# Using a Supramolecular Approach to Engineer Modular Hydrogel Platforms for Culturing Protoplasts – from General Tissue Engineering to Cellular Agriculture

## TL;DR

This paper explores using synthetic hydrogels to culture plant protoplasts in different formats, showing how these materials can influence cell growth and shape.

## Contribution

A supramolecular approach is introduced to create modular hydrogel platforms for protoplast culture, enabling control over cell behavior in 2D, 2.5D, and 3D systems.

## Key findings

- Bioactive hydrogels in 2D cultures caused protoplast enlargement after 11 days.
- 3D cultures with bioactive additives led to plasmolysis in protoplasts.
- 2.5D cultures with lower additive concentrations also stimulated protoplast enlargement.

## Abstract

Protoplast regeneration into plant cells and further into plants is an ongoing challenge in agricultural biotechnology. Inspired by mammalian tissue engineering, a strategic shift is proposed in plant tissue engineering to steer protoplast culture using fully synthetic materials‐based culture platforms. Here a supramolecular materials method to engineer modular culture methods for protoplasts is chosen to use. Supramolecular monomers as modular building blocks allow to make various hydrogel formulations and to study different protoplast cultures; including 2D cultures on top of supramolecular hydrogels, 2.5D cultures using supramolecular fibers in solution, and 3D cultures when encapsulated in bulk hydrogels or microgels. Importantly, the need is shown for bioactive functionalization of the supramolecular hydrogels with a peptide additive in 2D protoplast cultures. After 11 days, the bioactive hydrogel induced protoplast enlargement, which is absent on pristine hydrogels. The opposite effect is present for protoplasts cultured in 3D, showing plasmolysis as a result of the bioactive additive. Interestingly, in 2.5D lower bioactive additive concentrations in supramolecular fibers stimulated protoplast enlargement, demonstrated by similar morphological changes as in 2D. Finally, protoplast encapsulation in supramolecular microgels is showcased. This work demonstrates the potential to modularly engineer various synthetic platforms to facilitate cellular agriculture.

Using supramolecular monomers, various hydrogel culture systems were formulated to culture protoplasts; including 2D, 2.5D, and 3D hydrogels. Depending on the culture platform, bioactive functionalization led to protoplast enlargement (2D and 2.5D) or plasmolysis (3D). This work shows the potential to modularly engineer synthetic platforms for cellular agriculture.