The recent developments in tissue engineering, disease modelling or drug screening would not have been possible without 3D cell culture techniques. Using collagen gels, biologists have succeeded culturing various human cells in 3D environments to grow tissue models. But once cells cultured in 3D, it is challenging to precisely control cell functions due to the lack of suitable micro-engineering tools. In the past, multiphoton lithography (MPL) opened up new possibilities to mimic the native extracellular matrices (ECM) at micrometre-scale for precise control of cell-matrix interactions. Improving MPL and development of new hydrogel systems to account for fast writing speed as well as to guarantee stable structures with good cell-compatibility performance is therefore currently an agile research topic.

Collagen, which is often used as a 3D hydrogel, suffers from high cost, poorly defined composition and limited flexibility in design. Existing synthetic hydrogels for MPL are mostly based on acrylates, which are often severely cytotoxic and therefore incompatible with 3D cell culture applications.

Thus, despite the great research efforts, the current possibilities are limited and typically suffer from trade-offs, such as processing speed, cell adhesion capabilities, cell viability, and robustness of the hydrogels. This becomes even more complicated when attempting spatial and temporal control of cell function within a 3D microenvironment.

The challenges in bio-applications of MPL are therefore clear: the design of a fully synthetic, low cost and cell-compatible hydrogel, which is permissive for 3D cell growth and at the same time highly efficient in MPL is hard to achieve.

Xiao-Hua Qin and co-workers addressed this topic in a work published recently in Advanced Materials. [1] In their study, they used a novel cell-responsive polyvinyl alcohol (PVA) resin for rapid synthesis of 3D cellular environments as well as fast fabrication of reproducible microstructures, which can be digested by cell-secreted proteases. PVA was rendered photo-crosslinkable through a facile one-pot modification with norbornene anhydride. When combined with a dithiol crosslinker, PVA hydrogels form rapidly via thiol-ene photopolymerization. Using different crosslinking densities allows for tuning the mechanical properties of these gels to mimic different tissue environments. To promote cell-matrix interactions, protease-degradable and cell-adhesive peptides were incorporated into the network.

But how do the authors achieve microscale control of cell growth in their hydrogel?

Qin and colleagues demonstrated spatiotemporal control of cell invasion in a permissive hydrogel by multiphoton-triggered photo-grafting of adhesive cues in the hydrogel network which enable directed cell outgrowth.

Cell invasion requires not only degradable crosslinks, which allow the matrix to be remodelled by cells when needed, but also insoluble peptide ligands which control the cell adhesion.  The authors first photoencapsulated cell aggregates in a non-adhesive PVA hydrogel, using the protease-degradable dithiol peptides as crosslinker. Importantly, the degradable dithiol crosslinker was used in a sub-stoichiometric amount, thus part of the norbornene functionalities remain accessible after hydrogel formation. In a second step, they soaked the hydrogel in a solution containing the firbonectin-derived peptide (CGRGDS), which controls cell adhesion. Using MPL, the authors could then graft the adhesive cues within the PVA network on selected sites, pathing the way to direct cell growth in arbitrary 3D shapes.

Besides facile spatiotemporal control, thiol-ene photopolymerization also ensures fast gel formation of ca. 10 seconds by cytocompatible UV light, which becomes important for in-situ cell encapsulation applications. Additionally, the fast kinetics of the femtosecond laser induced thiol-ene reaction allows for high writing speeds of 50 mm/s which was only limited by the scanning speed of the MPL device at hand.

It is clear that such a cell-responsive, laser-tuneable hydrogel matrix which enables directed cell growth in 3D is a powerful platform for a variety of basic and translational research such as disease modelling or drug screening.

[1] Qin, X.-H., Wang, X., Rottmar, M., Nelson, B. J. & Maniura-Weber, K. Near-Infrared Light-Sensitive Polyvinyl Alcohol hydrogel Photoresist for Spatiotemporal Control of Cell-Instructive 3D Microenvironments.  Adv. Mater. 1705564 (2018)