Rat hepatocyte primary cell cultures

Summary The conditions for obtaining representative, adult rat hepatocyte primary cell cultures were improved such that viable yields of 50% of the liver were produced which gave rise to cultures representing 30% of the liver. The survival of the cultures in various media was compared revealing that in complex media, particularly containing galactose, survival was improved. This study was supported by Contract No. N01-CP-55705 from the National Cancer Institute and Research Grant No. BC-133B from the American Cancer Society.     

Fine structural identification of organoid mouse lung cells cultured on a pigskin substrate

Summary Mouse full-term embryonic lung tissue was cultured as organ bits using dead, sterile pigskin dermal collagen as a substrate. Explanted organ bits grew on the surface of, and into, the pigskin dermal collagen for at least 9 weeks after the initiation of culture. The out-growth consisted of a thick cellular sheet containing various sizes of ductular structures within a cellular matrix that did not show any particular structure. Electron microscopic observation revealed that the larger ductular structures consisted largely of ciliated cells. The smaller ductular structure consisted largely of Type II pneumocytes containing lamellar hodies. The cellular matrix consisted of Type II pneumonocytes and other cell types including fibroblasts and macrophages in the early stage of cultivation. Macrophages invaded the pigskin dermal collagen. An intermediate cell type, which has never been observed in vivo, possessing both cilia and lamellar bodies was identified in the larger ductular structures. Upon comparison of the ultrastructure of the organoid in vitro cultures in pigskin with the components and structure of the cultured cells more closely resembled adult lung than the fetal lung used to initiate the cultures. This work was supported by the Council for Tobacco Research Grant 1203M, American Cancer Society Grant RD-65 (for the equipment), and the National Cancer Institute Grant CA 25392.     

Simultaneous determination of ascorbic acid and dehydroascorbic acid in cultures of C3H/10T1/2 cells

Summary A reproducible method is described for the separation and quantification of ascorbic acid and dehydroascorbic acid by ion-pairing reverse-phase high performance liquid chromatography and detection by absorbance at 232 nm. Lowest detectable concentrations with a linear response of detection were 5 nmol for ascorbic acid and 50 nmol for dehydroascorbic acid. This method was applied to the analysis of C3H/10T1/2 cells and culture medium after influx or efflux experiments and single or multiple treatments with ascorbic acid. Subsequent measurement of the radioactivity in the eluted fractions increased the detectability of both ascorbic acid and dehydroascorbic acid to 10 to 20 pmol. This research was supported by grant CA 09320 and CA 31574 from the National Cancer Institute, Bethesda, MD, and grant BC441 from The American Cancer Society.     

Identification of hydrogen peroxide as a photoproduct toxic to human cells in tissue-culture medium irradiated with “daylight” fluorescent light

Summary Hydrogen peroxide, lethal for human cells, is produced in Dulbecco's modified Eagle's tissue culture medium when exposed to “daylight” fluorescent light. Addition of pure H₂O₂ and use of the enzyme catalase demonstrate that about 40% of the toxicity in irradiated medium is due to generated peroxide. Riboflavin and tryptophan, or riboflavin and tyrosine, are the components necessary for formation of lethal levels of H₂O₂ during light exposure. Supported by an American Cancer Society Research Grant and a Public Health Service Research Career Development Award to Richard J. Wang.     

Quantitation of human mammary epithelial antigens in cells cultured from normal and cancerous breast tissues

Summary A sensitive radioimmunoassay technique was developed to quantitatite the level of human breast celltype specific antigens on cells from normal breast and from various established cell lines of breast and nonbreast origins. Polyacrylamide gel electrophoresis revealed four major proteinaceous components (150,000; 75,000; 60,000; and 48,000) in human milk fat globule membranes that were used to immunize rabbits in order to elicit antimammary epithelial cell antibody. Antisera obtained were rendered specific by abosorptions and were able to recognize three specific mammary epithelial components of the breast epithelial cell. Human mammary epithelial (HME) antigen expression was highest (1290 ng/10⁶ cells) in normal breast epithelial cells from primary cultures of normal breasts. Lower levels (range: 955 to 330 ng/10⁶ cells) were found in breast epithelial cells from cell lines established from cancerous breast tissue. Cells of nonbreast origins as well as fibroblasts from breast gave much lower values (less than 30 ng/10⁶ cells). On treatment, with trypsin, of two breast epithelial cell lines (MDA-MB-157 and MCF-7) 80 to 85% of their HME antigen expression was lost, suggesting that a majority of these breast antigens reside on the cell surface. This work was Supported by Grant PTD-99 from the American Cancer Society, Grant CA19455 and CA20286 from the National Cancer Institute, and Biomedical Research Support Grant RR05467 from the National Institutes of Health. Most cells used in the present study were produced with support from National Cancer Institute Contract Y01-CP8-0500, Biological Carcinogenesis Branch, Division of Cancer Cause and Prevention, under the auspices of the Office of Naval Research and the Regents of the University of California.     

Influence of culture conditions on growth of FL-74 cells and feline oncornavirus cell membrane associated antigen production

Summary The FL-74 cell, a feline lymphoblastoid cell line derived from a tumor induced by leukemia virus, grows equally well in static suspension culture (plastic T-flask or silicone treated glass bottles) or in spinner culture. No growth was observed in unsiliconized glass bottles. Although feline leukemia virus production was nearly the same in FL-74 grown in each of the above types of vessel, the expression of the feline oncornavirus membrane associated antigen (FOCMA), as determined by membrane immunofluorescence, was more intense and more complete on cells grown in static suspension. Moreover, higher fluorescent antibody titer endpoints were observed with cells from static suspension cultures than with cells from spinner cultures. FL-74 cells grown in spinner culture, when subjected to partial synchrony by cold block or by deprivation of essential amino acids (arginine and/or isoleucine) for 12 hr, achieved a membrane fluorescent pattern for FOCMA similar to celsl grown in static suspension. It is proposed that the expression of FOCMA on the cell membrane surface is cell-cycle dependent, and that the rate at which a cell passes through the cell cycle determines the pattern and intensity of the fluorescence of the cell membrane. Supported in part by: American Cancer Society Grant IM-27 and NIH Contract NO1-CP-43217     

Effect of room fluorescent light on the deterioration of tissue culture medium

Summary A major cause of tissue culture medium deterioration is exposure to room fluorescent light. Riboflavin and tryptophan present in Dulbecco's modified Eagle's minimum essential medium, when exposed to light, yield toxic photoproducts responsible for loss of the ability of the medium to support clonal growth of human, mouse and Chinese hamster cell lines. Procedures for minimizing medium deterioration are discussed. This work was supported by American Cancer Society Research Grant No. VC-100B and US PHS Research Career Development Award No. 5 K04 GM70537 from the National Institute of General Medical Sciences.     

Liposomes as vehicles for cellular incorporation of biologically active macromolecules

Summary Lipid vesicles (liposome) have recently been shown to be a useful vehicle for the delivery of a variety of compounds to cultured cells. Using large unilamellar vesicles composed of phosphatidylserine [LUV(PS)] we were able to encapsulate poliovirus and purified poliovirus ribonucleic acid (RNA) and show that it can be delivered efficiently to cells in an infectious form. LUV-entrapped poliovirus RNA produced infectious titers 100-fold higher than comparable RNA preparations delivered to cells by other techniques. We have made a quantitative analysis of the uptake and infectivity of the vesicle-encapsulated RNA by using various ratios of RNA copies per vesicle and by determining the percentage uptake of labelled lipid and RNA by HeLa cells. Presented in the symposium on Gene Transfer, Differentiation and Neoplasia in Plant and Animal Cells at the 30th Annual Meeting of the Tissue Culture Association, Seattle, Washington, June 10–14, 1979. This symposium was supported in part by Grant CA 26748 from the National Cancer Institute, DHEW, and Grant RD-67 from the American Cancer Society. The research described here was supported by Grants AI-14042, CA-18527 and GM-18921 from the National Institute of Health and IN-54P-16 from the American Cancer Society.     

Methods for evaluating the morphological and immunohistochemical properties of human tumor colonies grown in soft agar

Summary Clonogenic assays have been widely adopted for the investigation of hematopoietic and human tumor stem cell biology. Inasmuch as specific, whole colonies need to be analyzed morphologically, we used various methods for fixing and embedding individual colonies in situ that allowed macroscopic, light microscopic (LM), immunofluorescence, and transmission electron microscopic (TEM) evaluation of the intact colony. Melanoma colonies stained with Masson’s Trichrome, hematoxylin and eosin (H&E), periodic acid-Schiff, Best’s carmine, Page-Green method for inclusion bodies, and Snook’s reticulum revealed cellular and extracellular components by LM. Ultrastructural studies revealed specific cellular organelles and extracellular components. Immunofluorescence studies demonstrated cell-surface fibronectin, a high molecular weight, adhesive glycoprotein. Myeloma colonies contained a heterogeneous cell population and produced amyloid fibers that were observed by TEM. Fixation and embedding the colonies in agar for TEM has several advantages over centrifugation methods and other conventional techniques for collecting cells in that (a) an entire specific colony can be studied, (b) there is excellent preservation of the cell and its spatial orientation in the colony, and (c) the extracellular matrix (ECM) of the colony is preserved for immunohistochemical analysis. This work was supported by Grant T32 CA09213, a National Research Service Award from the National Cancer Institute to B. P., by Grants PDT-205 (M. J. C. H.) and PDT-184 (F. M.) from the American Cancer Society, and from the National Cancer Institute (CA-17904).     

Cholera toxin stimulation of human mammary epithelial cells in culture

Summary Addition of cholera toxin to human mammary epithelial cultures derived from reduction mammoplasties and primary carcinomas greatly stimulated cell growth and increased the number of times the cells could be successfully subcultured. Other agents known to increase intracellular cAMP levels were also growth stimulatory. The increased growth potential conferred by cholera toxin enhances the usefulness of this cell culture system. This work was supported by USPHS Grant CA-24844 from the National Cancer Institute and Grant CD-61B from the American Cancer Society.     

Methods for the study ofin vitro immunization of mouse spleen cells

Conclusion We have reviewed some of our experiences in developing techniques for studying the functions of the cells of the immune system. It is quite clear that much remains to be done. Improvements in the culture system are needed to permit cells to be grown for longer durations and at lower cell concentrations. The important effects of fetal calf serum should be defined. More sophisticated methods for separating cells into distinct functional populations must be developed. New assays for identifying other functions of the cells, particularly a method for directly assaying the number of precursor cells in a population, are needed. When these techniques are applied to the study of immune cells, further facts should be learned which will permit the development of significant, testable hypotheses on the function and relationships of the cells of the immune system. This is publication No. 298 from the Department of Experimental Pathology, Scripps Clinic and Research Foundation, La Jolla, California 92037. This work was supported in part by U.S.P.H.S. Grant 7007 and in part by American Cancer Society Grant E-395. Dr. Mishell is supported by American Cancer Society Grant E-395. Dr. Dutton is supported by a Dernham Fellowship of the California Division, American Cancer Society (No. D-100). Dr. Raidt is supported by United States Public Health Service Postdoctoral Fellowship No. 7-F2-A1-31,590.     

Rat hepatocyte primary cultures

Summary Primary cultures of rat hepatocytes survived well for up to 4 days in defined medium in the presence of dexamethasone but not in its absence. The loss of viability was accompanied by a loss of ultrastructural features characteristic of hepatocytes. The cultures began producing plasminogen activator and a neutral protease after 24 hr in culture. Dexamethasone inhibited the production of both of these substances. The deterioration of the cultures appeared not to be related to plasminogen activator, but prolongation of survival by a variety of protease inhibitors suggested that the neutral protease might contribute to deterioration. Dr. Goldblatt was supported by Grant No. SG-87 from the American Cancer Society as an American Cancer Society Scholar while on sabbatical leave from the Department of Pathology, University of Connecticut Health Center, Farmington, Connecticut. This study was supported by Contracts NO1-CP-55705 from the National Cancer Institute and 68-02-2483 from the Environmental Protection Agency.     

Glycogen metabolism in human hormone-producing trophoblastic cells in continuous culture

Summary Glycogen metabolism was studied in human hormone-producing trophoblastic cells (BeWo line). Cells supplemented daily with high glucose (3 g per liter in medium) contained 5.5% glycogen and utilized glucose at an initial rate of 12.2 mμmoles per min per mg of protein. In cells supplemented daily with low glucose (1 g per liter), the initial rate of glucose consumption was 23 mμmoles per min per mg of protein and the glycogen content reached only 0.4% of wet weight 24 hr after medium replenishment. When glycogen-depleted cultures were refed glucose, an accumulation of glycogen was observed, with initial deposition occurring in areas near the cell surface. After exhaustion of extracellular glucose, cytoplasmic glycogen was utilized at a rate of 2.8 mμmoles per min per mg of protein. Addition of either low or high glucose to glycogen-depleted cells resulted in the same rate of glycogen synthesis (approximately 8 mμmoles per min per mg of protein). It was suggested that unique regulatory mechanisms function in the control of glycogen metabolism in glycoprotein hormone-producing cytotrophoblastic cells. This work was supported in part by Public Health Service Research Contract PH43-68-1010, Research Grant CA 05524 from the National Cancer Institute, and by grants from the Milwaukee Division of the American Cancer Society, Inc., and the Damon Runyon Memorial Fund for Cancer Research, Inc.     

Keratinization and the effect of vitamin A in aggregates of a squamous carcinoma cell line NBT II

Summary The terminal differentiation, keratinization, of a rat bladder tumor cell line, NBT II, occurred in multicellular aggregates. After aggregation, these cells did not undergo a round of mitosis before keratinization. 5-Bromodeoxyuridine added to the monolayer cell culture 2 days before aggregation completely prevented this differentiation; it was ineffective when added at the time of cell aggregation. Vitamin A prevented the keratinization of NBT II cells in aggregates but did not inhibit aggregate formation; it enhanced the number of cells engaged in DNA synthesis. This model appears to be very useful for analyzing the mechanisms of terminal differentiation and its modulation by vitamin A in tumor cells. This research was supported by Institutional Research Grant 731-01-E from the American Cancer Society and in part by Research Grant CA 14137 from the National Cancer Institute to Dr. J. Leighton.     

Continuous culture of normal adult mammalian hepatocytes exhibiting parenchymal functions

Summary Past in vitro studies of liver-cell functions have been performed on nonproliferating primary cells or serially propagated hepatic monolayers of neoplastic or fetal origin. We optimized conditions for the selective culture of adult rabbit and canine liver parenchymal cells and presently have four differentiated proliferating monolayer strains. At the 30th passage level these hepatic cultures still display the specific liver parenchymal functions of albumin and fibrinogen synthesis as well as tyrosine aminotransferase activity. Optimization of the conditions for hepatocyte culture was monitored by [³H]thymidine incorporation. Albumin and fibrinogen synthesis were measured by bioradioimmunoassay and tyrosine transaminase activity by a modification of Diamondstone's assay. Albumin and fibrinogen synthesis were correlated with hepatocyte growth kinetics. Supported by the Medical Staff Research and Education Fund, Wayne County General Hospital, Eloise, Michigan 48132, and American Cancer Society Institutional Grant No. 40M, The University of Michigan, Ann Arbor, Michigan 48109.     

Lack of evidence for activation of a serum factor in protease-induced differentiation of mouse erythroleukemia cells

summary The addition of certain proteases to cultures of Friend virus-infected mouse erythroleukemia cells can induced up to 90% of the cells in culture to become hemoglobin-containing, as assessed by positive staining for benzidine (B⁺). Because the mechanism of this protease action is unknown, media components were studied as possible targets for protease activity. Aliquots of medium plus serum were incubated for various times with levels of protease sufficient to induce approximately 50% of the cells to the B⁺ state. Cells were added to protease-pretreated serum either before or after inactivation of the protease. In all cases, enzymatically active protease had to be present with the cells to induce B⁺ cells to form. Serum and other components of the medium pretreated with protease were inactive. Mouse erythroleukemia cells grown in the absence of serum were also induced by proteases to form B⁺ cells. These data imply that the inducing action of proteases cannot be passively transferred by protease-pretreated serum or medium nor is serum required for protease-mediated induction of B⁺ cells. Taken together, these conclusions suggest that the protease action is on the cells or on cellular products intimately associated with cells. This study was supported by U.S. Public Health Service Grants CA 24403 and CA 37874; American Cancer Society Grant CH-303; the Spingold Foundation; the Chemotherapy Foundation, Inc; the Gar Reichman Foundation; an Irving Alpert Cancer Research Award; and institutional general research funds. Part of this work was presented at the 1984 meeting of the American Society of Biological Chemists and American Association of Immunologists, St. Louis, MO (21).     

In vitro stimulation of rat liver cells by homologous partial hepatectomy serum

Summary A low passage rat liver cell line demonstrated in vitro growth stimulation when cultured in the presence of serum of homologous, partially hepatectomized rats. After 4-day incubation a 3.25-fold increase in the cell population was observed in cultures supplemented with posthepatectomy serum at a dilution of 1∶10. No response was observed with sham-operated animal serum. Continous cultures of Chang human liver and Don hamster lung cells were not responsive to the posthepatectomy serum. The limitations of tetraphenylboron as a dispersing agent for primary rat liver cells are discussed. Supported by Grant 67-7 from the Illinois Division of the American Cancer Society.     

Monoclonal immunoglobulins in patients with carcinoma of the colon

Summary Structural studies were performed on five monoclonal immunoglobulins isolated from patients with carcinoma of the colon. Serologic analysis revealed that two of the five proteins shared idiotypic antigenic determinants; these two but none of the three others had V_HIII heavy chains. The results demonstrated a close structural similarity between the heavy chains of these two proteins both in their antigen-binding sites (the hypervariable region) and in their framework regions. Determination of the NH₂-terminal amino acid sequences indicated that the light chain variable regions of all five proteins were either V_xII or V_xIII. These data suggest that monoclonal immunoglobulins in patients with carcinoma of the colon have restricted heterogeneity and that, in some cases the production of monoclonal immunoglobulin(s) and the development of a solid tumor in a given patient may be related events.Publication no. 357 from the Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina. Research supported in part by National Science Foundation Research Grant PCM 79-24043, USPHS Grants HD-09938 and CA-25746, and by Medical University of South Carolina Biomedical Research Appropriations A911 and A912. A. C. W. was the recipient of American Cancer Society Faculty Research Award No. 125     

The contribution of cell death to medium-released fractions of cell cultures

Summary Cell death was estimated by prelabeling primary chick embryo skeletal muscle cell cultures with [³H]thymidine and by subsequently measuring the release of label into complete culture medium or serum-and embryo-extract-free medium for a 6 h period. Cultures of the established muscle cell line L6 and the fibroblastic cell line 3T3 were used for comparative purposes. Comparison of the nigrosin exclusion test with the thymidine release test shows that the former underestimates cell death because it measures only the instantaneously occurring cell death. The [³H]thymidine release test estimates the cumulative amount of cell death. From cumulative cell death estimates it is calculated that 12.0 and 17.8% of the³H-fucosylated medium-released fractions from primary cell cultures are the result of cell death contamination when release occurs in complete or macromolecule-free media, respectively. High speed centrifugation is shown to eliminate most contamination from cell death. Evidence is presented that the absence of macromolecules in the culture medium has little effect on the release process. Contamination of the released fraction resulting from cell death is much less in the established cell lines than in the primary cells. It is concluded that the release process can be studied in primary muscle cell cultures and especially in established cell lines if adequate precautions are taken and if corrections for cell death contamination are taken into account. This research benefited from use of the Cell Culture Facility supported by National Cancer Institute Grant CA 14733. This research was supported by a Muscular Dystrophy Association Grant to Dr. Heinz Herrmann and by American Cancer Society Grant RDP 8 and was submitted in partial fulfillmant of a Ph.D. degree at the University of Connecticut (T. C. Doetschman).     

Analysis of cellular heterogeneity in mouse thymus cultures

Summary Analysis of 5 to 6 d primary cultures of cells derived from murine thymus glands revealed a heterogeneous population of cells rather than “pure” reticuloepithelial cell cultures as was assumed previously by other investigators. The monolayer cultures consisted of at least three cell types: thymus epithelial cells, macrophagelike epithelioid cells, and fibroblasts. Surprisingly, about 50% of the cells had positive cytochemical staining reactions for acid phosphatase and nonspecific esterase. The same cells phagocytized carbon particles, latex beads, and yeast. Furthermore, these cells could be removed from the initial cell suspension by phagocytosis of carbonyl iron, followed by magnetic separation, but once they had adhered to the substratum they were resistant to trypsin removal. All of these findings supported the conclusion that about 50% of the cells in the monolayers were macrophages. The other cells present were thymus epithelial cells and a small number of fibroblasts. Both of the latter types of cell were cytochemically negative, did not phagocytize particulate material, and were not removed by carbonyl iron treatment, but were removed by treating the monolayer with trypsin. The findings in this report indicated that epithelioid morphology alone was inadequate to identify correctly the cell types found in thymus cultures and that the use of such cultures as a model to study in vitro the maturation of certain immunological functions has been based on assumptions here shown to be incorrect. This work was supported by the Graduate School, The Ohio State University, the Bremer Foundation, the American Cancer Society (IN-16R), and National Cancer Institute Grant 5 ROL CA-19346-03.