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3D-Bioprinted Cell Therapy and Disease Modeling Applications

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Precision medicine is, broadly speaking, a sweeping aspiration that will be achieved, or not, depending on the convergence of many different disciplines. The most important of these disciplines are molecular genetics and cell biology. Besides contributing to precision medicine directly, they also contribute through 3D bioprinting. In 3D bioprinting, molecular genetics and cell biology are teamed with engineering and robotics to fabricate physiologically accurate model organs for various purposes, such as advancing drug discovery, determining how individual patients will respond to different drugs, and even fabricating implantable therapeutics.

Building for function, not structure

Aspect Biosystems, which spun out of the University of British Columbia in 2013, takes a microfluidics approach to bioprinting. “What you get with microfluidics is extreme design flexibility,” says Tamer Mohamed, PhD, the company’s founder and CEO. “The analogy I like to give is that, instead of doing black-and-white bioprinting, we’re doing full-color-spectrum bioprinting, where we can access a whole palette of biological building blocks that we can combine and mix to engineer bioprinted therapeutics.”

Tamer Mohamed, PhD
Tamer Mohamed, PhD
Founder & CEO
Aspect Biosystems

To draw from the rich palette of materials, the bioprinter’s microfluidic printhead employs an integrated valving system that can precisely sequence between multiple different materials from various channels. “Right before these biomaterials are printed,” Mohamed explains, “they are chemically crosslinked on-chip into a solid material that we then pattern in 3D.”

Rather than creating exact replicas of human organs, Aspect designs biocompatible devices to provide specific therapeutic benefits. For instance, the company is developing an implant to perform the function of pancreatic beta cells, detecting glucose and producing insulin, in patients with type 1 diabetes.

“We focus on function over structure,” Mohamed points out. “We’re not necessarily creating organs and tissues that look identical to what we have inside the body.”

Aspect’s bioengineers design biomaterials to optimize the characteristics needed for each particular application. The bioprinted diabetes implant consists of 3D-printed pancreatic cells wrapped inside a protective layer that hides the foreign cells from the immune system. That layer needs to be non-immunogenic, as well as strong and durable enough to last for years inside the patient.

“We design the biomaterials to provide an environment for cells to thrive,” Mohamed adds. “And we also design these biomaterials to be printable, so we can print them in the appropriate shape for the particular surgical site that we’re going after.”

The RX1 bioprinter from Aspect Biosystems
The RX1 bioprinter from Aspect Biosystems is designed to provide microscale resolution, high-speed operations, and seamless cell and material patterning. These capabilities depend on the RX1’s microfluidic technology, which boasts precise motion and pressure control and on-printhead crosslinking. A protective coaxial sheath minimizes shear stress on cells, and a selection of microfluidic printheads provides functional flexibility. The RX1’s software facilitates the design of customized structures.

To make the pancreatic cells, Aspect is collaborating with Timothy Kieffer, PhD, professor of cellular and physiological sciences at the University of British Columbia. Kieffer has developed proven protocols for differentiating stem cells into pancreatic beta cells. Although starting with stem cells provides a source of scalable cells, there is always a risk that stem cell–derived pancreatic cells could turn cancerous. The implant’s design keeps all the cells set apart, rather than freely integrating with the host’s pancreas. According to Mohamed, this arrangement makes it easier to remove the cells if anything goes wrong.

“We’re the first and only group in the world to marry microfluidics to 3D printing and apply the combination to generate bioprinted therapeutics,” Mohamed asserts. “Our technology platform enables us to take a rational-design, first-principles approach to building implantable biological therapies.”

Testing drugs with an organ-on-a-chip

Printing organs for use outside the body is another way 3D printing can advance precision medicine. Swiss bioprinting company RegenHU is contributing its expertise to develop an organ-on-a-chip to test treatments for rheumatoid arthritis. The company has joined a European Union–funded research project called FLAMIN-GO, which brings together a consortium of academic and industrial partners to design a biological test bed for new drugs.

Simon MacKenzie, PhD
Simon MacKenzie, PhD
Chief Executive Officer
RegenHU

“Eventually, it would be patient specific,” maintains Simon MacKenzie, PhD, CEO of Regenhu. “You would take a patient’s own cells into this cell test system, and then you would be able to test the different drugs to see which one works best for that patient.”

Rheumatoid arthritis is an autoimmune disease that involves debilitating inflammation in the joints. The organ-on-a-chip will recapitulate the interactions between the many different elements in the joint environment, including synovial cells, leukocytes, blood vessels, cartilage, and bone. The 3D-printed multicompartment microfluidic platform allows for the movement of synovial fluid through the system, as would happen in the body.

RegenHU is providing a customized 3D bioprinter and design software for the project, as well as training and technical support for the researchers. The company’s newest printers, which constitute the R-Gen series, are designed to provide highly reproducible and accurate printing of different materials while maintaining cell viability.

RegenHU's e R-GEN 100,
RegenHU has developed the R-GEN 100, a tabletop 3D bioprinter. The base instrument accommodates up to five printing tools with individual temperature control capability, as well as four different printing workzones. Features include a vacuum sample mounting system, needle and substrate calibration systems, a process supervision system, and a light curing system for in-process material crosslinking.

R-Gen printers have five separate attachment slots for different printheads, and each can be loaded with a different material and kept at a different temperature. “The temperature that you keep the cells at is critical when you’re printing,” MacKenzie says. “We can now control the temperature of each of the printheads in our system.”

Printheads that use different extrusion technologies can be selected to optimize delivery for materials with different properties. For instance, a mechanical piston might be best for a sticky, viscous material, whereas a pneumatic printhead can reduce shear force and improve the survival of delicate cells. This flexibility, combined with the system’s high precision, enables users to create complex 3D structures involving multiple materials.

“We use laser calibration to define where the tip of the printer is,” MacKenzie notes. “You can change from one printhead to another and print exactly on top of where you printed earlier.”

Another improvement is the ability to stop a print run and adjust conditions for faster optimization. “We have new controls that allow you to change the parameters as the printer is running,” MacKenzie explains. “Instead of optimizating over a week, you can perform one long print run and optimize the pressures and the temperatures—even the printheads.”

Finally, the software has been updated to make it more accessible and user friendly. Previous versions of the system required some knowledge of coding, but the new software, called Shaper, includes design and process management tools that make it easier to customize each project.

“The vision is to go from research-based printing to real fabrication,” MacKenzie declares. “A lot of our customers are trying to do some really complex 3D cell models. We build instruments that have a broad range of applications that can be customized for each of the customers depending on what they want to do.”

Model organs need functional blood vessels

Ultimately, if you want to build a functioning organ, whether for research or biofabrication, that organ needs a blood supply. “Each tissue is different, each has unique cellular components,” says James (Jay) Hoying, PhD, chief scientist of Advanced Solutions Life Sciences (ASLS), a subsidiary of Advanced Solutions. “Also, nearly every tissue has a vasculature, and that’s where our focus has been. How can we build a vascular supply that will work with the cells that make the tissue?”

James Hoying, PhD
James Hoying, PhD
Chief Scientist, ASLS

To do this, Hoying developed Angiomics, a method of growing blood vessels from adipose tissue. “We start with fat and we strip away the fat cells,” Hoying explains. “Fat tissue is highly vascularized, with capillary beds distributed throughout. We get that microvasculature and fragment it.” The resulting microvessels retain the ability to regrow into larger blood vessels once they are back in a tissue environment.

With Angiomics, blood vessels grow in the dish just as they do inside the body. “This is how Mother Nature does it,” Hoying points out. “New vessels grow from existing vessels.” Bioprinting tissue cells adjacent to the microvessels creates an environment in which the new vessels will grow into the tissue, establishing a perfusable microvasculature. “We let the biology take over,” Hoying continues.

He emphasizes that creating lifelike vasculature is particularly important for modeling cancers: “To really capture as much of the tumor biology as you can outside the body, it’s important there’s a vascular supply there.”

ASLS has combined its Angiomics technology with its six-axis 3D-bioprinting hardware to develop an automated cancer tool called the Human Oncology Personal Evaluation (HOPE) platform. Beginning with a biopsy sample, a patient’s tumor can be replicated in the laboratory, complete with functional vasculature, allowing for personalized testing of various therapies to determine which will work best for that patient. When done by hand, this is a slow, labor-intensive process. The HOPE platform automates the system, taking advantage of the precision and reproducibility of robotic operations.

“We’re developing ways to integrate our Angiomics solution to recreate more of the biology, and then our bioassembly tissue fabrication platform is scaling out that activity,” informs Hoying. “Instead of one or two patients benefiting, we’ll be able to benefit maybe a thousand patients, each with their own personalized therapy.”

ASLS is also developing 3D-bioprinted bone grafts, called BioBone, complete with vasculature derived from the patient’s own cells. Starting with a computed tomography scan of the patient’s damaged bone, the platform enables the design of a 3D-bioprinted replacement that perfectly matches the patient’s anatomy. BioBone is in preclinical testing, and ASLS’s bioprinters are being used in laboratories around the world for fabrication of skin, cornea, pancreas, and other body structures.

Michael W. Golway
Michael W. Golway
Chief Executive Officer
ASLS

“That’s been part of our strategy, to get the platform out into the hands of smart, capable scientists who may have dedicated their entire careers to a given organ,” says Michael W. Golway, CEO of ASLS. “Our goal is to enable them with our platform, so they can accelerate the process from research and development to clinical applications.”

Printing with bioink

All of these 3D-printed wonders rely on bioink, which consists of natural or synthetic polymers, to support living cells through the printing process. Depending on what is being printed, a bioink may need to promote cell adhesion, proliferation, or differentiation.

In 2016, Cellink was the first company to commercialize a “universal bioink,” a cellulose-based technology for which the company was granted a U.S. patent last year. “For this field to really take off, there was a need for a standardized material that can ensure repeatable experiments for data collection,” says Erik Gatenholm, Cellink’s co-founder and CEO.

Cellulose was the perfect material to support cartilage cells, which are robust and fibrous. Other tissue types require different characteristics in a bioink, and Cellink now offers an array of bioinks customized to different cell types, including those for skin, bone, pancreas, vascular tissue, and other structures.

“What we’re experts at in our bioprinting division is to mix and match the synthetic and biological components to make an ideal environment for different human cells,” Gatenholm claims. “Today, we are the leading bioprinting company. We have systems in 65 countries, and we provide about 1,800 laboratories with our printers and bioinks.”

Cellink works closely with customers in industry and academia to develop applications. For example, the company has partnered with AstraZeneca for a variety of projects, including bioprinted liver organoids for drug discovery purposes. These organoids are printed with laminin-based bioinks that help create a naturalistic microenvironment to support tissue formation.

In another project, at Uppsala University, researchers are using a Cellink bioink formulation to facilitate bioprinting with human pancreatic beta cells. The bioink was formulated from extracellular matrix protein specific to human pancreas, and the bioprinted cells successfully developed into functional insulin-producing islets.

“Our business model is to be an expertise provider,” Gatenholm states. “We work with each customer to help it develop its specific applications. It’s very collaborative work.”

Now, Cellink is evolving into what Gatenholm calls “a bioconvergence company.” In addition to bioprinters and bioinks, the company is integrating a wider array of technologies to address healthcare challenges. For example, Gatenholm says that bioprinting a tumor would allow the testing of different therapies. But that is just a start, he continues. To really understand the tumor, that is, to know how it arose and how it is spreading, one would need technology that combines robotics, artificial intelligence, and big data tools.

Erik Gatenholm
Erik Gatenholm
Co-Founder & CEO
Cellink

One example of this is a live cell data analysis tool that can monitor tumor growth and make predictions about the effectiveness of different screening compounds. “We’ve developed a lot of different artificial intelligence algorithms,” Gatenholm notes. “We can use them to determine the growth rate for these tumors. Then, when we start screening with different compounds, the algorithms can predict different outcomes.”

As biofabrication technology advances, 3D-printed cells and organs for transplantation are moving from the realm of science fiction into clinical reality. “It’s exciting to see how this community is evolving,” Gatenholm remarks. “We’re proud to be part of that, and thankful to be able to drive that development.”



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