Culture of cells in beem capsules: a new technique for electron microscopic study of monolayer cultures.

A method is described for the monolayer cultivation of primary cell suspensions and established cell lines directly in carbon-coated BEEM capsules, BEEM capsules are routinely employed by electron microscopists in tissue embedding procedures; growing monolayer cultures directly on the lids of inverted BEEM capsules presents the obvious advantage of maintaining cell to cell and cell to substratum conthaets with a minimum of stress and damage in the preparative steps for electron microscopy.     

Monolayer culture of human endometrium: Methods of culture and identification of cell types

Summary Monolayer cultures can be established from human endometrial tissue after enzymatic dispersal into isolated glands or single cells. Three cell types that have distinct morphology by light and electron microscopy are observed in the resulting primary cultures. One cell type, an elongated spindle cell, is similar in appearance to fibroblasts derived from other tissues. A second cell type forms colonies of tightly cohesive cells, ranging in shape from oval to polygonal. These cells have typical organelles and junctional complexes characteristic of epithelial cells from the endometrium. The third cell type assumes a pavement-like appearance composed of polygonal cells when viewed by phase contrast microscopy, but lacks distinctive ultrastructural features of epithelial cells. These cells in culture resemble the endometrial stromal cell, the predominant cell type of the human endometrium in vivo. The epithelial cell does not survive subculturing but the other two cell types can be passaged through several generations and can be stored in liquid nitrogen and subsequently returned to culture. This work was supported by contract N01-CP75956 and grant R01-CA31733 from the National Cancer Institute. V. A. Varma is a recipient of an American Cancer Society fellowship; B. H. Dorman, a predoctoral fellowship from the Chemical Industry Institute of Toxicology; J. M. Siegfried, a training grant (CA09156) from the National Cancer Institute; and D. G. Kaufman, a Research Career Development Award (K04-CA-00431) from the National Cancer Institute.     

An Improved Method of Making Tissue Carriers for TEM and SEM Fixation

Hazards in fixing small pieces of tissue for electron microscopy include damage, drying, or loss. Over the years, microstrainer tissue carriers have been developed to minimize these problems. Construction materials have included glass tubing, copper grids for electron microscopy, stainless steel screen, and bolting silk (Padawer 1951, Friend 1963, Bronskill 1970). Carriers made from plastic embedding molds (e.g., BEEM capsules) with either TEM grids attached to the conical tip (Buchanan 1965) or Nitex screen cloth held to one end by a retaining ring have proven to be inexpensive and popular, though the former has a very small filtration area and in the latter small tissues may be lost or crushed between the screen cloth and the bottom rim of the carrier. This note describes a carrier in which Nitex is permanently sealed to the bottom edee of a BEEM capsule cylinder.     

Increased yield of mouse mammary tumor virus (MMTV) by cultivation of monolayer-derived mammary tumor cells in suspension

Summary The MJY-alpha epithelial-like mammary tumor cell line was adapted for cultivation in suspension using a shaker culture technique. Replication of suspension (MJY-beta) cells was more sensitive than monolayer cells to decreases in the concentration of serum in the medium. Comparison of amino acid incoerporation and lactate production rates revealed additional differences between monolayer and suspension cultures. In addition, growth in susfpension resulted in 10- to 400-fold increases in mouse mammary tumor virus (MMTV) production by the mammary tumor cells. Incrases in MMTV yield were detected within 48 h of culture initiation and MMTV production remained elevated throughout 20 cell passages in suspension. Exposure of MJY-beta cells to 14 μM hydrocorticone further increased MMTV yield two-to five-fold. The MJY-beta suspension cultures demonstrated that these epithelial-like cells do not require attachment to a solid substrate for replication or for MMTV production. Loss of structural polarization associated with growth as a monolayer resulted in stimulation of MMTV production greater than and independent of steroid exposure. This work was supported by the T. J. Martell Foundation for Cancer and Leukemia Research and by USPHS grant 5P-30CA23102. F. M. is a trainee on MSTP grant GM07280 from the National Institute of Health. This work was submitted in partial fullfillment of the requirements for the Ph. D. degree (F. M.).     

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Hazards in fixing small pieces of tissue for electron microscopy include damage, drying, or loss. Over the years, microstrainer tissue carriers have been developed to minimize these problems. Construction materials have included glass tubing, copper grids for electron microscopy, stainless steel screen, and bolting silk (Padawer 1951, Friend 1963, Bronskill 1970). Carriers made from plastic embedding molds (e.g., BEEM capsules) with either TEM grids attached to the conical tip (Buchanan 1965) or Nitex screen cloth held to one end by a retaining ring have proven to be inexpensive and popular, though the former has a very small filtration area and in the latter small tissues may be lost or crushed between the screen cloth and the bottom rim of the carrier. This note describes a carrier in which Nitex is permanently sealed to the bottom edee of a BEEM capsule cylinder.     

Immunofluorescent localization of secretin in pancreatic monolayer culture

Summary Immunofluorescent cells to synthetic secretin were identified in monolayer culture of neonatal rat pancreas. No cross reaction of anti-secretin was observed with either glucagon, somatostatin or gastrin. The presence of cells containing secretin or a secretin-like peptide adds a new cell type to the three already characterized (insulin, glucagon and somatostatin containing cells) in monolayer culture.This work was supported by a grant (no. 3.553.75) from the Fonds National Suisse de la Recherche Scientifique, and a grant for cancer research from the Ministry of Public Welfare of Japan     

Comparison of coulter volumes with radiometrically determined intracellular water volumes for cultured cells

Summary During methotrexate-induced differentiation of cultured human choriocarcinoma (BeWo) cells, proliferation is inhibited, morphologic and biochemical changes occur, and giant, often multinucleated, cells form. We have used the increase in cell volume as a marker of the mature syncytiotrophoblastlike phenotype. Uninduced and differentiated BeWo cells are not spherical, and theoretical considerations suggested that deviations in shape could result in significant errors in Coulter volume. To determine if the values obtained by electrical pulse sizing reflected the actual mass of BeWo cells, we have evaluated the relationship between Coulter volumes and intracellular water volumes obtained using a shape-independent estimate for eight cell types. A close correlation (r ²=0.97) was found, indicating that cell volume changes in populations of irregularly shaped cells can be accurately measured using a Coulter instrument. Supported by an operating grant from the National Cancer Institute of Canada. N.S.B. was a recipient of a studentship award from the Alberta Heritage Foundation for Medical Research. C.E.C. is a Senior Research Scientist of the National Cancer Institute of Canada. The McEachern Laboratory is a research facility of the Faculty of Medicine, University of Alberta, and the Cross Cancer Institute, Edmonton, Alberta.     

Development,characterization, and viral susceptibility of a feline (Felis catus) renal cell line (CRFK)

Summary Cell line CRFK, derived from kidney tissue of a normal domestic kitten, was initiated in 1964. With intermittent periods of storage in the frozen state, it has been grown in vitro during more than 200 passages, without apparent loss of susceptibility to selected viruses. Various herpesviruses and feline viruses belonging to differnet virus groups grow readily and with distinct, cytopathic features. The cells now grow as a smooth monolayer of epithelial-like cells; most have 37 chromosomes (2n−1) and are thus aneuploid for cat karyotype. Three distinct marker chromosomes are identified. The cell line, which is free of mycoplasmal contaimination, is useful in feline virus research and diagnostic medicine and has become of particular interest in cancer research. Supported in part by Contract E73-2001-NO1-CP-3-3237 within the Special Virus Cancer Program, National Cancer Institute, National Institutes of Health, Public Health Service and the Morris Animal Foundation and General Research Support funds of the New York State Veterinary College. CRFK cells from stock provided by C. G. Fabricant are available for distribution to investigators from the Cell Culture Laboratory, Naval Biomedical Research Laboratory.     

Serum-free culture conditions for the growth of normal rat mammary epithelial cells in primary culture

Summary A monolayer culture system has recently been developed for the extended growth and serial passage of normal rat mammary epithelial (RME) cells. In this system the cells undergo greater than 20 population doublings when grown on type I collagen-coated tissue culture dishes in Ham's F12 medium supplemented with insulin, hydrocortisone, epidermal growth factor, prolactin, progesterone, cholera toxin, and 5% fetal bovine serum (FBS). The purpose of the present studies was to define additional growth factors that would allow equivalent RME cell proliferation in serum-free medium. Ethanolamine (EA) was effective at reducing the FBS requirements for RME cell proliferation and at its optimum concentration did so by greater than 20-fold. Even with optimum levels of EA there was essentially no cell proliferation in the absence of FBS. However, addition of bovine serum albumin (BSA) to the hormone, growth factor, and EA-supplemented medium resulted in substantial proliferation in the absence of serum, and the further addition of transferrin (T) potentiated this effect. Thus, in this culture system, replacement of FBS with EA, BSA, and T resulted in RME cell proliferation in primary culture which was equivalent to that obtained in the 5% FBS-containing medium. This work was supported by grant RR-05529 from the Division of Research Resources, National Institutes of Health, Bethesda, MD, and by Public Health Service grant CA40064-01 from the National Cancer Institute, Bethesda, MD.     

Social interactions of monkeys reared in a nuclear family environment versus monkeys reared with mothers and peers

Four-year-old laboratory-born rhesus monkeys that had been reared in a nuclear family social environment consisting of mothers, fathers, siblings, peers, and other adults of both sexes were permitted to interact in various combinations with equal-aged monkeys that had been reared in an environment consisting of only mothers and peers. It was found that in most interaction sessions nuclear family subjects exhibited significantly higher levels of dominance and activity behaviors and significantly lower levels of submissive and passive behaviors than the mother-peer-reared subjects. These differences were not evident when subjects were tested within their own rearing groups. The significance of the results with respect to previous and future studies of social development in differential social environments is discussed.This research was supported by grant No. MH-11894 from the National Institute of Mental Health to the University of Wisconsin Primate Laboratory, by the Grant Foundation, and by grant No. RR-0167 from the National Institutes of Health to the University of Wisconsin Primate Research Center.     

Blood-brain barrier alterations in bacterial meningitis: Development of an in vitro model and observations on the effects of lipopolysaccharide

Summary To further examine the effects of purifiedHaemophilus influenzae type b lipopolysaccharide (LPS) on blood-brain barrier permeability, we have developed an in vitro model of the BBB. Microvascular endothelial cells were isolated from rat cerebral cortices by enzymatic digestion, dextran centrifugation, and separation on percoll gradients. The cells were determined to be endothelial in origin by positive fluorescent staining for Factor VIII-related antigen and the ability to take up acetylated low density lipoproteins, and their cerebral origin by the formation of junctional complexes in vitro. Cells were seeded onto semipermeable polycarbonate filters and permeability assessed by measuring traversal of radioactive albumin across the monolayer. Treatment of the cells with LPS at concentrations of 1.0μg/ml and 0.1μg/ml for 4 h led to statistically significant increases in albumin permeability of 4.6% (P=0.001) and 5.6% (P<0.001), respectively, without evidence of cell death as assessed by release of lactate dehydrogenase into the media. These results indicate that LPS significantly increases albumin permeability across a monolayer of cerebral microvascular endothelial cells in the absence of host inflammatory cells. Future studies on the effects of LPS on intracellular regulation will determine the mechanisms responsible for these alterations. Supported by a research grant (RO1-AI17904) and a training grant (T32-AI07046) from the National Institute of Allergy and Infectious Diseases, Bethesda, MD. W. Michael Scheld is an established investigator of the American Heart Association.     

A PLastic MIcrostrainer for HAndling MAmmalian BLastocysts

Friend (1963) has described a microstrainer for handling microscopic marine ova which permits direct transfer of batches of ova through various histological solutions without centrifugation or decanting. This strainer consists of a length of glass tubing with an electron microscope grid cemented over one end. In our laboratory, several modifications of this device have been made for use in handling mammalian ova and blastocysts. The most satisfactory model, which can be made easily, was contructed from BEEM plastic capsules (Better Equipment for Electron Microscopy, Inc., P.O. Box 132 Jerome Avenue Station, Bronx, New York 10468) and circular electron microscope grids of suitable mesh size.     

A Device for Flat-Face, Epoxy Resin Embedding of Tissues and Coverslip Cultures

An easily constructed device permits the flat-face embedding of four specimens in epoxy resins. Either pieces of tissue or cells grown on cover slips can be used. After polymerization, the flat-surfaced capsules may be examined under high magnification for selection of areas to be sectioned. Specimens difficult to orient can be cut out and re-embedded in the proper position. The use of thick-walled BEEM capsules and the clamping action afforded by four screws prevent leakage of the resin.     

Proteolipids. VI. the dual role of phospholipid in cytochrome oxidase reaction

After being deprived of solubilizing agent, the lipid-free cytochrome oxidase requires Triton X100 and additional phospholipid to obtain maximal activity. High levels of Triton X100 affect the interaction of phospholipid and cytochrome oxidase, thus decreasing the activity. In the terminal segment of the electron transport system, phospholipid serves not only to enhance the interaction between cytochromec and cytochromea, but also to maintain favorable molecular arrangements of reacting groups in both hemoproteins. The relationship between the enzyme activity and phospholipid content as well as the ultrastructure of the enzyme is discussed.Supported under a research grant from the National Institute for Arthritis and Metabolic Diseases AM04663.F. L. Crane is supported by career Grant K6-21, 839 from the National Institute for General Medical Research.     

Morphology and growth potential of stromal cell cultures derived from human endometrium

Summary Propagable cell cultures derived from human endometrial tissue were determined to contain cells predominantly of stromal cell origin based on their morphologic resemblance to endometrial stromal cells. These features included nexi, solitary cilia, and predecidual cytology. In addition to morphology the cell cultures retained a normal karyotype and responded to steroid hormones as evidenced by cellular aggregation. The stromal cells were evaluated for a variety of characteristics associated with transformed cells and seemed to be biologically normal without neoplastic phenotypes. Growth potential of the stromal cell cultures was also characterized in normal maintenance medium, in nutritionally depleted medium with reduced levels of calcium or serum, and in medium with increased levels of serum. The prolonged survival of the stromal cells in vitro coupled with the retention of in vivo characteristics and an absence of neoplastic phenotype provides a human cell system that is amenable to a variety of long-term experimental analyses. This work was supported by contract CP75956 and grant CA31733 from the National Cancer Institute. B. Hugh Dorman was the recipient of a predoctoral scholarship from the Chemical Industry Institute of Toxicology. Jill M. Siegfried was supported by National Research Service Award CA09156. David G. Kaufman is the recipient of a Research Career Development Award (CA00431) from the National Cancer Institute.     

A cloned rat thymic epithelial cell line established from serum-free selective culture

Summary A serum-free system has been developed for selective growth and long-term culture of rat thymic epithelial cells. The growth media is a modification of McKeehan's WAJC 404, plus insulin, cholera toxin, dexamethasone, and epidermal growth factor. Cultures have been continuously passaged and maintained for over 6 mo., and a cloned cell line, TEA3A1, has been established. These cells are epithelial, judging by morphology and ultrastructure, and are positive for A2B5 and thymosin α markers for thymic endocrine cells. This work was partly supported by grant PCM-834 0582 from the National Science Foundation, Washington, DC, and grant P01 CA 37589-2 from the National Cancer Institute, Bethesda, MD.     

Inhibition of collagen synthesis by α, α′-dipyridyl in human skin fibroblasts in culture

Summary In human diploid skin fibroblasts in culture we have shown that nonhydroxylated collagen precursors remain in the cell when proline hydroxylation is inhibited by α, α′-dipyridyl, a chelator of ferrous ions. The inhibition of proline hydroxylation is reversed by addition of fresh medium containing 50 μg per ml of sodium ascorbate, whereupon nonhydroxylated collagen precursors are hydroxylated within the cell and extruded into the medium. Extrusion of collagen already formed within the cell is not appreciably affected by α, α′-dipyridyl inhibition. Under normal conditions collagen is released from the monolayer into the medium within 3 hr of a pulse ofL[¹⁴C]proline. In the presence of α, α′-dipyridyl, about 35% of theL[¹⁴C]proline incorporated into protein is released into the medium within 8 hr as a proline-rich, hydroxyproline-deficient protein; at the same time, approximately 15% of the protein-boundl-[¹⁴C]proline remains in the cell for as long as 12 hr. When proline hydroxylation is restored after 2 and 12 hr of α, α′-dipyridyl inhibition, approximately the same amount of hydroxyproline is formed after each time interval in the monolayer. Therefore, nonhydroxylated collagen precursors retained in the cell are not appreciably degraded during at least 12 hr of inhibition by α, α′-dipyridyl and are extruded into the medium only upon restoration of hydroxylation. This work was supported in part by a grant from the Easter Seal Research Foundation, and by Project 236, Health Services and Mental Health Administration, Department of Health, Education and Welfare, Grant HD-03110 from the National Institute of Child Health and Human Development, an American Cancer Society Institutional Grant (1N 15-J), a General Research Support Award (5-S01-FR-05406) from the National Institutes of Health, a University Research Council Grant, a National Science Foundation Equipment Grant (GB-4577), and a Research Career Development Award (5-K3-AM-5058) from the National Institute of Arthritis and Metabolic Disease (G.K.S.).     

Light and electron microscopy of the leucocytes of Crassostrea virginica (Mollusca: Pelecypoda)

Summary The fine structure of oyster leucocytes resembles to a great extent, that of typical eucaryotic cells. Organelles which have been described for the first time in this report are light granules, dense granules, protocentriole and X structure. Light microscopy reveals two morphological types of oyster leucocytes: agranular and granular. Based upon nuclear morphology and cytoplasmic compositions revealed in electron microscopy, at least three types of agranular and one type of granular cells are recognized.In the Giemsa-stained preparations, granular leucocytes exhibit three distinct types of cytoplasmic granules: refractile, dark blue, and pink, which presumably correspond to light granules Type A, B, and C seen in the electron micrographs. A granular leucocyte may contain one or more types of granules. Cytochemical investigations show that oyster leucocytes contain at least three hydrolytic enzymes: non-specific esterases, acid, and alkaline phosphatase. The latter two enzymes constitute 63% of the enzyme activity detected. These intracellular enzymes may be associated with the light granules and/or lysosome-like bodies.It is also demonstrated that the granular leucocyte population is significantly higher (P<0.001) in the oysters experimentally infected with Bacillus mycoides (72.19±4.71%) as contrasted with that of the controls (37.18±4.48%).Leucocytes in progressive stages of degeneration are also described.Contribution No. 71 from Marine Research Laboratory, University of Connecticut.The initial phase of this investigation was carried out at the Department of Zoology, Rutgers, The State University, New Brunswick, New Jersey, and supported by Public Health Service Research Grant AI-00781 from the National Institute of Allergy and Infectious Diseases of the National Institute of Health, awarded to Dr. L. A. Stauber. Supported by a grant from the University of Connecticut Research Foundation and Faculty Summer Fellowship to S. Y. Feng.     

A time lapse cinemicrographic study of giant cells in populations of simian virus 40 transformed cells

This work was supported in part by grant CA 41608 from the National Cancer Institute.