Generation of Concentration Gradients by a Outer-Circumference-Driven On-Chip Mixer

The concentration control of reagents is an important factor in microfluidic devices for cell cultivation and chemical mixing, but it is difficult to realize owing to the characteristics of microfluidic devices. We developed a microfluidic device that can generate concentration gradients among multiple main chambers. Multiple main chambers are connected in parallel to the body channel via the neck channel. The main chamber is subjected to a volume change through a driving chamber that surrounds the main chamber, and agitation is performed on the basis of the inequality of flow caused by expansion or contraction. The neck channel is connected tangentially to the main chamber. When the main chamber expands or contracts, the flow in the main chamber is unequal, and a net vortex is generated. The liquid moving back and forth in the neck channel gradually absorbs the liquid in the body channel into the main chamber. As the concentration in the main chamber changes depending on the pressure applied to the driving chamber, we generated a concentration gradient by arranging chambers along the pressure gradient. This allowed for us to create an environment with different concentrations on a single microchip, which is expected to improve observation efficiency and save space.

Biopharmaceutical-Type Chinese Hamster Ovary Cell Cultivation Under Static Magnetic Field Exposure: A Study of Genotoxic Effect

The lack of a sufficient research base is the reason for the ongoing discussion regarding the genotoxic effect of magnetic field (MF) exposure on mammalian cell cultures. Chinese hamster ovary (CHO) suspension-type cells, which are widely used for biopharmaceutical production, are potentially subjected to an increased MF when cultivated in bioreactors equipped with bottom-placed magnetically coupled stirring mechanisms. The main challenge for conducting research in this field remains the availability of a suitable experimental setup that generates an appropriate MF for the raised research question. In the present study, a simple and cost-effective experimental setup was developed that generated a static MF, similar to what has been modeled in large-scale bioreactors and, at the same time, was suitable for experimental cell cultivation in laboratory conditions. The measured maximum magnetic flux density to which the cells were exposed was 0.66 T. To assess the possible genotoxic effect, cells were continuously subcultivated in laboratory petri dishes for a period of 14 days, corresponding to a typical duration of a biopharmaceutical production process in a conventional fed-batch regime. The genotoxic effect was assessed using the cytokinesis-block micronucleus assay with fluorescent staining. Results showed that a 0.66-T static MF exposure had no significant long-term effect on cell viability and chromosomal damage but demonstrated a short-term effect on cell apoptosis. Significant increase in nuclear bud formation was observed. These findings may encourage other researchers in future studies investigating cellular responses to MF exposure and contribute relevant data for comparison.

Protocol for chronic hepatitis B virus infection mouse model development by patient-derived orthotopic xenografts v1

This will be the first extensive comparative study of the main modern methods and protocols for isolation and cultivation primary hepatocellular carcinoma cells and tumor engraftment to the mice. All protocols will be optimized and characterized using the: (1) efficiency of the method for isolation cells from removed hepatocellular carcinoma in terms of their quantity and viability; (2) efficiency of the primary cell cultivation protocol in terms of the rate of monolayer formation and hepatitis B virus replication; (3) efficiency of the grafting method in terms of the growth rate and the possibility of hepatitis B virus persistence and replication in mice.The most effective methods will be recommended for use in translational biomedical research.

Extractive Fermentation for Recovery of Bacteriocin-Like Inhibitory Substances Derived from Lactococcus lactis Gh1 Using PEG2000/Dextran T500 Aqueous Two-Phase System

This work aimed to optimize the parameters affecting partitioning of a bacteriocin-like inhibitory substances (BLIS) from Lactococcus lactis Gh1 in extractive fermentation using polyethylene glycol (PEG)/dextran aqueous two-phase system (ATPS). This system was developed for the simultaneous cell cultivation and downstream processing of BLIS. Results showed that the molecular weight of PEG, PEG concentration, and dextran T500 affect the partition coefficient (K), purification factor (PF), and yield of BLIS partitioning. ATPS composed of 10% (w/w) PEG2000 and 8% (w/w) dextran T500, provided the greatest conditions for the extractive BLIS production. The K (1.00 ± 0.16), PF (2.92 ± 0.37) and yield (77.24 ± 2.81%) were increased at selected orbital speed (200 rpm) and pH (pH 7). Sustainable growth of the cells in the bioreactor and repeated fermentation up to the eighth extractive batch were observed during the scale up process, ensuring a continuous production and purification of BLIS. Hence, the simplicity and effectiveness of ATPS in the purification of BLIS were proven in this study.

Glycosaminoglycan-Based Cryogels as Scaffolds for Cell Cultivation and Tissue Regeneration

Cryogels are a class of macroporous, interconnective hydrogels polymerized at sub-zero temperatures forming mechanically robust, elastic networks. In this review, latest advances of cryogels containing mainly glycosaminoglycans (GAGs) or composites of GAGs and other natural or synthetic polymers are presented. Cryogels produced in this way correspond to the native extracellular matrix (ECM) in terms of both composition and molecular structure. Due to their specific structural feature and in addition to an excellent biocompatibility, GAG-based cryogels have several advantages over traditional GAG-hydrogels. This includes macroporous, interconnective pore structure, robust, elastic, and shape-memory-like mechanical behavior, as well as injectability for many GAG-based cryogels. After addressing the cryogelation process, the fabrication of GAG-based cryogels and known principles of GAG monomer crosslinking are discussed. Finally, an overview of specific GAG-based cryogels in biomedicine, mainly as polymeric scaffold material in tissue regeneration and tissue engineering-related controlled release of bioactive molecules and cells, is provided.

Ein Wechselbad für Mikroben: Wachsen unter dynamischen Umweltbedingungen

Microfluidic technologies are increasingly used within microbiology for screening and improved understanding of cellular behaviour. The presented system for dynamic microfluidic single-cell cultivation — dMSCC — offers a new cultivation method for microbial microcolonies at dynamic environments with oscillation between seconds to hours. This tool has a high potential to improve the understanding of cellular behaviour at dynamic environments as omnipresent in nature and lab-scale cultivation systems.

A novel scale-up strategy for cultivation of BHK-21 cells based on similar hydrodynamic environments in the bioreactors

The scale-up of animal cell cultivation is important but remains complex and challenging. In the present study, we propose a novel scale-up strategy for baby hamster Syrian kidney-21 (BHK-21) cell cultivation based on similar hydrodynamic environments. The hydrodynamic characteristics of the different scale bioreactors were determined by computational fluid dynamics (CFD) and further correlated with the agitation speed. The optimal hydrodynamic environment for cell cultivation and vaccine production was determined from the cultivation experiments of BHK-21 cells in 5-L laboratory-scale bioreactors equipped with different impellers at various agitation speeds. BHK-21 cell cultivation was scaled up from 5-L to 42-, 350-, and 1000-L bioreactors by adjusting the agitation speed to make the hydrodynamic features similar to those in the 5-L bioreactor, especially for the shear rate in the impeller zone (γimp) and energy dissipation rate in the tank bulk zone (εtan). The maximum cell density and cell aggregation rate in these scaled-up bioreactors were in the range of 4.6 × 106 ~ 4.8 × 106 cells/mL and 16 ~ 20%, which are comparable to or even better than those observed in the 5-L bioreactor (maximum cell density 4.8 × 106 cells/mL, cell aggregation rate 21%). The maximum virus titer of 108.0 LD50/mL achieved in the 1000-L bioreactor was close to 108.3 LD50/mL that obtained in the 5-L bioreactor. Hence, the scale-up strategy proposed in this study is feasible and can efficiently facilitate the scale-up processes of animal cell cultivation.