The effect of serum batch on the in vitro lifespans of cell cultures derived from old and young human donors

In a senescence study, skin fibroblast cultures grown in the presence of a second batch of fetal calf serum (FCS) revealed delayed onsets of cell culture senescence and prolonged in vitro lifespans when compared to cell cultures grown on the initial batch of serum. These statistically significant differences occurred despite the fact that both sera displayed equal growth promoting abilities as measured by cell culture growth curves performed on parallel cultures with the two sera. When cultures grown in either sera were analyzed separately, the onset of cell culture senescence was earlier and in vitro lifespan was shorter in those cultures derived from the old donor group (ages 63–92) when compared to cultures derived from young donors (ages 21–36).     

Immature embryo and anther culture of chromosome addition lines of rye in chinese spring wheat

Significant variation among Chinese Spring wheat (Triticum aestivum L.) and a set of seven addition lines in which chromosomes from rye (Secale cereale L.) were incorporated into the Chinese Spring background was observed for callus formation and plant regeneration from anther cultures and for plant regeneration from immature embryo cultures. Callus initiation from immature embryo cultures was uniformly high. Rye chromosome 4 contains factors which significantly increase both anther culture responses relative to Chinese Spring. Rye chromosomes 6 and 7 both contain positive factors for regeneration from immature embryo culture. While no correlation was found between anther culture and embryo culture responses, a positive correlation was observed between the two anther culture response variables.     

Polysaccharides of cell cultures of Silene vulgaris

Callus and suspension cultures of campion (Silene vulgaris) produced pectin polysaccharides, similar in structure to the polysaccharides of intact plants. The major components of the pectins were D-galacturonic acid, galactose, arabinose, and rhamnose residues. The maximum content of pectins was found in callus. The monosaccharide composition of arabinogalactans isolated from cells and a culture medium of callus cultures were similar, with the ratio between arabinose and galactose of 1: (2.3-6.5) being retained. The arabinogalactans from the cells and culture medium of the suspension cultures also had a similar structure, and the arabinose to galactose ratio was 1: (1.5-1.8). In contrast to the callus cultures, the suspension cultures produced arabinogalactans with an increased content of arabinose residues and a decreased content of galactose residues. The greatest content of arabinogalactan was detected in the culture medium of the suspension cultures.     

Comparative study of amylolytic enzymes production byAspergillus niger in liquid and solid-state cultivation

Summary Amylolytic enzymes produced by a strain ofAspergillus niger cultivated on cassava starch in liquid or solid culture were found to be mainly glucoamylases. For the same initial amount of substrate, the glucoamylase activity increased even after 60 h of culture on solid medium whereas it decreased in liquid culture. Some characteristics of the amylases produced in both culture conditions were compared. The pH optima for enzymes produced in solid and liquid cultures were 4.5 and 5.0 respectively. Glucoamylase synthetized in solid cultures was significantly more thermostable than that from liquid culture and was maximally active at 70°C compared to 50°C for the enzyme from liquid cultures. The Km values expressed as mg soluble starch/100 ml were 0.1% for crude enzyme from solid culture and 0.057% for crude enzyme from liquid culture.     

华根霉固态与液态培养产胞外蛋白的比较蛋白质组学分析

【目的】考察固态和液态两种培养方式对丝状真菌华根霉(Rhizopus chinensis)CCTCC M201021产胞外蛋白的影响,以加深对微生物固、液态培养特异性产酶的认识。【方法】利用成分相同的培养基对华根霉分别进行平板固态培养和液态培养,提取华根霉所产胞外蛋白,采用二维凝胶电泳(2-DE)结合质谱分析,分离鉴定差异蛋白。【结果】固态培养下华根霉所产胞外蛋白的蛋白酶活性是液态培养的9.2倍。胞外蛋白质组数据分析表明,华根霉固态和液态培养所产胞外蛋白存在显著差异,其中约70%是固态和液态培养下各自产生的特有蛋白。质谱鉴定进一步表明,固、液态培养对华根霉产胞外蛋白的种类和表达量都有显著影响,其中水解酶类所占比例较大,主要是与蛋白质降解相关的蛋白。【结论】固态和液态不同培养方式影响了华根霉产胞外蛋白的组成,有些基因只在特定培养方式下表达。固态培养下华根霉产胞外蛋白酶种类相对更多以及大部分蛋白酶的上调表达,可能是固态培养下胞外蛋白酶活性很高的原因。研究结果提示需要注意固液态不同培养方式下丝状真菌胞外酶的筛选和生产可能存在的不一致性。     

Dexamethasone stimulates osteogenic differentiation in vertebral and femoral bone marrow cell cultures: Comparison of IGF-I gene expression

Osteoblast-like cell cultures have been established from the marrow of adult rat vertebrae. We have simultaneously examined the response to dexamethasone (dex) treatment in cultures of young adult female vertebral and femoral marrow cells. Alkaline phosphatase (AP) activity was analyzed as well as the expression of mRNAs for osteocalcin (OC) and insulin-like growth factor I (IGF-I). The vertebral and femoral marrow cells were maintained for 7 days in primary culture with or without 10⁻⁸ M dex and then 6 days in secondary culture without dex or with 10⁻⁸ M or 10⁻⁷ M dex. All cells were examined on day 6 of secondary culture. Vertebral and femoral cultures each expressed the highest AP enzyme levels when grown with dex in primary culture (10⁻⁸ M) and secondary culture (10⁻⁷ M). Under all experimental conditions, vertebral cultures had lower AP enzyme activity than femoral cultures. When dex was omitted from secondary culture, OC gene expression was not detected in either vertebral or femoral passaged cells even if dex was present in primary culture. For dex conditions where OC was expressed, vertebral cultures had higher OC mRNA steady-state levels than femoral cultures. IGF-I gene expression was detected by Northern analysis in both vertebral and femoral secondary cultures. However, vertebral marrow cultures had much higher IGF-I mRNA levels compared to femoral cultures whether or not dex was present in primary culture. These findings demonstrate that dex supports the differentiation of both vertebral and femoral adult marrow osteogenic cells into osteoblasts. Our results support the hypothesis that osteoblastic marrow cultures differ depending upon which location in the skeleton they are from and that there are skeletal site–dependent differences in the insulin-like growth factor system components. J. Cell. Biochem. 71:382–391, 1998. © 1998 Wiley-Liss, Inc.     

Molecular and Genetic Characterization of Mu Transposable Elements in Zea Mays: Behavior in Callus Culture and Regenerated Plants

Active Mutator lines of maize (Zea mays L.) have a high mutation rate and contain multiple hypomethylated 1.4-kb and 1.7-kb Mu transposable elements. Correlated with the inactivation of the Mutator system, these Mu elements cease to transpose and become more methylated. To determine whether the shock of tissue culture can affect Mutator activities, F1 progenies of outcrosses between active or inactive Mutator stocks and inbred line A188 were used to initiate embryogenic callus cultures. HinfI restriction digestion of genomic DNA isolated from 3-5-month-old cultures demonstrated that there is a very good correlation between the modification state of Mu elements in the cultures and the Mutator parent. Despite the dedifferentiation and rapid proliferation characteristic of tissue culture, the Mutator activity state is relatively stable during an extended tissue culture period. Cultures established from inactive Mutator lines were not reactivated; cultures established from active lines maintained a high Mu copy number, and most Mu elements remained unmodified. In contrast, weakly active Mutator parents gave rise to cultures in which Mu element modification could switch between low and high methylation during the culture period. Evidence for transposition was investigated with EcoRI digestion of genomic DNA isolated at different times during culture. The appearance of novel Mu-hybridizing fragments and a strong background hybridization are interpreted as evidence that transposition events occur during culture. Plants regenerated from such active cultures transmitted Mutator activity to their progeny.     

Establishment of embryogenic callus and high protoplast yielding suspension cultures of sugarcane (Saccharum spp. hybrids)

For 18 sugarcane cultivars, four distinct callus types developed on leaf explant tissue cultured on modified MS medium, but only Type 3 (embryogenic) and Type 4 (organogenic) were capable of plant regeneration. Cell suspension cultures were initiated from embryogenic callus incubated in a liquid medium. In stage one the callus adapted to the liquid medium. In stage two a heterogeneous cell suspension culture formed in 14 cultivars after five to eight weeks of culture. In stage three a homogeneous cell suspension culture was developed in six cultivars after 10 to 14 weeks by selective subculturing to increase the proportion of actively dividing cells from the heterogeneous cell suspension culture. Plants were regenerated from cell aggregates in heterogeneous cell suspension cultures for up to 148 days of culture but plants could not be regenerated from homogeneous cell suspension cultures. High yields of protoplasts were obtained from homogeneous cell suspension cultures (3.4 to 5.2 × 10⁶ protoplasts per gram fresh weight of cells [gfwt^-1]) compared to heterogeneous cell suspension cultures (0.1 × 10⁶ protoplasts gfwt^-1). Higher yields of protoplasts were obtained from homogeneous cell suspension cultures for cultivars Q63 and Q96 after regenerating callus from the cell suspension cultures, then recycling this callus to liquid medium (S-cell suspension cultures). This process increased protoplast yield to 9.4 × 10⁶ protoplasts gfwt^-1. Protoplasts isolated from S-cell suspension cultures were regenerated to callus and recycled to produce SP-cell suspension cultures yielding 6.4 to 13.2 × 10⁶ protoplasts gfwt^-1. This recycling of callus to produce S-cell suspension cultures allowed protoplasts to be isolated for the first time from cell lines of cultivars Q110 and Q138.     

Changes in apoptotic rate and cell viability in three fish epidermis cultures after exposure to nonylphenol and to a wastewater sample containing low concentrations of nonylphenol

Three types of epidermal cultures of fish were used for toxicological investigations, a primary cell culture and a tissue culture prepared from the rainbow trout Oncorhynchus mykiss Walbaum and the cell line EPC, derived from a skin tumour of the carp Cyprinus carpio L. Two studies were carried out to compare the different culture systems. In the first cultures were incubated with nonylphenol and in the second set of experiments the cell cultures were exposed to a wastewater sample containing low concentrations of nonylphenol (NP). Both cell cultures were similarly sensitive to nonylphenol with respect to the endpoints cell viability (LC₅₀     

Variation in the life-span of clones derived from human diploid cell strains

The doubling potential of several hundred clones derived from WI-38 and WI-26 cell cultures has been determined. Clones were isolated at various population doubling levels (PDLs) during the finite in vitro life-span of the mass (uncloned) cultures. In all cases, there was a large variation in population doubling potential (or life-span) among the clones isolated from a single mass culture. When clones were isolated from mass cultures which had undergone eight or nine population doublings, only about 50% of the clones were capable of more than eight population doublings. This percentage was further reduced when clones were isolated from mass cultures at higher PDLs. Mass cultures appear to be composed of two subpopulation classes: one with a low population doubling potential, and the other with a higher population doubling potential. Nevertheless, the highest doubling potential observed in clones isolated from any single culture was about the same as the doubling potential of the mass culture from which single cells were taken.     

The Decline of Embryogenic Potential as Callus and Suspension Cultures of Carrot (Daucus carota L.) are Serially Subcultured

Callus and suspension cultures derived from seedling root segmentsof carrot can be assessed for their embryogenic potential (EP)by transfer of a standard culture inoculum to 25 ml culturemedium with 2,4-D omitted and incubation for a fixed period;the EP is expressed as the number of embryoids (0·5–2·5mm in length) developed per culture under these standard conditions.The initial decline in EP is indicative of increasing sensitivity,as culture proceeds, to inhibition by the auxin essential tocontinuing growth of the cultures. However, during culture changesoccur in the nuclear cytology of the cells leading to the appearanceof cells of impaired or nil totipotency and some such cellsare at a selective advantage so that eventually the cultures,as they are serially subcultured, no longer contain any totipotentcells. Normally the cells of such cultures have chromosome numbersin excess of the diploid complement. Evidence for the view thatthe cultures, as they exhibit declining EP, come to containa mixed population of cells comes from microscopic examinationof the cultures and from the isolation, cytological examinationand assessment of the EP of cell lines isolated by plating.Evidence that cells which lack totipotency and which in singleculture have similar growth rates to totipotent cells may neverthelessbe at a strong selective advantage in mixed culture is presentedfrom a study of the growth, and changing cellular compositionand EP during serial subculture of an artificially-preparedmixed culture initially containing equal numbers of diploidtotipotent cells and tetraploid cells lacking totipotency.     

Growth and differentiation of chicken embryo muscle cell cultures derived from fast- and slow-growing lines. Intrinsic differences in growth characteristics and insulin response

Primary myogenic cell cultures derived from 12-day embryos of genetically fast-growing chickens (fast cultures) and slow-growing chickens (slow cultures) were grown under identical conditions to examine differences in growth and differentiation at the cellular level. The two types of cultures exhibited significant (P less than 0.01) differences in proliferation, protein accumulation, response to the addition of insulin to the culture medium and the amount of insulin bound per nucleus. The fast cultures exhibited a larger number of both total nuclei and fused nuclei at 48, 72 and 96 h in culture, accumulated more protein per nucleus at 24, 48 and 72 h in culture and demonstrated a greater response to the addition of insulin to the culture medium, as reflected by increased fusion rate and protein accumulation at 24 h in culture. Maximal response to insulin in both types of cultures was obtained at 24 h to added insulin concentrations of 10(-10)-10(-9) M. Slow cultures bound more [125I]-insulin than fast cultures at 24 h in culture. These experiments suggest that different muscle growth potentials in animals of the same species are at least partly due to intrinsic cellular differences in the myogenic cells that give rise to adult muscle tissue.     

Physiological and molecular characterization of anaerobic benzene-degrading mixed cultures

Nine distinct anaerobic benzene-degrading cultures were enriched from sediment samples from four different sites. These cultures used nitrate, sulphate or CO2 as electron acceptors. The shortest doubling times were observed in nitrate-reducing cultures, although cell yield was lowest in these cultures. The highest substrate concentration utilized and maximum absolute rates of benzene degraded (in micro M day-1) were observed in methanogenic cultures. The microbial compositions of a methanogenic and nitrate-reducing culture were determined from a clone library of 16S rRNA genes. Five Bacterial 16S rRNA sequences, one of which resembled a clone previously found in a sulphate-reducing, benzene-degrading culture and four Archaeal 16S rRNA sequences were identified in a methanogenic culture. Four Bacterial and no Archaeal 16S rRNA sequences were identified in a nitrate-reducing culture. The relative abundance of the four nitrate-reducing putative species was determined by slot blot hybridization. Two green sulphur bacteria together formed 52% of the clone library, but were found to be less than 4% of the culture by slot blot analysis. One of the cloned 16S rRNA gene sequences comprised 70% of the culture and was phylogenetically 93% similar to both Azoarcus and Dechloromonas species, which have been shown to degrade aromatic compounds, including benzene, under nitrate-reducing conditions.     

The causes of competition between two cell lines ofDaucus carota in mixed culture

Summary Observations were made of the changes in frequency of two genetically different culture lines ofDaucus carota (designated DBB and DSL) during successive subcultures of mixed cultures. Under conditions of phosphate limited growth, one line (DSL) had a competitive advantage over the other. Under other cultural conditions, this competitive interaction was removed or reversed. Detailed comparisons of the growth patterns and phosphate uptake rates of the two culture lines grown separately allowed prediction of their behaviour in mixed cultures. Deviations of observed from predicted behaviour in mixed cultures demonstrated that the dominant culture line (DSL) could compete more effectively for the available phosphate, thereby modifying the growth of line DBB and causing its elimination from mixed cultures. It is concluded that the phosphate limited medium in which these culture lines were initiated had selected genotypes differing in their efficiencies of phosphate utilisation.     

Effects of Formate on Fermentative Hydrogen Production by Enterobacter aerogenes

This paper describes the effects of formate on fermentative hydrogen production by Enterobacter aerogenes by way of batch culture. When 20 mM formate was added to pH 6.3 and pH 5.8 E. aerogenes glucose cultures (formate culture) at the beginning of cultivation, hydrogen evolution through both glucose consumption and decomposition of the extrinsic formate occurred together, while hydrogen evolution occurred only through glucose consumption in the control cultures. The hydrogen evolution rates in the formate cultures were faster than in the control cultures, although cell growth and glucose consumption rates in the formate cultures were slower than the control cultures’. The decomposition rate of the extrinsic formate in the pH 5.8 formate culture was faster than in the pH 6.3 fomiate culture. The hydrogen yield from glucose in the pH 6.3 formate culture increased due to the increasing amount of the nicotinamide adenine dinucleotide for hydrogen production.     

Nitrogen metabolism in cultured cotyledon explants of Pinus radiata during de novo organogenesis

Nitrogen metabolism was investigated under shoot-forming (SF) and non-shoot-forming (NSF) conditions in cultured cotyledon explants of Pinus radiata by following the incorporation of [¹⁴C]-l,2-acetate into various metabolites. Early in culture, the lipid fraction contained the most ¹⁴C; however, this percentage decreased in favor of increased label in the amphoteric fraction. Label in the amphoteric fraction of SF cultures decreased by day 21 but plateaued in NSF cultures at this time. Radioactive labeling of the principle nitrogen metabolites, glutamate and glutamine, which made up the majority of the amphoteric fraction, paralleled labeling patterns in the amphoteric fraction. Percentage label in glutamate remained at similar levels throughout the 21-day culture period for both SF and NSF cultures. Specific activity of glutamate (kBq mg^-1) was significantly greater during promeristemoid formation in SF compared to that in NSF tissues. Glutamine labeling increased during shoot bud initiation in SF cultures, but dropped to lower levels during shoot bud development. In contrast, in NSF cultures, there was a continual and substantial increase in glutamine labeling throughout the 21-day culture period. These trends were similar when the specific activities of glutamine were determined, as there was a continual decrease from culture initiation to the end of shoot bud differentiation in SF cultures. In NSF cultures, in contrast, specific activity of glutamine increased substantially from day 5 to 21 relative to that in SF cultures. The nitrogen assimilation enzymes glutamate synthase and glutamine synthase increased in activity from day 0 to 21 for both SF and NSF tissues. Enzyme activities for glutamate dehydrogenase were similar in both treatments to day 10 in culture but subsequently diverged, with activities in NSF cultures being substantially greater than those of SF cultures by day 21. Taken together, labeling and enzyme data indicate that nitrogen metabolism is enhanced during culture, especially in SF tissues at the time of promeristemoid formation, and in non-organ-forming tissue senescence-like metabolism was exhibited later in culture.     

Growth and differentiation of chicken embryo muscle cell cultures derived from fast- and slow-growing lines

Abstract. Primary myogenic cell cultures derived from 12-day embryos of genetically fast-growing chickens (fast cultures) and slow-growing chickens (slow cultures) were grown under identical conditions to examine differences in growth and differentiation at the cellular level. The two types of cultures exhibited significant ( P <0.01) differences in proliferation, protein accumulation, response to the addition of insulin to the culture medium and the amount of insulin bound per nucleus. The fast cultures exhibited a larger number of both total nuclei and fused nuclei at 48, 72 and 96 h in culture, accumulated more protein per nucleus at 24, 48 and 72 h in culture and demonstrated a greater response to the addition of insulin to the culture medium, as reflected by increased fusion rate and protein accumulation at 24 h in culture. Maximal response to insulin in both types of cultures was obtained at 24 h to added insulin concentrations of 10⁻¹⁰−10⁻⁹ M . Slow cultures bound more [¹²⁵I]-insulin than fast cultures at 24 h in culture. These experiments suggest that different muscle growth potentials in animals of the same species are at least partly due to intrinsic cellular differences in the myogenic cells that give rise to adult muscle tissue.     

Cell attachment to microcarriers affects growth, metabolic activity, and culture productivity in bioreactor culture

It is not well understood how changes from suspension to microcarrier cultures affect cell growth, metabolism, and yield of recombinant proteins. To investigate the effects of culture conditions on cell characteristics, fed-batch bioreactor cultures were performed under different culture conditions (suspension cultures, cultures attached to Cytodex 3 and Cytopore 1 microcarriers) using two different Chinese hamster ovary cell lines producing either secreted human placental alkaline phosphatase (TR2-255) or tissue plasminogen activator (CHO 1-15-500). In controlled, agitated bioreactors, suspension cultures reached cell densities and product titers higher than those in microcarrier cultures, in contrast to the results in static flask cultures. Growth and metabolic activities showed similar trends in suspension and microcarrier culture regardless of cell line. However, the responses of the specific productivities to the different culture conditions differed significantly between the cell lines.     

Partial base-methylation and other structural differences in the 17 S ribosomal RNA of sycamore cells during growth in cell culture.

Sycamore (Acer pseudoplatanus L.) cytoplasmic rRNA was investigated in rapidly dividing cells, cells starting mitosis after the lag phase of growth (4 days) induced by deconditioning of the culture medium and also in growth-arrested cells from 10 day-old cultures deprived of exogenous auxin (i.e. exponential, early exponential and 2,4-dichlorophenoxyacetic acid (2,4-D)-deprived cultures). rRNA was extracted and purified from mixed 14C-labelled exponential cultures and 3H-labelled early exponential cultures. A 14C-labelled exponential culture and a 3H-labelled 2,4-D-deprived culture were analyzed in the same way. The 17 S rRNA molecules from both early exponential and 2,4-D-deprived cultures displayed a lower electrophoretic mobility on polyacrylamide gels than those from exponential cultures. Alkaline and acid hydrolysates of purified 17 S rRNA labelled on the phosphate groups or the methyl groups were analyzed on ion-exchange resins. There was no change in the extent of ribose methylation of the molecule from the three different cultures. However, the base methylation of the 17 S rRNA was decreased in early exponential cultures and in 2,4-D-deprived cultures. Part of the molecules synthesized in early exponential cultures specifically lacked 7-methylguanine, N6-methyladenine and N6,N6-dimethyladenine. The possible significance of these changes in the 17 S rRNA were discussed.     

Bovine generalised glycogenosis type II. Uptake of lysosomal alpha-glucosidase by cultured skeletal muscle and reversal of glycogen accumulation

acid alpha-glucosidase (EC 3.2.1.20) was purified from fetal bovine muscle by affinity chromatography on concanavalin A and Sephadex G-100 and added to the culture medium of mature muscle cultures from animals affected by glycogenosis type II. The enzyme activity in homogenates of treated cultures was significantly increased within 4 h of the addition of enzyme, was maximal by 18 h and the internalised activity was stable for at least 48 h after the removal of the enzyme from the culture medium. The acid alpha-glucosidase activity was internalised with an uptake constant of 300 nM and a Vmax of uptake of 133 nmol/h per mg protein. The glycogen concentration in affected cultures treated with exogenous acid alpha-glucosidase for 24 h had decreased by 20% and after a further 24 h of culture was comparable to the concentration observed in cultures from non-affected animals.