Immunohistochemical localizations of albumin and transferrin accumulated in Rhodamine fibrosarcoma tissue of rats for supporting growth of the tumor cells

Our recent studies indicated that Rhodamine fibrosarcoma (RdF) tissue of rats accumulated large amounts of albumin and transferrin and that these proteins were essential for growth of RdF cells in primary cultures in serum-free medium. Therefore, the localizations of albumin and transferrin accumulated in RdF tissue were examined by immunohistochemical stainings. Both anti-rat albumin IgG and anti-rat transferrin IgG stained the cell surface of the tumor cells strongly, but the intracellular area only weakly.     

Correlation of the inability to sustain growth in defined serum-free medium with the suppression of tumorigenicity in Wilms' nephroblastoma.

We report the investigation of the growth properties of tumorigenic and reverted nontumorigenic Wilms' nephroblastoma cells when cultured in serum-free medium. Wilms' tumor, a pediatric nephroblastoma, has been associated with deletions encompassing the p13 band of chromosome 11 and an independent loss of heterozygosity at 11p15. Weissman et al. (Science 236:175-180, 1987) transferred a human der(11) chromosome into the G401.6TG.6 Wilms' tumor cell line via the microcell-mediated chromosome transfer technique. The resulting microcell hybrids were nontumorigenic when assayed in nude mice; however these cells retained all of the in vitro growth and morphological characteristics of the tumorigenic parental cells in 10% fetal calf serum (FCS). Segregation of the der(11) chromosome from the nontumorigenic microcell hybrid cells resulted in the reappearance of the tumorigenic phenotype in vivo. In vitro culture of these cell lines in serum-free medium supplemented with 0.1% bovine serum albumin (BSA) and 10 ng/ml Na2O3Se resulted in sustained growth of both the tumorigenic parent and the tumorigenic segregant while the nontumorigenic microcell hybrids were unable to divide. The separate addition of either 10 ng/ml of epidermal growth factor (EGF) or 5 micrograms/ml of insulin did not alter this effect. However, the addition of 5 micrograms/ml of transferrin stimulated the nontumorigenic microcell hybrid cells to grow at a rate comparable to the tumorigenic cells. In addition, conditioned serum-free medium from the tumorigenic parental or tumorigenic segregant cell lines was able to stimulate the growth of the nontumorigenic microcell hybrid cells, whereas the reciprocal experiment had no effect on the growth of the tumorigenic cells. These data suggest that the inability of the microcell hybrid cells to grow in serum-free conditions is correlated with their genetic nontumorigenic phenotype and that a specific growth factor, transferrin, can bypass or alter this negative growth regulatory pathway(s) in vitro.     

Human recombinant insulin-like growth factor I. I. Development of a serum-free medium for clonal density assay of growth factors using balb/c 3T3 mouse embryo fibroblasts

Summary A serum-free clonal density growth assay was developed for the quantification of the biological activity of human recombinant insulin-like growth factor I (IGF-I). The assay measures IGF-I stimulated growth of Balb/c 3T3 cells cultured over 4 d on poly-d-lysine-coated plastic surfaces in a serum-free medium formulation composed of a 1∶1 (vol/vol) mixture of Ham's F12 and Dulbecco's modified Eagle's media, supplemented with 3.0 ng/ml bovine basic fibroblast growth factor (bFGF), 10 μg/ml human transferrin, 100 μg/ml ovalbumin, and 1.0 μM dexamethanose. Low-temperature trypsinization of serum-supplemented stock cultures combined with the use of poly-d-lysine-coated plates made it unnecessary to use serum or fibronectin to promote cell attachment and survival. Serum-free growth conditions were optimized with respect to the concentrations of the supplements. Addition of IGF-I resulted in 3.5-fold more cells than control cultures without IGF-I after 4 d. Deletion of bFGF resulted in no IGF-I stimulation of growth. The concentrations of various preparations of IGF-I required to achieve one-half maximal stimulation of cell number (ED₅₀), ranged between 1.25 and 4.7 ng/ml. In parallel assays, IGF-I was 6.6 times more potent than human recombinant insulin-like growth factor II and 32 times more potent than insulin. When cells were seeded into medium containing IGF-I, transferrin, ovalbumin, and dexamethasone but no bFGF, growth was minimal. Dose-response addition of bFGF showed an ED₅₀, of 0.9 ng/ml. The methods reported are useful to monitor the biological potency of recombinant and natural-source growth factors as well as providing a new means of studying the multiple growth factor requirements of Balb/c 3T3 cells in cultures. This work was supported by a contract from IMCERA Bioproducts, Inc.     

The growth requirements of BHK-21 cells in serum-free culture

A serum-free medium for serial culture of baby hamster kidney cell line 21 (BHK-21) as container-adherent cells was developed. The medium is a 1:1 (v/v) mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with fibroblast growth factor, fibronectin, insulin, oleic acid (preincubated with fatty-acid-free bovine serum albumin as carrier), and transferrin. The fibronectin was required for cell adherence, the other factors for optimal cell multiplication. When cell input was greater than about 1,900 cells/cm², this serum-free medium supported cell multiplication at a rate approximately equal to the rate in medium with 10% serum. At lower cell input, growth in the serum-free medium was poor unless it was supplemented with serum-free medium which had been conditioned by BHK-21 cells. The conditioned medium contained a factor(s) which enabled or stimulated cell multiplication.     

Fibronectin mediates cytokinesis and growth of rat follicular cells in serum-free medium.

In this study, the growth of rat follicular (RF-1) cells was severely depressed when the cells were subcultured by trypsinization directly into serum-free medium supplemented with insulin, transferrin and hydrocortisone, which are required for growth of these cells in vitro. Within 24 hr after plating, 50-65% of the cells became binucleated, indicating lack of cytokinesis. However, the addition of human plasma fibronectin (8 microgram/ml) to the serum-free medium eliminated cell binucleation and enhanced cell growth considerably. Fibronectin had the same effect when RF-1 cells were plated into tissue culture dishes on which fibronectin had been absorbed, and cells were inoculated into fibronectin-free medium. Cell binucleation and poor growth in serum-free medium occurred when the cells were subcultured by trypsinization, EGTA treatment or detachment of mitotic cells. Under some conditions, cells could be "rescued" if fibronectin was added soon after inoculation, indicating that fibronectin was needed mainly during a limited time when the subcultured cells were attaching to the tissue culture substratum. Our findings describe an adhesive activity for fibronectin which circumvents the serum preincubation usually needed after RF-1 cells are subcultured for growth in serum-free medium. They also indicate the importance of fibronectin-mediated adhesion for cytokinesis processes of these cells.     

Growth of human tumor cell lines in transferrin-free,low-iron medium

Summary Iron is essential for tumor cell growth. Previous studies have demonstrated that apart from transferrin-bound iron uptake, mammalian cells also possess a transport system capable of efficiently obtaining iron from small molecular weight iron chelates (Sturrock et al., 1990). In the present study, we have examined the ability of tumor cells to grow in the presence of low molecular weight iron chelates of citrate. In chemically defined serum-free medium, most human tumor cell lines required either transferrin (5 μg/ml) or a higher concentration of ferric citrate (500 μM) as an iron source. However, we have also found that from 13 human cell lines tested, 4 were capable of long-term growth in transferrin-free medium with a substantially lower concentration of ferric citrate (5 μM). When grown in medium containing transferrin, both regular and low-iron dependent cell lines use transferrin-bound iron. Growth of both cell types in transferrin medium was inhibited to a certain degree by monoclonal antibody 42/6, which specifically blocks the binding of transferrin to the transferrin receptor. On the contrary, growth of low-iron dependent cell lines in transferrin-free, low-iron medium (5 μM ferric citrate) could not be inhibited by monoclonal antibody 42/6. Furthermore, no autocrine production of transferrin was observed. Low-iron dependent cell lines still remain sensitive to iron depletion as the iron(III) chelator, desferrioxamine, inhibited their growth. We conclude that low-iron dependent tumor cells in transferrin-free, low-iron medium may employ a previously unknown mechanism for uptake of non-transferrin-bound iron that allows them to efficiently use low concentrations of ferric citrate as an iron source. The results are discussed in the context of alternative iron uptake mechanisms to the well-characterized receptor-mediated endocytosis process.     

Maintenance of growth transformation with Epstein-Barr virus is mediated by secretion of autocrine growth factors in two serum-free B-cell lines.

The characteristics of two tamarin (Saguinus oedipus) B-cell lines (sfBIT and sfBT) growth-transformed by Epstein-Barr virus (EBV) that proliferate continuously in serum-free medium are described. sfBIT was established by selecting cells for growth in RPMI 1640 supplemented with insulin, transferrin, and selenium (J. E. Shaw, R. G. Petit, and K. Leung, J. Virol. 61:4033-4037, 1987). sfBT, a subline of sfBIT cells reported here for the first time, required transferrin as the only protein supplement for continuous growth in RPMI 1640. Growth of sfBT cells was linear with human transferrin at 10(-2) to 10 micrograms/ml. Transferrin at 5 micrograms/ml yielded a culture density of 5 X 10(5) to 1 X 10(6) cells per ml, a cell doubling time of 2 to 3 days, and a culture viability greater than 95%. sfBIT and sfBT cells released transforming virus during continuous growth in serum-free culture medium without EBV-inducing agents. The spent medium of both serum-free lines supported cell growth at low culture density (1 x 10(4) to 5 X 10(4) cells per ml), but growth was arrested at low culture density with fresh serum-free medium. A procedure to measure growth-promoting activity (GPA) was established, and it revealed that the GPA of spent medium was greater than that of fresh medium for both serum-free cell lines. When fresh and spent media were dialyzed (molecular weight cutoff, 3,500) and subsequently concentrated by lyophilization, only the GPA of spent medium increased. We conclude that maintenance of growth transformation of tamarin cells latently infected with EBV is mediated by growth factors that are entirely autocrine in origin.     

Spontaneous interferon production and growth of lymphoblastoid cells in serum-free medium

Numerous lymphoblastoid cell lines were established from human adult peripheral blood and cord blood lymphocytes, using Epstein Barr virus, and most cell lines from cord blood lymphocytes spontaneously produced abundant interferon without induction with Sendai virus, whereas lymphoblastoid cells from adult peripheral blood lymphocytes did not. These potential cells grow well in a newly developed serum-free culture medium based on Dulbecco's modified Eagle medium supplemented with non-essential amino acid, vitamins, nucleic acid derivatives, metal compounds, human transferrin, insulin and bovine or human serum albumin (Chon Fr.V). In serum-free medium, as well as in serum-containing conventional medium (RPMI-1640), the cells could also spontaneously produce interferon. The cells in the serum-free, culture could produce about 10 000 U/ml of interferon every day, harvesting the culture fluid and refeeding the cells with the fresh medium at the saturation cell density (10⁷ cells/ml). The interferon proved to be α-type interferon on the basis of its physico-chemical and antigenic properties.     

The growth requirements of SV40 virus transformed Balb/c-3T3 cells in serum-free monolayer culture

The growth requirements of SV40 transformed Balb/c-3T3 cells have been studied in the absence of serum. For growth in serum-free medium, the cells require (i) insulin, (ii) transferrin, and (iii) cis-unsaturated fatty acids added in combination with fatty acid free bovine serum albumin. The growth rate, saturation density, and morphology of cells grown in this serum-free medium are the same as those of cells grown in serum supplemented medium. This mixture also supports the growth of SV40 transformed Swiss-3T3 cells and SV40 transformed primary mouse embryo cells, but does not support the growth of untransformed Balb/c-3T3 cells. The addition of fibronectin to this mixture allows routine subculture, repeated passage, and indefinite propagation of SV40 transformed Balb/c-3T3 cells. Cells grown in this medium for a period of two months retain their ability to induce tumors when injected into athymic nude mice.     

Development of a serum-free medium which supports the long-term growth of human and nonhuman primate lymphoid cells

The ability to grow lymphoid cells in serum-free media affords the advantage of separately analyzing those components found to be involved in proliferation and differentiation. Iscove's medium (IMDM) supplemented with bovine serum albumin or casein, cholesterol, ferrous chloride, insulin, β-mercaptoethanol, L-α-phosphatidylcholine, and transferrin supported the long-term proliferation of a gibbon ape lymphoma T-cell line, MLA144. These cells continue to produce Interleukin 2 (IL-2, T-cell growth factor) constitutively in the serum-free medium. IL-2-dependent human T cells initiated and maintained in culture in serum-free medium containing IL-2 have continued to replicate for over 3 months with two population doublings every 3 to 4 days. A normal, IL-2-dependent marmoset T-cell line, OH-1, also proliferated on the serum-free medium when supplemented with IL-2. Several established primate B-cell lines which do not require IL-2 for growth were able to proliferate in the serum-free medium. These B-cell lines included B95-8, an Epstein-Barr virus (EBV)-transformed marmoset cell line, HuCo/R-H, a human cord B-lymphocyte line transformed with EBV, and Namalwa, an EBV-positive B-cell line established from a Burkitt's lymphoma. B95-8 cells grown on serum-free medium showed high levels of EBV antigen-positive cells after induction with 12-O-tetradecanoyl-phorbol-13-acetate (TPA).     

Role of bovine albumin in a serum-free suspension cell culture medium.

A serum-free culture medium, supplemented with 1% bovine serum albumin, supported the growth of both primary and continuous suspension-type cultures of various mammalian tumor cells. The role of albumin added to the medium was also studied. Defatted albumin failed to support cell growth, unless reconstituted with its lipid extract. Similarly, defatted albumin when combined with oleic and linoleic acids, also supported cell growth. Therefore, albumin-bound fatty acids play an important growth-promoting role in serum-free medium.     

Establishment of a macrophagelike cell line derived from U-937, human histiocytic lymphoma,grown serum-free

Summary A human macrophagelike cell line which grows in serum-free medium was established from a histiocytic lymphoma cell line, U-937. U-937 cells failed to differentiate into macrophagelike cells in serum-free medium plus 12-O-tetradecanoyl phorbol 13-acetate (TPA). Fibronectin and albumin in serum were necessary for differentiation of U-937 ceds into macrophagelike cells in enriched RDF medium supplemented with insulin, transferrin, ethanolamine, selenite, egg yolk lipoprotein (eRDF-ITESL medium). The established cell line exhibited several characteristic properties of macrophage such as nitroblue tetrazolium reduction, phagocytic and α-naphthylbutyrate-esterase activities, and tumor necrosis factor and interleukin 1 production. At present the cells have been continuously maintained in eRDF-ITESL medium through over 150 passages.     

Growth of normal human amnion epithelial cells in serum-free medium

The serum-free growth of primary cultures of normal human epithelial-like cells from amniotic membranes was accomplished. The synthetic medium consists of a 1 : 1 basal nutrient mixture of Dulbecco's modified Eagle medium (DMEM) and Ham's F-12 supplemented with 2.5 μg/ml insulin, 50 ng/ml epidermal growth factor (EGF), 5 μg/ml transferrin, and 0.1 ng/ml triiodothyronine (T₃). EGF is the primary mitogen and is essential for cell proliferation in this system.     

Three-dimensional culture of human tumor cells under standardized medium conditions

The 3-dimensional culture of human tumor spheroids under standardized medium conditions may reveal information on specific biological parameters that could be masked in serum-supplemented media. Spheroids derived from human tumor cells are growth retarded in media free of serum. Ex-Cyte IV is a substance derived from human blood that can be used to improve growth in tissue culture. In this study the growth of spheroids from four different human tumor cell lines was studied when grown in medium free of serum, medium supplemented with varying concentrations Ex-Cyte IV, and medium supplemented with foetal calf serum (FCS). The parameters used for comparisons were growth rate, growth enhancement, clonogenicity and cell cycle distribution.The four cell lines showed different growth rates in serum-free medium, which were increased to different extents when Ex-Cyte IV or FCS were added. The growth enhancing effect induced by Ex-Cyte IV was differently concentration dependent for each cell line. The clonogenicity of cells grown as spheroids in serum-free medium was lower than in spheroids grown in supplemented media. There was no difference in clonogenicity between the differently supplemented media. All four cell lines responded to growth in serum-free medium with a drop in the S-phase and G2M phase.The present study provides a novel approach to the study of human tumor cells in 3-dimensional culture under defined conditions. The human serum derived substance Ex-Cyte IV may provide a method to obtain information on specific biological parameters that could be masked in serum-supplemented media.     

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.     

Serum-free primary culture of human normal mammary epithelial cells in collagen gel matrix

Collagen gel matrix has been used successfully to promote sustained growth of human normal mammary epithelial cells in primary culture using serum-containing medium supplemented with hormones and growth factors (Nandi et al., 1932). Sustained growth can now be accomplished in a serum-free medium consisting of a 1:1 mixture of Ham's F12 and DMEM supplemented with insulin, transferrin, epidermal growth factor, cholera toxin, cortisol, and BSA. Human normal mammary epithelial cells derived from reduction mammoplasties can be routinely propagated in this serum-free medium. The extent of growth and the resulting three-dimensional outgrowths in this serum-free medium, using the collagen gel matrix system, are comparable to those seen in serum-containing medium. This is the first demonstration of sustained growth of human normal mammary epithelial cells in serum-free primary culture.     

Continuous culture of rat C6 glioma in serum-free medium

In this communication we describe serum-free culture conditions for the serial propagation of the C6 glioma cell line. The growth rate, saturation density, and morphology of these cells are equivalent to those of their serum-grown counterparts when cultured in a 3:1 mixture of Dulbecco's modified Eagle's medium and Ham's medium F-12 supplemented with trace elements, insulin, transferrin, fibroblast growth factor, linoleic acid complexed to fatty acid-free bovine serum albumin, and a serum-spreading factor (SSF) partially purified from human plasma. The requirement for SSF in the medium can be satisfied by preincubating the tissue culture dishes with SSF. Tissue culture dishes sequentially pretreated with poly-D-lysine and purified cold insouluble globulin will also substitute for this requirement. The fatty acid-free bovine serum albumin/linoleic acid complex increases the growth rate of these cells but has no appreciable effect on their morphology, saturation density, or ability to grow with repeated subculture. The growth stimulation caused by this complex appears to be dependent on the fatty acid, as the fatty acid-free bovine serum albumin alone has no effect on the growth rate. Linoleic acid is cytotoxic in the absence of bovine serum albumin, and the fatty acid-free bovine serum albumin prevents this toxicity. Other fatty acids including oleic, arachidonic, and palmitic only partially substitute for the growth-promoting effect of linoleic acid.     

C6 glioma cells produce basic fibroblast growth factor that can stimulate their own proliferation

With purified preparations of basic fibroblast growth factor (bFGF), we studied the effect of its growth-promoting activity on C6 glioma cells. We also examined with its antibody whether the cultured glioma cells could produce it. It was shown that bFGF stimulated the DNA synthesis and proliferation of C6 glioma cells in serum-free medium, and that the activity was potentiated by heparin, the bFGF concentrations for half-maximal stimulation being 0.2 and 5 ng/ml in the presence and absence of heparin, respectively. This effect of heparin was dose-dependent and was half-maximal at 0.5 microgram/ml. Next, we raised the antiserum against bFGF and detected a single immunoreactive band from extracts of C6 glioma cells by immunoblot analysis. The immunoreactive substance was partially purified on a heparin-Sepharose column and was shown to stimulate the DNA synthesis of C6 glioma cells. On the basis of its immunoreactivity, molecular weight, affinity for heparin, and growth-promoting activity, this substance was identified as bFGF. The content of bFGF in the cells was elevated as the cell density increased, but no immunoreactivity was detected in the conditioned medium of the cells. These results suggest that C6 glioma cells produce and store bFGF which is potent in stimulating their own growth.     

Regulation of growth and differentiation of a rat hepatoma cell line by the synergistic interactions of hormones and collagenous substrata

Serum-free, hormonally defined media have been developed for optimal growth of a rat hepatoma cell line. The cells' hormonal requirements for growth are dramatically altered both qualitatively and quantitatively by whether they were plated onto tissue culture plastic or collagenous substrata. On collagenous substrata, the cells required insulin, glucagon, growth hormone, prolactin, and linoleic acid (bound to BSA), and zinc, copper, and selenium. For growth on tissue culture plastic, the cells required the above factors at higher concentrations plus several additional factors: transferrin, hydrocortisone, and triiodothyronine. To ascertain the relative influence of hormones versus substratum on the growth and differentiation of rat hepatoma cells, various parameters of growth and of liver-specific and housekeeping functions were compared in cells grown in serum-free, hormonally supplemented, or serum-supplemented medium and on either tissue culture plastic or type I collagen gels. The substratum was found to be the primary determinant of attachment and survival of the cells. Even in serum-free media, the cells showed attachment and survival efficiencies of 40-50% at low seeding densities and even higher efficiencies at high seeding densities when the cells were plated onto collagenous substrata. However, optimal attachment and survival efficiencies of the cells on collagenous substrata still required either serum or hormonal supplements. On tissue culture plastic, there was no survival of the cells at any seeding density without either serum or hormonal supplements added to the medium. A defined medium designed for cells plated on tissue culture plastic, containing increased levels of hormones plus additional factors over those in the defined medium designed for cells on collagenous substrata, was found to permit attachment and survival of the cells plated into serum-free medium and onto tissue culture plastic. Growth of the cells was influenced by both substrata and hormones. When plated onto collagen gel substrata as compared with tissue culture plastic, the cells required fewer hormones and growth factors in the serum-free, hormone-supplemented media to achieve optimal growth rates. Growth rates of the cells at low and high seeding densities were equivalent in the hormonally and serum-supplemented media as long as comparisons were made on the same substratum and the hormonally supplemented medium used was the one designed for that substratum. For a given medium, either serum or hormonally supplemented, the saturation densities were highest for tissue culture plastic as compared with collagen gels.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.