Effects of defined medium,fetal bovine serum,and human serum on growth and chemosensitivities of human breast cancer cells in primary culture: Inference for in vitro assays

Summary We compared the effects of defined medium, fetal bovine serum (FBS) and human serum (HuS) on the growth and responses to chemotherapeutic agents of human breast cancer cells in primary culture. Normal and tumor tissues were dissociated to small aggregates and single cells and seeded onto collagen-gel-coated wells in defined medium or medium supplemented with 5% FBS or 5% HuS. In all cases examined, defined medium and medium containing HuS were superior to medium containing FBS in supporting growth of both normal and tumor cell cultures. However, cultures in defined medium showed an initial cell loss. Cells from the same tumor cultured in different media varied in their responses to chemotherapeutic agents. In light of these results, medium supplemented with HuS, which promoted attachment of these cells in culture and stimulated their growth, should be the most appropriate nutrient environment for determining the effects of therapeutic agents on cells as it most closely resembles the in vivo situation. Because there were also variations in growth rates and chemosensitivities of tumor cells cultured in different human serum samples, we suggest that optimal conditions in which to culture these cells include the serum of the patient whose tumor is removed. This serum may provide host factors that influence cell growth and interact with exogenous factors. This work was supported by a grant from the National Cancer Institute of Canada and funds contributed by Mr. B. T. Wharton in memory of his wife, Nadia. J. T. Emerman is a research scholar of the National Cancer Institute of Canada.     

Rapid determination of ethylene glycol at toxic levels in serum and urine

The rapid gas chromatographic detection and determination of ethylene glycol in biological fluids is described. Phenylboronic acid in acetone was used for the esterification of glycol. The phenylboronates of ethylene glycol and 1,2-propylene glycol are not separated on a packed column of medium polarity (OV-17), but they can be separated on a non-polar column (OV-101). In both instances 1,3-propylene glycol can be used as an internal standard. The method requires only 100 μl of serum or urine and is suitable for trace analysis in an emergency toxicological laboratory. The utility of the method is demonstrated on two cases of human intoxication with ethylene glycol.     

Replacement of serum with a defined medium increases β-adrenergic receptor number in cultured glioma cells

β-Adrenergic receptors of C6 glioma cells were compared following growth in serum or in a serum-free defined medium. In the absence of serum there was an increase in β-receptor number by approx. 50% as measured by the binding of [¹²⁵I]iodohydroxybenzylpindolol. Other receptor properties, such as the affinities for β-adrenergic agonists and antagonists, were not different in the two growth conditions. The alteration in receptor number took over 3 days to occur and was not readily reversible. The increase in β-adrenergic receptor number of cells grown in a serum free medium indicates that replacement of serum with a defined medium is able to alter the expression of at least one cell surface marker, the β-adrenergic receptor. These results imply that growth of cells in defined media yields cells with properties of the plasma membrane that differ from those of cells grown in serum-containing media.     

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 Im-9 Human Lymphoblasts In Serum-Free Medium: Stimulation By Glucocorticoids

Abstract. 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 > hydrocortisone > 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.     

Direct analysis of growth factor requirements for isolated human fetal hepatocytes

Summary Hepatocytes were isolated from human fetal liver in order to analyze the direct effects of growth factors and hormones on human hepatocyte proliferation and function. Mechanical fragmentation and then dissociation of fetal liver tissue with a collagenase/dispase mixture resulted in high yield and viability of hepatocytes. Hepatocytes were selected in arginine-free, ornithine-supplemented medium and defined by morphology, albumin production and ornithine uptake into cellular protein. A screen of over twenty growth factors, hormones, mitogenic agents and crude organ and cell extracts for effect on the stimulation of hepatocyte growth revealed that EGF, insulin, dexamethasone, and factors concentrated in bovine neural extract and hepatoma cell-conditioned medium supported attachment, maintenance and growth of hepatocytes on a collagen-coated substratum. The population of cells selected and defined as differentiated hepatocytes had a proliferative potential of about 4 cumulative population doublings. EGF and insulin synergistically stimulated DNA synthesis in the absence of other hormones and growth factors. Although neural extracts enhanced hepatocyte number, no effect on DNA synthesis of neural extracts or purified heparin-binding growth factors from neural extracts could be demonstrated in the absence or presence of defined hormones, hepatoma-conditioned medium or serum. Hepatoma cell-conditioned medium had the largest impact on both hepatocyte cell number and DNA synthesis under all conditions. Dialyzed serum protein (1 mg/ml) at 10 times higher protein concentration had a similar effect to hepatoma cell-conditioned medium (100 μg/ml). The results suggest that hepatoma cell conditioned medium may be a concentrated and less complicated source than serum for purification and characterization of additional normal hepatocyte growth factors. This work was supported by NIH grant DK35310. Editor’s statement Many investigators have struggled with the special problems associated with culture of differentiated hepatocytes. In this paper attention is given to the specific growth factor requirements for fetal human hepatocytes. The observation that factors from hepatoma conditioned medium or neural extracts enhanced the growth of the cells may indicate that additional growth factors are to be identified that are important in the survival and proliferation of hepatocytes, and may also indicate that the malignant transformation of these cells may involve the production of autocrine growth stimulators.     

CULTIVATION OF MAMMALIAN CELLS IN DEFINED MEDIA WITH PROTEIN AND NONPROTEIN SUPPLEMENTS

An improved chemically defined basal medium (CMRL-1415) has been used to advantage in studying the effects on trypsinized, newly explanted mouse embryo cells of certain glycoproteins from plasma and serum, certain nonprotein macromolecules, and various combinations of these, in stationary cultures. When protein and nonprotein fractions were separated from fetal calf serum, the entire growth activity was found to be associated with the protein. When 100 mg % of dialyzed, freeze-dried, supernatant solution of Cohn's fraction V (method 6) from human plasma was used as a supplement for CMRL-1415, there was considerable improvement in the cultures; and seromucoid prepared from calf serum had a similar effect. Supernatant V was further fractionated by gel filtration to give a threefold concentration of growth activity in a single, highly purified α₁-acid glycoprotein (orosomucoid). Starch gel electrophoresis of horse serum that was used to supplement the basal medium revealed a decrease of both α₁-acid glycoprotein and α₂-macroglobulin during the cultivation of mouse embryo cells. When horse serum was fractionated on DEAE-cellulose columns, the only fraction that showed growth activity was a slow α₂-globulin. When the α₂-macroglobulin of Schultze was prepared from horse serum by salt precipitation, it was equally effective. When the α₂-macroglobulin from horse serum was tested (at 100 mg %) in combination with α₁-acid glycoprotein from Supernatant V, seromucoid from calf serum, or unfractionated Supernatant V, the growth response was greatly in excess of that produced by any of these supplements tested separately. The α₂-macroglobulin from horse serum could be replaced by certain nonprotein macromolecules (e.g., dextran or Ficoll). Thus, dextran (mol. wt. 100,000 to 200,000) had no visible effect on the cells when used alone at 0.1 or 1%. But when these levels of dextran were used in combination with low molecular weight glycoproteins (e.g., unfractionated Supernatant V at 100 mg %), the cultures remained active and healthy for unusually long periods.     

Improvement of a cytokine (TNF-α) bioassay by serum-free target cell (WEHI 164) cultivation

The elaboration of a sensitive bioassay for assessment of tumour necrosis factor alpha (TNF-α) in a defined medium is described. The assay is based on the cytotoxic effect of TNF-α on a target cell line, the murine fibrosarcoma WEHI 164 clone 13. Cytotoxicity was assessed by detecting the rate of tetrazolium salt reduction employing a spectrophotometer (ELISA-reader). A similar bioassay was used previously to assess TNF-α, though this was dependent on cell growth in a medium containing serum. By employing a synthetic serum replacement, the WEHI cells were adapted to growth in a defined medium which allowed both the propagation of the cell line and the assay to be performed under completely defined conditions. Thus, factors in serum that may influence the TNF-α assessment, such as growth factors, cytokines, soluble cytokine-receptors and macroglobulin, were avoided. The only protein required in this bioassay was insulin, while albumin was added as a carrier protein and to protect the cytokine against loss of biological activity during multiple freeze and thaw cycles. The present assay was optimised to achieve a high sensitivity and, by testing endogenous TNF-α originating from the macrophage-like cell line RAW in both the serum-free and serum-based assay, we found the highest sensitivity in the assay based on defined medium. The LC50 of recombinant mouse and human TNF-α were in the serum-free and serum-based assays considered to be 25 and 50 pg mL-1, respectively. The demonstration of a culture condition that enables long-term cultivation of target cells and a bioassay in a completely defined medium is in our opinion a substantial contribution to more reliable cytokine assessment. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Routine culturing of normal,dysplastic and malignant human mammary epithelial cells from small tissue samples

Summary We compared the growth and morphology of normal, dysplastic and malignant human mammary epithelial cells (HMEC) in medium containing 5% human serum, a serum-free medium (32) and serum-free medium with a low Ca⁺⁺ concentration. Tissues were dissociated and epithelial organoids or single cells were seeded onto collagen-coated dishes. The cells grew in serum-containing medium, but growth of fibroblasts was also stimulated. The serum-free medium consistently selected for and stimulated the growth of epithelial cells. There was little advantage in reducing the Ca⁺⁺ concentration to further increase cell yield. This serum-free primary culture system allows us to routinely prouce sufficient numbers of HMEC from small tissue samples for molecular biological investigations. Furthermore, the maintenance of cells in a defined medium can provide a system for evaluating the direct effects of factors on gene expression. This work was supported by a grant from the National Cancer Institute of Canada and funds contributed by Mr. B. T. Wharton in memory of his wife, Nadia.     

Anchorage-independent growth of normal human mesothelial cells: A sensitive bioassay for EGF which discloses the absence of this factor in fetal calf serum

Summary This laboratory recently reported that normal human mesothelial cells require epidermal growth factor (EGF) and hydrocortisone (HC), in addition to fetal calf serum and a complex defined medium component, in order to grow optimally in surface culture (9). We report here that this normal cell type also forms large colonies at high efficiency in semi-solid medium, but exhihits more stringent serum and EGF requirements for anchorage-independent than for surface growth. Mesothelial cells are unable to divide at all in semi-solid medium with added EGF or with less than 2% serum, whereas they grow slowly but progressively in surface culture under such conditions. In semi-solid medium containing 20% serum and HC, mesothelial cells are stimulated to divide by the addition of as little as 30 pg/ml purified EGF. Human urine or male mouse plasma could substitute for purified EGF, yielding growth commensurate with the levels of EGF in these biological fluids previously measured by others using radioreceptor and radioimmune assays. Thus growth of mesothelial cells in semi-solid medium can serve as a highly sensitive assay of EGF biological activity which is unaffected by the presence of serum proteins. In addition, our results demonstrate that fetal calf serum does not provide mitogenic levels of EGF to cultured cells, raising the question of the identity of plasma and serum mitogens. This work was supported by NIH grants RO1 AG02048 and RO1 CA26656 to James G. Rheinwald and by NIH postdoctoral fellowship F32 AG05303 to Paul J. La Rocca.     

Detection and partial purification of glial growth inhibitory factor(GGIF) in the conditioned medium of neuroblastoma cells

Glial growth inhibitory factor (GGIF) was detected in the culture medium of mouse neuroblastoma cells. The partial purification and characterization of GGIF revealed that it was an acidic and heat-unstable protein and separated into two molecular weight forms (GGIF1: 33.0 to 40.5 K Mr; and GGIF2: 31.0 to 34.0 K Mr) on DEAE. Sephacel column chromatography. GGIF inhibited the growth of neoplastic glial cells as well as normal glioblasts in monolayer culture. The inhibitory effect of GGIF on proliferation of glioblasts appeared even after the incubation for less than 1 h, and retained thereafter unless the factor was removed. Immobilized GGIF exposed to glioblasts was reusable, suggesting the existence of a certain component or receptor mediating GGIF action in the plasma membrane.     

Human megakaryocytic progenitors (CFU-M) assayed in methylcellulose: Physical characteristics and requirements for growth

The basic culture requirements and several physical characteristics were defined for megakaryocytic colony-forming cells (CFU-M) from normal human marrow growing in methylcellulose. Ficoll-hypaque separated mononuclear cells from human, marrow gave rise to megakaryocytic colonies in the presence of normal human plasma and phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM). Their identity as megakaryocytic colonies was confirmed by immunofluorescence staining with a monoclonal antibody to human factor VIII antigen and by electron microscopy of individually harvested colonies. Demonstration of the single-cell origin of the colonies was provided by analysis of the glucose-6-phosphate dehydrogenase (G-6-PD) enzyme type of individually harvested colonies grown from a G-6-PD heterozygote. The colonies grew best in heparinized or citrated plasma as opposed to serum. Detailed studies suggested that platelet-release products were responsible for this difference. Tritiated thymidine suicide studies showed that the percentage of CFU-M in DNA synthesis was 23 ± 8% (n = 10). The modal velocity sedimentation rate of CFU-M was 4.9 ± 0.6 mm/hr (n = 4) while that of concurrently studied granulocyte/macrophage colony-forming cells (CFU-GM) was 5.7 ± 0.5 mm/hr. Examination of the PHA-LCM dose-response characteristics suggested the presence in the conditioned medium of an inhibitor to megakaryocyte colony growth which was partially removed by chromatography of the medium on Sephadex G-100. The resulting conditioned medium increased the cloning efficiency for CFU-M compared with that with crude PHA-LCM (15.3 ± 7.0 and 8.2 ± 5.3/10⁵ marrow cells, respectively).     

Development of improved media and culture conditions for clonal growth of normal diploid cells

Multiplication of normal diploid cells in culture is controlled by a complex set of interacting extracellular variables. The amount of serum protein needed for colony formation by such cells is affected directly by many of the other variables, including the nature of the culture surface, the type of trypsinization procedure used, and the qualitative and quantitative composition of the culture medium. By a sequential process of adjusting all of these variables to optimum values for cellular multiplication with minimal amounts of serum protein, we have been able to obtain clonal growth of normal human and chicken cells with less than 500 microgram per ml dialyzed serum protein. Precise quantitative adjustment of nutrient concentrations is particularly important. The multiplication-promoting functions of serum can be classified operationally as "replaceable" (those that can be replaced by modifying the medium or the culture conditions) and "nonreplaceable" (those that we have not yet been able to replace). Elimination of the requirement for replaceable functions of serum has improved greatly the specificity and sensitivity of the bioassay for the nonreplaceable functions. The nonreplaceable multiplication-promoting activity from fetal bovine serum for human diploid fibroblasts has been separated from fetuin and serum albumin and purified approximately 15-fold.     

Growth limitation in hybridoma cell cultures: The role of inhibitory or toxic metabolites

Hybridoma cells usually grow to fairly low cell densities in batch cultures (1–3×10⁶ cells/ml). The reason for this is either that essential nutritional components of the medium are consumed, or that the cells produce some kind of inhibitory or toxic metabolite. This investigation presents evidence for the latter. Spent medium from cultures of hybridoma cells did not support growth of cells at lower cell densities (1–3×10⁵ cells/ml). The ability to support cell growth could not be restored by adding additional serum, energy sources (glucose, pyruvate) or L-glutamine. Furthermore, the consumption of amino acids could not account for this growth inhibition. On the contrary, the spent medium contained a substance that inhibited cell growth. This substance or metabolite was found in a fraction eluted from a gel filtration column when spent medium was applied to the column. This substance was found in the spent medium from all hybridoma and myeloma cell lines that were tested. The molecular weight of the substance was about 5 kD. Spent medium from two hybridoma cell lines also contained a substance that was eluted in the same fraction as albumin (67 kD). It is likely that this (or these) substance(s) is responsible for the growth limitation in hybridoma cell cultures.Abbreviations PBS phosphate buffered saline     

Theileria parva: isolation of macroschizonts from in vitro propagated lymphoblastoid cells of cattle

A method for purifying macroschizonts of Theileria parva from bovine lymphoblastoid cells, propagated in vitro, was developed. This method involved three steps. First, the macroschizonts were liberated by disrupting host cells suspended in growth medium at 4 × 10⁶ cells/ml at 300–400 psi, using the Stansted cell disrupter. This yielded 80–90% disrupted cells while causing minimum damage to the macroschizonts. Second, the host cell nuclei were separated by (a) centrifuging the lysate at 300g for 60 min, (b) resuspending the pellet in 0.02 times the volume of initial host cell suspension in Leibovitz's L15 growth medium, and (c) lysing the host cell nuclei by adding nucleus-lysing buffer (NLB, containing 0.14 M Tris, 0.1 M HCl, 0.12 M glucose, and 0.5 M NaCl adjusted with NaOH to pH 7) to 0.2 times the volume of initial host cell suspension. The resulting chromatin precipitate was removed by adding DE-52 cellulose equilibrated with NLB and allowing the precipitate to sediment. Lastly, the final suspension obtained in the second step was applied on a DE-52 cellulose column which was equilibrated with the elution buffer (NLB with 10% fetal, or newborn, bovine serum, pH 7). Macroschizonts free of intact host cells and naked host cell nuclei were collected in the eluate. The protein yield was 2.7 mg per 10⁹ starting undisrupted host cells, which was 1.7% of the total starting protein.     

Doubling potential, calendar time, and senescence of human diploid cells in culture

Human diploid cells (CF-1) derived from newborn foreskin tissue were maintained in a non-mitotic state for as long as 177 days by reducing the serum concentration of the incubation medium to 0.5%. The cells could be returned to the proliferative state by subcultivation with normal growth medium containing 10% serum. Cells treated in such a manner reached passage levels equivalent to controls that had been continuously cultured on growth medium, but they took a proportionately longer calendar time to achieve the equivalent passage levels. Also, by using ³H-thymidine incorporation, cells held in the non-mitotic conditions showed a longer ‘predictable life span’ than control cultures. During 21-day maintenance periods there was a 10–20% cell loss and ca 30% loss of protein per cell. The finite life span of these human diploid cells was clearly related to the number of cumulative population doublings rather than to the total calendar time in vitro.     

The effect of serum albumin and the effect of cell concentration on the in vitro growth of mouse and rat lymphocytes.

The growth of mouse and rat T and B lymphocytes, activated by concanavalin A or lipopolysaccharide, is increased over growth in protein-free medium 5–20-fold by human or bovine serum albumin. The growth is dependent on the concentration of albumin. At a concentration of 2–4 mg/ml the growth rate is the same as in the presence of 10% fetal bovine serum. Of the other serum proteins (Cohn fractions) only human fraction VI supports growth somewhat while human fractions II–IV and bovine fraction VI do not support growth. The growth of mouse and rat lymphocytes is greatly suppressed if lymphocytes are cultured at high cell concentrations, and the growth-promoting ability of serum albumin cannot be detected under such conditions. The growth rate can be improved by daily adjustment of the pH, by daily refeeding, and by daily change of medium. The growth inhibitory activity can be removed largely by dialysis. It is concluded that the suppression of growth at high cell concentrations is caused by a combination of effects, i.e., a shift of pH, lack of nutrients, and accumulation of cellular metabolites.     

Regulation of endothelial cell DNA synthesis and adherence

Summary A defined medium has been developed for primary culture of cells from human umbilical vein that will support maximal levels of cell division. The role of medium components in regulating the amount of thymidine incorporation has been assessed; insulin and to a lesser extent fibroblast growth factor (FGF) both increased the rate of incorporation when hydro-cortisone (HC) was present in the medium. Although these hormones in nonserum medium can stimulate incorporation, plating and maintenance of cells in serum medium for 12 h is necessary before transfer to defined medium. Without serum for this period, cells placed in defined medium, though well attached, did not divide. From the pulse: chase experiments it appears that more than one round of replication was supported by the 12-h period in serum. The role of various agents in regulating cell adhesion also was assessed. Factors precent in serum but not in platelets appear active. Cold insoluble globulin (CIG) is an active serum component inasmuch as it caused adherence when added to defined medium. However, other serum components were highly effective in promoting adhesion in the absence of CIG. Insulin also induced adhesion in nonserum medium though to a smaller extent; its effect was enhanced by plating cells on collagen. Hydrocortisone potentiated the effect of insulin and caused enhanced cell spreading in serum or CIG containing medium but not other medium. All well-spread cells were capable of fibronectin (FN) synthesis whether in serum or nonserum medium. Neither insulin nor HC stimulated fibronectin synthesis. This research was supported by a grant from the American Diabetes Association.     

Expression of human insulin-like growth factor I in bacteria: use of optimized gene fusion vectors to facilitate protein purification

Several fusions between the gene for human insulin-like growth factor I (IGF-I) and the genes for different IgG-binding fragments of staphylococcal protein A were assembled and compared regarding expression, secretion, and purification of the peptide hormone. After IgG affinity purification of the fusion proteins from the growth medium of Staphylococcus aureus or Escherichia coli, native IGF-I was released by cleavage of an Asn-Gly peptide bond with hydroxylamine. An optimized expression system based on a modified synthetic IgG-binding domain (z), resistant to hydroxylamine, gave the highest yield of fusion protein. After cleavage, the hormone could be separated from the IgG-binding moiety and from noncleaved fusion protein by a second passage through the IgG affinity column. The biological activity and the purity of the IGF-I obtained were confirmed by a radioreceptor assay, N-terminal sequence analysis, polyacrylamide gel electrophoresis, isoelectric focusing, and high-performance liquid chromatography.