Stem cell culture engineering - process scale up and beyond

Advances in stem cell research and recent work on clinical trials employing stem cells have heightened the prospect of stem cell applications in regenerative medicine. The eventual clinical application of stem cells will require transforming cell production from laboratory practices to robust processes. Most stem cell applications will require extensive ex vivo handling of cells, from isolation, cultivation, and directed differentiation to product cell separation, cell derivation, and final formulation. Some applications require large quantities of cells in each defined batch for clinical use in multiple patients; others may be for autologous use and require only small-scale operations. All share a common requirement: the production must be robust and generate cell products of consistent quality. Unlike the established manufacturing process of recombinant protein biologics, stem cell applications will likely see greater variability in their cell source and more fluctuations in product quality. Nevertheless, in devising stem cell-based bioprocesses, much insight could be gained from the manufacturing of biological materials, including recombinant proteins and anti-viral vaccines. The key to process robustness is thus not only the control of traditional process chemical and physical variables, but also the sustenance of cells in the desired potency or differentiation state through controlling non-traditional variables, such as signaling pathway modulators.     

The murine TRAIL receptor signals caspase-independent cell death through ceramide

Death receptors such as the 55 kDa tumor necrosis factor (TNF) receptor (TNF-R55) or Fas can initiate both apoptotic (caspase-dependent) and caspase-independent routes to programmed cell death (PCD). Here, we demonstrate for the first time that the single murine receptor for (TNF)-related apoptosis-inducing ligand (mTRAIL-R2) can induce a caspase-independent form of PCD with necrosis-like features in addition to apoptosis. Analysis of morphological and cellular features of caspase-independent PCD in response to TRAIL and TNF suggests that mTRAIL-R2 and TNF-R55 elicit caspase-independent PCD through similar pathways, although without participation of cathepsins. Cells overexpressing acid ceramidase (AC), an enzyme that metabolizes the sphingolipid ceramide, show enhanced survival from TRAIL-induced caspase-independent PCD but not from apoptosis, implicating a function of ceramide as a key mediator in caspase-independent PCD (but not apoptosis) induced by mTRAIL-R2. In concert with the enhanced resistance of AC-overexpressing cells against caspase-independent PCD induced by TNF, our results suggest that ceramide acts as a common mediator of caspase-independent PCD caused by death receptors such as mTRAIL-R2 and TNF-R55.     

Freeze-fracture and cytochemical studies on the in vitro cyst form of reptilian Blastocystis pythoni

After cultured cysts are osmotically shocked by treating with distilled water, there is an exponential increase in the cyst form of Blastocystis pythoni; this was demonstrated by an immunofluorescence antibody assay against the culture organisms. In 11-day-old cultures of B. pythoni, 68.8% of the organisms (= 2.2 x 10(8) cysts/ml) were in the cyst form. Examination of thin sections of cysts revealed many similarities to the cyst forms of Blastocystis obtained from fecal samples in previous investigations. Freeze-fracture images of the plasma membrane of non-cyst cells also revealed a similar distribution of the intramembrane particles (IMP) when compared to non-cysts of B. hominis, while the plasma membrane of the cyst form showed practically no IMP. The size and morphology of particle-rich small depressions and smooth small protrusions observed on the P face and E face of non-cyst cells, respectively, were similar to endocytic sites reported for B. hominis. In the present study glycogen was cytochemically demonstrated at the ultrastructural level by an alkaline bismuth staining method in both cyst and non-cyst cells.     

Irradiation of cells with ultraviolet-A (320-400 nm) in the presence of cell culture medium elicits biological effects due to extracellular generation of hydrogen peroxide

Biological effects of ultraviolet A (UVA) irradiation have been ascribed to the photochemical generation of singlet oxygen. Not all effects described in the literature, however, are explicable solely by the generation of singlet oxygen, but rather resemble effects elicited by hydrogen peroxide (H 2 O 2 ). Here, we show that when cells are kept in cell culture media during exposure to UVA, stress kinases, including ERK 1 and ERK 2 as well as Akt (protein kinase B), are activated, whereas there is no or only minor activation when cells are kept in phosphate-buffered saline during irradiation. Indeed, the exposure of cell culture media to UVA (30 J/cm 2 ) results in the generation of significant amounts of H 2 O 2 , with concentrations of about 100 μM. H 2 O 2 concentrations are at least three-fold higher in HEPES-buffered culture media after UVA irradiation. From experiments with solutions of riboflavin, tryptophan or HEPES, as well as combinations thereof, it is concluded that riboflavin mediates the photooxidation of either tryptophan or HEPES, resulting in the generation of H 2 O 2 . Thus, if signaling effects of UVA radiation are to be investigated in cell culture systems, riboflavin and HEPES/tryptophan should be avoided during irradiation because of artificial H 2 O 2 generation. It should be taken into account, however, that in vivo tryptophan and riboflavin might play an important role in the generation of reactive oxygen species by UVA as both substances are abundant in living tissues.     

Influence of culture time on the expression of drug-metabolizing enzymes in primary human hepatocytes and hepatoma cell line HepG2

Primary cultures of human hepatocytes and hepatoma cell line HepG2 are frequently used to evaluate the hepatic disposition of drugs and other xenobiotics. To check the variability of the expression of drug-metabolizing enzymes in these in vitro models, expression of genes coding for several cytochrome P450 isoforms and phase II enzymes was quantified during culture time by real-time RT-PCR. Gene expression was determined daily for primary hepatocytes maintained in a sandwich culture over 1 week and for HepG2, during the first 10 passages. In primary hepatocytes characteristic expression trends were observed which could be abstracted into three major classes of time curves. Genes of the first and the second class had an expression maximum around day 6 and day 4 in culture, respectively. The third class of genes had two expression peaks: at day 1 and 5 in culture. Surprisingly, also the cell line HepG2 showed significant expression changes during passages. For example, gene expression of cytochrome 1A1 varied 8-fold, that of cytochrome 2B6 30-fold, and that of NADP-quinone reductase 1 more than 200-fold within the first 10 passages. In conclusion, neither primary hepatocytes nor HepG2 cell line display a model for constant expression of drug-metabolizing enzymes.     

<Emphasis Type="Italic">Exobasidium symploci-japonicae</Emphasis> var. <Emphasis Type="Italic">carpogenum</Emphasis> var. nov. causing Exobasidium fruit deformation on <Emphasis Type="Italic">Symplocos lucida</Emphasis> in Japan

Exobasidium symploci-japonicae var. carpogenum, causing Exobasidium fruit deformation on Symplocos lucida collected in Fukuoka Prefecture, Japan, is newly described based on morphological observations of hymenial structure and mode of basidiospore germination. This new variety differs morphologically from the type variety, particularly in the septal number of basidiospores and in the shapes and sizes of conidia formed on the medium. Colonies of this new variety are also distinguishable from those of the type variety by yeast-like growth, morphology, and color of colonies.Contribution no.178, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

bullwinkle is required for epithelial morphogenesis during Drosophila oogenesis

Many organs, such as the liver, neural tube, and lung, form by the precise remodeling of flat epithelial sheets into tubes. Here we investigate epithelial tubulogenesis in Drosophila melanogaster by examining the development of the dorsal respiratory appendages of the eggshell. We employ a culture system that permits confocal analysis of stage 10-14 egg chambers. Time-lapse imaging of GFP-Moesin-expressing egg chambers reveals three phases of morphogenesis: tube formation, anterior extension, and paddle maturation. The dorsal-appendage-forming cells, previously thought to represent a single cell fate, consist of two subpopulations, those forming the tube roof and those forming the tube floor. These two cell types exhibit distinct morphological and molecular features. Roof-forming cells constrict apically and express high levels of Broad protein. Floor cells lack Broad, express the rhomboid-lacZ marker, and form the floor by directed cell elongation. We examine the morphogenetic phenotype of the bullwinkle (bwk) mutant and identify defects in both roof and floor formation. Dorsal appendage formation is an excellent system in which cell biological, molecular, and genetic tools facilitate the study of epithelial morphogenesis.     

A molecular systematic survey of cultured microbial associates of deep-water marine invertebrates

A taxonomic survey was conducted to determine the microbial diversity held within the Harbor Branch Oceanographic Marine Microbial Culture Collection (HBMMCC). The collection consists of approximately 17,000 microbial isolates, with 11,000 from a depth of greater than 150 ft seawater. A total of 2273 heterotrophic bacterial isolates were inventoried using the DNA fingerprinting technique amplified rDNA restriction analysis on approximately 750-800 base pairs (bp) encompassing hypervariable regions in the 5' portion of the small subunit (SSU) 16S rRNA gene. Restriction fragment length polymorphism patterns obtained from restriction digests with RsaI, HaeIII, and HhaI were used to infer taxonomic similarity. SSU 16S rDNA fragments were sequenced from a total of 356 isolates for more definitive taxonomic analysis. Sequence results show that this subset of the HBMMCC contains 224 different phylotypes from six major bacterial clades (Proteobacteria (Alpha, Beta, Gamma), Cytophaga, Flavobacteria, and Bacteroides (CFB), Gram + high GC content, Gram + low GC content). The 2273 microorganisms surveyed encompass 834 alpha-Proteobacteria (representing 60 different phylotypes), 25 beta-Proteobacteria (3 phylotypes), 767 gamma-Proteobacteria (77 phylotypes), 122 CFB (17 phylotypes), 327 Gram + high GC content (43 phylotypes), and 198 Gram + low GC content isolates (24 phylotypes). Notably, 11 phylotypes were < or =93% similar to the closest sequence match in the GenBank database even after sequencing a larger portion of the 16S rRNA gene (approximately 1400 bp), indicating the likely discovery of novel microbial taxa. Furthermore, previously reported "uncultured" microbes, such as sponge-specific isolates, are part of the HBMMCC. The results of this research will be available online as a searchable taxonomic database (www.hboi.edu/dbmr/dbmr_hbmmd.html).     

Hyperhydricity in shoot cultures of Scrophularia yoshimurae can be effectively reduced by ventilation of culture vessels

An effective procedure for obtaining healthy shoots from nodal segments of Scrophularia yoshimurae is described. Nodal segments cultured on Murashige and Skoog's (MS) basal medium supplemented with 1.0 mg L(-1) benzyladenine (BA) and 0.2 mg L(-1) alpha-naphthaleneacetic acid (NAA) induced multiple shoots in conical flasks that differed in the way they were closed and sealed. Hermitically sealed culture vessels resulted in high hyperhydricity/vitrification. High concentrations of ethylene and CO2 were found to accumulate in these vessels. The hyperhydricity of the shoot cultures could be decreased by progressively ventilating the vessels. Exchange of gases was achieved by removing the Parafilm sealing without affecting sterility. This reduced the hyperhydricity rate and gave a good recovery of vitrified shoots, but resulted in decreased proliferation and a dehydration of proliferating nodal segments and the culture medium. The best number of normal shoots was observed when the parafilm was removed for gaseous exchange after four weeks of culture incubation. The results show that hyperhydricity in shoot cultures of S. yoshimurae could be prevented by sufficient gas exchange during culture.