Self-organized biomolecular gradients for controlling cellular behaviour in cell culture

Project Information

SOCELL

Grant agreement ID: 795580

DOI

10.3030/795580

Project closed

EC signature date 26 April 2018

Start date 1 August 2018

End date 31 July 2020

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 173 076,00

EU contribution

€ 173 076,00

Coordinated by

SORBONNE UNIVERSITE
France

Periodic Reporting for period 1 - SOCELL (Self-organized biomolecular gradients for controlling cellular behaviour in cell culture)

Reporting period: 2018-08-01 to 2020-07-31

Summary of the context and overall objectives of the project

Programmed into their genetic code, cells are capable of knowing when and into what cell type they must differentiate into. While some factors are purely genetically programmed (senescence expression of genes), others are triggered by extracellular cues, such as by small biomolecules or forces. In particular, specific combination and concentration of biomolecules have shown to be very important during morphogenesis. As an example, the embryo of the fruit fly is capable to differentiate the anterior from the posterior region due to the gradient of a specific molecule released by the mother. Other more complex gradients of biomolecules have been described as the reason for other patterns observed in nature. For this reason, the scientific community has made efforts during the last century in developing methodologies to recreate biomolecular gradients. The achievement of these biomolecular gradients would not only help to better understand complex steps during embryogenesis, but would aid in developing better bioengineering tools and smart bandages.
In this project we have decided to approach the development of biomolecular gradients from a new perspective. While conventional methods rely on a passive mechanism (gradients disappear with time if not actively re-formed), we decided to develop active mediums, where there are capable of forming and keeping their own gradients (self-organized). Due to its programmability and robustness, we have decided in using DNA Nanotechnology as the machinery capable of forming and keeping the gradients. In order to do this, we have divided the project into 3 main objectives:
1. Functional DNA programs in the presence of living cells (due to their biocompability).
2. The DNA machinery has to alter the cellular behaviour.
3. The DNA machinery must have spatiotemporal control over the cellular fate.
We had foreseen this project to be laborious from the beginning, but we were capable of developing an active extracellular medium which had spatiotemporal control over living cells with the use of DNA programs. Being the first example of an out of equilibrium DNA program capable of working in the presence of living cells, we foresee it will be largely welcomed by the scientific community, due to its large possibilities and applications.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The first aim of the project was the achievement of a functional DNA machinery in the presence of in vitro cell cultures. To do this, we relied on enzyme-based DNA program. A compromise between the standard cell culture medium and the DNA machinery medium was developed to full-fill the desires of both without being deleterious for neither of them. With this new “cell-DNA buffer”, we could achieve the viability of both the cells and the DNA machinery for at least 48h. We observed that cells would internalize DNA with relatively easiness. We decided to mark the DNA with a fluorescent molecule, which made cells fluorescent upon its internalization (image 1 attached). With this approach, although simple, it satisfied the initial objective of having an interaction between the extracellular DNA machinery and the living cells. Once we had the interacting extracellular active medium, we were able to change the program of the DNA machinery so that it would respond in a temporal and spatial manner. For the spatial experiments, we had to come up with a rectangular device which allowed us to create a two-band pattern, such as a polish flag. In one side of the rectangular device, the fluorescence DNA was released and internalized by the cells, while on the other side there were no release, and hence no modification of the cells. We thus demonstrated that a synthetic extracellular program can transfer positional information to living cells, emulating an archetypal mechanism of pattern formation in embryo development. To our knowledge, this has not been accomplished without the use of microfluidics (and hence the introduction of shear stress). With these pioneer results, we foresee that active extracellular media could be advantageously applied to in vitro biomolecular tracking, tissue engineering or smart bandages.
These findings have been submitted for publication at a high impact factor journal. The first version of manuscript can be found in the open access repository biorxiv. Together with a member of the host group a side project reported on another golden access publication. To further exploit this work, the researcher has attended 3 congress, one of which has been invited by the organizers to give an oral contribution, to disseminate its work within the scientific community.
To help disseminate science, the researcher has been an organizer of the Paris Biological Physics Community Day 2019, where the idea is to promote divulgation by young scientist. At a more general public dissemination, the researcher achieved to encourage and establish the active participation of the host department into the “fête de la science”, a scientific demonstration for the general public with the idea of transmitting and encouraging the French young community into the scientific world. Furthermore, during the duration of the grant, several students from different schools and grades have attended the lab (and the host department) for traineeships of different durations, with the objective of transmitting them how the scientific community works and motivate them. In addition, the researcher was trusted with the supervision of a bachelor and a Master student. As a non-standard out-reach activity, two divulging videos explaining the basic mechanism of the DNA programs used in this project have been developed (YouTube).

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

With the achievement of this project we have shown the possibility of having active extracellular mediums guiding cellular fate with spatiotemporal control. To our knowledge, these results have surpassed the state of the art in three different aspects:
•While some relatively basic DNA programs have been shown to work in the presence of living cells, these results show that complex enzyme-based DNA programs can work in the presence of living cells.
•Our results show the first demonstration of programmable active medium in the presence of living human cells in vitro.
•The results of this project has shown the possibility of creating a stable pattern without the need of flow, which could be a promising alternative to the use of microfluidics.
With the development of active extracellular mediums, we foresee the expansion towards in vitro biomolecular tracking, tissue engineering or smart bandages. Although we don’t expect a fast implementation into the private sector, we do think that in 5-10 years this developed active extracellular mediums will be used to further control cellular behaviour.

The project has had a significant impact on the future of the researcher. With the successful application, it has encouraged him to apply for senior researcher position at the French National Centre for Scientific Research (CNRS), to other tenure track positions and to ERC starting grants. Furthermore, since the project has opened a new branch in the field of DNA nanotechnology and synthetic biology, it will not be difficult for the researcher to stablish his research domain, since other established researchers around the world have already shown their interest in starting a collaboration with the researcher.

fluorescent cells upon DNA internalization