Facilitated endospore detection for Bacillus spp. through automated algorithm-based image processing

Bacillus spp. endospores are important dormant cell forms and are distributed widely in environmental samples. While these endospores can have important industrial value (e.g. use in animal feed as probiotics), they can also be pathogenic for humans and animals, emphasizing the need for effective endospore detection. Standard spore detection by colony forming units (CFU) is time-consuming, elaborate and prone to error. Manual spore detection by spore count in cell counting chambers via phase-contrast microscopy is less time-consuming. However, it requires a trained person to conduct. Thus, the development of a facilitated spore detection tool is necessary. This work presents two alternative quantification methods: first, a colorimetric assay for detecting the biomarker dipicolinic acid (DPA) adapted to modern needs and applied for Bacillus spp. and second, a model-based automated spore detection algorithm for spore count in phase-contrast microscopic pictures. This automated spore count tool advances manual spore detection in cell counting chambers, and does not require human overview after sample preparation. In conclusion, this developed model detected various Bacillus spp. endospores with a correctness of 85–89%, and allows an automation and time-saving of Bacillus endospore detection. In the laboratory routine, endospore detection and counting was achieved within 5–10 min, compared to up to 48 h with conventional methods. The DPA-assay on the other hand enabled very accurate spore detection by simple colorimetric measurement and can thus be applied as a reference method

In vitro vascularization of hydrogel-based tissue constructs via a combined approach of cell sheet engineering and dynamic perfusion cell culture

The bioengineering of artificial tissue constructs requires special attention to their fast vascularization to provide cells with sufficient nutrients and oxygen. We addressed the challenge of in vitro vascularization by employing a combined approach of cell sheet engineering, 3D printing, and cellular self-organization in dynamic maturation culture. A confluent cell sheet of human umbilical vein endothelial cells (HUVECs) was detached from a thermoresponsive cell culture substrate and transferred onto a 3D-printed, perfusable tubular scaffold using a custom-made cell sheet rolling device. Under indirect co-culture conditions with human dermal fibroblasts (HDFs), the cell sheet-covered vessel mimic embedded in a collagen gel together with additional singularized HUVECs started sprouting into the surrounding gel, while the suspended cells around the tube self-organized and formed a dense lumen-containing 3D vascular network throughout the gel. The HDFs cultured below the HUVEC-containing cell culture insert provided angiogenic support to the HUVECs via molecular crosstalk without competing for space with the HUVECs or inducing rapid collagen matrix remodeling. The resulting vascular network remained viable under these conditions throughout the 3 week cell culture period. This static indirect co-culture setup was further transferred to dynamic flow conditions, where the medium perfusion was enabled via two independently addressable perfusion circuits equipped with two different cell culture chambers, one hosting the HDFs and the other hosting the HUVEC-laden collagen gel. Using this system, we successfully connected the collagen-embedded HUVEC culture to a dynamic medium flow, and within 1 week of the dynamic cell culture, we detected angiogenic sprouting and dense microvascular network formation via HUVEC self-organization in the hydrogel. Our approach of combining a 3D-printed and cell sheet-covered vascular precursor that retained its sprouting capacity together with the self-assembling HUVECs in a dynamic perfusion culture resulted in a vascular-like 3D network, which is a critical step toward the long-term vascularization of bioengineered in vitro tissue constructs

Microfluidic bifurcating networks for power-law fluids

Bifurcating networks are widely found in nature and are often responsible for controlling fluids that exhibit complex rheological behaviour. Examples are the vascular branching network that drives blood throughout the human body, the oxygen respiratory system in the human lungs and the bifurcating formations of xylem responsible for the distribution of water and other nutrients in plants and trees. Here, we take advantage of the biomimetic principles obtained by studying these natural systems to design fluid distribution networks for use in lab-on-a-chip devices. The novel biomimetic design rule we have recently proposed allows us to generate bifurcating microfluidic networks of rectangular cross-section for use with power-law and Newtonian fluids [1]. The design is based on Murray’s law, which was originally derived for blood flow in the vascular system, using the principle of minimum work. Murray [2] considered Newtonian fluid flows to predict the optimum ratio between the diameters of the parent and daughter vessels in networks with circular cross-section to obtain a uniform wall-shear stress along the network. In our study, we have extended the relationship to consider the flow of power-law fluids in planar geometries (i.e. geometries of rectangular cross-section with constant depth) typical of lab-on-a-chip applications. Furthermore, the design rule has been generalised to consider a range of shear-stress distributions via a branching parameter, offering the ability to precisely control the shear-stress distribution and predict the flow resistance along the bifurcating network

Thermal stability, phase decomposition, and micro-fatigue properties of pulsed electrodeposited nanocrystalline Co-Cu

Nanocrystalline (nc) immiscible Co-Cu alloys system is a promising material where the decomposition of the super-saturated solid solution can be used to obtain nano-structured materials. In this research, homogenous and solid nc Co-Cu thick films were synthesized through the pulsed electrodeposition technique in complex sodium tartrate electrolyte. Annealing procedures were conducted to evaluate its thermal stability and induce phase decomposition of cobalt and copper, which can be utilized to enhance mechanical properties and thermal stability. Initial cyclic micro-bending experiments were also conducted to observe micro-fatigue properties and structural evolution during mechanical loading

Efficient time splitting schemes for the monodomain equation in cardiac electrophysiology

Approximating the fast dynamics of depolarization waves in the human heart described by the monodomain model is numerically challenging. Splitting methods for the PDE-ODE coupling enable the computation with very fine space and time discretizations. Here, we compare different splitting approaches regarding convergence, accuracy, and efficiency. Simulations were performed for a benchmark problem with the Beeler–Reuter cell model on a truncated ellipsoid approximating the left ventricle including a localized stimulation. For this configuration, we provide a reference solution for the transmembrane potential. We found a semi-implicit approach with state variable interpolation to be the most efficient scheme. The results are transferred to a more physiological setup using a bi-ventricular domain with a complex external stimulation pattern to evaluate the accuracy of the activation time for different resolutions in space and time

Efficient time splitting schemes for the monodomain equation in cardiac electrophysiology

Approximating the fast dynamics of depolarization waves in the human heart described by the monodomain model is numerically challenging. Splitting methods for the PDE-ODE coupling enable the computation with very fine space and time discretizations. Here, we compare different splitting approaches regarding convergence, accuracy and efficiency. Simulations were performed for a benchmark configuration with the Beeler–Reuter cell model on a truncated ellipsoid approximating the left ventricle including a localized stimulation. For this benchmark configuration, we provide a reference solution for the transmembrane potential. We found a semi-implicit approach with state variable interpolation to be the most efficient scheme. The results are transferred to a more physiological setup using a bi-ventricular domain with a complex external stimulation pattern to evaluate the accuracy of the activation time for different resolutions in space and time

Flow-induced glycocalyx formation and cell alignment of HUVECs compared to iPSC-derived ECs for tissue engineering applications

The relevance of cellular in vitro models highly depends on their ability to mimic the physiological environment of the respective tissue or cell niche. Static culture conditions are often unsuitable, especially for endothelial models, since they completely neglect the physiological surface shear stress and corresponding reactions of endothelial cells (ECs) such as alignment in the direction of flow. Furthermore, formation and maturation of the glycocalyx, the essential polysaccharide layer covering all endothelial surfaces and regulating diverse processes, is highly dependent on applied fluid flow. This fragile but utterly important macromolecular layer is hard to analyze, its importance is often underestimated and accordingly neglected in many endothelial models. Therefore, we exposed human umbilical vein ECs (HUVECs) and human induced pluripotent stem cell-derived ECs (iPSC-ECs) as two relevant EC models in a side-by-side comparison to static and physiological dynamic (6.6 dyn cm−2) culture conditions. Both cell types demonstrated an elongation and alignment along the flow direction, some distinct changes in glycocalyx composition on the surface regarding the main glycosaminoglycan components heparan sulfate, chondroitin sulfate or hyaluronic acid as well as an increased and thereby improved glycocalyx thickness and functionality when cultured under homogeneous fluid flow. Thus, we were able to demonstrate the maturity of the employed iPSC-EC model regarding its ability to sense fluid flow along with the general importance of physiological shear stress for glycocalyx formation. Additionally, we investigated EC monolayer integrity with and without application of surface shear stress, revealing a comparable existence of tight junctions for all conditions and a reorganization of the cytoskeleton upon dynamic culture leading to an increased formation of focal adhesions. We then fabricated cell sheets of EC monolayers after static and dynamic culture via non-enzymatic detachment using thermoresponsive polymer coatings as culture substrates. In a first proof-of-concept we were able to transfer an aligned iPSC-EC sheet to a 3D-printed scaffold thereby making a step in the direction of vascular modelling. We envision these results to be a valuable contribution to improvements of in vitro endothelial models and vascular engineering in the future

Reversible male sterility in eggplant

SummarySince decades, plant male sterility is considered a powerful tool for biological containment to minimize unwanted self‐pollination for hybrid seed production. Furthermore, prevention of pollen dispersal also answers to concerns regarding transgene flow via pollen from Genetically Modified (GM) crops to traditional crop fields or wild relatives. We induced male sterility by suppressing endogenous general transcription factor genes, TAFs, using anther‐specific promoters combined with artificial microRNA (amiRNA) technology (Schwab et al., 2006). The system was made reversible by the ethanol inducible expression of an amiRNA‐insensitive form of the target gene. We provide proof of concept in eggplant, a cultivated crop belonging to the Solanaceae family that includes many important food crops. The transgenic eggplants that we generated are completely male sterile and fertility can be fully restored by short treatments with ethanol, confirming the efficiency but also the reliability of the system in view of open field cultivation. By combining this system with induced parthenocarpy (Rotino et al., 1997), we provide a novel example of complete transgene containment in eggplant, which enables biological mitigation measures for the benefit of coexistence or biosafety purposes for GM crop cultivation

Implementing a digital infrastructure for the lab using a central laboratory server and the SiLA2 communication standard

In this report, a fully integrated solution for laboratory digitization is presented. The approach presents a flexible and complete integration method for the digitally assisted workflow. The worker in the laboratory performs procedures in direct interaction with the digitized infrastructure that guides through the process and aids while performing tasks. The digital transformation of the laboratory starts with standardized integration of both new and “smart” lab devices, as well as legacy devices through a hardware gateway module. The open source Standardization in Lab Automation 2 standard is used for device communication. A central lab server channels all device communication and keeps a database record of every measurement, task and result generated or used in the lab. It acts as a central entry point for process management. This backbone enables a process control system to guide the worker through the lab process and provide additional assistance, like results of automated calculations or safety information. The description of the infrastructure and architecture is followed by a practical example on how to implement a digitized workflow. This approach is highly useful for – but not limited to – the biotechnological laboratory and has the potential to increase productivity in both industry and research for example by enabling automated documentation

Bringing IoT to the Lab : SiLA2 and Open-Source-Powered Gateway Module for Integrating Legacy Devices into the Digital Laboratory

In this article a gateway module to integrate legacy laboratory devices into the network of the digital laboratory in the 21st century is introduced. The device is based on ready to buy consumer hardware that is easy to get and inexpensive. Depending on the specific requirements of the desired application (bare embedded computer, RS232 serial port connector, IP65 certified casing and connectors) the needed investment ranges from about 95 € up to 200 €. The embedded computer runs an open source Linux operating system and can in principle be used to run any kind of software needed for communicating with the laboratory device. Here the open source SiLA2 standard is used for presenting the device’s functions in the network. As an example the digital integration of a magnetic stirrer is shown and can be used as a template for other applications. A method for easy remote integration of the device to ensure an easy and consistent workflow in development, testing and usage is also presented. This incorporates a method for remote installation of SiLA2 servers on the box as well as a web frontend for administration, debugging and management of those