Bioprinting plant cells to research cell viability and communication

A new study from North Carolina State University demonstrates a reproducible method for examining cellular communication across diverse plant cell types by “bioprinting” these cells using a 3D printer. Learning more about how plant cells communicate with each other—and with their environment—is essential to understanding plant cell functions and could lead to the development of superior crop varieties and optimal growth conditions.

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

Lisa Van den Broeck, a postdoctoral researcher at NC State and the lead author of a publication presenting the discovery, remarked, “A plant root contains many different cell types with specialized roles.” “There are also distinct sets of genes that are expressed, some of which are cell-specific. We wanted to discover what happens when you bioprint living cells and set them in an environment that you’ve designed: Are they alive and functioning as they should? ”

Mechanically, the process of 3D bioprinting plant cells is comparable to printing ink or plastic, with a few minor modifications.

Instead of using ink or plastic for 3D printing, Van den Broeck’s team uses “bioink,” or living plant cells. “The mechanics are the same in both procedures, with a few noticeable modifications for plant cells: a UV filter is used to maintain the atmosphere sterile and several print heads, as opposed to a single one, to print different bioinks simultaneously.”

Live plant cells without cell walls, or protoplasts, were bioprinted with nutrients, growth hormones, and agarose, a seaweed-derived thickening agent. Agarose helps give cells strength and structure, kind of like how mortar holds bricks together in a wall.

Professor of plant and microbial biology at NC State and co-corresponding author of the research, Ross Sozzani, stated, “We discovered that it is essential to employ correct scaffolding.” “When the bioink is printed, it must be liquid, but it must be solid when it exits the printer. By simulating the natural environment, biological signals and cues are maintained in the same manner as in soil.

More than half of the 3D bioprinted cells survived and proliferated over time, forming microcalli, or small cell colonies.

“We anticipated good cell viability on the day they were bioprinted, but we had never maintained cells for more than a few hours following bioprinting, so we had no idea what would happen days later,” Van den Broeck said. Comparable viability ranges are observed after manually pipetting cells, indicating that the 3D printing procedure does not appear to be hazardous to cells.

Sozzani explained that “this is a challenging manual process, but 3D bioprinting controls the pressure of the droplets and the speed at which they are created.” “Bioprinting makes it easier to do high-throughput processing and control the structure of the cells that are made after bioprinting, like putting them in layers or honeycomb shapes.”

Additionally, the researchers bioprinted individual cells to determine if they might regenerate or split and multiply. According to the research, for maximum survival, root and shoot cells of Arabidopsis require different nutrition and scaffolding combinations.

Furthermore, more than 40% of individual soybean embryonic cells remained alive two weeks after bioprinting and split to form microcalli.

As stated by Sozzani, this demonstrates that 3D bioprinting may be utilized to examine cellular regeneration in crop plants.

Finally, the scientists examined the cellular identification of the bioprinted cells. Arabidopsis root cells and embryonic soybean cells are noted for their high rates of proliferation and lack of permanent identities. In other words, these cells, like animal or human stem cells, are capable of transforming into many cell types.

“We discovered that bioprinted cells can acquire the characteristics of stem cells; they can divide, develop, and express certain genes,” stated Van den Broeck. “Bioprinting involves the reproduction of a whole population of cell types. “After 3D bioprinting, we were able to evaluate the genes expressed by individual cells to determine if cell identity was altered.

After 3D bioprinting, the researchers plan to keep studying how cells talk to each other, even at the level of a single cell.

Sozzani says that this study shows the great potential of 3D bioprinting for finding the right molecules that plant cells need to stay alive and talk to each other in a controlled environment.

The research, which is published in Science Advances, was financed by the National Science Foundation EAGER project MCB #203928 and by BASF Plant Sciences. The paper’s co-corresponding author is Tim Horn, assistant professor of mechanical and aerospace engineering at NC State.