Roles of various growth factors in growth of human osteosarcoma cells which can grow in protein-free medium.

The human osteosarcoma cell line (OST-1-PF) can grow in protein-free Coon's modified Ham's F12 medium. Growth of the cells in protein-free medium was partially density-dependent and partially depressed by medium change. An extract and conditioned medium of OST-1-PF cells contained high mitogenic activity for BALB/c3T3 cells. The growth factor in the cells was purified and identified as a basic fibroblast growth factor (bFGF)--like factor on the basis of its elution profile on heparin-affinity chromatography and the result of immunoblotting. An unidentified factor in a conditioned medium eliciting most of the DNA synthesis-stimulating activity showed a weak affinity for heparin. Various additions, including serum and growth factors, stimulated the growth of OST-1-PF cells in protein-free medium. Of these factors, epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), acidic fibroblast growth factor (aFGF) and bFGF were the most potent mitogens. High-affinity receptors of EGF and FGF were found on the surface of these cells. These results indicate that autonomous growth of OST-1-PF cells in protein-free medium is mainly controlled by an intracellular mechanism.     

Proteins secreted by Caco-2 cells support growth of other tumor cells in Chee's Essential Medium without supplements.

The growth promoting effect of several hormones and growth factors on two human colon tumor cell lines (Caco-2 and SW 48) was studied using six different chemically defined serum-free media (SFM). Caco-2 grew in a simple SFM [GF3: Chee's Essential Medium (CEM) plus insulin, transferrin and selenium], whereas, SW 48 cells did not grow in GF3 medium. This suggested that Caco-2 cells probably secrete proteins in SFM which influence attachment and growth of Caco-2 and other tumor cells. Lyophilized Caco-2 conditioned medium and substratum, when added to plain CEM, supported growth of SW 48 and SW 948 cells. The substratum material was more effective than conditioned medium in promoting growth of the cell lines. The substratum material helps attachment and spreading of the cells and, thus, improves growth of the cells over conditioned medium. Caco-2 conditioned medium and substratum were analyzed for their components using SDS-PAGE system and gel filtration chromatography. The substratum was analyzed for the presence of fibronectin and laminin by the ELISA technique. The conditioned medium does not contain TGF alpha and TGF beta. The growth stimulating activity of the conditioned medium is due to a protein component, approximately 58Kd in size.     

Cytochalasin B releases a major surface-associated glycoprotein, fibronectin, from cultured fibroblasts

BHK21 cells cultured in minimal essential medium (Eagle) supplemented with 10% dialyzed fetal calf serum did not grow as they did in whole serum containing medium. Logarithmic growth was, however, initiated after a lag period, the length of which was dependent upon the cell density: medium volume ratio. The quiescent cells conditioned the medium during this lag period, and growth stimulation was apparently due to the release of serine into the medium. Cells cultured in 10% dialyzed serum plus the low molecular weight fraction of serum (serum dialysate), grew with kinetics similar to cells cultured in serum containing medium. When serum dialysate was chromatographed on Bio-gel P-2 the growth promoting activity eluted with the amino acids. Each of the non-essential amino acids was tested for its ability to stimulate the growth of cells in 10% dialyzed serum. Serine was capable of stimulating cell growth to the same extent as 10% serum dialysate and its concentration optimum was similar to its concentration in 10% serum dialysate. The remaining non-essential amino acids were either slightly stimulatory or had no effect on cell growth. Shifting a logarithmically growing population of cells to serine-free medium resulted in the accumulation of 95% of the cells in the G1 phase of the cell cycle within 24 h. Escape from the G1 block could occur if serine was added to the medium or if the cells were allowed to condition the medium. Entry of cells into S phase after the addition of 0.05 μmoles/ml of serine followed a 4–6 h lag and 80% of the cells were synthesizing DNA 12 h after shift-up.     

Histochemical demonstration of an increase in acetylcholinesterase in established lines of human and mouse neuroblastomas by nerve growth factor.

Cells of three established lines of human neuroblastoma and an established line of C1300 mouse neuroblastoma were grown in control medium or in experimental medium containing mouse nerve growth factor (NGF). Cultures were stained histochemically for acetylcholinesterase (AChE) during log growth and at confluency. Human neuroblastoma cells grown in medium containing NGF were morphologically more differentiated and they were stained much more intensely for AChE during both phases of growth than were cells in control cultures. The enzyme was distributed over cell bodies and neurites. Neuroblastoma cells of the mouse line were not stimulated to form neurites by NGF, but they were more intensely stained for acetylcholinesterase than cells grown in control medium. These observations support earlier findings that NGF stimulates differentiation of human and mouse neuroblastoma cells in vitro.     

Fetal rhesus monkey lung cells can be grown in serum-free medium for the replication of dengue-2 vaccine virus

Summary Serum-free media were developed to grow diploid fetal rhesus monkey lung (DBS-FRhL-2) cells and to propagate dengue-type 2 virus vaccine strain PR-159 (dengue-2 vaccine virus). Vitamins, amino acids, growth factors, hormones and other organic compounds, and inorganic salts were substituted for fetal bovine serum. The composition of the medium that was optimal for growth of DBS-FRhL-2 cells differed from medium optimal for the propagation of dengue-2 vaccine virus. Insulin, epidermal growth factor, fibroblast growth factor, and platelet-derived growth factor were required for DBS-FRhL-2 cell proliferation in serum-free medium but were inhibitory for virus propagation. Adenosine, cytidine, guanosine, uridine, and thymidine, each at 0.01 mM concentration, were necessary as medium supplements to obtain a high yield of dengue-2 vaccine virus in DBS-FRhL-2 cells under serum-free conditions. DBS-FRhL-2 cells grown in serum-free medium produced dengue-2 vaccine virus with yields similar to those of cells grown in the presence of serum. Dengue-2 vaccine virus obtained under serum-free conditions retained its phenotypic markers such as temperature sensitivity and small plaque size. This investigation was supported by Contract DAMD-17-81-C1029 from the U.S. Army Medical Research and Development Command, and by The Hormel Foundation.     

Basic fibroblast growth factor,albumin, and transferrin purified from rat rhodamine fibrosarcoma tissue are all essential for growth of primary tumor cells from the same tissue in serum-free medium

Summary Primary Rhodamine fibrosarcoma (RdF) cells from rats were shown to grow in serum-free medium supplemented with basic fibroblast growth factor (bFGF), albumin, and transferrin, all of which were purified from RdF tissue. Their growth rate with these supplements was similar to that of cells in medium supplemented with calf serum. bFGF purified from RdF tissue (Rd-bFGF), which was previously designated as DNA synthesis factor, stimulated the growth of primary RdF cells maximally at 30 ng/ml in the presence of the other two proteins. Albumin and transferrin were separated from partially purified tumor growth stimulating activity which was previously shown to stimulate growth of primary RdF cells in serum-free medium. The albumin (RdA) and transferrin (RdT) found in the extract of RdF tissue were not due simply to contamination of the tissue with blood, but to their accumulations in the tissue. The growth stimulatory activities of RdA and RdT on primary RdF cells in serum-free medium were maximal at 30 and 10 μg/ml, respectively. These results suggest that Rd-bFGF, RdA, and RdT, all of which accumulate in the tumor tissue, are essential for growth of RdF cells in the tissue. This work was supported by a grant-in-aid for cancer research from the Ministry of Education, Science and Culture of Japan.     

兔胚胎神经干细胞的分离、培养和鉴别

目的：研究兔胎脑神经干细胞体外生长特性,为探讨神经干细胞的临床应用及神经系统的发育奠定基础。方法：采用含碱性成纤维细胞生长因子（bFGF）和表皮细胞生长因子（EGF）的N2无血清培养技术,取18天龄兔胚胎脑组织,分离神经干细胞,并观察分离的细胞体外培养、增殖、分化潜能,免疫组化鉴定。结果：从18天龄兔胎脑皮质和纹状体中成功分离出具有自我更新和多分化潜能的神经干细胞,在无血清培养时细胞呈半贴壁状态生长,形成神经球,可传代。细胞呈Nestin免疫反应阳性;在含血清培养基中培养时则分化,分化后的细胞表达神经元细胞、星形胶质细胞和少突胶质细胞的特异性抗原。结论：来自兔胎脑神经干细胞能在体外培养、增殖并保持传代能力。无血清N2EGF、bFGF培养基有利于兔胎脑神经干细胞的存活和增殖,含血清培养基能诱导兔胎脑神经干细胞分化。     

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)     

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.     

Utilization of glucose and amino acids in insect cell cultures: Quantifying the metabolic flows within the primary pathways and medium development

The current understanding of insect cell metabolism is very limited. In order to gain some insight into the growth and metabolism of insect cells Spodoptera frugiperda (Sf9), a comprehensive characterization of culture conditions for cells grown in the IPL-41 medium was made by measuring the amino acid composition of the growth medium and the cell extract, the macromolecular composition of the cells (DNA, RNA, and protein), medium concentrations of various metabolites and sugars, and the evolved CO(2). Since in the IPL-41-based serum-free medium all of the amino acids except cysteine are in great excess of what is needed by the cells for energy and protein production, a medium formulation with an osmolarity similar to the IPL-41 but with a lower amino acid content than IPL-41 was also developed. The new medium also lacks maltose and sucrose (contains only glucose), supported cell growth to a high cell density of 8 x 10(6) cells/mL. The cellular and energetic yields indicated that a tight coupling between the biosynthetic and energetic reactions was attained for cells grown in the new medium. Moreover, it was found that the intermittent feeding of glucose may not be required as the cell yield and growth rate were comparable whether the same total amount of glucose was provided intermittently or was included initially in the medium. The eventual cessation of growth in the new medium is believed to be due to the amino acid limitation because concentrations of both glutamine and glutamate were very low at the end of the growth phase. Thus, further optimization, which may include higher initial glutamine in the medium or its intermittent feeding, could lead to a further increase in the cell density. Finally, a stoichiometrically based analysis of metabolic reactions confirmed the operation of the key pathways and was used to quantify the distribution of metabolites among primary metabolic reactions. The quantitative flow values were used to highlight some key aspects of insect cell metabolism. (c) 1993 John Wiley & Sons, Inc.     

The role of intercellular contacts in the initiation of growth and in the development of a transiently nonculturable state by the cultures of Rhodococcus rhodochrous grown in poor media

It was found that the growth of Rhodococcus rhodochrous cells in modified Saton's medium strongly depends on the rate of culture agitation in the flask: an agitation at 250 rpm in flasks with baffles stops cell multiplication, whereas slight agitation leads to pronounced culture growth. The growth retardation phenomenon was reversible and did not manifest itself in exponential-phase cultures or when the cells were grown in a rich medium; furthermore, it was not connected with the degree of culture aeration. When agitated at a moderate rate, the bacterial cells formed aggregates in the lag phase, which broke up into single cells in the exponential phase. The inhibitory effect of vigorous agitation was removed by the addition to the medium of the supernatant (SN) of a log-phase culture grown in the same medium with moderate agitation. Vigorous agitation is thought to interfere with the cell contacts, whose establishment is necessary for the development of an R. rhodochrous culture in a poor medium, which occurs in the form of (micro) cryptic growth. When grown in modified Saton's medium, R. rhodochrous cells were capable of transition, in the prolonged stationary phase, to a resting and transiently nonculturable state. Such cells could be resuscitated by incubation in a liquid medium with the addition of the supernatant or the Rpf secreted protein. The formation of transiently nonculturable cells was only possible under the conditions of a considerable agitation rate (250-300 rpm), which prevented secondary (cryptic) growth of the culture. This circumstance indicates the importance of intercellular contacts not only for the initiation of growth but also for the transition of the bacteria to a dormant state.     

Identification of lactoferrin as an essential growth factor for human lymphocytic cell lines in serum-free medium

A serum-free medium supplemented with growth factor(s) was devised to grow human lymphocytic cell lines. The medium was developed using human lymphocytic cell line, Bri 7 cells. In the process of constructing the medium, human lactoferrin was found to be an essential growth factor for the cell line. Human lactoferrin has higher growth stimulatory activity than human transferrin, and was sensitive to heat. Long-term cultivation of the cells was achieved in the defined medium supplemented with human lactoferrin only. The defined medium specifically supported the growth of various other human B- and T-lymphocytic cell lines but not the growth of various mouse lymphocytic cell lines. In lactoferrin-supplemented medium, the growth of some human cell lines were further stimulated by the addition of a combination of insulin, ethanolamine and selenium, or another combination of 2-mercaptoethanol and the above three factors. Bovine lactoferrin could be substituted for human lactoferrin.     

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.     

Lack of evidence of chalone activity in used medium and extract of JB-1 tumor cells in vitro. A flow cytometry study.

In cell-free mouse ascites fluid from the JB-1 ascites tumor in the plateau phase of growth low-molecular chalone substances have been found which reversibly and specifically arrest JB-1 cells in the G1 and G2 phase of the cell cycle. The aim of this study was to investigate whether chalones were involved in the regulation of in vitro growth of JB-1 tumor cells. Used medium and cell extract from confluent, stationary JB-1 cell cultures were investigated for proliferation-inhibitory properties. JB-1 cells from stationary cultures were explanted in test cultures and the traverse of cells through the S phase was investigated by means of flow cytometry (FCM). Inhibition--expressed as a delay of the traverse of cells through the S phase--was not observed when a surplus of used medium, concentrated and fractionated used medium or concentrated and fractionated cell extract from JB-1 cells in vitro was added to test cultures. On the contrary, used medium and concentrated and fractionated used medium stimulated growth. Thus, no involvement of chalones in the growth regulation of JB-1 tumor cells in vitro was detected.     

Regulation of cell size in the yeast Saccharomyces cerevisiae.

For cells of the yeast Saccharomyces cerevisiae, the size at initiation of budding is proportional to growth rate for rates from 0.33 to 0.23 h-1. At growth rates lower than 0.23 h-1, cells displayed a minimum cell size at bud initiation independent of growth rate. Regardless of growth rate, cells displayed an increase in volume each time budding was initiated. When abnormally small cells, produced by starvation for nitrogen, were placed in fresh medium containing nitrogen but with different carbon sources, they did not initiate budding until they had grown to the critical size characteristic of that medium. Moreover, when cells were shifted from a medium supporting a low growth rate and small size at bud initiation to a medium supporting a higher growth rate and larger size at bud initiation, there was a transient accumulation of cells within G1. These results suggest that yeast cells are able to initiate cell division at different cell sizes and that regulation of cell size occurs within G1.     

On-chip single-cell microcultivation assay for monitoring environmental effects on isolated cells

We have developed a on-chip single-cell microcultivation assay as a means of observing the adaptation process of single bacterial cells during nutrient concentration changes. This assay enables the direct observation of single cells captured in microchambers made on thin glass slides and having semipermeable membrane lids, in which cells were kept isolated with optical tweezers. After changing a medium of 0.2% (w/v) glucose concentration to make it nutrient-free 0.9% NaCl medium, the growth of all cells inserted into the medium stopped within 20 min, irrespective of their cell cycles. When a nutrient-rich medium was restored, the cells started to grow again, even after the medium had remained nutrient-free for 42 h. The results indicate that the cell's growth and division are directly related to their nutrient condition. The growth curve also indicates that the cells keep their memory of what their growth and division had been before they stopped growing.     

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.     

Microcarrier culture of bowes melanoma cells in serum-free medium with Human plasma fraction IV-4+ V

Bowes melanoma cells were cultivated successfully in a serum-free medium which was constructed by the concept of maximum retention of proteins from fractionated human plasma having growth stimulatory activities. The cells could be cultivated in the serum-free medium without any adaptation period. The major serum-free component of the medium was the fraction IV-4 + V of the Cohn fractionation process of human plasma. Approximately six times increase of tissue-type plasminogen activator (t-PA) activity as compared with that in serum-free medium even though the cell growth was much slower. In addition, the growth stimulatory activities of thrombin and fibronectin were investigated during the cultivation of Bowes melanoma cells in this serum-free medium. These proteins contributed significantly to the enhanced growth of cells by reducing doubling time to 25 and 35 h as compared with 55 h in the serum-free medium without them. Especially, fibronectin supported cells to propagate near to the maximum cell density achieved in the medium with 10% FBS.     

Identification of lactoferrin as an essential growth factor for human lymphocytic cell lines in serum-free medium

A serum-free medium supplemented with growth factor(s) was devised to grow human lymphocytic cell lines. The medium was developed using human lymphocytic cell line, Bri 7 cells. In the process of constructing the medium, human lactoferrin was found to be an essential growth factor for the cell line. Human lactoferrin has higher growth stimulatory activity than human transferrin, and was sensitive to heat. Long-term cultivation of the cells was achieved in the defined medium supplemented with human lactoferrin only. The defined medium specifically supported the growth of various other human B- and T-lymphocytic cell lines but not the growth of various mouse lymphocytic cell lines. In lactoferrin-supplemented medium, the growth of some human cell lines were further stimulated by the addition of a combination of insulin, ethanolamine and selenium, or another combination of 2-mercaptoethanol and the above three factors. Bovine lactoferrin could be substituted for human lactoferrin.     

Effect of Rehydration on Recovery, Repair, and Growth of Injured Freeze-Dried Salmonella anatum

From 70 to 90% of the Salmonella anatum cells that survived freeze-drying in nonfat milk solids were injured. After rehydration, these injured survivors failed to grow on a selective plating medium containing deoxycholate but could form colonies on a nonselective medium. In a suitable environment after rehydration, injury disappeared in most of these cells. The rate of this repair at 25 C was very rapid initially and, in a medium containing milk solids, was completed within 1 hr after rehydration. The repaired cells initiated growth about 1 hr later than normal cells and grew at a slower rate. In a medium containing milk solids, initial recovery, extent of repair of injury, initiation of growth, and rate of growth were not influenced by supplementation with extra nutrients in other rehydration media. Rehydration controlled by modifying the concentrations of lactose, sucrose, or milk solids in the rehydration medium influenced the recovery of cells and the time that growth was initiated. Glycerol failed to increase recovery. Higher numbers of cells were recovered by rehydrating at 15 to 25 C, but an earlier initiation of growth and more rapid growth were observed at 35 C.