A quantitative approach for understanding small-scale human mesenchymal stem cell culture – implications for large-scale bioprocess development

Human mesenchymal stem cell (hMSC) therapies have the potential to revolutionise the healthcare industry and replicate the success of the therapeutic protein industry; however, for this to be achieved there is a need to apply key bioprocessing engineering principles and adopt a quantitative approach for large-scale reproducible hMSC bioprocess development. Here we provide a quantitative analysis of the changes in concentration of glucose, lactate and ammonium with time during hMSC monolayer culture over 4 passages, under 100% and 20% dissolved oxgen (dO₂), where either a 100%, 50% or 0% growth medium exchange was performed after 72h in culture. Yield coefficients, specific growth rates (h^-1) and doubling times (h) were calculated for all cases. The 100% dO₂ flasks outperformed the 20% dO₂ flasks with respect to cumulative cell number, with the latter consuming more glucose and producing more lactate and ammonium. Furthermore, the 100% and 50% medium exchange conditions resulted in similar cumulative cell numbers, whilst the 0% conditions were significantly lower. Cell immunophenotype and multipotency were not affected by the experimental culture conditions. This study demonstrates the importance of determining optimal culture conditions for hMSC expansion and highlights a potential cost savings from only making a 50% medium exchange, which may prove significant for large-scale bioprocessing.     

Alleviation of Cadmium Toxicity in Brassica juncea L. (Czern. & Coss.) by Calcium Application Involves Various Physiological and Biochemical Strategies

Calcium (Ca) plays important role in plant development and response to various environmental stresses. However, its involvement in mitigation of heavy metal stress in plants remains elusive. In this study, we examined the effect of Ca (50 mM) in controlling cadmium (Cd) uptake in mustard (Brassica juncea L.) plants exposed to toxic levels of Cd (200 mg L⁻¹ and 300 mg L⁻¹). The Cd treatment showed substantial decrease in plant height, root length, dry weight, pigments and protein content. Application of Ca improved the growth and biomass yield of the Cd-stressed mustard seedlings. More importantly, the oil content of mustard seeds of Cd-stressed plants was also enhanced with Ca treatment. Proline was significantly increased in mustard plants under Cd stress, and exogenously sprayed Ca was found to have a positive impact on proline content in Cd-stressed plants. Different concentrations of Cd increased lipid peroxidation but the application of Ca minimized it to appreciable level in Cd-treated plants. Excessive Cd treatment enhanced the activities of antioxidant enzymes superoxide dismutase, ascorbate peroxidase and glutathione reductase, which were further enhanced by the addition of Ca. Additionally, Cd stress caused reduced uptake of essential elements and increased Cd accumulation in roots and shoots. However, application of Ca enhanced the concentration of essential elements and decreased Cd accumulation in Cd-stressed plants. Our results indicated that application of Ca enables mustard plant to withstand the deleterious effect of Cd, resulting in improved growth and seed quality of mustard plants.     

Design and development of a new ambr250® bioreactor vessel for improved cell and gene therapy applications

The emergence of cell and gene therapies has generated significant interest in their clinical and commercial potential. However, these therapies are prohibitively expensive to manufacture and can require extensive time for development due to our limited process knowledge and understanding. The automated ambr250® stirred-tank bioreactor platform provides an effective platform for high-throughput process development. However, the original dual pitched-blade 20 mm impeller and baffles proved sub-optimal for cell therapy candidates that require suspension of microcarriers (e.g. for the culture of adherent human mesenchymal stem cells) or other particles such as activating Dynabeads® (e.g. for the culture of human T-cells). We demonstrate the development of a new ambr250® stirred-tank bioreactor vessel which has been designed specifically to improve the suspension of microcarriers/beads and thereby improve the culture of such cellular systems. The new design is unbaffled and has a single, larger elephant ear impeller. We undertook a range of engineering and physical characterizations to determine which vessel and impeller configuration would be most suitable for suspension based on the minimum agitation speed (N_JS) and associated specific power input (P/V)_JS. A vessel (diameter, T, = 60 mm) without baffles and incorporating a single elephant ear impeller (diameter 30 mm and 45° pitch-blade angle) was selected as it had the lowest (P/V)_JS and therefore potentially, based on Kolmogorov concepts, was the most flexible system. These experimentally-based conclusions were further validated firstly with computational fluid dynamic (CFD) simulations and secondly experimental studies involving the culture of both T-cells with Dynabeads® and hMSCs on microcarriers. The new ambr250® stirred-tank bioreactor successfully supported the culture of both cell types, with the T-cell culture demonstrating significant improvements compared to the original ambr250® and the hMSC-microcarrier culture gave significantly higher yields compared with spinner flask cultures. The new ambr250® bioreactor vessel design is an effective process development tool for cell and gene therapy candidates and potentially for autologous manufacture too.

     

Towards a comprehensive structural variation map of an individual human genome

Background

Several genomes have now been sequenced, with millions of genetic variants annotated. While significant progress has been made in mapping single nucleotide polymorphisms (SNPs) and small (<10 bp) insertion/deletions (indels), the annotation of larger structural variants has been less comprehensive. It is still unclear to what extent a typical genome differs from the reference assembly, and the analysis of the genomes sequenced to date have shown varying results for copy number variation (CNV) and inversions.

Results

We have combined computational re-analysis of existing whole genome sequence data with novel microarray-based analysis, and detect 12,178 structural variants covering 40.6 Mb that were not reported in the initial sequencing of the first published personal genome. We estimate a total non-SNP variation content of 48.8 Mb in a single genome. Our results indicate that this genome differs from the consensus reference sequence by approximately 1.2% when considering indels/CNVs, 0.1% by SNPs and approximately 0.3% by inversions. The structural variants impact 4,867 genes, and >24% of structural variants would not be imputed by SNP-association.

Conclusions

Our results indicate that a large number of structural variants have been unreported in the individual genomes published to date. This significant extent and complexity of structural variants, as well as the growing recognition of their medical relevance, necessitate they be actively studied in health-related analyses of personal genomes. The new catalogue of structural variants generated for this genome provides a crucial resource for future comparison studies.     

PCR-based restriction fragment length polymorphism and haplotype of the most common mutation L176F in the beta-glucuronidase gene

Mucopolysaccharidosis type VII or Sly syndrome is an autosomal recessive disorder of glycosaminoglycan storage leading to variable clinical symptoms, such as hepatosplenomegaly, bone deformities, hearing loss, corneal opacities, mental retardation, and hydrops fetalis in affected individuals. The disease is caused by approximately 40 different mutations in the beta-glucuronidase gene. Detection of the most common mutation L176F by single-strand conformation polymorphism (SSCP) was not always successful. Although DNA sequencing followed by PCR amplification can easily detect this mutation, accessibility to a DNA sequencer or useful reagents in the sequencing procedure is not readily available in many countries. A PCR-based restriction fragment length polymorphism (RFLP) developed in this report would allow rapid and easier detection of this mutation for screening new patients or neonates of heterozygous parents. Analysis of intragenic polymorphic sites in the L176F patients identified two distinct alleles; the predominant one probably originated in Spain.     

Blood glucose level and survival in streptozotocin-treated human chymase transgenic mice

A growing body of evidence suggests the potential role of chymase in organ injury in diabetes. We investigated blood glucose levels and survival in transgenic mice carrying the human chymase gene (Tg). Intraperitoneal injections of streptozotocin (STZ) (200, 100, 75 and 50 mg/kg in total, i.p.) were given to uninephrectomized Tg mice and wild-type C57BL/6 (BL) mice. Before STZ injection, the Tg mice had significantly lower body weights and slightly higher systolic blood pressure as compared with the BL mice. STZ-treated Tg mice showed significantly higher postprandial blood glucose levels as compared with the STZ-treated BL mice. The survival prevalence of STZ-treated Tg mice was zero, whereas BL mice showed a value of 40% until 42 days. STZ (100, 75 or 50 mg/kg, i.p.)-treated Tg mice also showed a similar pattern as compared with the STZ-treated BL mice. These data suggest that human chymase contributes to blood glucose levels and mortality during the progression of diabetes.     

Epileptogenesis induces long-term alterations in intracellular calcium release and sequestration mechanisms in the hippocampal neuronal culture model of epilepsy

Calcium and calcium-dependent processes have been hypothesized to be involved in the induction of epilepsy. It has been shown that epileptic neurons have altered calcium homeostatic mechanisms following epileptogenesis in the hippocampal neuronal culture (HNC) and pilocarpine models of epilepsy. To investigate the mechanisms causing these alterations in [Ca2+]i homeostatic processes following epileptogenesis, we utilized the HNC model of in vitro 'epilepsy' which produces spontaneous recurrent epileptiform discharges (SREDs). Using [Ca2+]i imaging, studies were initiated to evaluate the mechanisms mediating these changes in [Ca2+]i homeostasis. 'Epileptic' neurons required much longer to restore a glutamate induced [Ca2+]i load to baseline levels than control neurons. Inhibition of Ca2+ entry through voltage and receptor gated Ca2+ channels and stretch activated Ca2+ channels had no effect on the prolonged glutamate induced increase in [Ca2+]i in epileptic neurons. Employing thapsigargin, an inhibitor of the sarco/endoplasmic reticulum calcium ATPase (SERCA), it was shown that thapsigargin inhibited sequestration of [Ca2+]i by SERCA was significantly decreased in 'epileptic' neurons. Using Ca2+ induced Ca2+ release (CICR) cell permeable inhibitors for the ryanodine receptor (dantrolene) and the IP3 receptor (2-amino-ethoxydiphenylborate, 2APB) mediated CICR, we demonstrated that CICR was significantly augmented in the 'epileptic' neurons, and determined that the IP3 receptor mediated CICR was the major release mechanism altered in epileptogenesis. These data indicate that both inhibition of SERCA and augmentation of CICR activity contribute to the alterations accounting for the impaired calcium homeostatic processes observed in 'epileptic' neurons. The results suggest that persistent changes in [Ca2+]i levels following epileptogenesis may contribute to the long-term plasticity changes manifested in epilepsy and that understanding the basic mechanisms mediating these changes may provide an insight into the development of novel therapeutic approaches to treat epilepsy and prevent or reverse epileptogenesis.     

A note on the biology ofBracon gelechiae [Hym.: Braconidae] and augmentation of this parasite againstPectinophora Gossypiella [Lep.: Gelechiidae]

BioControl - Studies on the biology ofBracon gelechiae Ashm. and augmentation of this parasite againstPectinophora gossypiella (Saund.) were conducted. The development ofB. gelechiae from egg to...     

Seven new genera of dorylaimid nematodes from Colombian rain forest

Summary New genera described from soil in Colombian rain forest are Fuscheila (Thornenematidae), Capilonchus (Tylencholaiminae, Tylencholaimidae), Caveonchus (Tyleptinae, Leptonchidae), Coronatyleptus (Belonenchinae, Leptonchidae), Promumtazium (Mumtaziinae, Tylencholaimidae), Tantunema (Thorniinae, Tylencholaimidae) and Zalophidera (Xiphinemellinae, Tylencholaimidae). 12 new species belonging to these genera described and illustrated are: Fuscheila citrifera (type species), F. godmanae, Capilonchus lineatus (type-species), Caveonchus colombicus (type species), C. brevisaccus, Coronatyleptus barbarae, C. curvus, C. robustus, Promumtazium pyxidorum (type species), Tantunema bothriocephalum (type species), Zalophidera tylocephala (type species) and Z. idiostoma. Caveonchus saccatus (Goseco, Ferris & Ferris, 1981) n. comb. and Coronatyleptus coronatus (Siddiqi & Khan, 1965) n. comb. (type-species) are made for Basirotyleptus saccatus and B. coronatus, respectively. The composition of the families Leptonchidae and Tylencholamidae is discussed and Leptonchoidae Thorne, 1935 (Ferris, 1970) is made a junior synonym of Tylencholaimoidea Filipjev, 1934 (n.rank). ac]19810621