Rice Researchers 3D Print with Lasers and Sugar to Build Complex Vascular Networks

A team of researchers from Rice University has uncovered a promising strategy to generate vascular networks, one of the most daunting structures in the human body. Using powdered sugar and selective laser sintering, the researchers were able to build large structures from complex, branching, and intricate sugar networks that dissolve to create pathways for blood in lab-grown tissue.

This is the team’s latest effort to build complex vascular networks for engineered tissues to show that they could keep densely packed cells alive for two weeks. The findings of their study—published in the Nature Biomedical Engineering journal—prove that developing new technologies and materials to mimic and recapitulate the complex hierarchical networks of vessels gets them closer to providing oxygen and nutrients to a sufficient number of cells to get a meaningful long-term therapeutic function.

“One of the biggest hurdles to engineering clinically relevant tissues is packing a large tissue structure with hundreds of millions of living cells,” said study lead author Ian Kinstlinger, a bioengineering graduate student at Rice’s Brown School of Engineering. “Delivering enough oxygen and nutrients to all the cells across that large volume of tissue becomes a monumental challenge. Nature solved this problem through the evolution of complex vascular networks, which weave through our tissues and organs in patterns reminiscent of tree limbs. The vessels simultaneously become smaller in thickness but greater in number as they branch away from a central trunk, allowing oxygen and nutrients to be efficiently delivered to cells throughout the body.”

Overcoming the complications of 3D printing vascularization has remained a critical challenge in tissue engineering for decades, as only a handful of 3D printing processes have come close to mimic the in vivo conditions needed to generate blood vessels. Without them, the future of bioprinted organs and tissues for transplantation will remain elusive. Many organs have uniquely intricate vessels, like the kidney, which is highly vascularized and normally receives a fifth of the cardiac output, or the liver, in charge of receiving over 30% of the blood flow from the heart. By far, kidney transplantation is the most common type of organ transplantation worldwide, followed by transplants of the liver, making it crucial for regenerative medicine experts to tackle vascularization.

Ian Kinstlinger with a blood vessel template he 3D printed from powdered sugar (Credit: Jeff Fitlow/Rice University)

In the last few years, extrusion-based 3D printing techniques have been developed for vascular tissue engineering, however, the authors of this study considered that the method presented certain challenges, which led them to use a customized open-source, modified laser cutter to 3D print the sugar templates in the lab of study co-author Jordan Miller, an assistant professor of bioengineering at Rice.

Miller began work on the laser-sintering approach shortly after joining Rice in 2013. The 3D printing process fuses minute grains of powder into solid 3D objects, making possible some complex and detailed structures. In contrast to more common extrusion 3D printing, where melted strands of material are deposited through a nozzle, laser sintering works by gently melting and fusing small regions in a packed bed of dry powder. According to Miller, “both extrusion and laser sintering build 3D shapes one 2D layer at a time, but the laser method enables the generation of structures that would otherwise be prone to collapse if extruded.”

“There are certain architectures—such as overhanging structures, branched networks and multivascular networks—which you really can’t do well with extrusion printing,” said Miller, who demonstrated the concept of sugar templating with a 3D extrusion printer during his postdoctoral studies at the University of Pennsylvania. “Selective laser sintering gives us far more control in all three dimensions, allowing us to easily access complex topologies while still preserving the utility of the sugar material.”

Assistant professor of bioengineering at Rice University, Jordan Miller (Credit: Jeff Fitlow/Rice University)

Generating new 3D printing processes and biomaterials for vascularization is among the top priorities for the researchers at Miller’s Bioengineering Lab at Rice. The lab has a rich history of using sugar to construct vascular network templates. Miller has described in the past how sugar is biocompatible with the human body, structurally strong, and overall, a great material that could be 3D printed in the shape of blood vessel networks. His original inspiration for the project was an intricate dessert, even going as far as suggesting that “the 3D printing process we developed here is like making a very precise creme brulee.”

To make tissues, Kinstlinger chose a special blend of sugars to print the templates and then filled the volume around the printed sugar network with a mixture of cells in a liquid gel. Within minutes, the gel became semisolid and the sugar dissolved and flushed away to leave an open passageway for nutrients and oxygen. Clearly, sugar was a great choice for the team, providing an opportunity to create blood vessel templates because it is durable when dry, and it rapidly dissolves in water without damaging nearby cells.

A sample of blood vessel templates that Rice University bioengineers 3D printed using a special blend of powdered sugars. (Credit: B. Martin/Rice University)

In order to create the treelike vascular architectures in the study, the researchers developed a computational algorithm in collaboration with Nervous System, a design studio that uses computer simulation to make unique art, jewelry, and housewares that are inspired by patterns found in nature. After creating tissues patterned with these computationally generated vascular architectures, the team demonstrated the seeding of endothelial cells inside the channels and focused on studying the survival and function of cells grown in the surrounding tissue, which included rodent liver cells called hepatocytes.

The hepatocyte experiments were conducted in collaboration with the University of Washington (UW)’s bioengineer and study co-author Kelly Stevens, whose research group specializes in studying these delicate cells, which are notoriously difficult to maintain outside the body.

“This method could be used with a much wider range of material cocktails than many other bioprinting technologies. This makes it incredibly versatile,” explained Stevens, an assistant professor of bioengineering in the UW College of Engineering, assistant professor of pathology in the UW School of Medicine and an investigator at the UW Medicine Institute for Stem Cell and Regenerative Medicine.

The results from the study allowed the team to continue their work towards creating translationally relevant engineered tissue. Using sugar as a special ingredient and selective laser sintering techniques could help advance the field towards mimicking the function of vascular networks in the body, to finally deliver enough oxygen and nutrients to all the cells across a large volume of tissue.

Miller considered that along with the team they were able to prove that “perfusion through 3D vascular networks allows us to sustain these large liverlike tissues. While there are still long-standing challenges associated with maintaining hepatocyte function, the ability to both generate large volumes of tissue and sustain the cells in those volumes for sufficient time to assess their function is an exciting step forward.”

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Interview with Tamer Mohamed of Aspect Biosystems on Advancing Tissue Therapeutics

While attending The University of British Columbia (UBC), Tamer Mohamed, along with fellow graduate student Simon Beyer, began working at the Walus Laboratory on the development of a novel microfluidics-based bioprinting platform that could be used to fabricate human tissue constructs. One of the main reasons for their innovation was to potentially replace animal models in drug testing, which are costly, time-consuming and can have poor predictive accuracy. A few years went by and the two went on to win a MEMSCAP Design Award for their pioneering creation (the Lab-on-a-Printer Bioprinter) which would later become the basis for their startup, Aspect Biosystems. The UBC spinoff company was founded by Mohamed, Beyer, Konrad Walus (associate professor at UBC and head of the Walus Lab), and Sam Wadsworth, to turn their idea into a commercial product. The company quickly began providing pharmaceutical companies with high-efficacy tissue models that better mimic in vivo conditions, looking to improve the predictive accuracy of the front end drug discovery process. 3DPrint.com spoke to Mohamed to learn about his successful transition from graduate student to CEO of Aspect Biosystems.

Cofounders of Aspect Biosystems Tamer Mohamed and Simon Beyer at the Walus Lab when they were grad students

What was the inspiration behind Aspect Biosystems?

Aspect Biosystems was established with the vision of leveraging advancements in biology, microfluidics, and 3D printing to create technology-enabled therapeutics that will ultimately have a meaningful impact on patients. We are marrying our deep knowledge of human biology with cutting-edge 3D printing technology to create. Our story started almost a decade ago so we’ve spent years developing our foundational microfluidic bioprinting technology and are now applying our platform technology to create functional tissues, both internally through our proprietary programs, and with our partners around the world.

Can you tell me about the company’s growth model?

Platform technologies often have the advantage of flexibility, as they could allow you to pursue multiple applications. This also presents a challenge though, in that it is easy to become unfocused. At Aspect, we’ve built a strategy that allows us to both focus and diversify. Internally, we are advancing proprietary tissue programs in regenerative medicine. But we also recognize that to achieve our vision of enabling human tissues on demand, we can’t work alone. By providing access to our technology to partners around the world, we are able to create a network effect and tap into specific domain expertise. This allows our technology to be applied to a wide range of research purposes externally, without detracting resources or focus from our specific tissue programs internally. We collaborate with academia and industry on specific applications that allow us to fuel our growth and help generate revenue and a robust innovation pipeline.

How much has Aspect grown?

Aspect is the first and only company to leverage microfluidics to create functional tissue, and we are proud to pioneer this approach. Academically, we were one of the first groups in the world to print cells while at the UBC, so we see ourselves as pioneers in both bioprinting and platforms for creating tissue therapeutics. Five years ago, we had four full-time employees. Today we have a team of over 40 people focused on our mission and over 20 collaborations globally. We have attracted smart venture capital, partnered with some of the biggest names in our industry, and made major breakthroughs in applying our technology to create functional tissues. It is a great sign that, year-after-year, we continue to raise the bar. It is an even better sign that I believe the best is yet to come.

The Aspect Biosystem team celebrating Canada Day

What will the applications of this technology be in pharmaceutical research and drug trials?

I believe the opportunity with the highest value and best poised to make a significant impact on the pharmaceutical space is disease modeling. Using 3D bioprinting technology allows us to model diseases in a human-relevant system that would otherwise be difficult to study in animals or less sophisticated in vitro models. For example, working with GSK and Merck, we are leveraging our microfluidic 3D bioprinting platform to create physiologically-relevant 3D tissues containing patient-derived cells to assess the efficacy of anti-cancer drugs and to predict a patient’s response to treatment. This partnered program could unlock the discovery of novel therapeutic targets and the development of immuno-oncology therapeutics.

Would you tell us more about Aspect’s current and future work? 

Our current internal programs are focused on orthopedic and metabolic diseases. On the orthopedic side, we are leveraging our deep knowledge of musculoskeletal biology and biomaterials to create knee meniscal replacements. On the metabolic side, we are focused on liver tissue and creating a therapeutic tissue for Type 1 diabetes. Externally, our partners around the world are using our 3D bioprinting technology to advance research in the brain, lungs, heart, pancreas, and kidneys, just to name a few. By being both focused internally and diversified externally, we are building a robust pipeline for the future. Our end goal is to enable the creation of human tissues on demand, and we know that we can’t do it alone. Our network of academic researchers and industry partners are key to making our vision a reality.

How fast is the technology moving towards a future with lab-made functional organs?

Tamer Mohamed

We are focused on identifying specific diseases or biological malfunction inside the body and rationally designing advanced tissue therapeutics that address these areas of unmet medical need. So, while we may not actually be making something that looks exactly like an organ, we are recreating the biological function that has been lost or damaged to address the problem. For example, someone with Type 1 diabetes has a pancreas that is unable to perform the vital function of creating insulin. We don’t necessarily need to engineer something for them that looks exactly like a pancreas – instead, we are creating an implantable therapeutic tissue that replaces function that has been lost. In this case, that function is sensing glucose levels in the blood and biologically releasing insulin in response. This is an example of one of our internal programs – a bioengineered pancreatic tissue therapeutic that restores a critical function that been lost due to an autoimmune disease.

Is Canada a great place to develop a bioprinting company? 

Canada has a long and rich history in the field of regenerative medicine, going back to the discovery of stem cells in the 1960s. As a country, we have an opportunity to be a global leader in the field. At Aspect, we are proud to be part of these efforts. We are in ongoing discussions with different government groups as to how we can play a role in helping to lead the charge and the government has been embracing that. We have seen significant federal and provincial support for innovation and public/private partnerships, which definitely help stimulate growth in the field.

How disruptive is the technology you created?

By combining microfluidics with 3D printing, we are disrupting tissue engineering. We are able to programmatically process multiple cells and biologically-relevant materials in high-throughput to rationally design and produce functional tissues. We are constantly integrating new microfluidic processing units within our printhead technology and leveraging continuous advancements in the “lab-on-a-chip” space. With our microfluidic technology, we are generating a large amount of data. By using this data and machine learning, we are improving the quality and automation of the biomanufacturing process.

Ultimately, bioprinting is only as good as our understanding of biology – and our understanding of biology is growing wider and deeper. We are combining state-of-the-art stem cell science with our microfluidic 3D printing technology to create tissue therapeutics. For example, we are combining insulin-secreting cells derived from human embryonic stem cells (hESCs) with our printing technology to create therapeutic tissues for patients with Type 1 diabetes.

[Images: Aspect Biosystems and Tamer Mohamed]

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