Hydrogels, a “Jell-O”-like substance, are highly flexible and absorbent networks of polymer strings that are frequently used in tissue engineering to act as a scaffold to aid cellular growth and development.
The paper demonstrates for the first time that it is possible to immobilize different proteins simultaneously using a hydrogel. This is critical for controlling the determination of stem cells, which are used to engineer new tissue or organs.
Three-dimensional (3D) protein-patterned scaffolds provide a more biomimetic environment for cell culture than traditional two-dimensional surfaces, but simultaneous 3D protein patterning has proved difficult. We developed a method to spatially control the immobilization of different growth factors in distinct volumes in 3D hydrogels, and to specifically guide differentiation of stem/progenitor cells therein. Stem-cell differentiation factors sonic hedgehog (SHH) and ciliary neurotrophic factor (CNTF) were simultaneously immobilized using orthogonal physical binding pairs, barnase–barstar and streptavidin–biotin, respectively. Barnase and streptavidin were sequentially immobilized using two-photon chemistry for subsequent concurrent complexation with fusion proteins barstar–SHH and biotin–CNTF, resulting in bioactive 3D patterned hydrogels. The technique should be broadly applicable to the patterning of a wide range of proteins.
“We know that proteins are very important to define cell function and cell fate. So working with stem cells derived from the brain or retina we have demonstrated we can spatially immobilize proteins that will influence their differentiation in a three-dimensional environment,” explained Professor Molly Shoichet.
Immobilizing proteins maintains their bioactivity, which had previously been difficult to ensure. It is also important to maintain spacial control as the tissue and organs are three-dimensional. Therefore, being able to control cell fate and understanding how cells interact across three dimensions is critical.
“If we think about the retina, the retina is divided into seven layers. And if you start with a retinal stem cell, it has the potential to become all of those different cell types. So what we are doing is immobilizing a protein which will cause their differentiation into photoreceptors or bipolar neurons or other cell types that would make up those seven different cell types,” said Shoichet.
The end result is a new hydrogel that can guide stem cell development in three-dimensions.
Shoichet identifies two long-term outcomes from this discovery.
“We could use… it as a platform technology to look at the interaction of different cells and build tissues and organ,” Shoichet stated, while also noting that it could help lead to a more fundamental understanding of cellular interaction. “By growing cells in a 3D environment, similar to how they grow in our body, we can develop a better understanding of cell processes and interactions.”