Biomedically Engineered Spinach Transforms Lab-Grown Meat

Tissue Research Is Advancing Cellular Agriculture

While cutting into a prime rib or nibbling on a piece of gouda may seem like negligible contributions to the global climate crisis, the animal-based agriculture behind these tasty products is one of the world’s leading sources of greenhouse gas emissions. In the U.S. alone, the EPA estimates that over 11 percent of greenhouse gas emissions in 2020—including carbon dioxide and methane—were generated by agriculture. Animal-based agriculture, in particular, contributes vast amounts of nitrous oxide (N₂0) and methane from managed livestock manure, and N₂0 from grazed soils.

Exclusively plant-based diets do not appeal to everyone, so bioengineers are working on a new approach to create animal products such as milk, eggs, and meat in a lab environment: cellular agriculture. Meat grown using cell cultures and tissue engineering has yet to appear in most grocery stores, but lab-grown dairy products—marketed as vegan and lactose-free—are already for sale in the U.S. and other countries. Bioengineers believe the product could fit well into the diet of pescatarians and meat-eaters, who want the flavor and texture of real meat with a lessened environmental and ethical burden.

Animal products grown in labs are more common today, but they were cutting edge when Glenn Gaudette, a professor of engineering at Boston College, first learned about this emerging field. At the time, Gaudette was presenting groundbreaking work that he and colleagues from Worcester Polytechnic Institute (WPI) had published that focused on transforming ordinary spinach leaves into a natural substrate to grow human heart tissue. In this research, the WPI research team used MATLAB^® to analyze the muscle cell contractions.

He says it was barely of fleeting interest when a friend from the University of Bath told him about lab-grown animal products. But when the Good Food Institute, a nonprofit researching plant- and animal-based alternatives to meat, invited Gaudette to talk about his heart tissue engineering work several months later, he realized the implications that his work could have. In particular, he was interested in helping curb the environmental impact of animal-based agriculture.

“My research has always been about helping people with heart disease,” Gaudette says. “But while there are, unfortunately, many people that suffer from heart disease, the environment is something that every single one of us needs. It was a ‘wow’ moment for me. If we can make a slight difference here, I want to be part of that.”

“My research has always been about helping people with heart disease. But while there are, unfortunately, many people that suffer from heart disease, the environment is something that every single one of us needs. It was a ‘wow’ moment for me. If we can make a slight difference here, I want to be part of that.”

Animal-based agriculture is a significant contributor of greenhouse gases.

Simply Spectacular Spinach

Since his revelation, Gaudette, along with his WPI and Boston College colleagues, has been working to bring their tissue engineering insights to cellular agriculture. In new research, they have demonstrated how to use spinach leaves to create an edible surface on which to cultivate lab-grown beef—all without taking up an acre of land or killing a single cow. According to Gaudette, the same characteristics of spinach leaves that lend them to growing heart tissue also make them useful for cellular agriculture.

“The big advantage of spinach is that it already has a vascular network,” he explains. “If you ever hold spinach leaves up to the light, you can see the veins that branch off from the main stem.”

Gaudette says this vascular structure is very similar to how arteries and capillaries—biological pathways that cycle blood through our bodies to organs like the heart—appear in the human body.

“That’s a huge advantage in a material that already exists, which is part of why we looked at spinach,” Gaudette says. “It also provides a nice structure for these meat cells to grow on and spread out.”

“The big advantage of spinach is that it already has a vascular network. If you ever hold spinach leaves up to the light, you can see the veins that branch off from the main stem.”

Side-by-side comparison showing similarities of a mammalian vascular network and a spinach vascular network. — Comparison of animal and plant vascular network pattern branching and structures. (Image credit: Glenn Gaudette and WPI)

Three spinach leaves in a petri dish. The decellurized leaf in the center is transparent with the vascular structure still intact. — Spinach leaves were put through multi-day decellurization to remove any plant cells to isolate their vascular structure. (Image credit: Glenn Gaudette and WPI)

Before Gaudette can add bovine cells to spinach leaves, however, the leaves must undergo a decellularization process that uses chemical washes remove cells from living tissue. The researchers used boxes of spinach from the grocery store and put them through multi-day decellularization to remove any plant cells from the leaves and isolate their vascular structure.

The end product is a leaf that entirely lacks its green pigment and instead becomes semitransparent. At this point, Gaudette says the leaves are a scaffold ready to welcome bovine cell structures into their “veins.” The team also prepared a control group of bovine cells on a gelatin-coated glass scaffold to compare against their spinach model after a one-to-two-week growth period. By the end of their experiment, they found that the spinach scaffold worked just as well as existing methods, such as silk, collagen, and tissue culture–treated plastic, at promoting cell growth and viability.

Engineering Taste and Texture

Yet, while demonstrating that it is possible to grow bovine cells on a spinach substrate is a crucial breakthrough, Gaudette says that this innovation alone isn’t very useful for cellular agriculture. Instead, bovine cells must also be grown so that their texture and taste mimic that of naturally grown animal muscle.

“Texture comes from the alignment of the muscle cells,” he explains. “Those muscles are essentially a line, like a rope. That alignment is critical to give us the taste and texture of a steak or another meat product.”

Gaudette says he was excited when he saw that the spinach scaffolds naturally enabled the bovine cells to grow in a line. “Looking at how those cellular structures are aligning tells us that the muscle is essentially starting to form,” Gaudette explains.

However, the alignment of several thousand cells still needed to be closely monitored by studying the internal structure of each individual cell to determine its angular direction.

“Texture comes from the alignment of the muscle cells. Those muscles are essentially a line, like a rope. That alignment is critical to give us the taste and texture of a steak or another meat product.”

The decellurized leaf with bovine blood along the leaf’s vascular network. — A decellularized spinach leaf with bovine blood. (Image credit: Glenn Gaudette and WPI)

Image Processing to Perfect the Taste

While it is possible to do this kind of task by hand, Gaudette says it would have been extremely time-consuming and tedious. Instead, the team used MATLAB image processing and Circular Statistics Toolbox, CircStat, to study the cell alignment process. The first author of the research paper, Jordan Jones, explains that their program requested identifying data, such as the experiment number, and then used integrated image analysis software to read and analyze all the images collected. This took a little under an hour, Jones says, and was quicker and more hands-off than analyzing the images individually.

“Counting the features manually for alignment is simply not worthwhile and would introduce too much error,” Jones explains. “An automated program is necessary to produce useful data when it comes to alignment analysis.”

MATLAB was used to process the raw data collected from these images into meaningful quantitative metrics. CircStat toolbox then extracted characteristics that were used to create histograms and rose plots. These visuals helped the researchers identify minute-degree changes in cell alignment and count the number of cells and nuclei. Jones says that CircStat stood out to him as a convenient solution to a unique problem.

“CircStat allowed me to integrate circular statistics into my MATLAB code. Without it, I might have decided to use a less powerful analysis method, or there would be more variability from measurement to measurement.”

Phase one shows bovine satellite cells isolated from a cow’s muscle fiber that are then expanded. Phase two shows the cells are then seeded on the spinach scaffold that then form muscle tissue. — The steps researchers took to isolate and seed primary bovine satellite cells on a decellularized spinach leaf scaffold. (Image credit: Glenn Gaudette and WPI)

“CircStat allowed me to integrate circular statistics into my MATLAB code. Without it, I might have decided to use a less powerful analysis method, or there would be more variability from measurement to measurement.”

“MATLAB enabled us to analyze and visualize large amounts of data,” Gaudette says. “Researchers can use this analytical technique to answer essential questions about lab-grown meat such as what amount of working out or manually contracting of the meat’s muscle tissue, creates a better taste and texture in the final product.”

Gaudette sees a future where researchers can repurpose the team’s code for other biomedical research. For example, the team is currently collaborating on a project to study skin regeneration after a wound.

The Future of Lab-Grown Meat

Gaudette’s research into spinach scaffolds for cellular agriculture is a proof of principle, but the final products are not yet something you could cook up on a grill. That said, Gaudette and colleagues at Boston College are excited to continue exploring how to use natural scaffold techniques to develop even more realistic meat in the coming years.

Already, the team has released a new paper demonstrating how to use broccoli to cultivate meat cells. They are also looking at ways to use only food-safe chemicals in this work.

Another area that Gaudette says he’s investigating is how to use multiple scaffolds to create a more complex meat structure, such as the fat and muscle marbling of a high-quality steak.

“Eventually, we want to stack a bunch of those spinach leaves on top of each other,” he says. “If we could stack the leaves together such that some have muscle and some have fat cells, then, if you take a cross-section, you will have a marbled cut of steak.”

Most of all, Gaudette says he is excited to collaborate with nonengineers, including policymakers and ethicists, to transform how agriculture feeds the world.

“Animal agriculture has been around for centuries, but cellular agriculture is a brand-new industry,” Gaudette says.

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