Effects of antibiotics on protein synthesis and degradation in primary cultures of rat hepatocytes

Summary In primary hepatocyte cultures, maintained in a protein-free medium, streptomycin, penicillin, and Garamycin (gentamicin) all inhibited protein synthesis at concentrations above 0.1 mM. Some inhibition was also observed with the fungicide Mycostatin at 100 U/ml. Hepatocytic protein degradation was markedly inhibited by penicillin at concentrations above 0.1 mM, whereas streptomycin and Garamycin only showed slight inhibition at concentrations in excess of 1 mM. None of the antibiotics had any detectable effect on the structural integrity (viability) of the cells. The work was supported by grants from The Norwegian Cancer Society and The Norwegian Council for Science and the Humanities     

Egg yolk lipoprotein,a new supplement for the growth of mammalian cells in serum-free medium

Egg yolk lipoprotein promoted growth of a wide variety of mammalian cell lines, including plasma-cytomas and epithelial cell lines, in serum-free medium. The lipoprotein was active for cell growth when used with insulin, transferrin, ethanolamine and selenite. The most active lipoprotein fraction (YLP-pI7.5) was purified to give a single peak by chromatofocusing and gel filtration, and was homogeneous on a 0.35% agarose gel electrophoretogram. The lipoprotein was characterised as a very low density lipoprotein with a protein content of only 1.3%. This lipoprotein had an optimal concentration of 300 g/ml (4 g protein/ml). It was easily separable from proteinous molecules secreted into the serum-free medium by the cells, since it floated on the surface of the medium after addition of ammonium sulfate, to precipitate protein, and centrifugation. An associated structure of lipid and protein seemed to be still necessary for the lipoprotein to exhibit a growth promoting activity.     

Glycogen synthesis in serum-free cultured hepatocytes in response to insulin and dexamethasone

Summary Liver parenchymal cells cultured in serum-free medium may retain their ability to synthesize glycogen in response to insulin. Specific hormone requirements are needed by hepatocytesto retain the biochemical pattern of mature cells. Insulin supplementation of culture medium seems to be essential to maintain the glycogen synthesis rate of cultured hepatocytes. The continuous presence of dexamethasone amplified the insulin-induced glucogen synthesis. Cytophotometric analysis showed differences in the way that individual cells accumulate glycogen in response to insulin stimulus, which indicates that liver parenchymal cells in culture are functionally heterogeneous. The financial support for this work was from the Fondo de Investigationes Sanitarias de la Seguridad Social, grants 41/82 and 48/82.     

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.     

Improved maintenance of adult rat hepatocytes in a new serum-free medium in the presence or absence of barbiturates

Summary For serum-free primary culture of adult rat hepatocytes, a synthetic medium DM-160 and rat-tail collagen were selected for the basal medium and for the culture substratum, respectively. Barbiturates, such as phenobarbital and 1-ethyl-5-isobutylbarbiturate, efficiently supported survival of hepatocytes and maintained their morphologic features at lower concentrations under the serum-free conditions than under the serum-supplemented conditions. However, the hepatocyte survival rates under the serum-free conditions were lower than those under the serum-supplemented conditions in the presence or absence of barbiturates. Supplementation of the basal medium with a combination of five groups of factors (5Fs), such as eight amino acids (Ala, Arg, Gly, Ile, Met, Phe, Pro, and Trp), two unsaturated fatty acids (linoleate and oleate), a protease inhibitor (aprotinin), three vitamins (A, C, and E), and five trace elements (Mn, Fe, Cu, Zn, and Se), improved the hepatocyte survival under the serum-free conditions in the presence or absence of barbiturates. In other words, the serum could be completely substituted by the 5Fs. Hepatocyte cultures maintained in the 5Fs-suppelemented basal medium showed excellent induction of tyrosine aminotransferase activity in response to dexamethasone in the presence or absence of barbiturates. The efficiency of the 5Fs-supplemented basal medium for maintaining hepatocytes was not inferior to those of other media in common use with hepatocytes, such as Williams' medium E and Waymouth's medium MB-752/1. In conclusion, maintenance of functional hepatocytes in serum-free primary culture could be improved by use of the new medium preparation (the 5Fs-supplemented DM-160) in the presence of barbiturates. This work was supported by a grant no. 61771923 from the Ministry of Education, Science and Culture of Japan.     

A serum-free medium that supports the growth of piscine cell cultures

Summary An undefined, serum-free medium was developed for use with fish cell cultures. Lactalbumin hydrolyzate, trypticase-soy broth, Bacto-peptone, dextrose, yeastolate, and polyvinylpyrrolidone were initially combined in 100 ml of distilled H₂O, autoclaved, and added to 5% of the final volume of Medium 199. In addition, filter sterilized bovine pancreatic insulin, glutamine, and nonessential amino acids were added to the medium. The addition of insulin was observed to be unnecessary. Five fish cell lines [goldfish-derived CAR cells, fathead minnow (FHM) cells, epithelioma papillosum cyprini (EPC) cells, chinook salmon embryo (CHSE-214) cells, and a new cell line from goldfish air bladders (ABIII)] were all capable of growth in the serum-free medium at rates equivalent to cells grown in fetal bovine serum (FBS). The morphology of all cell lines, except CHSE-214 cells, was identical to cells grown in FBS. All cell lines were capable of long-term growth in the serum-free medium. The CAR, ABIII, EPC, and CHSE-214 cells in the serum-free medium supported the replication of goldfish virus-2 at levels equivalent to cells grown in FBS.     

Primary cultures of hepatocytes in serum and hormone-free medium: Identification of conditions which stimulate an in vivo-like induction of G6PD

Summary Recent results from several laboratories suggest that complex interactions between hormones and dietary carbohydrate may be responsible for regulating the induction of several hepatic lipogenic enzymes. Elucidation of these interactions requires the ability to culture hepatocytes for several days in serum-free medium where the hormones or carbohydrate or both present is strictly controlled. The functional response of primary adult rat hepatocytes was examined in a medium without exposure to serum, hormones, or carbohydrates and on three substrata commonly used to culture cells in a defined medium. Hepatocytes cultured on a floating collagen gel in which is embedded a nylon mesh possess cell attachment and morphologic characteristics superior to either cells cultured on a collagen-coated or fibronectin(Fn)-coated substratum. Cells cultured on the gel-mesh system retain insulin responsivity, as measured by protein synthesis rates and glucose-6-phosphate dehydrogenase (G6PD) induction, for at least 6 d in culture. Under these conditions, insulin, dexamethasone, and fructose increase G6PD specific activity to levels comparble to that seen in an induced animal. Hepatocytes cultured on the gel-mesh system tolerate restricted medium conditions better than cells cultured on collagen or Fn-coated substratum, and remain viable for sufficient times to allow, for the first time, full expression and maximal induction (i.e. like in vivo), of G6PD in cultured cells. This system represents a satisfactory model for in vivo liver metabolism and a superior system for studying the effects of hormones and metabolites on G6PD levels, as well as other nutritional-hormonally regulated enzymes.     

Expression of cathepsin B and aryl hydrocarbon hydroxylase activities,and of apolipoprotein B in human hepatoma cells maintained long-term in a serum-free medium

Summary We have established the human hepatoma cell line, HepG2, in a defined, serum-free medium. These cells were maintained and studied over a 100-generation period (i.e. 10 serial transfers). Cells maintained in serum-free medium exhibited growth parameters (i.e. saturation density, efficiency of plating, and population doubling time) similar to those obtained with HepG2 cells maintained in serum-supplemented medium. Serum-free cells were also similar to their serum-supplemented counterparts with respect to the expression of cathepsin B activity and the induction of aryl hydrocarbon hydroxylase by 2,3,7,8-tetra-chlorodibenzo-p-dioxin. Significantly, HepG2 cells maintained in serum-free conditions also retained the ability to synthesize and secrete proteins, including the liver plasma protein, apo-lipoprotein B. These results indicate that the serum-free medium used in this study supports the long-term growth and maintenance of human hepatoma, HepG2, cells in culture. Inasmuch as these cells retain phenotypes, including differentiated markers previously reported for their serum-supplemented counterparts, they may provide a more reliable, standardized culture system to study the expression, secretion, and regulation of proteins during biological and pathologic processes.     

A serum-free medium formulation supporting growth of human umbilical cord vein endothelial cells in long-term cultivation

A serum-free medium formulation – TUD-1 – was developed supporting growth of HUVEC in tissue culture. Special features of the basal medium formulation are highly elevated levels of glutamine and serine as well as the inclusion of N-acetylcysteine and phosphoascorbic acid. The cellular mitogenic needs are satisfied by bFGF, VEGF, EGF and liver growth factor. Further hormone supplementation consists of insulin and hydrocortisone. A protocoll for serum-free passage of HUVEC was established for serum-free long-term cultivation of freshly isolated HUVEC for up to 20 cumulative population doublings without significant differences in final cell density compared to controls cultivated with serum. This revised version was published online in July 2006 with corrections to the Cover Date.     

Growth requirements and long-term subcultivation of bovine granulosa cells cultured in a serum-free medium

Summary We have developed an improved serum-free medium to optimize the cell growth of bovine granulosa cells. The cells on collagen-coated culture plates proliferated extensively in a nutrient medium supplemented with insulin, heparin binding growth factor-2 (HBGF-2), lipoprotein, and bovine serum albumin (BSA). The cell doubling time at logarithmic phase and final cell density at confluent cultures were equal to those of cultures grown in the presence of medium supplemented with optimal concentration (10%) of fetal bovine serum (FBS). Whereas HBGF-2 or insulin alone had a small mitogenic effect of granulosa cells, lipoprotein or BSA did not. When lipoprotein, BSA, or insulin was added together with HBGF-2, synergistic cell proliferation was observed in all combinations. Insulin or lipoprotein had an additive mitogenic stimulation of these cells in the presence of BSA. After granulosa cells were subcultivated in a serum-containing medium until three generations [8.5 cumulative population doubling level (CPDL)], subsequent subcultivation of the cells in a complete serum-free medium could be achieved up to six generations (14.4 CPDL). These results demonstrate that this serum-free medium can support the optimal cell growth and long-term subcultivation of bovine granulosa cells.     

胰岛素对鹅原代肝细胞增殖及蛋白合成的影响

通过用不同浓度胰岛素培养天府肉鹅（Ansercygnoides）原代肝细胞探讨胰岛素对鹅原代肝细胞增殖及蛋白合成的影响。结果表明：与对照组相比,0—200nmol／L胰岛素对肝细胞上清液中谷草转氨酶（AST）、谷丙转氨酶（ALT）浓度没有显著影响,表明细胞功能正常;100、150和200nmol／L胰岛素显著增加细胞培养上清液中总蛋白（什）和白蛋白（ALB）浓度;Brdu．ELISA法检测DNA合成实验结果表明经胰岛素处理后显著增加鹅原代肝细胞增殖率。因此,胰岛素能促进鹅原代肝细胞的细胞增殖及蛋白合成。     

Receptors for epidermal growth factor (urogastrone) and insulin in primary cultures of rat hepatocytes maintained in serum-free medium

We have analyzed the receptors for epidermal growth factor (urogastrone) (EGF-URO) and insulin in primary cultures of adult rat hepatocytes maintained for up to 3 weeks on human placental cell matrix in serum-free defined medium. Cross-link labeling experiments revealed that the insulin receptor, partially damaged by the collagenase isolation procedure, was rapidly regenerated to yield an intact receptor. In contrast, cross-link labeling of the EGF-URO receptor revealed that, upon prolonged culture, there was a progressive disappearance of the high molecular mass (175 kilodaltons (kDa)) receptor form, and an appearance of low molecular mass receptor species (130 and 105 kDa). After 3 weeks of culture, the low molecular mass receptor forms accounted for all of the labeled EGF-URO receptor present in the cells. Measurements of EGF-URO binding indicated that the number of EGF-URO binding sites per cell (2.0 x 10(5) +/- 0.3 x 10(5)) did not change during the 3 weeks of culture. However, there was a decrease in EGF-URO binding affinity, reflected by an increase in the KD from 0.6 to 3.0 nM. At zero time and after 3 weeks in culture, Scatchard plots of the binding data were linear; at intermediate time points, the plots were curvilinear. Despite the changes in the EGF-URO receptor that occurred, cells were still responsive to EGF-URO in terms of the inhibition of acetate incorporation into lipid. The ED50 for EGF-URO (about 0.2 nM) was the same for short-term cultures (48 h) as for cells maintained in culture for 3 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adaptation of BHK cells producing a recombinant protein to serum-free media and protein-free medium

In this work a recombinant BHK21 clone producing a fusion protein with potential application in tumour target therapy was adapted to five different serum-free media (SFM) and to a protein-free medium (PFM). Only the PFM did not require a gradual adaptation to cell growth in the absence of serum. All tested SFM required a gradual adaptation (up to 35 days). For the majority of the SFM tested, cell specific productivity was not affected by the decrease in serum concentration during adaptation; however, cell growth was significantly affected by the serum decrease. Both cell growth and productivity were increased when PFM SMIF6 was used instead of the control medium. Long term measurements (approximately 100 days) of cell specific productivity for PFM and the two best SFM showed that productivity was maintained. This indicates the media capability to be used in long term production processes.     

A simple medium for the study of hepatocyte growth in culture under defined conditions

Summary The combination (1∶1) of Dulbecco's modified Eagle's medium and Waymouth's medium MAB 87/3 was found to provide favorable conditions for serum-free culture and growth of adult rat hepatocytes. In this simple medium, a majority of hepatocytes stimulated by epidermal growth factor plus insulin entered S phase and divided, with a normal (13 h) interval between DNA synthesis and cell division. The proliferative response did not require extra substratum or the presence of serum, even during cell isolation and plating. This work was supported by the Norwegian Cancer Society.     

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

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

Stimulation of DNA synthesis in primary cultures of adult rat hepatocytes by rat platelet-associated substance(s)

Summary Experiments in whole animals have shown that normally quiescent adult rat hepatocytes are induced to proliferate by blood borne substances, which we are now probing in primary monolayer cultures. Under our conditions, freshly isolated adult hepatocytes do not proliferate actively in a defined medium, but are stimulated to synthesize DNA — an essential first step — by either serum or an EGF-hormone combination. Stimulation of [³H]thymidine incorporation into hepatocyte DNA by addition of dialyzed mouse, human, horse, or bovine (fetal, newborn, or calf) serum, whose activities are all similar, is regularly surpassed by an EGF-insulin mixture without serum. This, in turn, is exceeded by dialyzed normal rat serum, which is several times more potent than the other sera tested. Removal of blood platelets reduces the activity of normal rat serum by over 50%. Heat inactivation (56° C) causes a similar loss, but heat treatment of platelet-poor serum fails to cause further reduction. The activity of mouse and human serum is not reduced by platelet removal. Serum from partially hepatectomized rats is not significantly more stimulatory than normal rat serum, and its activity is depressed in the same way by platelet deprivation and heat inactivation. Lack of enhancement by partial hepatectomy is not consonant with whole animal studies and requires further investigation. The heat-labile portion of the DNA synthesis-stimulating activity of rat serum appears to derive from platelets. This activity differs from the well-characterized heat-stable human PDGF. Its relation to other reported platelet-associated growth factors is still undetermined. This work was supported by USPHS Grants CA-02146 and AM-19435.     

The effect of amino acid composition of serum-free medium on DNA synthesis in primary hepatocyte cultures in the presence of epidermal growth factor

Summary The presence of optimal nutritional elements in cell culture medium is very important in studies of cultured cells. For this reason, several researchers have experimented with adding or increasing the concentration of one or more amino acids to the medium they were using to determine “essential” amino acids and optimal concentrations. We studied how leaving out one amino acid at a time from Dulbecco’s modified Eagle’s medium would affect epidermal growth factor-induced DNA synthesis in primary hepatocytes of the rat. Our “modified” DMEM contained only eight amino acids: arginine, cysteine, isoleucine, leucine, lysine, phenylalanine, tryptophan, and valine. Proline was found to be an essential amino acid in normal DMEM but not in the modified DMEM, and some other amino acids reduced DNA synthesis in this medium. This study showed that perhaps no single amino acid such as proline can be called “essential,” but rather an optimal balance of amino acids is required for each major function of each cell type cultured.     

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.     

Tissue engineering of cartilage with chondrocytes cultured in a chemically-defined, serum-free medium

Cell culture with serum-containing medium has potential problems associated with contamination of infectious agents. This study demonstrates for the first time the feasibility of regenerating cartilage tissues in vivo by implantation of chondrocytes cultured in vitro in a chemically-defined, serum-free medium. Chondrocytes cultured in the serum-free medium grew similarly to those in a serum-containing medium. Implantation of chondrocytes cultured in the serum-free medium and seeded on to polymer scaffolds resulted in the regeneration of cartilage tissues with histological aspects similar to those of cartilage tissues regenerated from chondrocytes cultured in serum-containing medium.     

A serum-free medium for the culture of insect cells and production of recombinant proteins

Summary A low protein aqueous lipid supplement (Ex-Cyte VLE), in combination with pluronic polyol, is an effective replacement for fetal bovine serum for insect Sf-9 cells. Serum-free medium with lipid supplement and pluronic (SFM-LP) supported higher cell viability and maximum cell populations than serum-supplemented medium. No adaptation procedures are required when switching cells from serum-containing medium to SFM-LP, and growth rates remain constant during continued passages in SFM-LP. The amounts of recombinant proteins produced, which is the major use for the Sf-9 cells, are better or equal in SFM-LP compared to serum-supplemented medium. SFM-LP also supports growth of the TN-368 cell line but IPLB-SF-21AE or IZD-Mb0503 lines grow poorly in this medium.