“Bio-printing” plant cells with a 3D printer to study cell function

Using a 3D printer to “bio-print” plant cells offers a reproducible way to study cell communication.

New research reveals a reproducible way to study cellular communication between various types of plant cells by “bio-printing” these cells via a 3D printer. Knowing more about how plant cells communicate with each other – and with their environment – ​​is essential to better understand plant cell functions. This could ultimately lead to the production of optimal growing environments and better crop varieties.

Published today (October 14) in the review Scientists progressthe study is North Carolina State University.

Scientists have bioprinted cells from the model plant Arabidopsis thaliana and soy. They wanted to study whether plant cells would live after being bioprinted – and for how long. Additionally, they also wanted to examine how they acquire and change their identity and function.

“A plant root has a lot of different cell types with specialized functions,” said Lisa Van den Broeck, an NC State postdoctoral researcher who is the first author of a paper describing the work. “There are also different sets of genes being expressed; some are cell-specific. We wanted to know what happens after you bioprint living cells and put them in an environment you design: are they alive and doing what they should? »

The plant cell 3D bioprinting process is mechanically similar to printing ink or plastics, with some necessary adjustments.

3D bio-printed plant cells

The plant cells are bio-printed by a 3D printer which has some necessary adjustments. Credit: Lisa Van den Broeck, NC State University

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

Living plant cells without cell walls, or protoplasts, were bioprinted with nutrients, growth hormones and a thickening agent called agarose – a compound made from seaweed. The agarose helps provide strength to the cells and a scaffolding, similar to the mortar that supports the bricks in the wall of a building.

“We found that using the proper scaffold is critical,” said Ross Sozzani, professor of plant and microbial biology at NC State and co-corresponding author of the paper. “When you print the bio-ink you need it to be liquid, but when it comes out it needs to be solid. Mimicking the natural environment helps maintain cell signals and signals as they would in soil .

The research 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 the day the cells were bioprinted, but we had never maintained the cells beyond a few hours after bioprinting, so we had no idea what was going on. would happen a few days later,” Van den Broeck said. “Similar viability ranges are shown after manually pipetting the cells, so the 3D printing process does not appear to be doing anything harmful to the cells.”

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

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

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

“This shows that 3D bioprinting can be useful for studying cell regeneration in crop plants,” Sozzani said.

Finally, the researchers studied the cellular identity of the bioprinted cells. Arabidopsis root cells and soybean embryo 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 types of cells.

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

The researchers plan to continue their work by studying cellular communication after 3D bioprinting, including at the single cell level.

“All told, this study shows 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.

Reference: “Establishing a reproducible approach to study cellular functions of plant cells with 3D bioprinting” by Lisa Van den Broeck, Michael F Schwartz, Srikumar Krishnamoorthy, Maimouna Abderamane Tahir, Ryan J Spurney, Imani Madison, Charles Melvin, Mariah Gobble, Thomas Nguyen, Rachel Peters, Aitch Hunt, Atiyya Muhammad, Baochun Li, Maarten Stuiver, Timothy Horn and Rosangela Sozzani, October 14, 2022, Scientists progress.
DOI: 10.1126/sciadv.abp9906

The search appears in Scientists progress and was supported by National Science Foundation EAGER grant MCB #203928 and BASF Plant Sciences. Tim Horn, assistant professor of mechanical and aerospace engineering at NC State, is co-corresponding author of the paper.

“Establish a reproducible approach to study cellular functions of plant cells with 3D bioprinting”

Abstract: Capturing cell-to-cell signals in a three-dimensional (3D) environment is essential to the study of cellular functions. A major challenge in current culture methods is the lack of accurate capture of multicellular 3D environments. In this study, we established a framework for 3D bioprinting plant cells to study cell viability, cell division, and cell identity. We have established the long-term cell viability of bioprinted Arabidopsis and soybean cells. To analyze the large image datasets generated, we developed a high-throughput image analysis pipeline. Moreover, we have shown re-entry into the cell cycle of bioprinted cells for which the timing coincides with the induction of central cell cycle genes and genes related to regeneration, ultimately leading to the formation of microcalli. Finally, the identity of bioprinted Arabidopsis root cells expressing endodermal markers was maintained for longer periods. The framework established here paves the way for the general use of 3D bioprinting to study cell reprogramming and cell cycle reentry towards tissue regeneration.