Influence of single amino acids on the development of hamster one-cell embryos in vitro

One-cell hamster embryos placed in culture have always shown a complete block to development at the two-cell stage. In a preliminary study using a chemically defined culture medium containing 20 amino acids (HECM-1), many one-cell embryos were able to escape the "two-cell block" and develop to the four-cell stage. Use of a simpler formulation containing only the amino acids hypotaurine and glutamine revealed marked inhibitory and stimulatory effects of adding the other amino acids. In the first experiment, 19 amino acids were separately examined for effects on one-cell embryo development. Six amino acids (phenylalanine, valine, isoleucine, tyrosine, tryptophan, and arginine) inhibited embryo development (reduced mean cell number; MCN), and three others (glycine, cystine, and lysine) stimulated development (increased MCN), compared with basic medium containing only glutamine and hypotaurine (low control). When the responses with the six inhibitory amino acids were totalled, only 3 of 185 (2%) one-cell embryos reached the six-or seven-cell stage compared to a total of 15 of 76 (20%) embryos that developed to these stages using the three stimulatory amino acids. When tested together in a second experiment, the six inhibitory amino acids significantly reduced the MCN, from 4.28 +/- 0.44 (low control) to 3.71 +/- 0.55. In this group, 17 of 117 (15%) of one-cell embryos reached more than four-cell and only 4 of 117 (3%) reached six- or 7-cell stages, compared with 39 of 117 (33%) and 12 of 117 (10%), respectively, for the basal medium group.(ABSTRACT TRUNCATED AT 250 WORDS)     

A serum-free system for culturing human placental trophoblasts

Summary We have compared hormone production by early gestation and term human placental trophoblasts cultured in Ham's F10 medium containing 10% fetal bovine serum with that by cells cultured in serum-free HB102 medium. Mean daily production of progesterone on Days 3 to 7 was approximately 25% less by both early gestation and term cells cultured in HB102 as compared to Ham's F10, but production was maintained at a stable level for at least 7 d longer than the cells in Ham's. Estradiol production from 10⁻⁶ M dehydroepiandrosterone by both early gestation and term cells was comparable in both media. Human placental lactogen production on Days 3 to 7 was 40% less by cells cultured in HB102. Human chorionic gonadotropin (hCG) output by early gestation cells was also 50% less in HB102 but term cells in HB102 produced twice as much hCG as those in Ham's F10. 3B-Hydroxysteroid dehydrogenase (3BHSD) activity in early gestation and term cells and 11B-hydroxysteroid dehydrogenase (11BHSD) activity of early gestation cultures was comparable in the two media. 11BHSD activity was decreased in the term cultures, and this decrease was more marked in Ham's than in HB102. Sulfatase and aromatase activities in the early gestation cultures were comparable in both media; sulfatase activity was comparable and aromatase activity only 20% less in the term cells cultured in HB102. These results indicate that serum-free HB102 supports differential function of human trophoblast cells and is useful for studies of placental activity for as long as 14 d in culture.     

A protein-free medium for the growth of hybridomas and other cells of the immune system

Summary A protein-free medium, termed ABC, has been developed which essentially eliminates the need for serum proteins. ABC supports the long-term growth of murine hybridomas as well as other transformed cells of the immune system. The requirement of hybridoma growth for transferrin has been met by substituting the soluble organo-iron compound, sodium nitroprusside. Substantial improvement in the growth of hybridomas was afforded by the inclusion of 18 trace elements complexed to disodium ethylene diaminetetraacetate (EDTA). The medium was further improved by the inclusion of components not found in Ham's F12 medium or by raising the concentrations of existing low molecular weight components. Murine hybridomas can be cultured routinely in this protein-free medium in an anchorage-independent manner with doubling times generally under 24 h. Visualized on electrophoretic gels, levels of monoclonal antibody taken from those cultures often exceeded 80% of the total protein. The medium was also able to support the growth of HuT 78 and H9 cells as well as certain other transformed cells of the immune system. In addition, normal human peripheral blood lymphocytes, activated with phytohemagglutinin and cultured with 50 U/ml recombinant interleukin 2, could be grown for 2 wk with a 50-fold expansion over input cell number.     

Growth of the malignant and nonmalignant human squamous cells in a protein-free defined medium

Summary A novel protein-free synthetic medium has been developed for the culture of human squamous cell carcinoma cells. This medium, designated PF86-1, supports the serial subcultivation of six out of nine human squamous cell carcinoma cell lines in a protein-free, chemically defined condition without the adapting culture from serum-containing conditions. These cell lines growing in PF86-1 exhibited nearly equal potency to grow in massive culture without noticeable changes in morphology but presented a significantly decreased level of colony forming efficiency when compared with the cells cultured in serum-containing media, suggesting the implication of some autocrine mechanism. Interestingly, this medium supported the growth of normal human squamous cells of oral mucosa and skin for more than 2 mo. in the primary explant culture in spite of high levels of calcium ion concentration, where the overgrowth of fibroblasts as contaminant was not observed. These results suggest that PF86-1 supports the growth of cells derived from epidermal tissues selectively and provides the same defined condition for growth of malignant and nonmalignant human squamous cells. It seems, therefore, that PF86-1 allows investigations on the products of squamous cell carcinoma cells or on the differences of growth mechanisms between normal and neoplastic human squamous cells.     

THE EFFECTS OF PREDATION ON THE AGE AND SIZE OF MATURITY OF PREY

The effects of nonselective predation on the optimal age and size of maturity of their prey are investigated using mathematical models of a simple life history with juvenile and adult stages. Fitness is measured by the product of survival to the adult stage and expected adult reproduction, which is usually an increasing function of size at maturity. Size is determined by both age at maturity and the value of costly traits that increase mean growth rate (growth effort). The analysis includes cases with fixed size but flexible time to maturity, fixed time but flexible size, and adaptively flexible values of both variables. In these analyses, growth effort is flexible. For comparison with previous theory, models with a fixed growth effort are analyzed. In each case, there may be indirect effects of predation on the prey's food supply. The effect of increased predation depends on (1) which variables are flexible; (2) whether increased growth effort requires increased exposure to predators; and (3) how increased predator density affects the abundance of food for juvenile prey. If there is no indirect effect of predators on prey food supply, size at maturity will generally decrease in response to increased predation. However, the indirect effect from increased food has the opposite effect, and the net result of predation is often increased size. Age at maturity may either increase or decrease, depending on functional forms and parameter values; this is true regardless of the presence of indirect effects. The results are compared with those of previous theoretical analyses. Observed shifts in life history in response to predation are reviewed, and the role of size-selective predation is reassessed.     

Micropropagation of Sterculia urens Roxb. — an endangered tree species

An in vitro procedure for large scale multiplication of Sterculia urens Roxb. (Gum Kadaya Tree) has been developed using cotyledonary node segments. An average of 4.0 shoots per node were obtained on Murashige and Skoog's (MS) medium containing 2.0 mgl^–1 6-benzyl amino-purine (BAP) within 21 days of initial culture. Upon subsequent subculture 16 shoots/node could be harvested every three weeks and upto three times. Sixty per cent of the shoots were successfully rooted. Rooted plantlets were transferred to plastic pots containing soil under mist house conditions before they were finally exposed to an external environment. Fifty seven per cent of the plantlets survived in nursery sheds.     

In vitro multiplication in liquid culture of Syngonium contaminated with Bacillus spp. and Rathayibacter tritici

Contaminated Syngonium clusters were multiplied in an air lift bioreactor in liquid medium containing sucrose with the medium being circulated through a sterilizing filter. After 30 days, the culture in filtered medium produced 19.5 shoot initials per gram fresh weight of inoculum compared to 8.7 shoot initials produced in unfiltered medium. Transfer to an elongation medium with 30 mg l^-1 Rifampicin produced shoots on 67% of the clusters, while transfer to elongation medium without Rifampicin poduced shoots on 40% of the clusters. Clusters grown for three subcultures in a reactor without medium filtration had lost their multiplication ability. Clusters grown for three subcultures in a reactor with filtration, however, continued to show a two-three fold increase in fresh weight and shoot production.Abbreviations MS Murashige and Skoog     

A medium optimization strategy for improvement of growth and methane production by Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum, strain Hveragerdi, has been cultivated in a completely defined mineral salts medium, under strictly anaerobic conditions with CO₂ and H₂ as sole carbon and energy source, respectively. During optimization of the medium an iron limitation was identified that could not be overcome by the simple addition of iron—or iron complexed with nitrilotriacetate (NTA)—to the medium, due to the formation of insoluble FeS complexes. In order to define a medium optimization strategy, and to avoid laborious empirical optimization procedures, a theoretical model has been developed in order to describe the solubility of iron and other mineral species in the medium as a function of the concentration of sulfur and NTA. This model may be applied for the optimization of any medium component. With this information sulfide has been replaced by a combination of cysteine and thiosulphate in conjunction with a non-toxic reducing agent (titanium (III) citrate). Using this defined medium precipitation was avoided and an iron limitation was overcome resulting in a 5-fold improvement of the final biomass concentration from 2–3 g l⁻¹ to 11.2 g l⁻¹ together with a 2-fold increase (from 45 to almost 100%) in the conversion of CO₂ and H₂ to CH₄, even at gas flow rates as high as 6 l min⁻¹.     

Stimulation of the growth and solamargine production by Solanum paludosum multiple shoot cultures using a new culture medium

Organogenic callus cultures of Solanum paludosum were obtained from root, hypocotyle and cotyledon explants of plantlets cultured in sterile conditions. These callus cultures developed multiple shoots which proliferated in Murashige and Skoog basal liquid medium. These multiple shoots produced solamargine, the main steroidal glycoalkaloid present in the unripe fruits.The optimization of the macronutrient composition of the liquid medium was performed by a method derived from the plant composition. This approach results in the establishment of an appropriate medium (SPOM medium) suitable for the improvement of both growth and solamargine production by multiple shoot cultures of S. paludosum.     

Continuous,high capacity reconstitution of nutrient media from concentrated intermediates

We designed an Integrated Media Preparation System (IMPS) for continuous, on-line preparation of cell culture media and delivery to intermediate storage vessels or directly to a bioreactor. Key components of the IMPS include: a high precision, continuous fluid mixing device; formulation-specific liquid medium concentrates; validated process controls and membrane filtration; and automated dispensing into large volume flexible plastic containers. The IMPS system is designed to produce sterile, single-strength liquid medium from common raw materials at a delivery rate of 1000–3000 liters per hour and will manufacture homogenous batches from several thousand liters to over 60,000 liters. Fortified nutrient media prepared from multi-component 50X concentrates have been demonstrated to accelerate bioreactor seed chains, increase product yield, and reduce the overall manufacturing cost of nutrient medium. A productivity matrix will analyze the fully-loaded costs and contrast alternative methods for media preparation against projected biological yield.Abbreviations IMPS Integrated Media Preparation System - 50X Nutrient fluid components formulated at fifty-fold final use concentration - 1X Nutrient fluid formulated at final, single-strength use concentration - cGMP Current Good Manufacturing Practices - SCADA Supervisory Control and Data Acquisition - PLC Process Logic Controller - LTI Life Technologies, Inc. - WFI Water for Injection - CIP Clean in place - SIP Sterilize in place - HPLC High performance liquid chromatography - DMEM Dulbecco's Modified Eagle's Medium