Manually and automatically produced pellet cultures of human primary chondrocytes: A comparative analysis

Cartilage defects are often associated with restriction of the locomotor system. New methods are required to investigate cartilage tissue and for the repair of cartilage tissue. 3D cultures are promising due to better simulation of in vivo conditions. The aim of this study was to provide a model system for studying cartilage tissue. We solved this problem by automated production of pellet cultures of human primary chondrocytes in media with and without antibiotics using the Biomek® Cell Workstation and consequent automated bioscreening with a high‐throughput screening system, and compared with the regular manual processes. The Biomek® Cell Workstation allows the cultivation of different cell types (suspensions cells and adherent cells) and 3D cell cultures (pellet cultures, alginate beads and spheroid cultures). The proliferation was analyzed by DNA quantification and compared with the EZ4U proliferation assay as a new tool for pellet cultures. The toxicity was evaluated by the detection of ubiquitous adenylate kinases. The proliferation increased from day 14 until day 35 and was associated with a decrease in the cytotoxicity. The comparative analysis showed similar results for manual and automated processes. We concluded that the manual methods can be replaced by automated processes (pellet manufacturing and screening), which would allow large‐scale procedures to support studies on cartilage regeneration.     

A micromethod for the quantitation of cellular proteins in Percoll with the Coomassie brilliant blue dye-binding assay

A simple procedure for the determination of cellular proteins in Percoll-containing samples is described. Percoll precipitated when particulate proteins were solubilized by dilution of the samples in a NaOH-Triton X-100 mixture. After centrifugation at high speed (12,000g), the supernatant was assayed for proteins with the Coomassie brilliant blue dye-binding assay. With an automatic spectrophotometer, 50-microliter aliquots gave a linear response between 0 and 3 micrograms of bovine serum albumin. After a fivefold dilution in the alkali-detergent mixture, proteins in samples containing up to at least 60% Percoll can be accurately quantitated on a standard curve prepared in the absence of Percoll. Because the sensitivity of the assay was better than 100 ng, the procedure outlined in this paper can also be used as a general protein micromethod.     

Engineering aspects of solid state fermentation

Engineering aspects of solid state fermentation have not been available in spite of the recent interest in the technique and the appearance of a number of reviews on this subject. The present state of the art regarding substrate uptake, oxygen transfer, growth characteristics, growth estimation, control systems for maintaining parameters, mathematical models, design of fermenters and automation of fermentations does not provide a detailed insight. The work carried out at the Central Food Technological Research Institute, Mysore, on the development of a large-scale solid state fermenter, comparative cost estimation of SSF and submerged fermentation as well as development of know-how for production of a variety of food enzymes, has led to the commercial exploitation of the technology by industry. Future R&D needs in this area, neglected at present but yet so promising, are indicated.     

Invasive Snake Activity Before and After Automated Aerial Baiting

Aerially delivered toxic baits have proven effective for landscape-level control of numerous invasive vertebrate populations with major benefits for conservation and ecosystem function, but this technique has not been broadly adapted for control of invasive reptiles. Nonnative brown treesnakes (Boiga irregularis) on the Pacific island of Guam have caused severe ecological and economic damage and pose an invasion risk on other islands, making them a high-profile candidate for application of aerial baiting methods. Although terrestrial applications of traps, toxicants, and hand-removal are standard brown treesnake management practices, these methods are not cost-effective for control in the island's large tracts of remote, rugged forest. In 2016, the first major in situ evaluation of a helicopter-borne automated aerial bait delivery system applied snake-targeted toxic baits at an effective rate of approximately 120 baits/ha over a 110-ha forested test plot on Guam. We evaluated the extent and duration of the suppressive effect of this toxic bait application on brown treesnakes by measuring nontoxic bait take rates as a proxy index of relative snake abundance before and after toxic bait application in a treatment plot and surrounding reference area. We placed 4,420 nontoxic baits in random transects at georeferenced locations, from 1 month before until nearly 12 months after toxic bait application, allowing temporal analysis of the suppressive effect and spatial analysis of treatment plot reinvasion. Over the first 30 days after toxic bait application, average nontoxic bait take rate in the treatment plot was 41.2% lower than the pre-application rate, and there was no immediate decrease in bait take in the reference area. Reduced snake activity was still evident nearly 12 months after bait application. Roads forming a portion of the treatment boundary appeared to slow snake movement between treated area and surrounding untreated area. Trail cameras monitoring a subset of bait tubes showed that 97.5% of baits removed were taken by snakes rather than nontarget species. We indexed rodent abundance in the treatment plot and reference area, and found no indication of a rodent population increase following toxic bait application. Our results show that automated aerial bait applications can suppress brown treesnake abundance over a large area and that reinvasion from surrounding untreated habitat occurs over several months. We anticipate that repeated bait applications could achieve and maintain greatly reduced brown treesnake abundance on a landscape scale, potentially improving biosecurity and enabling experimental reintroduction of native birds extirpated by brown treesnake predation. Published 2019. This article is a U.S. Government work and is in the public domain in the USA. The Journal of Wildlife Management published by Wiley Periodicals, Inc. on behalf of The Wildlife Society.     

Scientific discovery as a combinatorial optimisation problem: How best to navigate the landscape of possible experiments?

A considerable number of areas of bioscience, including gene and drug discovery, metabolic engineering for the biotechnological improvement of organisms, and the processes of natural and directed evolution, are best viewed in terms of a 'landscape' representing a large search space of possible solutions or experiments populated by a considerably smaller number of actual solutions that then emerge. This is what makes these problems 'hard', but as such these are to be seen as combinatorial optimisation problems that are best attacked by heuristic methods known from that field. Such landscapes, which may also represent or include multiple objectives, are effectively modelled in silico, with modern active learning algorithms such as those based on Darwinian evolution providing guidance, using existing knowledge, as to what is the 'best' experiment to do next. An awareness, and the application, of these methods can thereby enhance the scientific discovery process considerably. This analysis fits comfortably with an emerging epistemology that sees scientific reasoning, the search for solutions, and scientific discovery as Bayesian processes.     

Automated,scalable culture of human embryonic stem cells in feeder‐free conditions

Large‐scale manufacture of human embryonic stem cells (hESCs) is prerequisite to their widespread use in biomedical applications. However, current hESC culture strategies are labor‐intensive and employ highly variable processes, presenting challenges for scaled production and commercial development. Here we demonstrate that passaging of the hESC lines, HUES7, and NOTT1, with trypsin in feeder‐free conditions, is compatible with complete automation on the CompacT SelecT, a commercially available and industrially relevant robotic platform. Pluripotency was successfully retained, as evidenced by consistent proliferation during serial passage, expression of stem cell markers (OCT4, NANOG, TRA1‐81, and SSEA‐4), stable karyotype, and multi‐germlayer differentiation in vitro, including to pharmacologically responsive cardiomyocytes. Automation of hESC culture will expedite cell‐use in clinical, scientific, and industrial applications. Biotechnol. Bioeng. 2009;102: 1636–1644. © 2008 Wiley Periodicals, Inc.     

How to track and assess genotyping errors in population genetics studies

Genotyping errors occur when the genotype determined after molecular analysis does not correspond to the real genotype of the individual under consideration. Virtually every genetic data set includes some erroneous genotypes, but genotyping errors remain a taboo subject in population genetics, even though they might greatly bias the final conclusions, especially for studies based on individual identification. Here, we consider four case studies representing a large variety of population genetics investigations differing in their sampling strategies (noninvasive or traditional), in the type of organism studied (plant or animal) and the molecular markers used [microsatellites or amplified fragment length polymorphisms (AFLPs)]. In these data sets, the estimated genotyping error rate ranges from 0.8% for microsatellite loci from bear tissues to 2.6% for AFLP loci from dwarf birch leaves. Main sources of errors were allelic dropouts for microsatellites and differences in peak intensities for AFLPs, but in both cases human factors were non-negligible error generators. Therefore, tracking genotyping errors and identifying their causes are necessary to clean up the data sets and validate the final results according to the precision required. In addition, we propose the outline of a protocol designed to limit and quantify genotyping errors at each step of the genotyping process. In particular, we recommend (i) several efficient precautions to prevent contaminations and technical artefacts; (ii) systematic use of blind samples and automation; (iii) experience and rigor for laboratory work and scoring; and (iv) systematic reporting of the error rate in population genetics studies.     

The Oompa-Loompas of the genome factories

Automation Technologies for Genome Characterization

edited by Tony J. Beugelsdijk, John Wiley and Sons, 1997. UK£55.00 hbk (xvi +306 pages) ISBN 0 471 12806 6