Looking for extracellular electron transfers with human cells – TECH

? The cells will be cultured on electrodes with controlled potential, as for microbial biofilms. We will use MRC5 lung tissue cells that have given promising preliminary results, and then progenitor cells from adipose tissue for their ability to differentiate into adipocytes, chondrocytes or osteoblasts. Electrochemical tests will be coupled with biochemical and structural characterization of cells. The objective is to design electrochemical reactors and electrode surfaces dedicated to cell culture to determine any electrical signal related to a metabolic process or, conversely, any biochemical change due to EET.

The first tests used MRC5 cells. The presence of suspended cells in a buffered medium catalysed the electrochemical reduction of oxygen. This observation, made in the few preliminary experiments that made it possible to defend the project, is now reproduced and confirmed. This catalytic process was then observed with other cell lines. Although this catalysis does not necessarily imply EETs, it is to our knowledge an original result.
Tests on various types of electrode materials and surfaces (carbon fabric, carbon felt, gold, surface modified with carbon nanotubes, etc.) led to the choice of the most suitable surface.
The experimental system was designed to allow the implementation of electrochemical techniques during the growth of cells on the surface of the electrodes. To our knowledge, this type of experimentation has never been described in the litterature yet. The implementation of the experimental system required solving a number of practical problems related to the introduction of a three-electrode electroanalytical system in a sterile incubator and the maintenance of the experiment over several decades in a hot, humid and controlled atmosphere. The experimental system is now under control and allows reproducible experiments. A microscopy protocol has been set up for the characterization of cellular carpets.
These experimental protocols have shown that the potential applied to a carbon surface is a key parameter in cell growth. This is one of the major results that was hoped for in the initial project. In addition, it appears that cell growth is correlated with the appearance of an oxidation current, but this observation remains to be confirmed and work is progressing in this direction.

? As planned in the project, the electrochemical cell culture protocol that is now well established will be applied to the differentiation of human cells from adipose tissue.
We have also implemented an electrochemical protocol that allows the continuous monitoring of EET mediated by a redox mediator in solution. This protocol applied to different cell lines confirms the occurrence of EET with very different intensities depending on the mediator ability to penetrate or not into the cells. The tests have so far only been carried out in buffered solutions that ensured the survival of the cells but did not allow them to proliferate. In this respect, the objective is now to transpose the protocol using a culture medium in order to correlate cell growth with the electrochemical monitoring of EETs via redox mediators.

The results must be strengthened befor to be dissemated

Many bacterial cells are able to connect their metabolism with an electrode. Since the publication of the pioneering articles of 2002, the variety of bacterial species that have proved to be capable of extracellular electron transfer (EET) has been constantly expanding, and it now seems to be a current and ubiquitous lifestyle for microbial cells. EETs are most often correlated with essential metabolic pathways, particularly the respiratory metabolism of bacteria. The electrochemical approach of EETs is thus revolutionizing the understanding of microbial processes in most ecosystems.

Numerous elements of the biomedical literature show that similar EETs, i.e. involving metabolic pathways, exist with human cells. For example, stem cells are commonly grown on conductive materials whose electrochemical properties drive differentiation into the different phenotypes. The exchange of extracellular electrons, in the form of "bioelectricity", is known to play essential roles in tissue repair or organ morphogenesis. In addition, human cells are eukaryotic cells and EETs associated with metabolic pathways have already been evidenced with yeasts, which are also eukaryotic cells. Despite the accumulation of indirect evidence of the occurrence of EETs with human cells, electrochemical approaches have never been implemented in this context before.

Using the experience gained on microbial EETs by the consortium, the TECH project aims to build the electrochemical devices that will bring to light metabolic EETs with human cells. Three types of cells will be implemented: i) MRC5 lung cells, which have already given promising preliminary results, ii) progenitor cells from human adipose tissue, whose differentiation into white or beige adipocytes is related to the status of the respiratory metabolism and, iii) different cancer cell lineages, as the cancerous state of the cells could be correlated with dysfunction of the respiratory metabolism.

The objective is to create the electrochemical reactors and electrode surfaces and to design the operating and analytical procedures that will allow EETs to be characterized during the development and/or differentiation of cells. The project will build synergy between two research groups belonging to the same laboratory, one mastering the EETs of microbial cells and the other the culture of human cells. In addition, two experts in the cellular metabolism of stem cells and cancer cells are associated as consultants. An interdisciplinary group will thus be created to combine skills in bioelectrochemistry, electroanalysis, cell adhesion, culture and cellular metabolism, and to couple electrochemical analysis methods with biochemical, biological and imaging techniques by microscopy.

The project is submitted to the “Défi des autres savoirs” because the objective is to bring experimental evidence of a new paradigm. The project is applying for a modest budget because the objective is not to push investigations towards fundamental biology. This is a seminal project that intends to prove the concept of EET with human cells in order to convince the biomedical communities and to build further projects with them, having larger budgets on specific applied objectives. Demonstrating the occurrence of EETs with human cells will pave the way to new stem cell culture techniques, new tools for investigating cellular metabolisms and the pathologies, such as obesity and cancer, which are associated with their dysfunction and, we hope, will bring to light new therapeutic pathways.