Measuring the adhesion limit of fibronectin for fibroblasts with a narrow-gap rotational rheometer.

We study the adhesion limit of 3T6 fibroblasts, cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum at 37 °C and 5% CO2, with a narrow-gap rotational rheometer in the parallel-disk configuration. Reducing the uncertainty in gap width to about 1 µm allows studying the cells at narrow gaps, which enables to study the critical shear stress of the cells in low-viscous media. The adhesion limit on fibronectin-coated glass plates is determined as a function of concentration and adhesion time. We found that cells in groups have a tendency to detach at slightly higher shear stresses than single cells. Moreover, 60 min after the settling phase are enough for the cells to adhere to the coated plate at maximum strength. We show that the setup may also be used for cells that are not adhered from suspension, but are grown directly on the substrate.

Basic Colorimetric Proliferation Assays: MTT, WST, and Resazurin.

This chapter describes selected assays for the evaluation of cellular viability and proliferation of cell cultures. The underlying principle of these assays is the measurement of a biochemical marker to evaluate the cell's metabolic activity. The formation of the omnipresent reducing agents NADH and NADPH is used as a marker for metabolic activity in the following assays. Using NADH and NADPH as electron sources, specific dyes are biochemically reduced which results in a color change that can be determined with basic photometrical methods. The assays selected for this chapter include MTT, WST, and resazurin. They are applicable for adherent or suspended cell lines, easy to perform, and comparably economical. Detailed protocols and notes for easier handling and avoiding pitfalls are enclosed to each assay.

Quantification of oxygen production and respiration rates in mixotrophic cultivation of microalgae in nonstirred photobioreactors.

Online monitoring and controlling of different cellular parameters are key issues in aerobic bioprocesses. Since mixotrophic cultivation, in which we observe a mixture of cellular respiration and oxygen production has gained more popularity, there is a need for an on-process quantification of these parameters. The presented and adapted double gassing-out method applied to a mixotrophic cultivation of Galdieria sulphuraria, will be a tool for monitoring and further optimization of algal fermentation in nonstirred photobioreactors (PBR). We measured the highest net specific oxygen production rate (opr net) as 5.73 · 10-3 molO2 g-1 h-1 at the lowest oxygen uptake rate (OUR) of 1.00 · 10-4 molO2 L-1 h-1. Due to higher cell densities, we also demonstrated the increasing shading effect by a decrease of opr net, reaching the lowest value of 1.25 10-5 molO2 g-1 h-1. Nevertheless, with this on process measurement, we can predict the relation between the zone in which oxygen is net produced to the area where cell respiration dominates in a PBR, which has a major impact to optimize cell growth along with the formation of different products of interest such as pigments.

Prolonged Ex vivo expansion and differentiation of naïve murine CD43(-) B splenocytes.

Ex vivo expansion of naive primary B cells is still a challenge, yet would open new possibilities for in vitro studies of the immune response or the production of monoclonal antibodies. In our hands, unstimulated murine B cells did not expand in significant numbers, while culture viability decreased rapidly within a few days. Activation mimicking in vivo stimulation through either T cell-independent or T-cell dependent signaling, led to several division cycles, albeit accompanied by irreversible differentiation. By co-culturing B cells under moderate hypothermia (30°C) on live feeder fibroblasts expressing recombinant CD40 ligand (CD154) and by repeatedly transferring cultured B cells to new feeder cell cultures, we could extend the growth of primary mouse B cells compared to cultures maintained at 37°C. B cells under these conditions showed an activated phenotype as shown by the presence of AID and IRF4, two factors required for IgH class switch recombination in antigen-activated B cells. In contrast to cells cultured at 37°C, B cells under hyperthermia did surprisingly not differentiate into Blimp-1 expressing plasmablasts. Thus, the repeated batch process under hyperthermic conditions represents a first step towards the development of a continuous cultivation system for the expansion of primary B cells. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:978-989, 2016.

Online oxygen measurements in ex vivo perfused muscle tissue in a porcine model using dynamic quenching methods.

INTRODUCTION: Transplantation of autologous free tissue flaps is the best applicable technique for treating large and complex tissue defects and still has one major failure criterion. Tissue--and in particular muscle tissue--is strongly sensitive to ischemia, thus after a critical period of oxygen depletion the risk of a partial or total flap loss is high. MATERIALS AND METHODS: For that reason a miniaturized ex vivo perfusion system has been developed, that supplies the tissue during operational delays. The purpose of this study was to determine the oxygenation levels during such a perfusion using different perfusates and therefore to objectify if a complementary oxygenation unit is required to improve perfusion quality. The oxygen levels of the tissue, as well of the perfusate, were measured by using minimal invasive optical oxygen sensors that are based on dynamic quenching. The ex vivo perfused tissue was the porcine rectus abdominis muscle. RESULTS: Results show, that during perfusion with heparinized crystalloid fluid (Jonosteril) and heparinized autologous whole blood, additional oxygenation of the perfusion reactor led to different ex vivo oxygen tissue saturations, which can be detected by dynamic quenching. CONCLUSION: Dynamic quenching methods are a promising and valuable technique to perform online oxygen measurements in ex vivo perfused muscle tissue in a porcine model.