Growth of kidney epithelial cells in hormone-supplemented,serum-free medium

Madin Darby canine kidney cells can grow in synthetic medium supplemented with 5 factors – insulin, transferrin, prostaglandin E₁, hydrocortisone and triiodothyronine – as a serum substitute. These 5 factors permit growth for one month in the absence of serum, and a growth rate equivalent to that observed in serum-supplemented medium. Dibutyryl cAMP substitutes for prostaglandin E₁ in the medium, suggesting that increased growth of Maden Darby canine kidney cells results from increased intracellular cAMP. Potential applications of the serum-free medium are discussed. The medium permits the selective growth of primary epithelial cell cultures in the absence of fibroblast over-growth, and a defined analysis of the mechanisms by which hormones regulate hemicyst formation.     

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.     

Growth of distal fetal rat lung epithelial cells in a defined serum-free medium

Summary Fetal rat distal lung epithelial cells, in contrast to adult type II pneumocytes, will divide readily in culture in the presence of 10% (vol:vol) fetal bovine serum. The presence of serum makes purification of uncontaminated cell-derived growth factors difficult and modifies cellular responses to oxidant injury. We report the development of a defined serum-free medium that will support growth of fetal distal lung epithelial cells in primary culture. Initial studies used a low-serum (2%; vol:vol) to determine the effect of basal media, substrata, and various additives. Subsequent studies demonstrated growth on a poly-d-lysine substratum under serum-free culture conditions in Dulbecco’s modified minimal essential medium with insulin (50 μg/ml), endothelial cell growth supplement (20 μg/ml), bovine pituitary extract (100 μg/ml), bovine serum albumin (50 μg/ml), selenous acid (4 ng/ml), reduced glutathione (500 ng/ml), soybean trypsin inhibitor (100 μg/ml), transferrin (5 μg/ml), HEPES buffer (2.6 mg/ml), and cholera toxin (5 μg/ml). Growth was enhanced by reducing the gas phase oxygen concentration from 21 to 3%. The undefined components of this medium, bovine pituitary extract and endothelial cell growth supplement, could be replaced by platelet-derived growth factor (20 ng/ml) with prostaglandin E₁ (25 nM). The response of fetal distal lung epithelial cells to known growth factors differs substantially from that observed with type II pneumocytes from adult lung and is similar in many, though not all, respects to the responses reported for proximal airway cells from adult lung. Supported by grants MT-7867 and PG-42 from the Medical Research Council of Canada, HL-40458 from the National Heart, Lung and Blood Institute, Bethesda, MD, and an equipment grant from the Ontario Thoracic Society. Dr. Caniggia is a recipient of a research fellowship from the Italian Ministry of Education.     

Culture of fetal alveolar epithelial type II cells in serum-free medium

Summary A serum-free culture medium (defined medium = DM) was elaborated by adding to Eagle’s minimum essential medium (MEM), non-essential amino acids, transferrin, putrescine, tripeptide glycyl-histidyl-lysine, somatostatin, sodium selenite, ethanolamine, phosphoethanolamine, sodium pyruvate, and metal trace elements. This medium was tested for its ability to support sustained surfactant biosynthesis in fetal alveolar epithelial type II cells. For up to 8 days, ultrastructure was maintained with persistance of lamellar inclusion bodies. Thymidine incorporation into DNA was enhanced about 50% in DM as compared with MEM, whereas it was enhanced 300% in 10% fetal bovine serum. With DM, the incorporation of tritiated choline into phosphatidylcholine (PC) of isolated surfactant material was about twice that with MEM. Deletion experiments evidenced the prominent role of pyruvate, transferrin, and selenium in the stimulation of surfactant PC biosynthesis. The addition of biotin to DM enhanced surfactant PC biosynthesis slightly and nonsurfactant PC biosynthesis markedly. The presence of nucleosides seemed unfavorable to the synthesis of surfactant PC. Type II cells responded to the addition of epidermal growth factor and insulinlike growth factor-I both by increased thymidine incorporation into DNA and choline incorporation into PC. It is concluded that DM represents a useful tool for cultivating type II cells without loss of their specialized properties and for studying the regulation of cell proliferation and surfactant biosynthesis in a controlled environment.     

Growth of human malignant lymphoid cell lines in serum-free medium

Human T lymphoid cell lines (MOLT-4f, MOLT-3, HSB-2, CEM) and human B lymphoid cell lines (BJAB, RAJI, WIL-2) were grown longterm (up to 8 months) in serum-free medium. This medium consisted of Iscove's modified Dulbecco's medium (IMDM), supplemented with bovine serum albumin (BSA) and transferrin (TF). This serum-free medium containing albumin and transferrin is designated AT-IMDM. Lipids were not essential. Cell viability remained high, greater than 80%, in the serum-free medium and the cells maintained their distinctive characteristics. Interleukin-2 (IL-2) production capacity was maintained by the human T lymphoid cell lines JURKAT-77 and MO in short term culture. This simple medium composed of relatively inexpensive and readily available components should be useful for studies of lymphoid cell growth and differentiation and lymphoid cell products.     

Growth of human malignant lymphoid cell lines in serum-free medium

Summary Human T lymphoid cell lines (MOLT-4f, MOLT-3, HSB-2, CEM) and human B lymphoid cell lines (BJAB, RAJI, WIL-2) were grown longterm (up to 8 months) in serum-free medium. This medium consisted of Iscove's modified Dulbecco's medium (IMDM), supplemented with bovine serum albumin (BSA) and transferrin (TF). This serum-free medium containing albumin and transferrin is designated AT-IMDM. Lipids were not essential. Cell viability remained high, greater than 80%, in the serum-free medium and the cells maintained their distinctive characteristics. Interleukin-2 (IL-2) production capacity was maintained by the human T lymphoid cell lines JURKAT-77 and MO in short term culture. This simple medium composed of relatively inexpensive and readily available components should be useful for studies of lymphoid cell growth and differentiation and lymphoid cell products. This research was supported by a grant from the National Institutes of Health, CA 12800, and the Concern Foundation of Los Angeles, and CA 09120 (C. U.)     

Characterization and serial propagation of mouse prostate epithelial cells in serum-free medium

Epithelial cell cultures derived from the ventral prostate of normal adult mice have been propagated in serum-free medium. The cultures were initiated and maintained in Ham's F-12 nutrient mixture supplemented with insulin (5 micrograms/ml), EGF (10 ng/ml), hydrocortisone (0.5 micrograms/ml), cholera toxin (10 ng/ml), bovine pituitary extract (25 micrograms protein/ml) and antibiotics. The cells exhibited microvilli on cell surfaces, interdigitations and junctional complexes including desmosomes between cells, and cytokeratins in cytoplasm which are characteristic of epithelial cells. In addition, the cells exhibited the tissue-specific markers, prostatic acid phosphatase and prostate-specific antigen.     

Growth of seminal vesicle epithelial cells in serum-free collagen gel culture

Summary Epithelial cells from mouse seminal vesicles were enzymatically dissociated enriched by gradient centrifugation, and maintained in collagen gel cultures with defined (serum-free) media. The epithelial origin of the cells was determined morhologically, immunocytochemically, and biochemically. Cells formed three-dimensional colonies with a lumen in collagen gels. Cell number was increased eight-fold within a 8 to 12-d culture period in a medium supplemented with epidermal growth factor (EGF) (10 ng/ml), insulin (10 μg/ml), transferrin (10 μg/ml), cholera toxin (10 ng/ml), and hydrocortisone (0.1 μg/ml). The cells required eGF and insulin; the growth-promoting effects of these two peptide hormones were optimized by transferrin, cholera toxin, and hydrocortisone. Fetal bovine serum did not support growth; rather, it suppressed the stimulated growth observed in serum-free media. A time-course study revealed that a lag period preceded rapi growth. The collagen gel, serum-free culture provides a powerful tool to study the effects of hormones on proliferation and differentiation of androgen sensitive cells.     

Culture of newborn monkey liver epithelial progenitor cells in chemical defined serum-free medium

Studies with hepatic progenitor cells from non-human primates would allow better understanding of their human counterparts. In this study, rhesus monkey liver epithelial progenitor cells (mLEPCs) were derived from a small piece of newborn livers in chemical defined serum-free medium. Digested hepatic cells were treated in Ca²⁺-containing medium to form cell aggregates. Two types of cell aggregates were generated: elongated spindle cells and polygonal epithelial cells. Elongated spindle cells were expressed as vimentin and brachyury, and they were disappeared within 5 d in our cultures. The remaining type consisted of small polygonal epithelial cells that expressed cytokeratin 7 (CK7), CK8, CK18, nestin, CD49f, and E-cad, the markers of hepatic stem cells, but were negative for α-fetoprotein, albumin, and CK19. They can proliferate and be passaged, if on laminin or rat tail collagen gel, to initiate colonies. When cultured with dexamethasone and oncostatin M, the expression of mature hepatocyte markers, such as α-1-antitrypsin, intracytoplasmic glycogen storage, indocyanine green uptake, and lipid droplet generation, were induced in differentiated cells. If transferred onto mouse embryonic fibroblasts feeders, they gave rise to CK19-positive cholangiocytes with formation of doughnut-like structure. Thus, mLEPCs with bipotency were derived from newborn monkey liver and may serve as a preclinical model for assessment of cell therapy in humans.     

Secretion of lysosomal hydrolases by cultured human amnion epithelial cells

Primary microcultures of human amnion epithelial cells were established, starting from sterile term placentae. Over a period of 1 week in culture, the epithelial cells release into the extracellular medium substantial amounts of some lysosomal hydrolases, such as sphingomyelinase, N-acetyl-beta-glucosaminidase, alpha-fucosidase, beta-glucuronidase, alpha-mannosidase, and arylsulfatase. Judging from experiments conducted with the protein synthesis inhibitor, cycloheximide, the enzymes released are not newly synthesized forms, but very likely derive from lysosomes. The constitutive secretion of lysosomal enzymes, coupled with lack of immunogenicity, makes amnion epithelial cells a convenient source of enzymes for implantation in attempts of enzyme replacement therapies.     

Chicken egg yolk-supplemented medium and the serum-free growth of normal mammalian cells

Summary Supplementation of tissue culture medium with chicken egg yolk can support the proliferation of low density bovine vascular and corneal endothelial cells and vascular smooth muscle cells maintained on basement lamina-coated dishes. The optimal growth-promoting effect was observed at concentrations of 7.5 to 10% egg yolk (vol/vol). The average doubling time of bovinn vascular endothelial cells during their logarithmic growth phase when exposed to egg yolk-supplemented medium was longer than that of their counterparts grown in serum-supplemented medium (21 versus 15 h, respectively). Cultures grown in egg yolk-supplemented medium on basement lamina-coated dishes could be serially passaged, but their in vitro life span (15 generations) was less than that of serum-grown cultures (50 generations). The egg white was devoid of any grwoth-promoting activity. This work was supported by Grants HL 20197 and HL 23678 from the National Institutes of Health, Bethesda, MD.     

Growth characterizations of a human monocytic leukemia cell line in a serum-free medium

A clone, AH-01S, derived from a human monocytic leukemia cell line, THP-1, grew rapidly in a serum-free medium containing insulin, transferrin, ethanolamine, and sodium selenite. In batch culture using the serum-free medium, the AH-01S cells proliferated at a specific growth rate (μ) of 0.30 to 0.50 (1/day) from a cell concentration of 1 × 10⁴ cells/ml to 1.6 × 10⁶ cells/ml, an increase of 160 times. A higher cell concentration of 0.45 × 10⁷ cells/ml (cell volume ratio was 0.5%) was obtained in spinner flask culture using the serum-free medium. A mean specific growth rate 0.50 (1/day) was also observed in a culture in a fully instrumented cell culture fermentor. However, μ decreased drastically after the cell concentration reached 1.5 × 10⁶ cells/ml. Analyses of medium composition during cultivation revealed that under lower cell concentration, l-glutamine was the main carbon source while glucose was converted to lactate almost stoichiometrically, and that the production of lactate from glucose decreased at higher cell concentrations. To obtain cultures of 1 × 10⁹ cells, 1,200 to 1,300 mg of a carbon source (glucose) and 400 to 500 of amino acids were consumed during high cell concentration cultivation of the AH-01S cells in the serum-free medium.     

Growth of IM-9 human lymphoblasts in serum-free medium: stimulation by glucocorticoids

The IM-9 human B-lymphoblast cell line grows well in a completely defined serum-free medium containing insulin, transferrin, low density lipoprotein and oleic acid in complex with fatty acid-free bovine serum albumin. Growth of the IM-9 cells is stimulated by addition of physiological concentrations of hydrocortisone to this medium. The order of growth stimulatory potency of several steroids is dexamethasone greater than hydrocortisone greater than aldosterone, whereas testosterone does not stimulate growth of the IM-9 cells. This order of potency suggests that the effect is mediated by binding to glucocorticoid receptors. Growth of the IM-9 cells is also stimulated by the neuropeptide substance P. The defined serum-free medium described in this report will be useful for further studies of the biological responses of the IM-9 cells to other hormones in the absence of interference from hormones and growth factors present in serum.     

Iron absorption by CaCo 2 cells cultivated in serum-free medium as in vitro model of the human intestinal epithelial barrier

A cell culture system consisting of confluent monolayer of human enterocyte-like CaCo 2 cells, cultivated in a serum-free nutritive medium, on microporous synthetic membranes has been used as an in vitro model of the intestinal epithelial barrier. The uptake of ⁵⁵ferric citrate, as well as the transepithelial passage from the apical to the basolateral pole, have been studied. CaCo 2 cells accumulate iron in a time- and concentration-dependent process, largely specific from the apical pole. When ⁵⁵ferric citrate is added at the apical pole, radioiron appears at the basal pole and the clearance rate is ～four times higher than in the opposite direction; the amounts of ⁵⁵Fe increase with the concentration in iron citrate and the duration of incubation. At least two concurrent mechanisms could be involved in iron absorption across monolayers of CaCo 2 cells. A first route would correspond to a paracellular passage of the metal from the apical to the basal pole. The second route would involve a selective intake of iron at the apical pole and could require a reduction of ferric iron, prior to the entry. Iron accumulated by the cells would, for a minor part, be stored within ferritin, whereas the major part would be excreted at the basolateral pole, either as low molecular weight material of undetermined chemical composition but from which iron is easily mobilized by apotransferrin or associated with neosynthesized apotransferrin. Vesicular transport and protein synthesis seem to be required. © 1994 Wiley-Liss, Inc.     

Biosynthesis of proteoglycans by proliferating and differentiating normal human keratinocytes cultured in serum-free medium

Normal human keratinocytes (NHK) were cultured in serum-free medium, containing low (0.1 mM) or high (2 mM) calcium, to obtain proliferating and differentiating cultures, respectively. Proteoglycan (PG) synthesis of proliferating and differentiating NHK was investigated. Cultures were labeled with 35S-sulfate, and the PGs were extracted from medium and cell layer. The newly synthesized PGs were isolated by ion-exchange chromatography on a column of DEAE-Sephacel. The molecular properties of the PGs and the size and composition of glycosaminoglycans (GAGs) were determined. In general, the PGs are relatively small size (Mr 70,000-120,000). The PGs of proliferating cultures are larger in molecular size than the PGs of differentiating cultures, and this is due to the degradation of the GAG chains. The molecular weight of the GAG chains of proliferating NHK ranged from 4,800 to 22,000, and the range for GAGs from differentiating cultures varied from 2,800 to 9,600. By compositional analysis, these PGs proved to contain heparan sulfate, chondroitin sulfate, and dermatan sulfate as determined by nitrous acid degradation, and chondroitinase ACII and ABC digestion. No significant differences were found in the overall GAG composition of the medium secreted PGs of proliferating and differentiating cultures. In contrast, cell-associated PGs of differentiating cells had higher levels of heparan sulfate than those of proliferating cells.     

Production of human immunodeficiency virus from T-lymphoblastoid cells using serum-free medium

Summary A serum-free medium was constructed for the suspension culture of mammalian cells. Effects of the serum-free medium on the cultivation of T-lymphoblastoid cells secreting human immunodeficiency virus (HIV) were observed. The cell growth and HIV production in the serum-free medium were comparable to those in the medium supplemented with 10% fetal bovine serum (FBS), which showed a possibility of economical industrial application.