A modular microfluidic bioreactor to investigate plant cell–cell interactions

Abstract

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.

Cell type matters: competence for alkaloid metabolism differs in two seed-derived cell strains of Catharanthus roseus

“…The interaction between different cell types might extend beyond mere passing on of metabolites for downstream processing; there might be regulatory interactions as well that steer the metabolic competence. We have recently addressed this possibility using a modular chip system, where different cell types can interact by a microfluidic flow without the need of physical contact (Finkbeiner et al 2021 ). When we placed C1 cells upstream of a chip with C4 cells, we were able to obtain the desired vindoline while simultaneously tabersonine was depleted.…”

Cell Type Matters: Competence For Alkaloid Metabolism Differs In Two Seed-Derived Cell Strains of Catharanthus Roseus.

“…The interaction between different cell types might extend beyond mere passing on of metabolites for downstream processing, there might be regulatory interactions as well that steer the metabolic competence. We have recently addressed this possibility using a modular chip system, where different cell types can interact by a micro uidic ow without the need of physical contact (Finkbeiner et al 2021). When we placed C1 cells upstream of a chip with C4 cells, we were able to obtain the desired vindoline while simultaneously tabersonine was depleted.…”

Genetic Manipulation and Bioreactor Culture of Plants as a Tool for Industry and Its Applications

“…Such bioreactors can be very structurally diverse. Laboratory conditions most often require the use of reactors with a smaller volume and simpler structure, these are often tightly closed vessels with a stirrer, sprinkler, or aeration nozzle, while the industrial ones are complex devices in which most parameters are controlled digitally [ 79 , 80 ]; they are most often equipped with a microprocessor control unit to monitor the pH, temperature, and density of the cultured cells. The selection of the bioreactor and the process itself depend on the method of production, i.e., secretion into the culture medium or accumulation in biomass.…”

“…In addition, intensive technical progress, consisting of increasing the computational capacity of microprocessors supervising the work of fully automated breeding systems, as well as the progressive miniaturization and reduction of power consumption thanks to the use of nanotechnology, will allow, in the near future, for the further improvement of the processes and increase their profitability and environmental friendliness. The new technical solutions will probably be based on natural cell–cell interactions in order to maximize the imitation of those occurring in living tissues, which is ensured, for example, by a microfluidic bioreactor [ 79 ]. Moreover, the constantly increasing number of products, such as dietary supplements or cosmetics containing components derived from plant in vitro cultures, will, in the future, further limit the exploitation of natural resources, allowing the preservation of biodiversity while increasing the supply of the desired phytochemicals.…”