A fluid dynamics approach to bioreactor design for cell and tissue culture

The problem of controlling cylindrical tank bioreactor conditions for cell and tissue culture purposes has been considered from a flow dynamics perspective. Simple laminar flows in the vortex breakdown region are proposed as being a suitable alternative to turbulent spinner flask flows and horizontally oriented rotational flows. Vortex breakdown flows have been measured using three-dimensional Stereoscopic particle image velocimetry, and non-dimensionalized velocity and stress distributions are presented. Regions of locally high principal stress occur in the vicinity of the impeller and the lower sidewall. Topological changes in the vortex breakdown region caused by an increase in Reynolds number are reflected in a redistribution of the peak stress regions. The inclusion of submerged scaffold models adds complexity to the flow, although vortex breakdown may still occur. Relatively large stresses occur along the edge of disks jutting into the boundary of the vortex breakdown region.     

The social function of tail up in the domestic cat (Felis silvestris catus)

We investigated the social function of tail up in order to verify its possible relationship with the hierarchical organization of a social group. Domestic cats live at higher densities than their ancestor which is a solitary species. Since the signals needed by solitary animals have different properties than those needed by group-living individuals, signalling pattern utilised by the domestic cat has inevitably changed. Kittens displayed the tail up when greeting their mother; this behaviour can also be observed in wild species. But, in domestic cat the tail up can be also observed when an adult individual meets another one and it signals the intention to interact amicably. Rank order affected the display of tail up posture: it was more frequently displayed by low-ranking cats, and high-ranking individuals received it more often than other members of the social group. Then, tail up seems to be a signal by means of which a cat shows the recognition of the higher social status of the individual to whom is directed. We confirmed the association between tail up and other affiliative behavioural patterns and the individual variability in displaying them. Considerations on the evolution of the tail up as a visual signal will be discussed.     

Single-layer centrifugation with Androcoll-E can be scaled up to allow large volumes of stallion ejaculate to be processed easily

The objective of the current study was to optimize the volumes of Androcoll-E and sperm sample used in various sizes of centrifuge tube to scale up single-layer centrifugation (SLC) for routine use in the field. Although sperm suspensions of equivalent quality were produced using Androcoll-E in small and large tubes, the sperm yield was much lower in the latter (P < 0.001). In contrast, in 200-mL tubes (XL), the yields were approximately 25% higher than those for the small tubes. An increased volume (4.5 mL) of extended ejaculate in small tubes (SLC-Inc) or 15 to 18 mL extended ejaculate on 15 mL of colloid of a reduced density, Androcoll-E-Large (SLC-Large), in 50-mL tubes were both found to give similar yields of motile spermatozoa as that of the SLC-Small method (SLC-Small, 49.7 ± 18.6%; SLC-Inc, 53.3 ± 17.1%; SLC-Large, 44.9 ± 18.3%) and were found to be equivalent in quality (motility: 88.0 ± 8.8%, 84.0 ± 3.5%, 90.0 ± 5.4%; normal morphology: 69.4 ± 17.0%, 69.4 ± 12.7%, 63.9 ± 15.6%; viability: 78 ± 16.7%, 83.8 ± 12.5%, 80.05 ± 14.6%; DNA fragmentation index: 14.7 ± 10.9%, 12.8 ± 8.1%, 11.6 ± 7.6%, respectively). The processing of an “average” stallion Equus caballus ejaculate in approximately twenty-seven 10-mL tubes (SLC-Inc) or eight 50-mL tubes (SLC-Large) is feasible, the latter being considered more practical for on-stud use.     

Comparison of embryo yield and pregnancy rate between in vivo and in vitro methods in the same Nelore (Bos indicus) donor cows

To investigate why the preferred means to produce bovine embryos in Brazil has changed from in vivo to in vitro, we compared these two approaches in the same Nelore cows (n = 30) and assessed total embryo production and pregnancy rates. Without a specific schedule, all cows were subjected to ultrasound-guided ovum pick up (OPU)/in vitro production (IVP) and MOET, with intervals ranging from 15 to 45 d between procedures, respectively. To produce in vivo embryos, cows were superovulated and embryos were recovered nonsurgically from 1 to 3 times (1.4 ± 0.6), whereas OPU/IVP was repeated from 1 to 5 times (3.2 ± 1.2) in each donor cow during a 12-mo interval. Embryos obtained from both methods were transferred to crossbred heifers. On average, 25.6 ± 15.3 immature oocytes were collected per OPU attempt. The average number of embryos produced by OPU/IVP (9.4 ± 5.3) was higher (P < 0.05) than the MOET method (6.7 ± 3.7). However, pregnancy rates were lower (P < 0.05) following transfer of IVP (33.5%) versus in vivo-derived embryos (41.5%) embryos. Embryonic losses between Days 30 and 60 and fetal sex ratio were similar (P > 0.05) between in vivo and in vitro-derived embryos. We concluded that in Nelore cows, with an interval of 15 d between OPU procedures, it was possible to produce more embryos and pregnancies compared to conventional MOET.     

Actinomycetes scale‐up for the production of the antibacterial,nocathiacin

An Amycolatopsis fastidiosa culture, which produces the nocathiacin class of antibacterial compounds, was scaled up to the 15,000 L working volume. Lower volume pilot fermentations (600, 900, and 1,500 L scale) were conducted to determine process feasibility at the 15,000 L scale. The effects of inoculum volume, impeller tip speed, volumetric gas flow rate, superficial gas velocity, backpressure, and sterilization heat stress were examined to determine optimal scale‐up operating conditions. Inoculum volume (6 vs. 2 vol %) and medium sterilization (R_o of 68 vs. 92 min^?1) had no effect on productivity or titer, and higher impeller tip speeds (2.1 vs. 2.9 m/s) had a slight effect (20% decrease). In contrast, higher backpressure, incorporating increased head pressure at the 15,000 L scale (1.2 vs. 0.7 kg/cm²) and low gas flow rates (0.25 vs. 0.8 vvm), appeared to be problematic (40–50% decrease). High off‐gas CO₂ levels were likely reasons for observed lower productivity. Consequently, air flow rate for this 25‐fold scale‐up (600–15,000 L) was controlled to match off‐gas CO₂ profiles of acceptable smaller scale batches to maintain levels below 0.5%. The 15,000 L‐scale fermentation achieved an expected nocathiacin I titer of 310 mg/L after 7 days. Other on‐line data (i.e., pH, oxygen uptake rate, and CO₂ evolution rate) and off‐line data (i.e., analog production, glucose utilization, ammonium production, and dry cell weight) at the 15,000 L scale also tracked similarly to the smaller scale, demonstrating successful fermentation scale‐up. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Separation of product associating E. coli host cell proteins OppA and DppA from recombinant apolipoprotein A‐IMilano in an industrial HIC unit operation

We have shown how product associating E. coli host cell proteins (HCPs) OppA and DppA can be substantially separated from apolipoprotein A‐I_Milano (apo A‐I_M) using Butyl Sepharose hydrophobic interaction chromatography (HIC). This work illustrates the complex problems that frequently arise during development and scale‐up of biopharmaceutical manufacturing processes. Product association of the HCPs is confirmed using co‐immunoprecipitation and Western blotting techniques. Two‐dimensional gel electrophoresis and mass spectrometry techniques are used to confirm the identity of OppA and DppA. In this example, clearance of these difficult to separate HCPs decreased significantly when the process was scaled to a 1.4 m diameter column. Laboratory‐scale experimentation and trouble shooting identified several key parameters that could be further optimized to improve HCP clearance. The key parameters included resin loading, peak cut point on the ascending side, wash volume, and wash salt concentration. By implementing all of the process improvements that were identified, it was possible to obtain adequate HCP clearance so as to meet the final specification. Although it remains speculative, it is believed that viscosity effects may have contributed to the lower HCP clearance observed early in the manufacturing campaign. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Scale‐up of Oldenlandia affinis suspension cultures in photobioreactors for cyclotide production

Cyclotides are a family of backbone‐cyclized cystine‐knot‐containing macrocyclic peptides from plants that possess extremely interesting biological activities. Suspension cultures of Oldenlandia affinis, a model plant containing cyclotides, were scaled‐up from shake flask to photobioreactor operation in order to produce these plant peptides under controlled conditions. Cell growth was highly dependent on inoculation culture; cell density as well as culture age had an effect on the growth rates and thus affected the kalata B1 productivity of the bioprocess. In a 25 l scale bioreactor the maximum doubling time was about 1.12 days compared to 2.24 days in shake flasks. The accumulation of kalata B1 of 0.09 mg g^?1 DW and 0.07–0.10 mg g^?1 DW respectively, however, was on a similar level during the corresponding stationary growth phases in both bioreactor and flask processes. An adjustment of cell culture growth via culture preparation and inoculum density to high cyclotide accumulation results in an estimated output during the most productive retardation phase of about 21 mg kalata B1 per day in the 25 l system. This makes the biotechnological cyclotide synthesis under GMP conditions a competitive production tool compared to field cultivation, chemical, and recombinant synthesis in drug discovery for structure analysis and bioactivity assays.     

Plant glandular trichomes mediate protective mutualism in a spider–plant system

1. Although several species of Peucetia (Oxyopidae) live strictly in association with plants bearing glandular trichomes worldwide, to date little is known about whether these associations are mutualistic. 2. In this study we manipulated the presence of Peucetia flava on the glandular plant Rhynchanthera dichotoma in the rainy and post‐rain season, to test the strength of its effects on leaf, bud, and flower damage and plant reproductive output. In addition, we ran independent field experiments to examine whether these sticky structures improve spider fidelity to plants. 3. Peucetia suppressed some species of foliar phytophages, but not others. Although spiders have reduced levels of leaf herbivory, this phenomenon was temporally conditional, i.e. occurred only in the post‐rain but not in the rainy season. Floral herbivory was also reduced in the presence of spiders, but these predators did not affect plant fitness components. 4. Plants that had their glandular trichomes removed retained fewer insects than those bearing such structures. Spiders remained longer on plants with glandular trichomes than on plants in which these structures had been removed. Isotopic analyses showed that spiders that fed on live and dead labelled flies adhered to the glandular hairs in similar proportions. 5. Spiders incurred no costs to the plants, but can potentially increase individual plant fitness by reducing damage to reproductive tissues. Temporal conditionality probably occurred because plant productivity exceeded herbivore consumption, thus dampening top‐down effects. Specialisation to live on glandular plants may have favoured scavenging behaviour in Peucetia, possibly an adaptation to periods of food scarcity.     

Large-scale in vitro embryo production and pregnancy rates from Bos taurus, Bos indicus, and indicus-taurus dairy cows using sexed sperm

Herein we describe a large-scale commercial program for in vitro production of embryos from dairy Bos taurus, Bos indicus, and indicus-taurus donors, using sexed sperm. From 5,407 OPU, we compared the number of recovered oocytes (n = 90,086), viable oocytes (n = 64,826), and embryos produced in vitro from Gir (Bos indicus, n = 617), Holstein (Bos taurus, n = 180), 1/4 Holstein × 3/4 Gir (n = 44), and 1/2 Holstein-Gir (n = 37) crossbred cows, and the pregnancy rate of recipient cows. Viable oocytes were in vitro matured (24 h at 38.8 °C, 5% CO₂ in air) and fertilized by incubating them for 18 to 20 h with frozen-thawed sexed sperm (X-chromosome bearing) from Gir (n = 8) or Holstein (n = 7) sires (2 × 10⁶ sperm/dose). Embryos were cultured in similar conditions of temperature and atmosphere as for IVM, with variable intervals of culture (between Days 2 and 5) completed in a portable incubator. All embryos were transferred fresh, after 24 to 72 h of transportation (up to 2,000 km). On average, 16.7 ± 6.3 oocytes (mean ± SEM) were obtained per OPU procedure and 72.0% were considered viable. Total and viable oocytes per OPU procedure were 17.1 ± 4.5 and 12.1 ± 3.9 for Gir cows, 11.4 ± 3.9 and 8.0 ± 2.7 for Holstein cows, 20.4 ± 5.8 and 16.8 ± 5.0 for 1/4 Holstein × 3/4 Gir, and 31.4 ± 5.6 and 24.3 ± 4.7 for 1/2 Holstein-Gir crossbred females (P < 0.01). The mean number of embryos produced by OPU/IVF and the pregnancy rates were 3.2 (12,243/ 3,778) and 40% for Gir cows, 2.1 (2,426/1,138) and 36% for Holstein cows, 3.9 (1,033/267) and 37% for 1/4 Holstein × 3/4 Gir, and 5.5 (1,222/224), and 37% for 1/2 Holstein-Gir. In conclusion, we compared oocyte yield from two levels of indicus-taurus breeds and demonstrated the efficiency of sexed sperm for in vitro embryo production. Culturing embryos during long distance transportation was successful, with potential for international movement of embryos.