INHIBITION OF MALIGNANT CELL PROLIFERATION BY CULTURE MEDIA CONDITIONED BY CARDIAC OR SKELETAL MUSCLE

The present work is an attempt to explain the high resistance of muscles to cancer development. We used primary cultures of rat skeletal and cardiac muscle, and examined the effect of the supernatant of these cultures (conditioned medium; CM) on proliferation of cancer cells. The results demonstrated that CM inhibited the proliferation of several types of malignant cells by more than 50%, without a significant inhibition on normal cells. Cell cycle analysis revealed that CM increased the number of cells in S and G2 phases, suggesting a cytostatic effect of CM. For defining the biological properties of the factor(s) which are present in the CM, skeletal muscle cultures were grown in chemically defined medium (serum free medium). The concentrated sample was applied to a Sephadex G-50 column and three fractions were obtained. Only one fraction showed inhibitory activity. Four protein bands were observed in this fraction, as revealed by SDS-PAGE. We suggest that some, or all of these proteins are responsible for inhibition of tumor cell replication.     

A serum-free, chemically-defined medium for function and growth of primary neonatal rat heart cell cultures

A serum-free, chemically defined medium for supporting rhythmic contraction, maximum survival, and moderate growth of cardiac cells was achieved by using a combination of hormones and growth supplements in a mixture of equal volumes of Ham's F12 and Dulbecco's modified Eagle's medium. The hormones and growth supplements included insulin, transferrin, selenium, fetuin, bovine serum albumin, hydrocortisone (HC), L-thyroxine (T4), and epidermal growth factor (EGF). Cardiac cells were grown on fibronectin-precoated plates using the above serum-free medium. Cells grown in this medium exhibited a higher beating rate and were maintained for a longer time compared to those cells grown in serum. The effects of T4, EGF, and HC on beating rate and survival time of both cultures of mixed cell population and enriched myoblast cell population were studied. In the enriched myoblast cell cultures grown in serum-supplemented medium, the beating rate ranged from 40 to 200 beats/min, and these cultures survived for 30 d. When these enriched cell cultures were grown in serum-free hormone-supplemented medium, the beating rate ranged from 190 to 240 beats/min, and these cultures survived for more than 90 d. These results show that some hormones affect growth, whereas others affect function.     

A serum-free,chemically-defined medium for function and growth of primary neonatal rat heart cell cultures

Summary A serum-free, chemically defined medium for supporting rhythmic contraction, maximum survival, and moderate growth of cardiac cells was achieved by using a combination of hormones and growth supplements in a mixture of equal volumes of Ham’s F12 and Dulbecco’s modified Eagle’s medium. The hormones and growth supplements included insulin, transferrin, selenium, fetuin, bovine serum albumin, hydrocortisone (HC),l-thyroxine (T₄), and epidermal growth factor (EGF). Cardiac cells were grown on fibronectin-precoated plates using the above serum-free medium. Cells grow in this medium exhibited a higher beating rate and were maintained for a longer time compared to those cells grown in serum. The effects of T₄, EGF, and HC on beating rate and survival time of both cultures of mixed cell population and enriched myoblast cell population were studied. In the enriched myoblast cell cultures grown in serum-supplemented medium, the beating rate ranged from 40 to 200 beats/min, and these cultures survived for 30 d. When these enriched cell cultures were grown in serum-free hormone-supplemented medium, the beating rate ranged from 190 to 240 beats/min, and these cultures survived for more than 90 d. These results show that some hormones affect growth, whereas others affect function.     

Cytotoxicity of thermally oxidized fats

Summary The effects of oxidized fat components (free fatty acids from the distillable nonurea adductable fraction) isolated from heated corn oil or heated olive oil on the morphology and growth of heart cells in primary culture were investigated. The free fatty acid fractions isolated from the fresh fats served as controls. Different concentrations of the fat fractions (20, 60, and 100 μg/ml) were added to the medium in the form of an emulsion with bovine serum albumin (Fraction V, poor in unesterified fatty acids). In the cell cultures treated with heated fats, intracellular lipid accumulation, increased cytoplasmic vacuolization, mitotic aberrations, pyknotic cells, and decreased mitosis were observed and were more pronounced in the case of the heated olive oil. These cytotoxic effects increased with higher concentrations of heated fats in the medium. The fresh fats also produced intracellular lipid accumulation, reductions in mitosis, and changes in the nucleus and cytoplasm, at the higher levels. These effects were greater in fresh olive oil-treated cultures. These observations indicate that oxidized fat components interfere physically or biochemically with normal cell functions resulting in pathological changes.     

Formulation and production of a blood-free and chemically defined virus production media for VERO cells

Vaccines provide effective protection against many infectious diseases as well as therapeutics for select pathologies, such as cancer. Many viral vaccines require amplification of virus in cell cultures during manufacture. Traditionally, cell cultures, such as VERO, have been used for virus production in bovine serum-containing culture media. However, due to concerns of potential adventitious agents present in fetal bovine serum (FBS), regulatory agencies suggest avoiding the use of bovine serum in vaccine production. Current serum-free media suitable for VERO-based virus production contains high concentrations of undefined plant hydrolysates. Although these media have been extensively used, the lack of chemical definition has the potential to adversely affect cell growth kinetics and subsequent virus production. As plant hydrolysates are made from plant raw materials, performance variations could be significant among different lots of production. We developed a chemically defined, serum-free medium, OptiVERO, which was optimized specifically for VERO cells. VERO cell growth kinetics were demonstrated to be equivalent to EMEM-10% FBS in this chemically defined medium while the plant hydrolysate-containing medium demonstrated a slower doubling time in both two-dimensional (2D) and 3D cultures. Virus production comparisons demonstrated that the chemically defined OptiVERO medium performed at least as good as the EMEM-10%FBS and better than the plant hydrolysate-containing media. We report the success in using recombinant proteins to replace undefined plant hydrolysates to formulate a chemically defined medium that can efficiently support VERO cell expansion and virus production.     

Extrusion of DNA from the macronucleus of Tetrahymena pyriformis GL.

Administration of the thymidine analog 5-bromodeoxyuridine to exponentially growing cultures of Tetrahymena pyriformis GL in chemically defined medium results in inhibition of cell multiplication by at least one generation before DNA synthesis stops. Cell multiplication can be restored in these cultures, if they are transferred to fresh growth medium, but although most of the cells in the culture contain close to a G2-amount of DNA, a full DNA replication round is a prerequisite for renewed cell multiplication. Large extrusion bodies are found at the first division after transfer to fresh growth medium. Autoradiographic analysis has revealed that the DNA in the extrusion body is a representative of the DNA in the macronucleus indicating a random distribution of DNA between daughter nuclei and extrusion body.     

Leakage and delivery of liposome-encapsulated methotrexate-gamma-aspartate in a chemically defined medium

A chemically defined medium was developed to study liposome-mediated delivery of methotrexate-gamma-aspartate to cells under conditions where dilute suspensions of negatively charged liposomes to not leak extensively. The defined medium induced 14% leakage of methotrexate-gamma-aspartate from egg phosphatidylglycerol/cholesterol (67:33) liposomes diluted to 53 nM lipid. In contrast, commercially available serum replacements induced up to 91% leakage from the same liposomes. The growth inhibitory properties of non-loaded phosphatidylglycerol liposomes were greater in the chemically defined medium that they were in medium supplemented with 10% serum. Egg phosphatidylglycerol, dioleoylphosphatidylglycerol and dilaurylphosphatidylglycerol liposomes inhibited cell growth more than dimyristoylphosphatidylglycerol and dipalmitoylphosphatidylglycerol liposomes. In 10% serum, phosphatidylglycerol liposomes with widely varying phase-transition temperatures were nearly equally effective to deliver drug to CV1-P and L929 cells, despite great differences in liposome stability. Liposome encapsulated methotrexate-gamma-aspartate was more potent when the cells were grown in the defined medium, and the increase in drug delivery was observed from phosphatidylglycerol liposomes of different phase-transition temperatures. The minimum fraction of negatively charged phospholipid required for optimal liposome-mediated drug delivery varied between cell types and among growth media. The growth inhibitory effects of liposome-encapsulated methotrexate-gamma-aspartate was also determined under conditions where the cells were exposed to drug for periods shorter than the entire growth assay. Reduction of the exposure time decreased the potency of both encapsulated and free drug in medium containing 10% serum, and decreased the potency of free drug in the defined medium. However, the potency of encapsulated drug in the defined medium was similar for all exposure lengths between 1 and 48 hours.     

Action of DL-alpha-difluoromethyl ornithine on Acanthamoeba culbertsoni.

The multiplication of A. culbertsoni in the peptone medium was not inhibited by 10-20 mM concentration of alpha-difluoromethyl ornithine (DMFO) while a partial and transient inhibition of cell multiplication was observed by 10-20 mM DFMO in proteose peptone, yeast extract, glucose (PYG) medium. Ornithine decarboxylase (ODC) activity in the cells and cell free extracts was strongly inhibited by DFMO, excluding enzyme refractoriness and impermeability of cells for DFMO as the possible causes of DFMO resistance. The presence of polyamines in the peptone and PYG media as well as uptake of polyamines by the amoebae has been demonstrated. The growth and multiplication of A. culbertsoni in chemically defined medium was not affected by 1-5 mM DFMO while 10-20 mM DMFO yielded partial inhibition. A lowering of diaminopropane levels and enhancement of spermidine levels was observed in DFMO inhibited cells and level of ODC was drastically reduced in the inhibited cultures. Uptake of polyamines from the growth media may partly account for DFMO resistance of A. culbertsoni. Alternative mechanisms for DFMO resistance are indicated.     

The effect of the dimeric and multimeric forms of fibronectin on the adhesion and growth of primary glomerular cells

Primary glomerular cells placed in a chemically defined medium containing Waymouth's medium MB 752/1 supplemented with insulin, transferrin, fibroblast growth factor, nonessential amino acids, sodium pyruvate, and antibiotics showed rapid outgrowth of cells which morphologically resembled well differentiated visceral epithelial cells followed by outgrowth of poorly differentiated cells; morphologic evidence suggests these latter cells are precursor cells of the epithelial cell lineage. Whereas the well differentiated glomerular epithelial cells were never observed to divide by sequential phase microscopic observations, a chemically defined medium was developed for optimal growth of the poorly differentiated cell type. This serum-free medium contained Waymouth's medium MB 752/1 supplemented with insulin, transferrin, selenium, and fibronectin (plus non-essential amino acids, sodium pyruvate, and antibiotics). Using this chemically defined medium, we have compared the effects of dimeric and multimeric fibronectin (high molecular weight disulfide-bonded fibronectin produced by incubation of dimeric fibronectin with 3 M guanidine followed by dialysis against 0.05 M cyclohexylaminopropane sulfonic acid (CAPS) buffer, pH 11) on the adhesion and growth of the poorly differentiated primary glomerular cell type. Dimeric fibronectin (FN) was twice as effective as multimeric FN in promoting glomerular cell adhesion, although both forms of FN promoted cell adhesion better than an uncoated substratum. In contrast, cell growth studies demonstrated that multimeric FN was a more potent growth stimulant than dimeric FN. The differential effects of dimeric and multimeric forms of FN in vitro suggests that these molecules may have different functions in vivo.     

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.     

Extrusion of DNA from the Macronucleus of Tetrahymena pyriformis GL

SYNOPSIS Administration of the thymidine analog 5-bromodeoxyuridine to exponentially growing cultures of Tetrahymena pyriformis GL in chemically defined medium results in inhibition of cell multiplication by at least one generation before DNA synthesis stops. Cell multiplication can be restored in these cultures, if they are transferred to fresh growth medium, but although most of the cells in the culture contain close to a G2-amount of DNA, a full DNA replication round is a prerequisite for renewed cell multiplication. Large extrusion bodies are found at the first division after transfer to fresh growth medium. Autoradiographic analysis has revealed that the DNA in the extrusion body is a representative of the DNA in the macronucleus indicating a random distribution of DNA between daughter nuclei and extrusion body.     

Amino Acid Requirements and Proteolytic Activity of Streptococcus sanguis

The growth response of Streptococcus sanguis groups 1:A and 1:B in a complete chemically defined medium was not influenced by the oxygen concentration of the growth atmosphere. All of the cultures required cysteine and arginine; tyrosine and branched-chain amino acids were frequently required. Proteolysis of casein, mucin, and the anionic proteins of germfree rat saliva by S. sanguis was demonstrated. Hydrolytic activity toward casein was found in the soluble contents of the cells and in the cellular debris after disruption of the cells, with the soluble fractions exhibiting greater proteolytic activity toward casein. The soluble fractions from S. sanguis did not hydrolyze mucin, but this substrate was hydrolyzed by the cell debris fraction. When the amino acid requirements and proteolytic activity of S. sanguis and S. mutans were compared, these two oral streptococcal species exhibited distinct and characteristic differences.     

Effect of fractions of horse and fetal bovine serum on neoplastic conversion of C3H mouse cells in tissue culture

Summary Cultures derived from C3H/He mouse embryos were grown in medium NCTC 135 supplemented with horse serum, fetal bovine serum, or various combinations of large and small molecule fractions of horse and fetal bovine serum. Cultures in medium NCTC 135 alone or in medium 135 supplemented with the small molecule fraction of either horse or fetal bovine serum did not grow as continuous long term lines. The best growth was obtained when the cultures were in medium containing the large molecule fraction of fetal bovine serum either alone or in combination with a small molecule fraction. Cells grown in the presence of the low molecular weight fraction of horse serum invariably produced tumors on injection into syngeneic animals. Cells in the small molecular weight fraction of fetal bovine serum combined with the large molecular weight fraction of horse serum produced tumors after a prolonged period in vitro. *** DIRECT SUPPORT *** A00S8010 00003     

Effect of change in growth environment on cultured myocardial cells investigated in a standardized medium

Summary Neonatal rat heart cells cultivated in either of two different media which varied only in their serum supplements were transferred to chemically defined medium (Ham's F10) for 24 h before measuring a variety of parameters. The 24-h period of exposure to chemically defined medium was not sufficient to reverse the effects imposed on the cells by the serum used in the first phase of growth. The cells differed in rate and duration of action potentials and contractions. The initial serum composition affected the response of the cells to calcium deficiency. Studies involving the effects of pharmaceutical reagents such as isoproterenol were also influenced by the serum. In attempting to determine the cause and possible mechanism, it was found that mitochondrial membrane permeability for nitroblue tetrazolium (NBT) was unchanged. Although the serum supplements differed in fatty acid composition, the fatty acid profiles of the cell phospholipids were relatively constant. We conclude that (a) the function of the cells is affected by the growth environment, particularly serum; (b) that a short exposure to a uniform chemically defined medium is not sufficient to reverse these effects; and (c) that the differences in effects are not the result of changes in the fatty acid composition of the whole cell phospholipids nor in mitochondrial membrane permeability as measured by NBT.     

Control of multiplication of uninfected rat cells and rat cells converted by murine sarcoma virus

The rate of multiplication of rat embryo fibroblasts in monolayer culture depends upon the amount of multiplication-stimulating activity in the culture medium, as well as the efficiency of stimulation by and utilization of this activity. Multiplication-stimulating activity is defined by its capacity to stimulate DNA synthesis and cell division in stationary populations of cells. Usually, multiplication stimulating activity is supplied as serum in cell culture media, but rat cells also produce it. A comparison of multiplication of uninfected and Murine Sarcoma virus-converted rat cells showed that converted cells multiplied at a greater rate than did uninfected cells, with the use of less or the same amount of multiplication-stimulating activity; the converted cells produced cells produced an inhibitor of multiplication-stimulating activity, and the efficiency of stimulation of DNA synthesis was similar for uninfected and converted cells. It appears that in the presence of serum the efficiency of utilization of multiplication-stimulating activity is greater for converted cells than for uninfected rat cells.     

Clonal growth and self-renewal of human myeloid leukemia cell lines (BRM and DD) in serum-free semi-solid culture

Primary and secondary colony formation of two new human myeloid leukemia cell lines (BRM and DD) were studied in serum-free semisolid cultures. The results indicate that bovine serum albumin and transferrin were essential for clonal growth in chemically defined medium. Insulin contributed only moderately beneficial effects. Initial cell density was also a major modulator of plating efficiency. Positive cooperation between the leukemia cells was shown by using autologous conditioned media. This is the first serum-free culture method that allows self-renewal of human myeloid leukemia cell lines in terms of secondary colony formation in methylcellulose cultures.     

Incorporation of radioactive precursors into glycosaminoglycans by rat muscle fibroblasts exposed to a solubilized rat bone matrix fraction

Confluent cultures of rat muscle fibroblastic cells respond by increased glycosaminoglycan (GAG) synthesis when cultured in medium containing a solubilized bone matrix fraction (SBM) at a concentration of 100 micrograms/ml. The metabolism of the GAG associated with the cell pellet, the cell surface and the tissue culture medium fractions was studied, in the presence and absence of SBM, by measuring the incorporation of radioactivity from [3H]glucosamine and [35S]SO4 into the isolated GAG. Net synthesis of hyaluronic acid and of chondroitin sulfate in the medium fraction increased more rapidly in cultures containing SBM compared to controls, and the accumulation of labelled GAG in the medium of the treated cultures was approximately linear with respect to the length of incubation. The addition of SBM also resulted in increased incorporation of 3H and of 35S into the GAG of the cell surface and cell pellet fractions. In these fractions, stimulation of incorporation of radioactivity occurred in two waves: an early, relatively minor increase and a later relatively major increase. The relatively major stimulation of radioactivity into the GAG of the cell surface fraction occurred between 24 and 48 h and was independent of any apparent effect of serum.     

Autocrine factor participation in defence of cytotoxic CTLL-2 cells from oxidative stress

The role of autocrine factors (AF) secreted by cytotoxic IL-2-dependent CTLL-2 cells along with pyruvate in cell defense from oxidative stress was investigated. The addition of conditioned medium (CM) containing pyruvate and AF into CTLL-2 cell cultures increased significantly cell survival under oxidative stress condition. The kinetics of hydrogen peroxide removal from cell cultures under oxidative stress in the case of CM addition has been obtained. The removal of H2O2 mostly by means of its reaction with pyruvate that is contained in CM has been shown at the beginning of oxidative stress (up to 15 min). Pyruvate content in CM was determined as 138 +/- 7 microM. Cell filtration on column with Bio-Gel P-10 was used for removal pyruvate from CM. Three fractions of CM (A, B and C) were obtained as a result of gel filtration. Pyruvate was not detected in any fraction. The fraction A was eluted from column as the first one and contained the largest molecules. Cell survival test showed the fraction B to have the highest ability to protect CTLL-2 cells under oxidative stress. The fraction A supported cell survival to a less degree and fraction C was shown to have no protective ability. The addition of the fraction B to the cell cultures resulted in preservation of significantly higher intracellular ATP level in the cells under oxidative stress than in the control ones. Moreover, AF of the fraction B was shown to react directly with hydrogen peroxide and inactivate it in the absence of cells. AF of the fraction A did not have such properties.     

Proteoglycan biosynthesis by chick embryo retina glial-like cells

In this report we present biochemical evidence that purified cultures of chick embryo retina glial-like cells actively synthesize heparan sulfate (HS) and chondroitin sulfate/dermatan sulfate (CS/DS) proteoglycans as well as hyaluronic acid. Glial-like cell cultures were metabolically labeled with [3H]glucosamine and 35SO4, and the medium, cell layer, and substratum-bound fractions were analyzed separately. Proteoglycans were characterized according to charge, apparent molecular size, and glycosaminoglycan (GAG) composition and were found to be differentially distributed among the cellular compartments. HS was the predominant GAG overall and was the major species found in the cell layer and substratum-bound fractions. CS/DS was also present in each fraction and comprised the largest proportion of GAGs in the medium. The major GAG-containing material resolved into three different size classes. The first, found in the cell layer and substratum-bound fractions, contained both CS/DS and HS and was of large size. A second, intermediately sized class with a higher CS/DS:HS ratio was found in the medium. The smallest class was found in the cell layer fraction and comprised HS, most likely present as free GAG chains. In addition, each fraction contained hyaluronic acid. Characteristics of these macromolecules differ from those produced by purified cultures of chick embryo retina neurons and photoreceptors in terms of size, compartmental distribution, and presence of hyaluronic acid.