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Two‐photon patterning guides cells in the right direction

13 May 2020

ETH Zurich uses light to create precisely positioned patterns of molecules.

Research into the development of cells and the biochemical signals that pass between them involves constructing artificial structures within which those cells can be precisely located and studied.

A project at Swiss research institute ETH Zurich has now developed a new method for constructing these experimental structures, using a laser to create patterns which then guide cell development. The work was published in Advanced Materials.

In particular, the technique makes it possible to investigate the action of morphogens, chemicals which act as signals to cells and control their subsequent position and development. Studying this process requires a way to distribute the signal molecules within a hydrogel with a high degree of precision, which the ETH Zurich technique has achieved.

The breakthrough involves two-photon patterning (2PP), in which the polymerization of a hydrogel can be carefully controlled. The non-linear two-photon process ensures that absorption only takes place at high photon densities, and is limited to the focal volume of an ultrafast pulsed laser. EZH Zurich used the technique to attach a protein termed nerve growth factor (NGF) onto the substrate.

"Wherever the light is focused in the material, it triggers a chemical reaction that anchors NGF to the hydrogel," commented Nicolas Broguiere of ETH Zurich. "We carefully optimized the design of the photosensitive hydrogel so that the signal molecules attach only in the areas exposed to the laser, and nowhere else."

The KTH technique "offers unprecedented orthogonality and signal‐to‐noise ratio in both inert hydrogels and complex mammalian matrices," according to the project's published paper. "Importantly, the method enables 2PP in a single step in the presence of fragile biomolecules and cells, and is compatible with time‐controlled growth factor presentation."

Showing neurons the right path

This approach can create what KTH terms "paintings of morphogens with details one thousand times smaller than a millimeter," approximately the size of a single nerve fibre.

In trials, a 2PP system was applied to a number of different hydrogel formulations, and used to encourage peptide synthesis and assist the monitoring of protein function, as well as guide the growth of axons through patterns of NGF proteins. The researchers could then observe microscopically how the neurons followed the mapped-out pattern, effectively steering the neurons in 3D using their own biochemical language.

Research at the ETH Zurich Tissue Engineering and Biofabrication group under Marcy Zenobi-Wong has previously investigated how the principles of two-photon absorption can allow the fabrication and manipulation of photosensitive hydrogels in 3D with feature sizes down to the nanoscale. This technology shows promise for opening new possibilities in several research fields, including tissue engineering and stem cell production.

The new breakthrough now develops this approach as a way to interrogate the role of complex signaling molecules in 3D matrices, helping to better understand biological guidance in tissue development and regeneration. This could ultimately assist in the control of nerve regrowth after an accident, when reconnection can happen haphazardly and without the restoration of full function.

"I don't want to give the impression that we're ready to start treating patients with this method," said Zenobi-Wong. "But in the future, a refined version of our approach could help show neurons the right path directly in the body, thereby improving recovery from neural injuries."

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