Novel micro‐bioreactor high throughput technology for cell culture process development: Reproducibility and scalability assessment of fed‐batch CHO cultures

With increasing timeline pressures to get therapeutic and vaccine candidates into the clinic, resource intensive approaches such as the use of shake flasks and bench‐top bioreactors may limit the design space for experimentation to yield highly productive processes. The need to conduct large numbers of experiments has resulted in the use of miniaturized high‐throughput (HT) technology for process development. One such high‐throughput system is the SimCell? platform, a robotically driven, cell culture bioreactor system developed by BioProcessors Corp. This study describes the use of the SimCell? micro‐bioreactor technology for fed‐batch cultivation of a GS‐CHO transfectant expressing a model IgG4 monoclonal antibody. Cultivations were conducted in gas‐permeable chambers based on a micro‐fluidic design, with six micro‐bioreactors (MBs) per micro‐bioreactor array (MBA). Online, non‐invasive measurement of total cell density, pH and dissolved oxygen (DO) was performed. One hundred fourteen parallel MBs (19 MBAs) were employed to examine process reproducibility and scalability at shake flask, 3‐ and 100‐L bioreactor scales. The results of the study demonstrate that the SimCell? platform operated under fed‐batch conditions could support viable cell concentrations up to least 12 × 10⁶ cells/mL. In addition, both intra‐MB (MB to MB) as well as intra‐MBA (MBA to MBA) culture performance was found to be highly reproducible. The intra‐MB and ‐MBA variability was calculated for each measurement as the coefficient of variation defined as CV (%) = (standard deviation/mean) × 100. The % CV values for most intra‐MB and intra‐MBA measurements were generally under 10% and the intra‐MBA values were slightly lower than those for intra‐MB. Cell growth, process parameters, metabolic and protein titer profiles were also compared to those from shake flask, bench‐top, and pilot scale bioreactor cultivations and found to be within ±20% of the historical averages. Biotechnol. Bioeng. 2010; 106: 57–67. © 2010 Wiley Periodicals, Inc.     

Synthesis of novel europium-labeled estradiol derivatives for time-resolved fluoroimmunoassays.

The O-(5-carboxypentyl)-, O-(4-aminobutyl)-, O-(6-aminohexyl)oximes of 2- and 4-formylestradiol as well as the 4-carboxyethylthioether derivative of estradiol were synthesized. These estradiol derivatives were characterized using IR-, 1H-, and 13C NMR spectroscopy. All synthesized estradiol derivatives were labeled with europium chelates. These "tracers" were purified and tested in a competitive time-resolved fluoroimmunoassay with monoclonal antibody (SSI 57-2) raised against the 6-O-(carboxymethyl)oxime-bovine serum albumin derivative of estradiol. All the tested europium-labeled estradiol-4-derivatives were bound by the antibody, whereas tracers linked via position 2 were not recognized by this antibody. It was observed that tracers conjugated via C-4 gave more sensitive standard curves than tracers conjugated via C-6. Especially, the estradiol-4-thioether derivative was found to be highly useful in time-resolved fluoroimmunoassays of estradiol while using this antibody.     

Leaf litter decomposition differs among genotypes in a local <Emphasis Type="Italic">Betula pendula</Emphasis> population

Ecosystem processes, such as plant litter decomposition, are known to be partly genetically determined, but the magnitude of genetic variation within local populations is still poorly known. We used micropropagated field-grown saplings of 19 Betula pendula genotypes, representing genetic variation in a natural birch population, to examine (1) whether genotype can explain variation in leaf litter decomposition within a local plant population, and (2) whether genotypic variation in litter decomposition is associated with genotypic variation in other plant attributes. We found that a local B. pendula population can have substantial genotypic variation in leaf litter mass loss at the early stages of the decomposition process and that this variation can be associated with genotypic variation in herbivore resistance and leaf concentrations of soluble proteins and total nitrogen (N). Our results are among the first to show that fundamental ecosystem processes can be significantly affected by truly intraspecific genetic variation of a plant species.     

Silvicultural practices near the tree-line

The close correlation between climatic fluctuation and forest growth near the tree-line is discussed. Depending on climatic conditions, the opinions of foresters on the yield potential of the northernmost pine forests have varied greatly from time to time. In Finland the Zone of Protection-Forests is defined by law (1922) and the Forest Research Institute has prepared practical instructions for the silvicultural management of the Protection-Forests (1959). These instructions are rather conservative. Thus, radical measures, such as clear-felling and artificial reforestation, are avoided whereas, in the first place, silvicultural thinnings and removal of over-mature standards in younger stands are recommended. This kind of management allows the rational utilization of the yield capacity of the forests, without endangering their future existence. The scenic value and multiple use of these forests also are considered in the management instructions.     

Growth and genotype × environment interactions in Betula pendula: can tree genetic variation be maintained by small-scale forest ground heterogeneity?

Forest ground heterogeneity can affect interactions among tree species and control the assembly of local forest communities. Less is known of the effects of spatial heterogeneity on the maintenance of tree genetic variation through small-scale genotype × environment (G × E) interactions. We measured growth variation within and among 17 Betula pendula genotypes, planted in a clear-cut forest site through the summers 2009–2011. We assessed the spatial heterogeneity at two scales: among forest stands having history of the same or different tree species combinations (treated as replicate blocks), and along a gradient of within-block forest density, revealed by the stump density. To add a temporal perspective, we distinguished between old (cut 50 years earlier) and new stumps (cut one year earlier). The broad-sense heritabilities for growth were 0.093–0.055 and the coefficients of genotypic variation 0.37–0.21 in 2009–2011. The growth difference among the genotypes was 3.5–5.5 fold, significant in all years, and the rank of genotype means correlated positively between the years. The most favourable block had 106 % higher growth than the least favourable block and the amount of total variation explained by block increased from 0.4 % in 2009 to 6.9 % in 2011. Genotype × block interaction was marginally significant in 2009, but not later. Similarly, the response of growth to old stump density in sapling vicinity varied among the genotypes in 2009, but not later. In 2010 and 2011, the mean growth increased by 50–91 % along the old stump density gradient. Our results suggest that despite creating significant variation in sapling growth the small-scale forest ground heterogeneity, which reflects the recent forest history, may not significantly contribute to the maintenance of genetic variation in B. pendula populations.     

Role of Soil Organisms in the Maintenance of Species-Rich Seminatural Grasslands through Mowing

To preserve species-rich grasslands, management practices such as mowing are often required. Mowing is known to promote aboveground conditions that help to maintain plant species richness, but whether belowground effects are important as well is not known. We hypothesized that if mowing decreases belowground carbon transfer by reducing root mass, this will reduce the abundance and activity of soil decomposers and lead to diminished nutrient availability in soil. In grasslands, this would provide a means to mitigate the negative effects of nitrogen enrichment on plant species richness. We established experimental plots on grassland with one-third of plots growing untouched, one-third mowed once a summer, and one-third mowed twice a summer for three growing seasons. Root and soil attributes were measured at each plot 1 month after each mowing. Mowing decreased root mass but had few effects on belowground biota and soil NH₄-N, NO₃-N, and PO₄-P concentrations. Mowing did not affect root N concentrations. Our results show that despite reducing root mass, mowing may have few effects on those soil biota that control nutrient supply and, as a result, have no clear-cut effects on nutrient availability in soil. This suggests that the effects of mowing on soil decomposers and soil nutrient availability may not provide an effective means to mitigate N enrichment and enhance plant species richness in grasslands in a timescale of a few years. Whether such effects could, however, be achieved with longer lasting mowing remains open.     

Soil feedback does not explain mowing effects on vegetation structure in a semi-natural grassland

Due to its ability to create aboveground conditions that favour plant diversity, mowing is often used to preserve the high conservation value of semi-natural species-rich grasslands. However, mowing can also affect belowground conditions. By decreasing plant carbon supply to soil, mowing can suppress the activity of soil decomposers, diminish plant nutrient availability and thus create a feedback on plant growth. In this study, we first documented the effects of three-year mowing on plant community structure in a species-rich grassland. We found that mowing decreased the total areal cover of woody plants and increased the total cover of leguminous forbs. At the species level, mowing further increased the cover of two non-leguminous forbs, Prunella vulgaris and Sagina procumbens. Mowing did not affect the species number, diversity or evenness of the plant community. To study whether any of these effects could be explained by mowing-induced changes in the soil, and particularly by reduced nutrient availability, we then collected soil from different treatment plots and monitored the growth of nine plant species in these soils in a greenhouse. Plant growth did not differ between soils collected from mowed and unmowed plots, suggesting that our mowing regimes did not impose such changes in soil decomposer activity and nutrient supply that would feedback on plant growth. Moreover, each of the nine species responded equally to the different nutrient availability in different parts of the grassland, which indicates that even if mowing had reduced plant nutrient supply, this would not have led to changes in plant community structure. It appears that those changes in aboveground vegetation that we recorded after three years of mowing were purely due to the aboveground effects, such as frequent cutting of woody plants and enhanced light availability for low-growing forbs.     

In vitro establishment of lytic and nonproductive infection by herpes simplex virus type 1 in three-dimensional keratinocyte culture.

The F strain of herpes simplex virus type 1 (HSV-1) was tested for its ability to produce lytic or nonproductive infection in squamous epithelial cells cultured in a three-dimensional organotypic tissue culture. For the tissue culture, we used HaCat cells (immortalized skin keratinocytes) and normal fibroblasts derived from the skin. The cultures were infected with HSV-1 (5 PFU) either when the epithelial cells had grown as a monolayer with a confluence of 80% on the collagen fibroblast gel or 30 min after lifting of the epithelial cells into the air-liquid interface. The cultures were collected 1 week after inoculation. Typical cytopathic effects of HSV infection (ballooning and reticular degeneration with multinucleate giant cells) were seen only in those cultures in which the epithelial cells were infected before lifting. The presence of HSV was confirmed by DNA and RNA in situ hybridization and PCR. No morphological changes were found in cultures infected after lifting into the air-liquid interface. No infectious virus was recovered either from cells or culture supernatant. However, these cultures were positive for HSV DNA on PCR and showed expression of the LAT gene by in situ hybridization and Northern blot (RNA) hybridization. The present results indicate that both nonproductive and lytic HSV infection can be produced in vitro and the outcome of the infection depends on the time of viral inoculation in relation to epithelial maturation.     

Time-resolved fluoroimmunoassay for equol in plasma and urine

We present a method for the determination of the isoflavan equol in plasma and urine. This estrogenic isoflavan, which is formed by the action of the intestinal microflora, may have higher biological activity than its precursor daidzein. High urinary excretion of equol has been suggested to be associated with a reduction in breast cancer risk. The method is based on time-resolved fluoroimmunoassay, using a europium chelate as a label. After synthesis of 4′-O-carboxymethylequol the compound is coupled to bovine serum albumin (BSA), then used as antigen to immunize rabbits. The tracer with the europium chelate is synthesized using the same 4′-O-derivative of equol. After enzymatic hydrolysis (urine) or enzymatic hydrolysis and ether extraction (plasma) the immunoassay is carried out. The antiserum cross-reacted to variable extent with some isoflavonoids. For the plasma method the cross-reactivity does not seem to influence the results, which were highly specific. The overestimation of the values using the urine method (164%) compared to the results obtained by a gas chromatography–mass spectrometry (GC–MS) method is probably due to some influence of the matrix on the signal, and interference of structurally related compounds. It is suggested that plasma assays are used but if urine samples are measured a formula has to be used to correct the values making them comparable to the GC–MS results. The correlation coefficients between the time-resolved fluoroimmunoassay (TR-FIA) methods and GC–MS methods were high; r-values for the plasma and urine method, were 0.98 and 0.91, respectively. The intra-assay coefficient of variation (CV%) for the TR-FIA plasma and urine results at three different concentrations vary between 5.5–6.5 and 3.4–6.9, respectively. The inter-assay CV% varies between 5.4–9.7 and 7.4–7.7, respectively. The working ranges of the plasma and urine assay are 1.27–512 and 1.9–512 nmol/l, respectively.