ISCRM Researchers Recover Cells from Hydrogels with Minimal Disturbance

UW researchers developed a new technique to recover cells from composite sortase-degradable hydrogels without altering their function.

Not far from the Montlake Campus of University of Washington Medical Center, Cole DeForest, PhD leads a thriving tool development lab where teams of researchers design biomaterial platforms that recreate living systems with full 4D complexity. Just a few miles away, at UW Medicine South Lake Union, Jennifer Davis, PhD studies cardiac fibrosis, the process by which the heart replaces damaged or lost muscle tissue with scar tissue as a result of injury and disease.

DeForest is the Weyerhaeuser Endowed Associate Professor of Chemical Engineering and Bioengineering. Davis is the Eva & Ara Woods Associate Professor of Bioengineering and Lab Medicine and Pathology. Both DeForest and Davis are faculty members in the Institute for Stem Cell and Regenerative Medicine (ISCRM).

The two labs, separated by a short shuttle drive, are bound by one at least one common factor. Ross Bretherton, a graduate student in Bioengineering is co-mentored by DeForest and Davis, putting him at the nexus of collaboration between engineers and biologists – the sweet spot in regenerative medicine.

“I’m in a unique place where I can be part of one team making materials that provide precise control over cellular space and time and another team actively applying these types of tools to study cardiovascular disease,” says Bretherton, whose research is supported by a state-funded ISCRM fellowship. “There are enormous possibilities in the field of biomaterials. We are engineering materials that can control cells with increasing specificity, which means we can model diseases more accurately than ever.”

The authors of the study including Bretherton (top left), Davis (bottom, second from right), and DeForest (bottom right). Other authors include Ashleigh Theberge, PhD, Darrian Bugg, PhD, Amanda Haack, Jonah Kern, Irina Kopyeva, and Fariha Rahman.

Improving Outcomes for Heart Disease Patients

The human stakes are high. The Davis Lab is on a mission to understand and harness the cellular mechanisms of scarring to improve outcomes for millions of heart disease and heart attack patients. But, in order to control scarring, the Davis and her team need to understand what makes fibrotic cardiac cells tick – and, to do that, they need a way to observe, manipulate, and evaluate the cells in their natural state.

Biomaterials, as a field, offers a sophisticated modeling option that combines principles of engineering and biology. In this approach, different cell types, encased in fluid-based microenvironments, coexist just as they would in the body. Cell behavior can be coaxed, tracked, and measured across space and time and in response to varying stimuli. For researchers like Davis, the bio-inspired mini worlds have obvious appeal.

“It’s very important to have a material that evolves in terms of its properties,” says Davis. “We need to be able to track cells across different regions and at varying timepoints – and we need control over when and where we extract them. In fibrosis, for example tissues become progressively collagenous – or you might want to model something that is more instantaneous, like the release of a protein that influences development.”

However, biomaterials, like any modeling tool, have limitations. “We have very well-established chemistries for getting cells into our materials,” explains Bretherton. “What’s lacking now is the ability to remove the cells with as little disturbance as possible so that we can do the actual biological deep dives that lead us to translational insights.”

The DeForest Lab has previously used light as one such tool to release cells from materials. However, there are limitations. In order for a material to be responsive to light, it has to absorb light, which restricts how thick it can be. And, performing sophisticated experiments involving and cells requires specialized equipment. Protease enzymes can also be used to remove cells from materials, but most proteases can strip proteins off the surface of cells and corrupt their genetic wiring.

Now a paper in the journal Advanced Materials describes a new method for cell extraction using engineered versions of an enzyme called sortase that have evolved to recognize and break specific peptide sequences. Bretherton is the first author of the study. DeForest is the corresponding author in close collaboration with co-authors Davis and Ashleigh Theberge, PhD, an Associate Professor in the Department of Chemistry.

An Exciting Strategy to Extract Cells

To evaluate whether sortase could be used to extract cells from materials without changing their state, Bretherton treated cardiac fibroblasts with various enzymes and used RNA sequencing to evaluate the impact of the enzymes on gene expression. The investigators found only a tiny number of genes out of 40,000 possible were being expressed differently, demonstrating the usefulness of the approach.

“This work presents an exciting strategy to get cells out of engineered 3D biomaterials without inadvertently impacting their function in the process,” says DeForest. “With this cell-recovery method, we can readily apply advanced single-cell tools (flow cytometry, RNAseq, etc) that are not compatible with encapsulated cells but are required to fully understand their function. Since the treatment does not significantly altering cell state during their release, the approach provides a uniquely powerful route to understand how cells are behaving in 3D, a requirement for downstream success in engineering functional tissues/organs for clinical transplantation.”

Using patterning devices developed by the Theberge Lab researchers were able to sculpt hydrogels containing cells into user-defined shapes, such as the University of Washington logo depicted here (scale bar 1mm)

According to Bretherton, the usefulness of the enzymes has to do with the architecture of the biomaterials favored by the DeForest Lab. “The materials we make are formed of very long flexible polymers, which start as a viscous liquid. We use crosslinks to solidify the polymers, bring them together, and arrange them into a continuous network. These crosslinks are encoded with recognition sequences to allow the sortase enzymes to cut the crosslinks and turn the material back into a liquid when we want to remove cells from the materials.”

Jen Davis speaks to the translational potential of the research described in the paper. “By putting fibroblasts into these materials, we can study the conditions which produce scar, and perhaps whether reversing those conditions could calm the cells back down,” says Davis. “These material systems have the potential to unlock our understanding of scarring in the heart, enabling us to develop the next generation of treatments for this deadly disease process.”