Homogenisation and oxygen transfer rates in large agitated and sparged animal cell bioreactors: Some implications for growth and production

Because of concern for cell damage, very low agitation energy inputs have been used in industrial animal cell bioreactors, typical values being two orders of magnitude less than those found in bacterial fermentations. Aeration rates are also very small. As a result, such bioreactors might be both poorly mixed and also unable to provide the higher oxygen up-take rates demanded by more intensive operation. This paper reports experimental studies both of K _L a and of mixing (via pH measurements) in bioreactors up to 8 m³ at Wellcome and of scaled down models of such reactors at Birmingham. Alongside these physical measurements, sensitivity of certain cell lines to continuously controlled dO₂ has been studied and the oxygen up-take rates measured in representative growth conditions. An analysis of characteristic times and mixing theory, together with other recent work showing that more vigorous agitation and aeration can be used especially in the presence of Pluronic F-68, indicates ways of improving their performance. pH gradients offer a special challenge.     

Engineering challenges in high density cell culture systems

High density cell culture systems offer the advantage of production of bio-pharmaceuticals in compact bioreactors with high volumetric production rates; however, these systems are difficult to design and operate. First of all, the cells have to be retained in the bioreactor by physical means during perfusion. The design of the cell retention is the key to performance of high density cell culture systems. Oxygenation and media design are also important for maximizing the cell number. In high density perfusion reactors, variable cell density, and hence the metabolic demand, require constant adjustment of perfusion rates. The use of cell specific perfusion rate (CSPR) control provides a constant environment to the cells resulting in consistent production. On-line measurement of cell density and metabolic activities can be used for the estimation of cell densities and the control of CSPR. Issues related to mass transfer and mixing become more important at high cell densities. Due to the difference in mass transfer coefficients for oxygen and CO₂, a significant accumulation of dissolved CO₂ is experienced with silicone tubing aeration. Also, mixing is observed to decrease at high densities. Base addition, if not properly done, could result in localized cell lysis and poor culture performance. Non-uniform mixing in reactors promotes the heterogeneity of the culture. Cell aggregation results in segregation of the cells within different mixing zones. This paper discusses these issues and makes recommendations for further development of high density cell culture bioreactors.     

Procedural improvements for in vitro production of viable uterine stage caprine embryos

Efforts to improve proportions of caprine immature oocytes developing into viable uterine-stage embryos in vitro involved study of 1924 oocytes in experiments designed to examine influences of fertilization media, sperm incubation temperatures, sperm treatment procedures, different protein supplementations, and different insemination intervals. Oocytecumulus complexes (OCCs) were matured during 27 h in TCM-199 supplemented with 20% FBS, 100 μg LH ml⁻¹, 0.5 μg FSH ml⁻¹, and 1 μg Estradiol-17-β ml⁻¹at 38.5 °C in a humidified 5% CO₂, 5% O₂, and 90% N₂ atmosphere. Freshly collected sperm were washed and incubated at either 22 °C or 38.5 °C for 5 h and then treated with either 0.1 μM calcium ionophore A23187 for 1 min, or with 7.35 mM calcium lactate in the presence of oocytes during the insemination interval, or with 100 μg heparin +2 mM caffeine ml⁻¹ for 15 min. The interval for insemination was experimentally varied i.e. 14 or 24 h. Results showed that: (a) when used as a fertilization medium mDM supported more blastocyst development than TALP (10.5% vs. 0%, P < 0.05); (b) incubation temperatures of 22 °C or 38.5 °C prepared goat spermatozoa equally for capacitation in mDM containing 20% FBS; (c) when oocytes were inseminated with sperm incubated in mDM with 20% FBS and capacitated with calcium lactate more embryos reached the blastocyst stage (P < 0.05) than after incubation in the same conditions but after sperm capacitation with heparin, and A23187 (31.8% vs. 24.2% and 10.2%, respectively; (d) a 24 h insemination interval was not superior to 14 h when sperm were incubated with either 20% FBS or 6 mg BSA ml⁻¹ and capacitated with calcium lactate (P > 0.05). Three morulae resulting from the best conditions in this work (FBS, calcium lactate, 14 h insemination) were transferred into the uterine horn ipsilateral to the corpus luteum of a recipient and two normal female kids were born after normal gestation. This is the first report in which it has been possible to consistently take caprine development to the blastocyst stage in vitro, and to obtain offspring following uterine transfer. Methodology reported here should facilitate implementation of new reproductive and genetic strategies in goat breeding.     

Effect of anti-herpes specific transfer factor

Using a blood cell separator, lymphocytes were collected from otherwise healthy convalescents suffering from herpetic infections. A specific anti-herpes dialysate (AH-DLE) was prepared from the lymphocytes, using standard procedures. Patients with recurrent herpetic infections were treated with a single dose of the dialysate, at the initial signs of herpetic infection (group A), with two doses (group B) or with three doses (group C). A total number of 37 patients (29 women, 8 men, age range 15–73 years) were treated. No improvement was observed in 7 patients (18.9%), whilst 7 patients did not manifest any exacerbation of their herpetic infection in the course of the one-year follow-up. The remaining 62.2% of the patients showed a marked improvement: decrease of the frequency and/or duration or relapses. Before AH-DLE administration, the mean number of herpes relapses in this group of patients was 12 p.a.. After therapy, the number of relapses decreased to 3.5 p.a.. No statistically significant difference was observed between groups A and B. The least favourable results were registered in group C. However, this group included 6 female patients extremely resistant to the previously therapeutic attempts, including inosiplex, non-specific DLE or acyclovir. Thus, even in this group, the therapy was successful in 50% of the patients.     

Dominance, competition, and energetic reserves in the European starling, Sturnus vulgaris

We investigated the relationships between social dominance,competition for food, and strategies of body mass and fat regulationin the European starling (Sturnus vulgaris). In birds housedin groups of three, subdominant birds stored more fat than dominants.A removal experiment established a causal link between socialdominance and fat reserves; in groups that had the dominantindividual removed, the remaining birds reduced body mass andfat, relative to control groups that had the subordinate removed.In a second experiment, we investigated the influences of degreeof competition for food and dominance on body mass and fat reserves.Birds under high competition increased fat reserves and tendedto have higher body mass than birds under low competition. Theincrease in fat reserves was higher in the subdominants thanin the dominants. These results are consistent with hypothesesconcerning dominance-dependent access to food; subdominant birds,or birds under increased competition, may store more fat asan insurance against periods when food cannot be obtained. However,relations between dominance, body mass, and fat reserves mayalso arise through other proximate factors relating to dominance-dependentcosts and benefits of fat storage, such as predation risk andenergetic expenditure.     

Litter fall and decomposition in a Pinus halepensis forest on Mallorca

Abstract. Litter fall and decomposition in a Pinus halepensis forest was studied in order to help understand nutrient cycles in this ecosystem, threatened as it is by fire and tourism. The study was done over two years in an experimental forest stand at Cap des Pinar on Mallorca, Spain. The woodland area has not been disturbed for about 40 yr. Total litter fall amounted to 3.44 ton ha^-1 yr^-1 and 2.52 ton ha^-1 yr^-1 in the first and second year, and leaf fall to 2.00 ton ha^-1 yr^-1 and 1.93 ton ha^-1 yr^-1 respectively with a maximum in July. As to litter fall, there was a summer maximum for brown needles and kernels, a spring maximum for inflorescences and bud scales, and an autumn maximum for bark. Erratic maxima occurred for fall of green needles, cones and branches, linked to strong winds in winter. The total amount of litter mass on the forest soil reached 12.68 ton/ha: 5.75 ton/ha in the L organic horizon, 3.46 ton/ha needles, and 6.93 ton/ha in the F organic horizon. Weight loss from annual decomposition, measured using litter bags, was 18.1 % in year 1 and 26.8% in year 2. Over 365 days, an Olson (1963) decomposition rate of 0.045 %/day was found in year 1 and of 0.084 %/day for year 2. Decomposition half-time was 1529 for year 1 and 827 days for year 2.     

Application of the multiple antigenic peptides (MAP) strategy to the production of prophormone convertases antibodies: Synthesis,characterization and use of 8-branched immunogenic peptides

Antiserum against an N-terminal sequence of murine prohormone convertase-1 (mPC1) incorporating the sequence immediatley following the junction between the putative pro-region and the active enzyme was obtained. This was accomplished using the multiple antigenic peptide (MAP) approach whereupon an 8-branched polylysine core to which are grafted multiple copies of a 16 amino acid peptide representing the N-terminal sequence of mPC1 (positions 84–99) was synthesized by solid-phase Fmoc chemistry. The ensuing peptide was purified and fully characterized by RP-HPLC, ¹H-NMR, amino acid composition, peptide sequencing and ion-spray mass spectrometry. The immunological properties of the resulting antibodies in detecting recombinant PC1 in both crude and purified preparations were compared with antibodies raised against a similar N-terminal segment of PC1 but using the conventioanl method of peptide–carrier protein conjugation and also developed against a C-terminal fusion protein of PC1. Our data indicate that the MAP antibody was as efficient as both the amino and carboxy-terminal antibodies in qualitative as well as quantitative analysis of PC1 encoded protein by radioimmunoassay. Following an identical approach, antibodies against other prohormone convertases like furin, PC5/6 and PACE4 were also developed and subsequently applied to a number of biochemical and immunological studies. In each case, the ease of preparation and high immunogenicity of the MAP approach were confirmed and reside in the simplicity and rapidity with which a potent and useful antiserum is obtained.     

Incorporation of two18O atoms into a peptide during isoaspartyl repair reveals repeated passage through a succinimide intermediate

To study the mechanism of protein carboxyl methyltransferase-driven repair of age-damaged sites in polypeptides, a modell-isoaspartyl peptide,l-isotetragastrin, was enzymatically repaired to normall-tetragastrin in the presence of¹⁸O-enriched water. By this design, the enrichment of¹⁸O atoms in the peptide would reflect the number of passages through a hydrolyzable succinimide intermediate during formation of the repaired product. Mass determinations by FAB mass spectrometry revealed repaired peptide with two¹⁸O atoms incorporated, demonstrating that more than a single cycle of methylation and demethylation is necessary to ensure stoichiometric repair.Abbreviations HPLC high-pressure liquid chromatography - FAB fast atom bombardment - TFA trifluoroacetic acid - PCM proteind-aspartyl/L-isoaspartyl carboxyl methyltransfer-ase - l-Normal [l-Asp³]tetragastrin - l-Iso [L-isoAsp³]tetragastrin - d-Normal [d-Asp³]tetragastrin - d-Iso [d-isoAsp³]tetragastrin     

NAD Glycohydrolases: A possible function in calcium homeostasis

NAD glycohydrolases are the longest known enzymes that catalyze ADP-ribose transfer. The function of these ubiquitous, membrane-bound enzymes has been a long standing puzzle. The NAD glycohydrolase are briefly reviewed in light of the discovery by our laboratory that NAD glycohydrolases are bifunctional enzymes that can catalyze both the synthesis and hydrolysis of cyclic ADP-ribose, a putative second messenger of calcium homeostasis.Abbreviations NADase nicotinamide adenine dinucleotide glycohydrolase - NAD nicotinamide adenine dinucleotide - ADP-ribose adenosine diphosphoribose - cADPR cyclic adenosine diphosphoribose