Human macrovascular endothelial cells: Optimization of culture conditions

Summary The purpose of this study is to identify optimal culture conditions to support the proliferation of human macrovascular endothelial cells. Two cell lines were employed: human saphenous vein endothelial cells (HSVEC) and human umbilical vein endothelial cells (HUVEC). The influence of basal nutrient media (14 types), fetal bovine serum (FBS), and mitogens (three types) were investigated in relation to cell proliferation. Additionally, a variety of extracellular matrix (ECM) substrate-coated culture dishes were also tested. The most effective nutrient medium in augmenting cell proliferation was MCDB 131. Compared to the more commonly used M199 medium, MCDB 131 resulted in a 2.3-fold increase in cell proliferation. Media containing 20% FBS increased cell proliferation 7.5-fold compared to serum-free media. Among the mitogens tested, heparin (50 μg/ml) and endothelial cell growth supplement (ECGS) (50μg/ml) significantly improved cell proliferation. Epithelial growth factor (EGF) provided no improvement in cell proliferation. There were no statistical differences in cell proliferation or morphology when endothelial cells were grown on uncoated culture plates compared to plates coated with ECM proteins: fibronectin, laminin, gelatin, or collagen types I and IV. The culture environment yielding maximal HSVEC and HUVEC proliferation is MCDB 131 nutrient medium supplemented with 2 mM glutamine, 20% FBS, 50 μg/ml heparin, and 50 μg/ml ECGS. The ECM substrate-coated culture dishes offer no advantage.     

Rat pituitary tumor cells in serum-free culture. II. Serum factor and thyroid hormone requirements for estrogen-responsive growth

Summary The effect of 17β-estradiol (E₂) on growth of GH₄C₁ rat pituitary tumor cells was investigated under serum-free conditions and with medium containing charcoal-extracted serum. Serum-free TRM-1 medium was a 1∶1 (vol/vol) mixture of F12-DME supplemented with 50 μg/ml gentamicin, 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 10 μg/ml insulin, 10 μg/ml transferrin, 10 ng/ml selenous acid, 10 nM 3,5,3′-triiodothyronine (T₃), 50 μM ethanolamine, and 500 μg/ml bovine serum albumin. The cells grew continuously in TRM-1 but were E₂ responsive only when growth was retarded by reducing the T₃ concentration to 10 pM (TRM-MOD). Addition of 1 to 10 nM E₂ to TRM-MOD increased growth by 0.3 to 0.9 cell population doublings over controls in 9 d. By using medium supplemented with charcoal-extracted sera, basal growth became 1 to 1.5 cell population doublings in 9 d. Addition of 0.1 pM E₂ to medium containing charcoal-extracted serum caused a significant increase in cell number whereas pM-nM concentrations stimulated 200 to 570% increases over controls. The effect of steroid hormone was the same in phenol-red-containing and indicator-free medium. The data presented confirm that the major requirements for demonstration of estrogenic effects in culture were optimum concentrations of thyroid hormones and the presence of yet-to-be-characterized serum factors. This work was supported by National Cancer Institute (Bethesda, MD) grants CA-26617 and CA-38024, American Cancer Society grant BC-255, and grant 2225 from The Council for Tobacco Research, U.S.A., Inc.     

A new serum-free method of measuring growth factor activities for human breast cancer cells in culture

Summary Growth of the MCF-7, T47D, and ZR-75-1 human breast cancer cells was established in a serum-free defined medium (MOM-1) composed of a 1∶1 (vol/vol) mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium containing 15 mM HEPES (pH 7.2), 2 mM 1-glutamine, 20 μg/ml glutathione, 10 μg/ml insulin, 10 μg/ml transferrin (Tf), 10 ng/ml selenous acid, 0.3 nM triiodothyronine, 50 μg/ml ethanolamine, 20 ng/ml epidermal, growth factor, 2.0 nM 17β-estradiol, and 1.0 mg/ml bovine serum albumin (BSA). Proliferation in MOM-1 was 50 to 70% of the serum stimulated rate. Deletion of components from MOM-1 gave a medium (Tf-BSA) containing only HEPES, 10 μg/ml Tf, and 200 μg/ml BSA, which sustained MCF-7 and T47D cells in a slowly dividing and mitogen responsive state; ZR-75-1 cells required Tf plus 1.0 mg/ml BSA. In Tf-BSA, insulin and insulin-like growth factor I(IGF-I) were mitogenic with ED₅₀ values of 2 to 3 ng/ml and 30 to 150 pg/ml, respectively, with MCF-7 cells. The T47D cells were responsive to these factors in Tf-BSA but required 10-fold higher concentrations for ED₅₀. At saturating concentrations, insulin and IGF-I promoted 1.5 to 3.5 cell population doublings over controls in 8 d. At≤ng/ml concentrations, epidermal growth factor, insulin-like growth factor II, and basic fibroblast growth factor were mitogenic for human breast cancer cells in Tf-BSA. Mitogen activities in uterus and pituitary extracts were assayed readily in Tf-BSA. This new method offers a convenient means of comparing the potencies of growth-promoting factors on human breast cancer cells without interfering activities known to be present in serum. This work was supported by grants CA-38024 and CA-26617, from the National Cancer Institute, Bethesda, MD, and by American Cancer Society grant BC-255 and grant 2225 from the Council for Tobacco Research, USA, Inc.     

Proliferation and differnetiation of human squamous carcinoma cell lines and normal keratinocytes: Effects of epidermal growth factor,retinoids, and hydrocortisone

Summary Exposure of squamous carcinoma cell (SCC) lines, exhibiting high levels of epidermal growth factor (EGF) receptors, to EGF for 6 d caused a dose-dependent inhibition of cell proliferation. This EGF-induced inhibition of cell proliferation occurred under both low (0.06 mM) and normal (1.6 mM) Ca²⁺ concentrations. Furthermore, the extent of EGF-induced inhibition of cell proliferation seemed to be independent of the number of EGF-receptors. This conclusion is based on the notion that the various SCC lines exhibited an increasing number of EGF receptors accompanied by a decreasing ability to differentiate, whereas no relationship was observed with the EGF-induced inhibition of cell proliferation in these cell lines. Retinoids caused also a dose-dependent inhibition of cell proliferation. The effects of EGF and retinoids were additive, indicating that different regulatory mechanisms are involved. On the other hand, hydrocortisone caused a stimulation of SCC-proliferation, also independent of EGF. In contrast to SCC cells, EGF did not affect significantly the rate of proliferation of normal keratinocytes. However, the simultaneous addition of EGF and hydrocortisone resulted in a significant increase in the rate of keratinocyte proliferation only in cells grown under normal calcium conditions. Differentiation capacity of normal keratinocytes and SCC lines was not affected by EGF. Furthermore, the retinoid-induced decrease and hydrocortisone-induced increase of competence of cells to form cornified envelopes was not affected by EGF. These observations suggest that the action of retinoids and hydrocortisone on both cell proliferation and cell differentiation occurs independently of EGF receptors. This work was partly supported by The Netherlands Cancer Foundation (Koningin Wilhelmina Fonds), grant IKW 85–71.     

Optimized medium for clonal growth of human microvascular endothelial cells with minimal serum

Summary An optimized basal nutrient medium, MCBD 131, has been developed that supports clonal growth of human microvascular endothelial cells (HMVEC) with as little as 0.7% dialyzed fetal bovine serum (dFBS) when also supplemented with 10 ng/ml epidermal growth factor (EGF) and 1 μg/ml hydrocortisone. An extensive initial survey of available media showed that MCDB 402, a medium optimized for low-serum growth of Swiss 3T3 cells, supported the best clonal growth of HMVEC with 10% dFBS. Quantitative adjustment of the composition of MCDB 402 for improved clonal growth of HMVEC with reduced amounts of dFBS resulted in development of MCDB 131. Although many different adjustments contributed to the optimal properties of MCDB 131 for growth of HMVEC, the most unusual feature of this medium is its high magnesium concentration. A major benefit was achieved by increasing Mg²⁺ from 0.8 mM in MCDB 402 to 10.0 mM in MCDB 131. In the absence of defined supplements, MCDB 131 supports good clonal growth of HMVEC with 2% dFBS. This can be reduced to 0.7% by adding EGF and hydrocortisone, which act synergistically to improve growth with low levels of dFBS. This research was supported by grant CA 15305 from the National Cancer Institute, Bethesda, MD.     

Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. I. Regulation of proliferation by hormones and growth factors

Summary A serum-free primary culture system has been developed which allows for three-dimensional growth and differentiation of normal rat mammary epithelial cells (RMECs) within an extracellular matrix preparation. RMECs were isolated from mamary glands of immature 50- to 60-d-old rats and the organoids embedded within a reconstituted basement membrane matrix prepared from the Engelbreth-Holm-Swarm sarcoma. Cells grown in a serum-free media consisting of phenol red-free Dulbecco's modified Eagle's medium-F12 culture medium containing 10 μg/ml insulin, 1 μg/ml prolactin, 1 μg/ml progesterone, 1 μg/ml hydrocortisone, 10 ng/ml epidermal growth factor (EGF), 1 mg/ml fatty-acid-free bovine serum albumin (BSA), 5 μg/ml transferrin, and 5 μM ascorbic acid proliferated extensively (15- to 20-fold increase in cell number as quantitated using the MTT dye assay) over a 2- to 3-wk culture period and remained viable for months in culture. Several types of colonies were observed including the alveolarlike budding cluster which predominates at later times in culture, units with no or various degrees of ductal-like projections, stellate colonies, and two-and three-dimensional web units. Optimal proliferation required insulin, prolactin, progesterone, EGF, and bovine serum albumin. Hydrocortisone was not required for proliferation, but the colonies developing in its absence were morphologically altered, with a high frequency of colonies that formed an extensively branched network with many fine projections. Cell proliferation was also dependent on substratum, with significantly less growth and development occurring in RMECs grown within a type I collagen gel matrix compared to RMECs grown within the reconstituted basement membrane. In conjunction with other studies demonstrating extensive differentiation as well as proliferation, it is concluded that this model should prove to be an improtant tool to study the hormonal regulation of the growth and development of rat mammary cells. This work was supported by grants CA 33240 and CA 35641 and by core grant CA 24538 from the National Institutes of Health, Bethesda, MD.     

Reinitiation of DNA synthesis in quiescent mouse keratinocytes; Regulation by polypeptide hormones,cholera toxin,dexamethasone, and retinoic acid

Summary Cloned mouse keratinocytes (MK-1 cells) display density-dependent growth arrest when reaching confluency in a serum-free medium with a calcium concentration <0.1 mM, supplemented only with insulin and transferrin. In this quiescent state, greater than 95% of the cell population is in the G_0/1 phase of the cell cycle. Treatment of quiescent MK-1 cells with 1 to 10 ng/ml epidermal growth factor (EGF) resulted in a sharp burst of DNA synthetic activity. Both insulin and cholera toxin potentiated the mitogenic effect of EGF, but neither agent was necessary or sufficient to induce thymidine incorporation into DNA. Dexamethasone abolished the effect of insulin, but not the mitogenic effect of EGF alone. In contrast, retinoic acid (RA) did not possess any mitogenic effect for quiescent MK-1 cells, nor did it modulate the actions of EGF or dexamethasone. A number of commercially available crude extracts of bovine brain and pituitary were also capable of initiating DNA synthesis in resting MK-1 cells. Finally, transforming growth factor type beta (TGFβ) proved to be a potent inhibitor of the mitogen-induced DNA synthesis in MK-1 cells (IC₅₀∶10pM). This defined culture system is eminently suited to study the regulation of DNA synthesis of epidermal cells. In addition, it can be used as a sensitive bioassay for the detection of epidermal mitogens, as well as inhibitors of DNA synthesis such as TGFβ. Supported by PHS Award CA-41556 from the National Cancer Institute, Bethesda, MD.     

Growth of MTW9/PL2 estrogen-responsive rat mammary tumor cells in hormonally defined serum-free media

Summary Growth of the estrogen-responsive MTW9/PL2 rat mammary tumor cells was demonstrated in serum-free defined medium (designated DDM-1) formulated with F12-DME (1:1 vol/vol) supplemented with 15 mM HEPES pH 7.4 insulin 10 μg/ml, transferrin 10 μg/ml, sodium selenite 10 ng/ml, triiodo-l-thyronine 0.3 nM, phosphoethanolamine 5 μM, epidermal growth factor (20 ng/ml), 17 β-estradiol 2 nM, and bovine serum albumin 20 μg/ml. In DDM-1, the growth rate was about one-half that seen in serum-containing medium. When ethanolamine (50 μM), glutathione (20 μg/ml), and linoleic acid/bovine serum albumin (150 μg/ml) were added (formulation DDM-2), the growth rate was 80% of serum-containing medium and not seed-density dependent. Deletion of estradiol from DDM-1 or DDM-2 had no effect on growth rate. Also, cells grown in steroid hormone deficient medium for 4 mo. continued to form estrogen-responsive tumors in rats as did cells cultured for the same period in 2 nM estradiol. To investigate autocrine growth factor secretion, a third medium (DDM-3) was prepared by deleting insulin, epidermal growth factor, phosphoethanolamine, estradiol, and both forms of bovine serum albumin from DDM-2. Growth in mitogen-free medium equaled 86% of the serum-stimulated rate and was seed-density dependent; phenol red deletion from DDM-3 had no effect on growth rate. Evidence presented suggests that autocrine factors stimulate growth of the MTW9/PL2 cells in DDM-3, and that this secretion may support the growth of estrogen-responsive cells in culture in the absence of steroid hormone. This work was supported by National Cancer Institute grants CA-38024 and CA-26617 and American Cancer Society grant BC255.     

Growth and differentiation of human keratinocytes without a feeder layer or conditioned medium

Summary An improved procedure has been developed for clonal growth of normal human epidermal keratinocytes (HK) without feeder cells or conditioned medium. The use of medium 199, supplemented with 0.4 μg/ml hydrocortisone (HC) and 20% (v/v) whole fetal bovine serum (wFBS) and conditioned overnight by 3T3 cells, eliminated the need for a feeder layer of lethally irradiated 3T3 cells for HK growth. Several other media with equivalent conditioning and supplementation failed to support satisfactory multiplication of HK, including Dulbecco's modified Eagle's medium, which is normally used for growth of HK with a feeder layer. Increasing the concentration of HC to 10 μg/ml (2.8×10⁻⁵ M) made possible clonal growth of HK without any conditioning of the medium. The addition of 10⁻⁵ M putrescine, 10⁻⁵ M vitamin B₁₂, or 3.7×10⁻⁶ M β-estradiol further enhanced growth in unconditioned medium. Substantially greater improvement was obtained by the addition of pituitary extract or fractions prepared from pituitary extract. In medium 199 supplemented with 10 μg/ml HC, 20% (v/v) wFBS, and 0.15 mg/ml each of two pituitary fractions, single HK attach with a colony-forming efficiency equal to that in conditioned medium and form stratified, keratinized colonies that grow to confluency and can be subcultured. These results make it clear that HK do not require special “conditioning factors” from fibroblasts for clonal growth and differentiation in culture. Thus, factors directly involved in growth and the expression of differentiation can be analyzed without the interfering effects of any other type of cell. Preliminary studies with epidermal growth factor (EGF), which stimulates growth and extends life span of HK grown in the presence of fibroblasts, have shown that, in the absence of fibroblasts, EGF has no effect either on clonal growth or on cumulative multiplication potential of HK. This paper contains material from a thesis submitted to the Graduate School of the University of Colorado, Boulder, by Donna M. Peehl in partial fulfillment of the requirements for the Ph.D. degree. This work was supported by Grant CA 15305 from the National Cancer Institute and Grant AG 00310 from the National Institute on Aging.     

Primary and long term epithelial cell cultures from human fetal normal colonic mucosa

Summary Primary and passaged cultures of normal colon epithelial cells, derived from human fetuses (13 to 17 wk of conceptual age) have been established. These cultures have been passaged 16 times thus far. The cultures have been initiated and maintained in medium consisting of 50% Dulbecco's minimum essential medium and 50% Ham's F12 medium and supplemented with antibiotics (penicillin, 100 U/ml; streptomycin, 100 μg/ml); ascorbic acid, 40 μg/ml;l-isoleucine, 50 μg/ml; epidermal growth factor, 20 ng/ml; insulin, 5 μg/ml; cholera toxin, 5 ng/ml; transferrin, 1 μg/ml; fetal bovine serum (10%); and HEPES, 25 mM final concentration, and incubated at 37°C in humidified gas containing 5% CO₂: 95% air. The cellular and subcellular characteristics of primary and passaged cultures were defined using light microscopy and scanning and transmission electron microscopy. The cells exhibited microvilli on cell surfaces and showed junctional complexes and interdigitations between cells. Indented nuclei with dense chromatin and marginated heterochromatin, numerous mitochondria, rough endoplasmic reticulum, polysomes, and extensive Golgi zones were conspicuous. Also, periodic acid Schiff's reagent-positive staining of the cells suggests the active synthesis of complex mucopolysaccharides in the cytoplasm. This study was supported by USPHS Grant CA-30185 from the National Large Bowel Cancer Project, National Cancer Institute.     

Growth and differentiation in cultured human thyroid cells: Effects of epidermal growth factor and thyrotropin

Summary Human thyroid cells were grown and subcultured in vitro to examine their responses to known hormones and growth factors, and to serum. The cells were obtained from surgical specimens and were either neoplastic or nonneoplastic. The effects of culture conditions on cell growth were measured by changes in cell numbers and by stimulation of [³H]thymidine incorporation. The results showed that serum (0.5%) was essential for cell proliferation, and that a mixture of insulin (10 μg/ml), transferrin (5 μg/ml), hydrocortisone (10 μg/ml), somatostatin (10 ng/ml), and glycyl-histidyl-lysine (10 ng/ml) enhanced the effect of serum. Maximum growth of the cells was obtained when epidermal growth factor was present at 10⁻⁹ M. Differentiation was measured by production of thyroglobulin, which was found to be stimulated by thyrotropin. This system provides a means to study the hormonal control of growth and differentiation in human thyroid cells. This work was supported by grants from the Medical Research Council of Canada; the Department of Medicine, University of Toronto; and the National Cancer Institute of Canada. J. E. E. is a C.H. Best Foundation and Department of Medicine postdoctoral fellow.     

Enhancement of pancreatic islet cell monolayer growth by endothelial cell matrix and insulin

Summary The roles of glucose and insulin in the promotion of DNA synthesis in pancreatic islet cell monolayers were assessed using a variety of in vitro conditions. Several substrates including collagen, poly-l-lysine, Matrigel, and the extracellular matrix produced by cultured bovine endothelial cells (BCEM) were compared for their ability to promote monolayer growth. Islets grown on BCEM in combination with medium RPMI 1640 supplemented with 22.2 mM glucose or 10 μg/ml insulin gave the best results as determined by new DNA synthesis. The new-form monolayers were free of contaminating, fibroblasts. These results suggest that insulin is critical to pancreatic islet growth when the cells are attached to biocompatible matrices.     

Duct,exocrine, and endocrine components of cultured fetal mouse pancreas

Summary Twenty to twenty-two days postcoitum mouse fetal pancreas organ bits were cultured on the dermal surface of irradiated pigskin as a substrate. The medium used for long term culture consisted of Eagle’s Minimum Essential Medium with the addition of 10% bovine serum, 0.02 U/ml insulin, 0.025 μg/ml glucagon, 3.63 μg/ml hydrocortisone, 100 μg/ml soybean trypsin inhibitor or 10⁻⁸ M atropine. When the medium lacked trypsin inhibitor or atropine but contained the three hormones, the pigskin support began to be destroyed after 2 to 4 wk in culture. Thereafter, the cultured cells could not grow and survive on the digested pigskin. When 10⁻⁶ M atropine was added to the medium, amylase secretion from cultured cells and destruction of pigskin were inhibited completely but pancreas cells could not grow or survive. In contrast, 100 μg/ml soybean trypsin inhibitor or 10⁻⁸ M atropine permitted cell growth, permitted amylase secretion from the cultured acinar cells, and prevented the destruction of pigskin. Under these conditions pancreas cells migrated or grew or both from the organ bits onto the surface of the pigskin dermis and organoid aggregations formed. Hydrocortisone was needed to permit growth for more than 2 wk. Glucagon and insulin had additive effects. Light and electron microscopic observations indicated the culture of at least five kinds of cells, i.e., duct, acinar, centroacinar, endocrine, and mesenchymal. The majority of cultured cells were duct cells and acinar cells. There were few mesenchymal cells. Mouse pancreas cells were cultured for at least 12 wk by this method. This investigation was supported by PHS Grant CA 30220 awarded by the National Cancer Institute, DHHS, Grant 1203M awarded by the Council for Tobacco Research, Inc., and Grant RD-65 (for equipment) awarded by the American Cancer Society. Nude mice were provided by Dr. Wendall M. Farrow of Life Sciences, Inc., Resource Laboratory N01, CP6-1005 of the National Cancer Institute.     

Proliferation of epithelial cells derived from rat dorsolateral prostate in serum-free primary cell culture and their response to androgen

Summary Primary cultured epithelial cells derived from the rat dorsolateral prostate proliferated in serum-free nutrient medium WAJC 404 supplemented with mitogens: insulin (650 nM), cholera toxin (120 pM), epidermal growth factor (EGF) (2.5 nM), dexamethasone (300 nM), and bovine pituitary extract (25 μg/ml). The culture consisted of two types of epithelial cell colonies: one originated from single cells or small cell aggregates and the other was epithelial cell outgrowth from small tissue fragments attached to a substratum. There were differences in requirements for the mitogens between the two types of colonies. Requirements for cholera toxin, bovine pituitary extract, and dexamethasone were higher in the former type of colonies, and those for EGF were higher in the latter type of colonies. Proliferation of the epithelial cells in either type, of colony was suppressed more than 50% by 1 nM dihydrotestosterone. This suppressive effect was not mediated by stromal component in the tissue fragments, and was counteracted by cyproterone acetate, indicating specific and direct action of the androgen on prostate epithelial cells. The results suggest that there is discrete participation of polypeptide growth factors and androgen in proliferation and differentiation, respectively, of prostate epithelial cells in vivo.     

Continuous growth of human tumor cell lines in serum-free media

Summary Five human tumor cell lines were studied for growth factor requirements and for replication in serum-free media. Of the five tumor lines HT-29 (colon carcinoma), TWI (melanoma), A-549 (lung carcinoma), Panc-1, (carcinoma of the pancreas) and EJ, (bladder carcinoma) only HT-29 and TWI grew in the serum-free medium (SFM). In a series of additional experiments, a combination of transferrin (5 μg/ml), insulin (5 μg/ml), triiodothyronine (2×10⁻¹⁰ M), epidermal growth factor (20 ng/ml), and selenium (5 ng/ml) was added to Chee’s essential medium (CEM) without serum (C-TITES medium). The C-TITES modification of CEM was found to allow optimal replication of HT-29 and TWI cells. Both HT-29 and TWI cells have replicated continuously in C-TITES medium for periods of more than 15 mo. These cells replicate with slightly lower doubling times than in CEM supplemented with 10% fetal bovine serum. Deletion of insulin or transferrin from the C-TITES medium resulted in cessation of cell growth of HT-29 and TWI. HT-29 assumed a somewhat rounded morphology, whereas TWI grew with the characteristic fibroblastic morphology in C-TITES medium. Cell line EJ did not grow in C-TITES medium. The other two cell lines, A-549 and Panc-1, grew in C-TITES medium but their growth rate was much slower than that in SSM. Availability of cell lines that can be propagated in serum-free, hormone-supplemented medium may aid in the study of the mechanisms by which hormones influence cell growth. This work was supported by Veterans Administration Research Awards to two of the authors (Karimullah A. Zirvi and George J. Hill) and grant no. CA-37138 from the National Cancer Institute.     

Long-term multiplication of the Chinese hamster ovary (CHO) cell line in a serum-free medium

Summary A new synthetic medium (referred to as GC₃) that supports the growth of the Chinese hamster ovary cell line has been developed. It is composed of a 1∶1 mixture of Ham's F12 and modified Eagle's minimum essential (MEM.S) mediums supplemented with transferrin (10 μg/ml), insulin (80 mU/ml), and selenium (1×10⁻⁷ M). Other more simple supplementations of our basal medium MEM.S/F12 (transferrin+insulin, transferrin+selenium, ferrous iron+selenium) also give good cell growth responses. Fibronectin or serum pretreatment is not needed for cellular attachment and spreading. Our culture system is characterized by a continuous serum-free cultivation (more than 200 doublings), a clonal growth, a high density proliferation, and a rapid growth rate near that of cells in serum-supplemented medium.     

Clonal growth of human keratinocytes with small amounts of dialyzed serum

Summary A survey of commercially availabla media revealed that medium F-12 was superior to medium 199 for clonal growth of human epidermal keratinocytes (HK) when supplemented with 10 μg/ml hydrocortisone (HC) plus dialyzed fetal bovine serum protein (FBSP), rather than the whole serum used in previous studies. Qualitative and quantitative adjustment of the medium composition for optimal clonal growth with minimal amounts of FBSP generated a new medium, MCDB 151, which supports clonal growth of HK with 10 μg/ml HC and as little as 1 mg/ml FBSP (equivalent in protein concentration to 2.0% whole serum). MCDB 151 differs significantly from MCDB 105, previously developed in this laboratory for normal human fibroblasts, and each medium selectively favors growth of its own type of cell in primary cultures of disaggregated human neonatal foreskin cells. Differences in the amounts of calcium and adenine in the two media appear to be among the most influential factors mediating the selective growth. Optimal growth of HK occurs at a very low level of Ca²⁺ that causes the colonies to remain as monolayers rather than stratifying as they do in the presence of higher levels of calcium. However, keratin synthesis, which was examined through use of highly specific fluorescent antibodies, is not affected by the Ca²⁺ concentration. Agents that increase intracellular cyclic AMP levels appear to have no effect on HK growth in MCDB 151 with 10 μg/ml HC and 1.0 mg/ml FBSP. This paper contains material from a thesis submitted to the Graduate School of the University of Colorado, Boulder, by Donna M. Peehl in partial fulfillment of the requirements for the Ph.D. degree. This work was supported by Grant CA 15305 from the National Cancer Institute and Grant AG 00310 from the National Institute on Aging.     

Effects of iron,copper, zinc,calcium, and magnesium on human lymphocytes in culture

The effects of simultaneous changes of calcium, magnesium, iron, copper, and zinc concentrations were evaluated in normal human T and B lymphocytes, cultured in cation-depleted media. Optimal concentrations for thymidine incorporation (TI) in both cell populations were Fe and Zn 15 μM and Cu 5 μM; for t cells Ca 2 mM and Mg 4 mM; for B cells Ca 4 mM and Mg 6 mM. TI decreased with increasing molarity of cations and the decrease was particularly apparent with Cu. Minimal amounts of Ca and Mg (0.5 mM) were necessary for growth, even in presence of optimal concentrations of Fe, Cu, and Zn. Fe and Cu showed synergistic stimulatory effects at low concentrations and synergistic inhibitory effects at high concentrations. Antagonism between Fe and Zn, Cu and Zn, and Ca and Zn was also demonstrated. CD4/CD8 increased with PHA stimulation in presence of Zn, and decreased with ConA stimulation in presence of Zn or Fe. The results demonstrate: (1) the relationship and interdependence of Fe, Cu, and Zn concentrations in modulating the growth of normal lymphocytes; (2) the stimulatory effects of Fe on B cells and Zn on CD8 positive cells; (3) the inhibitory effect of Cu at concentrations lower than those of Fe and Zn; (4) the requirement of Ca and Mg in certain concentration and ratio for the action of the other cations; and (5) the Ca and Mg requirement for the growth of B cells higher than T cells.     

Yersinia intermedia: A new species of enterobacteriaceae composed of rhamnose-positive,melibiose-positive,raffinose-positive strains (formerly calledYersinia enterocolitica orYersinia enterocolitica-like)

The drugs griseofulvin (10 μg/ml), nalidixic acid (0.05 μg/ml), quinine dihydrochloride (50 μg/ml), quinine ethylcarbonate (50 μg/ml), quinine urea hydrochloride (50 μg/ml), quinine lactate (50 μg/ml), and pamaquine (50 μg/ml) were chosen for laboratory studies. The minimal inhibitory concentration of the drug was used for determining the range of drug concentration needed to produce “mutational synergism” with ultraviolet radiation. Forward mutation from streptomycin sensitivity to resistance was used as a marker for mutagenicity. No stimulatory or inhibitory effects were noted on viable counts and mutation frequency, when the drugs were added (20–60 μg/ml) to the growth medium of unirradiatedEscherichia coli HCR⁺, HCR⁻, and irradiated HCR⁻ strains. These drugs increased mutation frequency and lethality of irradiated HCR⁺ bacteria. Incorporation of adenine (6 μm) into the minimal expression medium reverses the mutagenic effect of chloroquine. Chloroquine (50 μg/ml) did not interfere with the photoactivation of irradiated HCR⁺ cells. Our findings suggest that these chemicals selectively interfere with excision-repair.     

Selective growth of normal adult human urothelial cells in serum-free medium

Summary A serum-free medium (HMRI-2) has been developed for the outgrowth and subculture of epithelial cells from normal adult human ureter and bladder. Medium HMRI-2 consists of Ham’s MCDB 152 with double the amounts of the essential amino acids in Stock 1, low Ca²⁺ (0.06 mM) and is supplemented with epithelial growth factor, 5 ng/ml; transferrin, 5 μg/ml; insulin, 5 μg/ml; ethanolamine and phosphoethanolamine, 0.1 mM each; hydrocortisone, 2.8×10⁻⁶ M; and bovine pituitary extract, 126 μg protein/ml. The cultured cells showed ultrastructural markers of epithelial cells (prekeratin fibers, tonofilaments, surface microvilli with glycocalyx), exhibited ABO antigens, and had a normal human diploid karyotype. Primary cultures could be subcultured and also cryopreserved in HMRI-2 in liquid nitrogen. Cells in mass cultures showed a population doubling time of 40.5±4.5 h and had a maximum in vitro life span of 20 to 25 population doublings. It was observed that primary outgrowths, secondary cultures, and even cryopreserved cells all retained the capacity to respond to high Ca²⁺ and serum by differentiation and desquamation. This study has resulted in the availability of easily obtainable serum-free epithelial cultures from normal adult human ureter and bladder. The useful in vitro life span of these cultures may be important in future studies of carcinogenesis. This work was supported by a grant from the National Cancer Institute (R01CA25089), Bethesda, MD.