Imagine going to a surgeon to have a diseased or injured organ replaced to get a fully functional lab replacement. This is still science fiction and not reality, because researchers today struggle to organize cells into the complex 3D arrangements that our bodies can master on their own.
There are two major obstacles to overcome on the way to organically grown organs and tissues. The first is to use a biologically compatible 3D scaffold in which cells can grow. The second is to decorate this scaffold with biochemical messages in the correct configuration to trigger the formation of the desired organ or tissue.
Top view of a collagen hydrogel that the researchers decorated with immobilized mCherry proteins, which glow red under fluorescent light. The team shone the ultraviolet light on the hydrogel using a cut-out mask in the shape of an old University of Washington logo. The black regions were masked in the light, so the mCherry protein did not adhere to those portions of the hydrogel. The scale bar is 50 micrometers.Batalov et al., PNAS, 2021
In a major step toward transforming that hope into reality, researchers at the University of Washington have developed a technique for modifying natural biological polymers with protein-based biochemical messages that affect cellular behavior. His approach, published the week of Jan. 18 in the Proceedings of the National Academy of Sciences, uses a near-infrared laser to cause chemical adhesion of protein messages to a scaffold made from biological polymers such as col. lagen, a connective tissue found throughout our country. bodies.
Mammalian cells responded as expected to the protein signals attached within the 3D scaffold, according to lead author Cole DeForest, an associate professor of chemical engineering and bioengineering at UW. The proteins in these biological scaffolds caused changes in the messaging pathways within cells that affect growth, signaling, and other cellular behaviors.
These methods could form the basis of biological-based scaffolding that could one day make functional tissues grown in the laboratory a reality, said DeForest, who is also a faculty member at the UW Institute of Engineering and Molecular Sciences. the Institute of Regenerative and Stem Cell Medicine (UW).
“This approach provides us with the opportunities we have been waiting for to exercise greater control over the function and fate of cells in naturally derived biomaterials, not only in three-dimensional space, but also over time.” , said DeForest. “In addition, it makes use of exceptionally accurate photochemicals that can be controlled in 4D while uniquely preserving the function and bioactivity of proteins.”
Top view of two collagen hydrogels that the researchers decorated with immobilized mCherry proteins, which glow red under fluorescent light. The team scanned monster-shaped nearby infrared lasers (left) and the space needle (right) to create these patterns. The black regions were not scanned with the laser, so the mCherry protein did not adhere to these portions of the hydrogel. The scale bar is 50 micrometers.Batalov et al., PNAS, 2021
DeForest’s colleagues on this project are lead author Ivan Batalov, a former UW postdoctoral researcher in chemical engineering and bioengineering, and co-author Kelly Stevens, adjunct professor of bioengineering and laboratory medicine and pathology at UW.
His method is the first for the field, spatially controlling the function of cells within natural biological materials rather than synthetically derived ones. Several research groups, including those at DeForest, have developed light-based methods for modifying synthetic scaffolds with protein signals. But natural biological polymers may be a more attractive scaffold for tissue engineering, as they innately possess biochemical characteristics on which cells are based for structure, communication, and other purposes.
“A natural biomaterial like collagen inherently includes many of the same signs of signaling as those found in native tissue,” DeForest said. “In many cases, these types of materials keep cells ‘happier’ by providing them with signals similar to those found in the body.”
They worked with two types of biological polymers: collagen and fibrin, a protein involved in blood clotting. They gathered each in scaffolds full of liquid known as hydrogels.
The team used near-infrared lasers to create this intricate human heart-shaped pattern of immobilized mCherry proteins, which glow red under fluorescent light, inside a collagen hydrogel. On the left is an image composed of 3D slices of ice. To the right are cross-sectional views of the mCherry patterns. The scale bar is 50 micrometers.Batalov et al., PNAS, 2021
The signals the team added to the hydrogels are proteins, one of the main messengers of cells. Proteins come in many forms, all with their own chemical properties. As a result, the researchers designed their system to use a universal mechanism for attaching proteins to a hydrogel: the bond between two chemical groups, an alkoxyamine and an aldehyde. Prior to mounting the hydrogel, they decorated the collagen or fibrin precursors with alkoxyamine groups, all physically blocked with a “cage” to prevent the alkoxyamines from reacting prematurely. The cage can be removed with ultraviolet light or with a nearby infrared laser.
Using methods previously developed in the DeForest lab, the researchers also installed aldehyde groups at one end of the proteins they wanted to bind to the hydrogels. They then combined the aldehyde-carrying proteins with the alkoxyamine-coated hydrogels and used a brief light pulse to remove the cage covering the alkoxyamine. Exposed alkoxyamine reacted easily with the aldehyde group of the proteins, binding them within the hydrogel. The team used masks with cut patterns, as well as changes in laser scanning geometries, to create intricate patterns of protein arrangements in the hydrogel, including an old UW logo, the Seattle space needle, a monster and the 3D layout of the human. color.
This is a top view of a cylindrical fibrin hydrogel. By design, the right side of the hydrogel contains immobilized Delta-1 proteins, which activate Notch signaling pathways within cells. The left side does not contain immobilized Delta-1 (see tab). The team introduced human bone cancer cells into the hydrogel, designed to glow when Notch signaling pathways are activated. The right side of the hydrogel glows brightly, indicating that cells in this region have activated their Notch signaling pathways. The cells on the left side of the hydrogel have none. The scale bar is 1 millimeter.Batalov et al., PNAS, 2021
The bound proteins were fully functional, providing the desired signals to the cells. Rat liver cells, loaded with collagen hydrogels that carry a protein called EGF, which promotes cell growth, showed signs of DNA replication and cell division. In an independent experiment, the researchers decorated a fibrin hydrogel with patterns of a protein called Delta-1, which activates a specific pathway in cells called Notch signaling. When human bone cancer cells were introduced into the hydrogel, cells in regions with Delta-1 patterns activated Notch signaling, while cells in areas without Delta-1 did not. .
These experiments with multiple biological scaffolds and protein signals indicate that their approach could work for almost any type of protein signal and biomaterial system, DeForest said.
“We can now begin to create hydrogel scaffolds with many different signals, using our understanding of cell signaling in response to specific protein combinations to modulate critical biological function in time and space,” he added.
With the more complex signals loaded on hydrogels, scientists could try to control stem cell differentiation, a key step in turning science fiction into science facts.
The research was funded by the National Science Foundation, the National Institutes of Health and Gree Real Estate.