Over the last few decades, the pursuit of human tissue engineering has moved steadily closer to clinical reality. The potential implications are profound. The ability to regenerate diseased or injured tissues – or even entire organs – using a combination of biological and synthetic materials could transform patient care, shorten transplant waiting lists, and accelerate drug discovery.
Kelly Stevens, PhD is a UW associate professor of bioengineering and lab medicine and pathology and a faculty member in the Institute for Stem Cell and Regenerative Medicine (ISCRM). The Stevens Lab is on a mission to build 3D printed human organs, starting with the immensely complex liver.
Dr. Stevens speaks to the momentum she is seeing in her lab and across the field. “For the first time, technologies and approaches that we once considered to be science fiction, like 3D printing organs, are moving beyond proof of principle. Our challenge now is to eliminate the guesswork that is slowing down the development of human tissue therapies.”
In practical terms, that guesswork has to do with the composition of the bioink that engineers like Stevens use to print tissues and organs. Any bioink will likely contain a mix of multiple types of living cells and some variety of plastic, polymer, or other human-made materials. Because there are so many possible permutations, and because there is still so much to learn about the interplay of biological and synthetic materials, and indeed about human biology itself, the process of testing the safety and functionality of each bioink remains expensive and laborious, especially in animal trials.
Stevens believes one solution to the bottleneck may be at-hand. “We already have a 3D printer we are using for printing organs. So we decided to put the technology to work in a new system that allows us to cut down the trial and error and screen dozens of different formulations at the same time.”
3D Printed Device Accelerates Comparison of Potential Bio-Inks
The new system, dubbed PHAST (Parallelized Host Apposition for Screening Tissues in vivo) is described in a new paper in the journal Cell Stem Cell. Stevens is the senior author of the article. Fan Zhang, PhD, a postdoctoral researcher in the Stevens Lab, and Colleen O’Connor, PhD, a former member of the lab, are co-first authors. ISCRM faculty members Cole DeForest, PhD and Elizabeth Wayne, PhD are also authors of the paper. Daniela Witten, PhD, a professor of Statistics and Biostatistics, performed the statistical modeling studies in the paper.
The goal of PHAST, as pronunciation of the acronym suggests, is to take a task that has been prohibitively slow and make it fast (and accurate). In fact, Stevens emphasizes that PHAST enables researchers to produce results in one to two months that would have previously required years to achieve – or been outright impossible.
It is an engineering feat designed to solve a biological conundrum. Any human patient or animal subject is made up of many cell types that respond to many different biological and biochemical cues inside the body. How these resulting cellular behaviors – which are confoundingly mysterious to begin with – will interact with synthetic materials in unfamiliar habitats (for example, human endothelial cells relocated to the blood vessel of a mouse) has to be understood for human tissue engineering to be useful in the clinic. The Stevens Lab designed PHAST with that “black box” of questions in mind.
In the investigation, the researchers injected different combinations of microtissue formulations with varying cellular and material compositions into 43 wells of a 3D printed device. The device was implanted into mice so that Stevens and her team could examine which combinations best supported engraftment between the implanted cells and the blood vessels of the host.
Variables under scrutiny include the type of cell populations and the material in which the cells are encased. Each option has its own pros and cons and even slight variations can have an impact on cell behavior. Stevens says that just a subtle adjustment to the concentration of the material changed how well implanted cells engrafted to blood vessels in an animal, even when her team used the same cells and same materials.
Finding the Sweet Spots in Bioink Formulation
One of the game-changing features of PHAST is that the system does not just indicate that one formulation is better than another. Rather, PHAST can help researchers identify the optimal point on a continuum for a particular variable. One important factor is stiffness.
Cells may prefer softer scaffolds, while stiffer constructs are easier to print. The parallelized design of PHAST makes the search for the sweet spot much more efficient. Stevens credits the DeForest Research Group for permitting measurements that allowed them to determine the stiffness of each material.
While PHAST can be used to speed up research, Stevens adds that it could also be used as a personalized medicine tool. “You can just imagine a medical team putting cells from different patients into a 3D-printed array to understand how effective a given drug might be for different individuals. That’s a long-term application for this technology.”
In the meantime, Stevens stresses that PHAST must also check another box. It has to be practical. “The idea is to make a technology that is as simple as possible so that is useful for as many people as possible. For example, the wells are designed so that they can be filled with standard equipment that would be available in any lab.”
The researchers faced another engineering challenge: how to prevent 43 populations of cells living microns apart from interacting with one another. In the spirit of good fences making good neighbors, the team expanded the distance between the wells. They then introduced reporter cells, engineered in the lab of Dr. Wayne, to turn green in the presence of specific proteins, into the wells. No flashing lights confirmed a lack of crosstalk.
Looking ahead, Stevens is excited to improve on the technology, to see it adapted for other species, and to have it put to work in other tissue engineering labs. “We’re going to use it here to 3D print a liver, but I hope others will use it for their own goals, to answer their own questions, and to advance their programs in regeneration, repair, screening, and therapies for patients.”
Acknowledgements
This research was supported by the NIH R01DK128551 (to K.R.S.), Wellcome Leap as part of the HOPE program (to K.R.S.), W.M. Keck Foundation (to K.R.S.), Allen Distinguished Investigator Award (to K.R.S.), a Paul G. Allen Frontiers Group advised grant of the Paul G. Allen Family Foundation (to K.R.S.), NIH Maximizing Investigators’ Research Award (R35GM138036, to C.A.D.), NSF CAREER Award (DMR 1652141, to C.A.D.), NSF Graduate Research Fellowships (to C.E.O., S.P.S., N.E.G., and I.K.)