3D printing inside cells: new method inserts lasers and an elephant directly into cytosol

A novel method has enabled scientists to 3D-print structures into live cells, paving the way for a new class of intracellular bioengineering tools and applications.

In a first, scientists from the Jožef Stefan Institute (Ljubljana, Slovenia) have developed a universal 3D printing method for delivering larger objects directly into living cells. Putting their technique into practice, the team printed numerous intracellular structures, including barcodes, lasers and, bizarrely, an elephant.

3D printing is now an invaluable weapon in many a scientist’s arsenal – its uses are widespread, ranging from electronics and robotics to biology and biomedicine. The best resolution is achieved using a technique called two-photon polymerization (TPP), which involves a photo-sensitive resin (a photoresist) illuminated by a femtosecond laser to enable printing features down to 100 nm in size.

This type of 3D printing has previously been demonstrated in living organisms and, in one instance, inside a synthetic cell – but never in living cells. Instead, micro-scale objects are embedded via endocytosis, phagocytosis, microinjection and membrane poration, while larger objects can only be internalized using phagocytosis, and even then, they are not deposited directly inside the cytosol. As a result, there is no method to deliver micron-scale objects into non-phagocytic cells, where they could be useful as probes, sensors or in barcoding.

The researchers, therefore, developed a 3D printing technique to overcome this, allowing them to insert custom micrometer-sized structures directly into the interior of living cells.

Their breakthrough used TPP, and involved injecting a droplet of a negative-tone photoresist into a live HeLa cell. The photoresist was then selectively polymerized in a designed pattern by a femtosecond laser – only material in the laser focal spot was polymerized and as the laser moved layer by layer along the pattern’s path, it formed an intracellular object with submicron resolution. The unpolymerized photoresist then slowly dissolved, leaving behind complex microstructures in various shapes.

These included barcodes for cell tracking, diffraction gratings for remote readout, microlasers and even a 10 μm elephant.

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The team also investigated the effects of 3D printing on the cells: time-lapse imaging revealed they had normal morphology and continued to divide, with the structure passed on to one of the daughter cells. Meanwhile, confocal and fluorescence imaging showed that the interior of the cells, in particular the nucleus, deformed to accommodate the printed objects.

“Our method provides a new tool to manipulate living cells from the inside,” co-author Maruša Mur commented, “enabling a new approach to studying their mechanical and biological responses.”

For now, the research is preliminary, however, it could set the stage for novel intracellular bioengineering tools to come to the fore, with a whole host of potential applications, such as intracellular sensing, biomechanical manipulation, bioelectronics and targeted drug delivery.