Plants produce a wide variety of secondary metabolites, which often are of interest to pharmaceutical and nutraceutical industry. Plant-cell cultures allow producing these metabolites in a standardised manner, independently from various biotic and abiotic factors difficult to control during conventional cultivation. However, plant-cell fermentation proves to be very difficult, since these chemically complex compounds often result from the interaction of different biosynthetic pathways operating in different cell types. To simulate such interactions in cultured cells is a challenge. Here, we present a microfluidic bioreactor for plant-cell cultivation to mimic the cell–cell interactions occurring in real plant tissues. In a modular set-up of several microfluidic bioreactors, different cell types can connect through a flow that transports signals or metabolites from module to module. The fabrication of the chip includes hot embossing of a polycarbonate housing and subsequent integration of a porous membrane and in-plane tube fittings in a two-step ultrasonic welding process. The resulting microfluidic chip is biocompatible and transparent. Simulation of mass transfer for the nutrient sucrose predicts a sufficient nutrient supply through the membrane. We demonstrate the potential of this chip for plant cell biology in three proof-of-concept applications. First, we use the chip to show that tobacco BY-2 cells in suspension divide depending on a “quorum-sensing factor” secreted by proliferating cells. Second, we show that a combination of two Catharanthus roseus cell strains with complementary metabolic potency allows obtaining vindoline, a precursor of the anti-tumour compound vincristine. Third, we extend the approach to operationalise secretion of phytotoxins by the fungus Neofusicoccum parvum as a step towards systems to screen for interorganismal chemical signalling.
We introduce a variety of biocompatible fluidic connectors that can be integrated into microfluidic chips by ultrasonic welding. Commercially available barbed fittings and dispensing needles with Luer lock fittings were integrated between two chip components ensuring a fluidic in-plane contact. In addition, straight Luer lock fittings in combination with ultrasonic hot embossing, 3D printed thermoplastic connectors with Luer lock and barbed fittings were integrated out-of-plane. The integration was successful without clogging any fluidic channels.
Depending on the connector type, the pressure tightness differs. Dispensing needles showed the lowest pressure tightness of only 1.14 bar. However, all other connector types were pressure tight to at least 3.75 bar.
The main advantage of the integration technique of ultrasonic welding is the rapid implementation of individual connectors adapted to the required situation—for prototypes as well as for large-scale production. Moreover, multiple connectors can be integrated simultaneously in just one single step. This provides a user-friendly and stable connection of commonly used connector types such as barbed or Luer lock fittings for microfluidic applications.
A microtiter plate-based assay was developed to evaluate the ability of lipases to perform transesterifications when employed in different organic solvents. A 4-nitrophenol assay was carried out employing seven different lipase formulations and two fatty acid methyl esters with different chain lengths in a total of six organic solvents with logP values approximately between 1 and −1. This assay delivered results within comparatively short times measured by a color reaction and thus facilitates the choice of an enzyme-solvent combination for the synthesis of glycolipids. To validate the findings, glycolipid syntheses were performed using the same lipase formulation in the same solvents. When comparing the results obtained using the microtiter plate-based assay to the results of the glycolipid syntheses using the same lipases and solvents, matching results were obtained.
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