3D printing plant cells shows promise for studying cell function

A new study from North Carolina State University shows a reproducible way to study cellular communication between different types of plant cells by “bioprinting” these cells via a 3D printer. Learning more about how plant cells communicate with each other — and with their environment — is key to understanding plant cell functions and could ultimately lead to better crop varieties and optimal growing environments.

The researchers bioprinted cells from the model plant Arabidopsis thaliana and from soybeans to study not only whether plant cells would live after they were bioprinted — and for how long — but also to investigate how they acquire and change their identity and function.

“A plant root has many different cell types with specialized functions,” said Lisa Van den Broeck, an NC State postdoctoral researcher who is the lead author of a paper describing the work. “Different sets of genes are also expressed, some are cell-specific. We wanted to know what happens after you bioprint living cells and put them in an environment that you design: do they live and do what they’re supposed to do?”

The process of 3D bioprinting plant cells is mechanically similar to printing ink or plastic, with a few necessary adjustments.

“Instead of 3D printing ink or plastic, we use ‘bioink’ or living plant cells,” says Van den Broeck. “The mechanics are the same in both processes, with a few notable differences for plant cells: an ultraviolet filter used to keep the environment sterile and multiple printheads — rather than just one — to print different bio-inks simultaneously.”

Living plant cells without cell walls, or protoplasts, were bioprinted along with nutrients, growth hormones and a thickening agent called agarose – a seaweed-based compound. Agarose helps provide cells with strength and scaffolding, similar to mortar that supports bricks in a building’s wall.

“We found that using the right scaffolding is critical,” said Ross Sozzani, a professor of plant and microbial biology at NC State and a co-corresponding author of the paper. “When you print bioink, it should be liquid, but when it comes out, it should be solid. By mimicking the natural environment, cellular signals and signals continue to occur as they do in the soil.”

The study showed that more than half of the 3D bioprinted cells were viable and divided over time to form microcalli or small colonies of cells.

“We expected good viability on the day the cells were bioprinted, but we never maintained the cells for more than a few hours after bioprinting, so we had no idea what would happen days later,” said Van den Broeck. “Similar viability ranges are shown after manually pipetting cells, so the 3D printing process doesn’t seem to do anything harmful to cells.”

“This is a manually difficult process and 3D bioprinting controls the pressure of the droplets and the speed at which the droplets are printed,” Sozzani said. “Bioprinting offers better possibilities for high throughput processing and control over the architecture of the cells after bioprinting, such as layers or honeycomb shapes.”

The researchers also bioprinted individual cells to test whether they can regenerate, or divide and multiply. The findings showed that the root and shoot cells of Arabidopsis required different combinations of nutrients and scaffolds for optimal viability.

Meanwhile, more than 40% of individual soybean embryonic cells remained viable two weeks after bioprinting and also distributed over time to form microcalli.

“This shows that 3D bioprinting can be useful to study cellular regeneration in crops,” Sozzani said.

Finally, the researchers studied the cellular identity of the bioprinted cells. Arabidopsis root cells and embryonic soybean cells are known for their high proliferation rates and lack of fixed identities. In other words, like animal or human stem cells, these cells can become different cell types.

“We found that bioprinted cells can take on the identity of stem cells; they divide and grow and express specific genes,” said Van den Broeck. “When you bioprint, you print a whole population of cell types. We were able to examine the genes expressed by individual cells after 3D bioprinting to understand any changes in cell identity.”

The researchers plan to continue their investigation of cellular communication beyond 3D bioprinting, including at the single-cell level.

“All in all, this study demonstrates the powerful potential of using 3D bioprinting to identify the optimal compounds needed to support plant cell viability and communication in a controlled environment,” Sozzani said.

The research appears in Scientific progress. Tim Horn, assistant professor of mechanical and aerospace engineering at NC State, is a co-corresponding author of the article.