Maciej Pilarek

Enhanced growth and recombinant protein production of Escherichia coli by a perfluorinated oxygen carrier in miniaturized fed-batch cultures

BackgroundLiquid perfluorochemicals (PFCs) are interesting oxygen carriers in medicine and biotechnology with a high solubility for oxygen. They have been repeatedly used for improving oxygen transfer into prokaryotic and eukaryotic cell cultures, however their application is still limited. Here we show the great benefit of air/oxygen saturated perfluorodecalin (PFD) for high cell density cultivation of Escherichia coli in microwell plates and their positive effect on the soluble production of a correctly folded heterologously expressed alcohol dehydrogenase.ResultsIn EnBase® cultivations the best effect was seen with PFD saturated with oxygen enriched air (appr. 10 μM oxygen per ml) when PFD was added at the time of induction. In contrast the effect of PFD was negligible when it was added already at the time of inoculation. Optimisation of addition time and content of loaded oxygen into the PFD resulted in an increased the cell density by 40% compared to control cultures, and correspondingly also the product yield increased, demonstrated at the example of a recombinant alcohol dehydrogenase.ConclusionsPFCs are a valuable additive in miniaturized cell culture formats. For production of recombinant proteins in low cell density shaken cultures the addition of oxygen-enriched PFD makes the process more robust, i.e. a high product yield is not any more limited to a very narrow cell density window during which the induction has to be done. The positive effect of PFD was even more obvious when it was added during high cell density cultures. The effect of the PFD phase depends on the amount of oxygen which is loaded into the PFD and which thus is a matter of optimisation.

Mini-scale cultivation method enables expeditious plasmid production in Escherichia coli

The standard procedure in the lab for plasmid isolation usually involves a 2‐mL, 16 h over‐night cultivation in 15‐mL bioreaction tubes in LB medium. This is time consuming, and not suitable for high‐throughput applications. This study shows that it is possible to produce plasmid DNA (pDNA) in a 1.5‐mL microcentrifuge tube with only 100 μL cultivation volume in less than 7 h with a simple protocol. Compared with the standard LB cultivation for pDNA production reaching a final pDNA concentration range of 1.5–4 μg mL–1, a 6‐ to 10‐fold increase in plasmid concentration (from 10 up to 25 μg mL–1 cultivation volume) is achieved using an optimized medium with an internal substrate delivery system (EnBase®). Different strains, plasmids, and the applicability of different inoculation tools (i.e. different starting ODs) were compared, demonstrating the robustness of the system. Additionally, dissolved oxygen was monitored in real time online, indicating that under optimized conditions oxygen limitation can be avoided. We developed a simple protocol with a significantly decreased procedure time, enabling simultaneous handling of more samples, while a consistent quality and a higher final pDNA concentration are ensured.

Liquid perfluorochemical-supported hybrid cell culture system for proliferation of chondrocytes on fibrous polylactide scaffolds

CP5 bovine chondrocytes were cultured on biodegradable electrospun fibrous polylactide (PLA) scaffolds placed on a flexible interface formed between two immiscible liquid phases: (1) hydrophobic perfluorochemical (PFC) and (2) aqueous culture medium, as a new way of cartilage implant development. Robust and intensive growth of CP5 cells was achieved in our hybrid liquid–solid–liquid culture system consisting of the fibrous PLA scaffolds in contrast to limited growth of the CP5 cells in traditional culture system with PLA scaffold placed on solid surface. The multicellular aggregates of CP5 cells covered the surface of PLA scaffolds and the chondrocytes migrated through and overgrew internal fibers of the scaffolds. Our hybrid culture system simultaneously allows the adhesion of adherent CP5 cells to fibers of PLA scaffolds as well as, due to use of phase of PFC, enhances the mass transfer in the case of supplying/removing of respiratory gases, i.e., O2 and CO2. Our flexible (independent of vessel shape) system is simple, ready-to-use and may utilize a variety of polymer-based scaffolds traditionally proposed for implant development.

Cultivation of Marine Microorganisms in Single-Use Systems

: Marine cultures are an important source of novel substances and enzymes. As efforts to isolate strains from (deep) sea environments increase, the demand for methodology platforms to cultivate these organisms is also rising. Due to the high salt concentration and the shear sensitivity exhibited by some heterotrophic microalgae, single-use systems originally designed for the cultivation of mammalian cell lines can be a valuable alternative. Using the cultivation of the heterotrophic marine microalgae Crypthecodinium cohnii as an example, this chapter makes suggestions for experimental design, for improving process development by integrating parallel experiments, and for scaling-up and scaling-down methodologies. It describes how to identify suitable single-use systems and how to integrate a two-layer system with perfluordecalin to improve the gas transfer in deep-well plates. The process is also scaled up in several single-use systems. We also describe challenges in the process development to achieve sufficient oxygen transfer, monitoring, and control, and we discuss limitations such as corrosion, long-term stability, and leachables in single-use systems. Finally, we demonstrate a method for cheap, fast, and consistent process development for marine microorganisms.

Comparative Studies of Electrospinning and Solution Blow Spinning Processes for the Production of Nanofibrous Poly(L-Lactic Acid) Materials for Biomedical Engineering

Abstract Comparative statistical analysis of the infiuence of processing parameters, for electrospinning (ES) and solution blow spinning (SBS) processes, on nanofibrous poly(L-lactic acid) (PLLA) material morphology and average fiber diameter was conducted in order to identify the key processing parameter for tailoring the product properties. Further, a comparative preliminary biocompatibility evaluation was performed. Based on Design of Experiment (DOE) principles, analysis of standard effects of voltage, air pressure, solution feed rate and concentration, on nanofibers average diameter was performed with the Pareto’s charts and the best fitted surface charts. Nanofibers were analyzed by scanning electron microscopy (SEM). The preliminary biocompatibility comparative tests were performed based on SEM microphotographs of CP5 cells cultured on materials derived from ES and SBS. Polymer solution concentration was identified as the key parameter infiuencing morphology and dimensions of nanofibrous mat produced from both techniques. In both cases, when polymer concentration increases the average fiber diameter increase. The preliminary biocompatibility test suggests that nanofibers produced by ES as well as SBS are suitable as the biomedical engineering scaffold material.

Design of experiments‐based high‐throughput strategy for development and optimization of efficient cell disruption protocols

Efficient and reproducible cell lysis is a crucial step during downstream processing of intracellular products. The composition of an optimal lysis buffer should be chosen depending on the organism, its growth status, the applied detection methods, and even the target molecule. Especially for high‐throughput applications, where sample volumes are limited, the adaptation of a lysis buffer to the specific campaign is an urgent need. Here, we present a general design of experiments‐based strategy suitable for eight constituents and demonstrate the strength of this approach by the development of an efficient lysis buffer for Gram‐negative bacteria, which is applicable in a high‐throughput format in a short time. The concentrations of four lysis‐inducing chemical agents EDTA, lysozyme, Triton X‐100, and polymyxin B were optimized for maximal soluble protein concentration and ß‐galactosidase activity in a 96‐well format on a Microlab Star liquid handling platform under design of experiments methodology. The resulting lysis buffer showed the same performance as a commercially available lysis buffer. The developed protocol resulted in an optimized buffer within only three runs. The established procedure can be easily applied to adapt the lysis buffer to other strains and target molecules.

Enzymatic Activity of Some Industrially-Applied Cellulolytic Enzyme Preparations

Abstract Enzymatic hydrolysis is the essential step in the production of 2nd generation biofuels made from lignocellulosic biomass, i.e. agricultural or forestry solid wastes. The enzyme-catalysed robust degradation of cellulose and hemicellulose to monosaccharides requires the synergistic action of the independent types of highly-specific enzymes, usually offered as ready-to-use preparations. The basic aim of the study was to experimentally determine the enzymatic activity of two widely industrially-applied, commercially available cellulolytic enzyme preparations: (i) Cellic® CTec2 and (ii) the mixture of Celluclast® 1.5L and Novozyme 188, in the hydrolysis of pre-treated lignocellulosic biomass, i.e. (a) energetic willow and (b) rye straw, or untreated (c) cellulose paper as well, used as feedstocks. Before the hydrolysis, every kind of utilized lignocellulosic biomass was subjected to alkaline-based (10% NaOH) pre-treatment at high-temperature (121°C) and overpressure (0.1 MPa) conditions. The influence of the type of applied enzymes, as well as their concentration, on the effectiveness of hydrolysis was quantitatively evaluated, and finally the enzyme activities were determined for each of tested cellulolytic enzyme preparations.