Systematic microcarrier screening and agitated culture conditions improves human mesenchymal stem cell yield in bioreactors

Production of human mesenchymal stem cells for allogeneic cell therapies requires scalable, cost‐effective manufacturing processes. Microcarriers enable the culture of anchorage‐dependent cells in stirred‐tank bioreactors. However, no robust, transferable methodology for microcarrier selection exists, with studies providing little or no reason explaining why a microcarrier was employed. We systematically evaluated 13 microcarriers for human bone marrow‐derived MSC (hBM‐MSCs) expansion from three donors to establish a reproducible and transferable methodology for microcarrier selection. Monolayer studies demonstrated input cell line variability with respect to growth kinetics and metabolite flux. HBM‐MSC1 underwent more cumulative population doublings over three passages in comparison to hBM‐MSC2 and hBM‐MSC3. In 100 mL spinner flasks, agitated conditions were significantly better than static conditions, irrespective of donor, and relative microcarrier performance was identical where the same microcarriers outperformed others with respect to growth kinetics and metabolite flux. Relative growth kinetics between donor cells on the microcarriers were the same as the monolayer study. Plastic microcarriers were selected as the optimal microcarrier for hBM‐MSC expansion. HBM‐MSCs were successfully harvested and characterised, demonstrating hBM‐MSC immunophenotype and differentiation capacity. This approach provides a systematic method for microcarrier selection, and the findings identify potentially significant bioprocessing implications for microcarrier‐based allogeneic cell therapy manufacture.     

Polymorphism and complexity of HLA-DR: evidence for intra-HLA-DR region crossing-over events

HLA-DR molecules were isolated from HLA-DR3, –5, and –w6 positive homozygous B-cell lines by immunoprecipitation with monoclonal antibodies and analyzed by gel electrophoretic techniques. DNA isolated from the same cell lines was digested with the restriction enzyme Taq I and hybridized with a DR beta full-length cDNA probe. We demonstrated that certain DR I alleles are found in combination with different DR III alleles as defined by Southern blotting, protein chemistry, a functional assay using purified protein derivative-specific T-cell lines, and, in one case, also alloreactive T-cell reagents. Our results indicate that within the family of HLA-DRw52-associated haplotypes DR beta chain genes may have been transferred from one haplotype to another. The implications of these findings are discussed.     

Ultrastructure and adrenergic innervation of preglomerular arterioles in the euryhaline teleost,Salmo gairdneri

Summary The opisthonephric kidney of the rainbow trout was investigated by light- and electron microscopy and a fluorescent-histochemical technique for biogenic amines was used. Preglomerular sphincters at the origin of afferent arterioles are present in this euryhaline teleost. The branching point of the afferent arteriole is characterized by (i) the formation of a right angle with the parent vessel, (ii) circularly arranged smooth muscle cells of the tunica media, (iii) additional circularly arranged smooth muscle cells intercalated between endothelium and tunica media, and (iv) a collar-like arrangement of several large endothelial cells with elaborate marginal folds and abundant myoendothelial junctions. A dense adrenergic innervation displaying specific fluorescence was found along the terminal arterioles and afferent arterioles, and conspicuously at the preglomerular sphincters. These results are suggestive of a neural participation in kidney function. They are discussed on the basis of recent evidence from pharmacological and physiological experiments for neural involvement in glomerular intermittency.     

Pollination Biology of Piperaceae Species in Southeastern Brazil

Phenology, floral biology and breeding system are describedfor 14 Piperaceae species at two sites covered by semi-deciduousforest in southeastern Brazil. Flowering occurred continuouslythroughout the year, with a peak during the windy months. Allspecies had minute odorous and nectarless flowers visited byinsects; five of them showed a substantial degree of self-compatibility,and one was andromonoecious. Seven species were wind pollinated,and three were exclusively pollinated by insects. Flower visitorswere mainly hoverflies and bees. Copyright 2000 Annals of BotanyCompany Piper spp., Ottonia spp., Pothomorphe umbellata, pepper family, phenology, floral biology, breeding system, ambophily, semi-deciduous forest     

Four thousand years of plant exploitation in the Chad Basin of northeast Nigeria I: The archaeobotany of Kursakata

This paper discusses archaeobotanical remains from the settlement mound of Kursakata, Nigeria, comprising both charred and uncharred seeds and fruits as well as charcoal. In addition, impressions of plant tempering material in potsherds were analysed. The late Stone Age and Iron Age sequence at Kursakata is date from 1000 cal. B.C. to cal. A.D. 100. DomesticatedPennisetum (pearl millet), wild Paniceae and wild rice are the most common taxa. Kernels from tree fruits were regularly found including large numbers ofVitex simplicifolia—a tree which is absent from the area today. A distinct change in plant spectra can be observed between the late Stone Age and the Iron Age. Although domesticated pearl millet was already known at the beginning of the settlement sequence of Kursakata, it only gained greater economic importance during the Iron Age. Besides farming, pastoralism and fishing, gathering of wild plants always played a major role in the subsistence strategy of the inhabitants of Kursakata. The charcoal results show that firewood was mainly collected from woodlands on the clay plains, which must have been more diverse than today. The end of the late Stone Age in the Chad Basin was presumably accompanied by the onset of drier environmental conditions from ca. 800 cal. B.C. onwards.     

In vitro desiccation tolerance of the epiphytic Ghost Orchid,Dendrophylax lindenii (Lindl.) Benth x. Rolfe

The Ghost Orchid, Dendrophylax lindenii (Lindl.) Benth x. Rolfe, is a rare and endangered epiphytic orchid native to south Florida and Cuba. The orchid is considered difficult to propagate under greenhouse conditions, requiring high humidity and low air movement. In contrast, the orchid’s native habitat seasonally dries out with decreased precipitation and humidity. This suggests some level of desiccation tolerance. Ghost Orchid plants were assessed for potential desiccation tolerance and ability to recover from desiccation stress under in vitro conditions. In vitro-derived plants were placed into sterile baby food jars and transferred to chambers maintained at 10% relative humidity, which is extremely low compared to relative humidity levels (40–100%) recorded under natural field conditions. Plants were removed every week for 4 wk and recovered on P723 medium supplemented with banana powder for 4 wk. Data were collected at the initiation of the experiment, after the desiccation periods, and after 4 wk in vitro recovery. Ghost Orchid plants demonstrated extremely high desiccation tolerance. Even after 4 wk desiccation, plant survival was observed at 79.2% after recovery. Desiccated plants exhibited significant decreases in tissue water potential (− 18.44 MPa), fresh weight (65.5% loss), and water content (14.2%); however, high plant survival was still observed under these conditions similar to poikilohydric plants. Overall, the Ghost Orchid demonstrated high desiccation tolerance, which should be considered for future greenhouse culture and for its application in the direct field establishment of in vitro-derived plants without greenhouse acclimatization.

     

Ca<Subscript>v</Subscript>1.3 and BK Channels for Timing and Regulating Cell Firing

L-type Ca²⁺ channels (LTCCs, Ca_v1) open readily during membrane depolarization and allow Ca²⁺ to enter the cell. In this way, LTCCs regulate cell excitability and trigger a variety of Ca²⁺-dependent physiological processes such as: excitation–contraction coupling in muscle cells, gene expression, synaptic plasticity, neuronal differentiation, hormone secretion, and pacemaker activity in heart, neurons, and endocrine cells. Among the two major isoforms of LTCCs expressed in excitable tissues (Ca_v1.2 and Ca_v1.3), Ca_v1.3 appears suitable for supporting a pacemaker current in spontaneously firing cells. It has steep voltage dependence and low threshold of activation and inactivates slowly. Using Ca_v1.3^−/− KO mice and membrane current recording techniques such as the dynamic and the action potential clamp, it has been possible to resolve the time course of Ca_v1.3 pacemaker currents that regulate the spontaneous firing of dopaminergic neurons and adrenal chromaffin cells. In several cell types, Ca_v1.3 is selectively coupled to BK channels within membrane nanodomains and controls both the firing frequency and the action potential repolarization phase. Here we review the most critical aspects of Ca_v1.3 channel gating and its coupling to large conductance BK channels recently discovered in spontaneously firing neurons and neuroendocrine cells with the aim of furnishing a converging view of the role that these two channel types play in the regulation of cell excitability.