His delicate filament scaffolds allow cells to grow perfectly

• Biology
• Research

In brief

• ETH Zurich doctoral student Hao Liu uses lasers to create microfilament structures from a special type of gelatine. In the lab, the structures serve as a scaffold for growing cells.
• The method opens up new possibilities for the production of lifelike tissue models for biomedical research, and could reduce the number of animal experiments in drug development.
• Patented by ETH Zurich, the technology is also of interest for surgery, transplantation medicine and the production of cultivated meat.

It was in Japan that Hao Liu first came into contact with the production of biological tissue in the cell culture dish. "During my Master's studies at Osaka University, I worked on a project in which we used a 3D printer to cultivate meat from Wagyu cattle," Liu says. Wagyu beef is considered to be some of the tenderest, juiciest and most expensive meat in the world. The researchers therefore tried to recreate it in the lab. "Back then, I learnt that you can develop something relevant and make a difference by growing tissue."

Liu began his studies in China. He moved to Osaka for his Master's degree and has been a doctoral student at ETH Zurich since 2020. He has just completed his doctorate. He has already secured an ETH Pioneer Fellowship, which he intends to use to develop a new device that manufactures tissue with microfilament structures and ready it for the market.

Tissue consists of microstructures

Such microstructures are found throughout our body. The cells in our muscles, tendons, connective tissue and nervous system are not arranged randomly, but follow clear patterns. These give the tissue both stability and flexibility and help it to carry out its various functions. For example, the cells and fibres of muscle tissue are highly aligned so that the muscles can contract. In tendons, which connect muscle to bone, the cells must be organised in such a way that the tendons can withstand enormous tensile forces. And nerve tissue also needs to be aligned so that signals can be transmitted between the cells.

When researchers fabricate such tissues in the laboratory, they have to replicate such alignments. In many cases, they achieve this by first producing an artificial but biocompatible 3D scaffold with aligned microstructures. The researchers then grow cells on and in this scaffold to form perfectly structured tissue. In the future, this can be used as a substitute material in surgery - for example in peripheral nerve regeneration after serious injuries. In addition, such tissue constructs can be used to research diseases and test drugs as in vitro tissue models, thereby reducing animal testing. Or they could be used to produce cultivated meat in the laboratory, as Liu did in Japan.

A fortunate decision to keep workpieces

At ETH, it was Liu's hard work, plus a little bit of luck, that led to his discovery of a new method for producing a tissue scaffold with highly aligned and extremely fine filaments. He built upon a well-known process and used a chemically modified gelatine that reacts to light. The gelatine starts off as a liquid. "Where we expose it with a laser, it solidifies into hydrogel. Wherever the laser can't reach, the gelatine remains liquid," Liu explains. Targeted application of the laser can produce customised three-dimensional hydrogel structures.

Liu went on to test this printing process. He almost threw some of the hydrogel workpieces away, but instead put them aside. When he took them out again later, he first noticed something with the naked eye, and then confirmed it under the microscope: rather than being uniform, the hydrogel structures consisted of extremely fine filaments. "Marcy Zenobi-Wong, the professor who supervised my doctoral thesis, and I were delighted," Liu recalls. He had created microfilaments in the hydrogel with diameter similar to fiber components found in manybody tissues. He then grew cells in this hydrogel scaffold in order to produce aligned tissue constructs. "If I had thrown the workpieces away back then, I wouldn't be where I am today."

The new process can be used to produce microstructures (left) that are similar to those of muscle fibres (right). (Visualisations: Liu H et al. Advanced Materials, 2022)

Liu began to study the literature on physics and realised that a well-known optical phenomenon was creating the microfilaments in his hydrogel scaffolds. The light in a laser beam is not equally intense throughout. Analysing the cross-section of a laser beam with microscopic resolution reveals that the light intensity resembles a spot pattern: in some places it's extremely high, in others quite low. If the light-sensitive material is solidified with a laser beam, it doesn't harden evenly, but instead a construct of parallel thread-like gel structures emerges. Between these gel filaments exist channel-like spaces. Both the filaments and the channels have a diameter of around 2 to 20 micrometres. If cells are encapsulated in this hydrogel scaffolds, they can grow in the channels. The result is an aligned tissue construct that is very similar to the natural structure of many body tissues.

The microfilaments are created because laser light is inhomogeneous. (Visualisations: Liu H et al. Advanced Materials 2022, CC-BY 4.0, modified)

"The optical phenomenon that creates the filament microstructures in the gel has long been known to physicists and material scientists," Liu says. "But it hadn't yet been used in biology; we're the first."

The filamented light 3D bioprinter can be used to produce aligned tissue constructs. (Visualisations: Zurich University of the Arts / Samuel Thalmann)

Together with industrial design students from the Zurich University of the Arts, Liu's team finished a design for a prototype printer to produce such filamented hydrogel scaffolds for aligned tissues. With the help of the Pioneer Fellowship, Liu now wants to bring a compact bioprinter to market.

Drug development and nerve regeneration

"As a first step, we want to make the technology and the printer available to other scientists so that they, too, can produce such aligned tissues and use them in their research," Liu says. "Several labs have already expressed interest." At the same time, he wants to develop various tissue models, such as muscle tissue or tendons. "Our aim is to create human tissue models for high-throughput drug screening and other applications." As a result, he sees future business potential not only in selling the device but also in the development and sale of tissue for research and medicine.

With the technology, Liu's lab has already succeeded in producing muscle, tendon, nerve and cartilage tissue constructs. The technology has been patented by ETH Zurich. "Our technology is suitable for a wide range of applications," Liu says. "It's even conceivable that this could be used in the future to produce nerve conduits that can be transplanted into patients suffering from nerve injuries." Or to produce lab-grown meat, as he learnt in Japan.

Hydrogel scaffolds and cells to produce connective tissue, nerve tissue and muscle tissue (from left to right, microscopy images). (Photograph: ETH Zurich)

The widely travelled scientist definitely wants to stay in Switzerland for the next few years to see the growth of the technology. And after that? Is he looking to move on to somewhere else? It's certainly a possibility. Perhaps he'll go to the US. "In each country, I've learnt about different research focuses and different research cultures. Going to a new environment motivates me a lot. I think it helps you to question what you've done so far and to develop as a person," he explains.

Japan is known for its stem cell research, Liu says. There he saw how the government commissions research projects and how research groups then realise those projects according to strict specifications. In Switzerland, he experienced exactly the opposite: a great deal of academic freedom, which his supervisor Zenobi-Wong also gave him during his doctoral thesis. This allowed him to easily adjust the focus of his work after his discovery. He also appreciates the European scientific culture and ETH in particular for the engineering approach that it emphasises. From his point of view, these are excellent conditions for developing a technology together with his team and partners and bringing it to market, as he's now doing now.

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