Multi-photon lithography: printing cells with micrometer accuracy

How do cells react to certain drugs? And how exactly is new tissue created? This can be analyzed by using bioprinting to embed cells in fine frameworks. However, current methods are often imprecise or too slow to process cells before they are damaged. At the TU Vienna, a high-resolution bioprinting process has now been developed using a new bio-ink.

In an interview with MEDICA-tradefair.com, Prof. Aleksandr Ovsianikov talks about what makes the new process different from previous ones, why the choice of material for bio-inks is so difficult and to what extent bioprinting is already used for printing organs.

Prof. Ovsianikov, how does your new bioprinting technology work?

Prof. Aleksandr Ovsianikov: The technology is based on multi-photon lithography (MPL) and so we use photosensitive materials, which can for example be cross-linked when exposed to light. In most cases the result is that a liquid photopolymer solidifies. In contrast to conventional lithography we employ ultra-short pulsed lasers as a light source. They allow to confine this solidification to a tiny volume at the focus of the beam. When this beam is moved through the material is leaves a thin trace of solid material behind, so 3D structures can be "written" directly in the volume of the material. In the last step, called development and appropriate solvent is used to wash out the material that was not exposed to light and stayed liquid. In case of bioprinting the materials are water-based and are washed with aqueous solution at the end.

What advantages does this have compared to other methods?

Ovsianikov: The majority of bioprinting processes really deposit cells and so the features produced by these methods cannot be smaller than a single cell. For example, extrusion bioprinting is often just some kind of automated syringe, which deposits the strands of cell-containing material in a certain pattern. These strands are usually at least 100 µm wide. MPL can produce features which are a nearly thousand times smaller than that. We do not deposit a material, but produce structures in the volume of a photosensitive material already containing cells instead. This has three important advantages: First, the resolution can even be smaller than that of a single mammalian cell. Secondly, highly complex 3D structures can be produced. And third, by controlling the light intensity, the properties of the resulting material can also be controlled "on the fly".

What materials do you use for printing? What is the challenging part?

Ovsianikov: For a process using live cells one needs materials with high water content, usually such so-called bio-inks are over 80 percent cell culture medium. One has to also make sure that these materials are not toxic. Finally, the combination of the materials and the process has to stay cell friendly. Unfortunately, in case of MPL there is not really a catalogue of materials or components which one can select from, in accordance to such a "wish list". Therefore, we went forward and developed such materials, for example specialized gelatin-based bio-inks.

In case of MPL, since the resolution is so high, it is also important that the process is fast. Otherwise it might take very long to produce structures of relevant size. Processing speed relies on the material sensitivity. We have developed specialized bio-inks that can be processed with a laser scanning speed of up to one meter per second in the presence of cells. This is an absolute record.

Where is MPL used?

Ovsianikov: MPL itself is quite universal and is used wherever precise 3D structures are needed: in micro-optics and microfluidics, microneedles for transdermal drug delivery and biodegradable scaffolds for tissue engineering.

One of the important areas of application of bioprinting is realization of 3D tissue models. Among other things, these are highly relevant for drug development in the form of organs-on-a-chip. The idea is to create a mini version of different human organs in a microfluidic chip format, in order to for example test safety of different drugs in a high throughput manner. We have already used the MPL to produce a barrier membrane for a placenta-on-a-chip. A recent work demonstrated a possibility to produce branching vascular structures with diameters in the sub-50 micrometer range.

What further development possibilities do you see in the technology?

Ovsianikov: We have just really introduced MPL in the domain of Bioprinting very recently. In addition to further technical improvements I expect that the material portfolio will be massively expanded in the upcoming years. Specialized bio-inks will be developed for different cell and tissue types.

My group began commercializing this technology a couple of years ago in order to make it more accessible to other users. With the NanoOne, our start-up UpNano GmbH has developed the fastest device in the world that is also capable of embedding cells. In November we won two awards in the Austrian "GEWINN JUNGUNNTERNEHMER 2020" competition. We are very proud of and happy to see high interest from all around the world.

One hope is that at some point in the future it will be possible to print entire organs using bio-printing. But that is still a long way off.