultures is dimorphic. Explants from early stages behave similarly to wild-type when cultured, but differences between wild-type and the mutant behaviors in culture arise at the time when the stigmatoid growths begin to appear in vivo in the mutants. These results imply that ovule primordia pass through stages of distinct sequential restrictions of fate, first to growth as gynoecia, and then second to growth as ovules. The mutant strains described here perturb the commitment to differentiation as ovules.     

Explant-cell culture of primary mammary tumors from MMTV-c-Myc transgenic mice

We have established an explant-cell culture system for mammary gland tumors from c-myc oncogene-expressing transgenic mice and potentially other transgenic strains. By coating culture dish surfaces with fetal bovine serum and using culture media supplemented with low serum and growth factors, the mammary tumor specimens could be maintained in culture for over 3 mo. Throughout the culture period, the explants produced abundant outgrowths of epithelial cells. As the outgrowths of epithelial cells filled the dishes, the explants were serially transferred from one dish to another-a process that could be repeated at least six times, thus providing a continuous supply of primary tumor cells. This culture system provides a useful tool for studying the biology of mouse mammary gland tumors and possibly tumors from other organ sites.     

Prolonged expression of differentiated phenotype by chondrocytes cultured at low density on a composite substrate of collagen and agarose that restricts cell spreading

The dedifferentiation of chondrocytes in culture is frequently associated with transition from a rounded to a spread morphology. A number of culture methods which prevent cell spreading have been described; however, all have disadvantages that limit their widespread use. In this paper we describe a new technique which allows prolonged cultivation of attached chondrocytes at low density while inhibiting spreading: the cells are grown on a composite substrate of agarose and collagen. By varying the ratio of agarose to collagen in the gel, the degree of spreading can be varied. The cultures are suitable for ultrastructural and immunofluorescence analysis and for studies of the synthesis and secretion of macromolecules. In order to determine whether the differentiated phenotype was maintained on composite gels, we compared the levels of messenger RNAs for cartilage-specific proteoglycan, link protein, alpha 1 (II) and alpha 1 (I) collagens in chondrocytes grown at low density on composite gels or at high or low density on tissue culture plastic for up to 21 days. The rate of decline in the level of mRNAs encoding the cartilage-specific products and the rate of increase in the level of alpha 1 (I) collagen mRNA were slower in the composite cultures than in the cultures on plastic. This culture technique may, therefore, prolong expression of the differentiated phenotype of chondrocytes relative to cultivation on plastic and will be useful for further studies on the role of cell shape in regulating differentiated gene expression.     

Separation and culture of ovine endometrial epithelial and stromal cells: evidence of morphological and functional polarity.

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.     

The distribution of colony-forming cells in the kidney

Abstract. A method is described for producing outgrowths of small nephron segments (average 24 cells) in culture. The method was used to estimate an overall colony-forming efficiency of 4.6% for cells constituting the segments. Efficiency was found to be lower for thick segments (1%) than for thin segments (6%) from Henle's loop. The latter higher level indicates that precursor cells are concentrated near the middle of the nephron. For comparison, a two-dose irradiation technique was used to calculate a mean number of 5 ± 2 (SE) clonogens per segment producing outgrowths. This tended to be higher than the value of about 1 calculated from the 65% of segments producing outgrowths, as expected if the remaining segments contained no clonogens.     

Culture of human main pancreatic duct epithelial cells

Summary Attempts to grow human pancreatic duct epithelial cells in long-term culture have proven difficult. We have developed a system of growing these cells for several passages by adapting methods used to culture dog pancreatic duct cells. Epithelial cells were enzymatically dissociated from the main pancreatic duct and plated onto collagen-coated culture inserts suspended above a human fibroblast feeder layer. After primary culture, the cells were either passaged onto new inserts or plastic tissue culture plates in the absence of collagen. Cells grown on the latter plates were maintained in a serum-free medium. Primary pancreatic duct epithelial cells grow steadily to confluence as a monolayer in the feeder layer system. After primary culture, cells passaged onto new inserts with fresh feeder layer or plastic plates and fed with serum-free medium continued to develop into confluent monolayers for up to four passages. The cells were columnar with prominent apical microvilli, sub-apical secretory vesicles, and lateral intercellular junctions resembling the morphology of normal in vivo epithelial cells. These cells were also positive for cytokeratin 19, 7, and 8 and carbonic anhydrase II, as measured by immunohistochemistry. Metabolically, these cells synthesized and secreted mucin, as measured by incorporation of tritiated N-acetyl-d-glucosamine. In conclusion, we demonstrated that human pancreatic epithelial cells from the main duct can be successfully grown in culture and repeatedly passaged using a feeder layer system, with serum-free medium, and in organotypic cultures.