3D printing plant cells helps to study cell function, researchers suggest

A North Carolina State University study demonstrates how "bioprinting" plant cells using a 3D printer can be a reproducible method of studying cellular communication among various plant cells.

Published very recently in Science Advances, understanding how plant cells interact with one another and their surroundings are essential for understanding how they function.

This knowledge will help us develop better crop kinds and ideal growing conditions.

As stated in the release, to analyze whether and how long plant cells would survive after being bioprinted and how they would acquire and alter their identity and function, the researchers bioprinted cells from the model plant Arabidopsis thaliana and soybeans.

“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 live cells and place them into an environment that you design: Are they alive and doing what they should be doing?”

Mechanically similar to printing ink or plastics

3D bioprinting plant cells are mechanically similar to printing ink or polymers with a few minor adjustments.

“Instead of 3D printing ink or plastic, we use ‘bioink,’ or living plant cells. 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 print heads – rather than just one – to print different bioinks simultaneously,” Van den Broeck said.

Protoplasts, living plant cells without cell walls, were bioprinted with nutrients, growth hormones, and agarose, a thickening agent derived from seaweed. Agarose aids in giving cells stability and scaffolding, much like mortar do for bricks in a building's wall.

“We found that it is critical to use proper scaffolding,” said Ross Sozzani, professor of plant and microbial biology at NC State and a co-corresponding author of the paper.

“When you print the bioink, you need it to be liquid, but when it comes out, it needs to be solid. Mimicking the natural environment helps keep cellular signals and cues occurring as they would in soil.”

Cells were viable and divided over time

According to the study, more than half of the 3D bioprinted cells were alive and divided into small cell colonies called microcalli as a result of time.

“We expected good viability on the day the cells were bioprinted, but we had never maintained cells past a few hours after bioprinting, so we had no idea what would happen days later,” Van den Broeck said.

“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 provides a better opportunity for high throughput processing and control over the architecture of the cells after bioprinting, such as layers or honeycomb shapes.”

Power of Arabidopsis

Additionally, the researchers bioprinted individual cells to see if they might split and grow or regenerate. The results demonstrated that Arabidopsis root and shoot cells required varied nutrients and scaffolding for optimum survival.

The researchers also looked into the cellular makeup of the bioprinted cells. High proliferation rates and the lack of stable identities are characteristics of embryonic soybean cells and Arabidopsis root cells. In other words, these cells can differentiate into many cell types, like an animal or human stem cell.

Abstract:

Capturing cell-to-cell signals in a three-dimensional (3D) environment is key to studying cellular functions. A major challenge in current culturing methods is the lack of accurately capturing 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 established long-term cell viability for bioprinted Arabidopsis and soybean cells. To analyze the generated large image datasets, we developed a high-throughput image analysis pipeline. Furthermore, we showed the cell cycle reentry of bioprinted cells for which the timing coincides with the induction of core cell cycle genes and regeneration-related genes, ultimately leading tomicrocallus formation. Last, 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 for studying cellular reprogramming and cell cycle reentry toward tissue regeneration.