3D-printed blood vessels bring artificial organs closer to reality

Growing functional human organs outside the body is a long-sought "holy grail" of organ transplantation medicine that remains elusive. New research from Harvard's Wyss Institute for Biologically Inspired Engineering and John A. Paulson School of Engineering and Applied Science (SEAS) brings that quest one big step closer to completion.

A team of scientists created a new method to 3D print vascular networks that consist of interconnected blood vessels possessing a distinct "shell" of smooth muscle cells and endothelial cells surrounding a hollow "core" through which fluid can flow, embedded inside a human cardiac tissue. This vascular architecture closely mimics that of naturally occurring blood vessels and represents significant progress toward being able to manufacture implantable human organs. The achievement is published in Advanced Materials.

"In prior work, we developed a new 3D bioprinting method, known as "sacrificial writing in functional tissue" (SWIFT), for patterning hollow channels within a living cellular matrix. Here, building on this method, we introduce coaxial SWIFT (co-SWIFT) that recapitulates the multilayer architecture found in native blood vessels, making it easier to form an interconnected endothelium and more robust to withstand the internal pressure of blood flow," said first author Paul Stankey, a graduate student at SEAS in the lab of co-senior author and Wyss Core Faculty member Jennifer Lewis, Sc.D.

The key innovation developed by the team was a unique core-shell nozzle with two independently controllable fluid channels for the "inks" that make up the printed vessels: a collagen-based shell ink and a gelatin-based core ink. The interior core chamber of the nozzle extends slightly beyond the shell chamber so that the nozzle can fully puncture a previously printed vessel to create interconnected branching networks for sufficient oxygenation of human tissues and organs via perfusion. The size of the vessels can be varied during printing by changing either the printing speed or the ink flow rates.

To confirm the new co-SWIFT method worked, the team first printed their multilayer vessels into a transparent granular hydrogel matrix. Next, they printed vessels into a recently created matrix called uPOROS composed of a porous collagen-based material that replicates the dense, fibrous structure of living muscle tissue. They were able to successfully print branching vascular networks in both of these cell-free matrices. After these biomimetic vessels were printed, the matrix was heated, which caused collagen in the matrix and shell ink to crosslink, and the sacrificial gelatin core ink to melt, enabling its easy removal and resulting in an open, perfusable vasculature.

Moving into even more biologically relevant materials, the team repeated the printing process using a shell ink that was infused with smooth muscle cells (SMCs), which comprise the outer layer of human blood vessels. After melting out the gelatin core ink, they then perfused endothelial cells (ECs), which form the inner layer of human blood vessels, into their vasculature. After seven days of perfusion, both the SMCs and the ECs were alive and functioning as vessel walls -- there was a three-fold decrease in the permeability of the vessels compared to those without ECs.

Finally, they were ready to test their method inside living human tissue. They constructed hundreds of thousands of cardiac organ building blocks (OBBs) -- tiny spheres of beating human heart cells, which are compressed into a dense cellular matrix. Next, using co-SWIFT, they printed a biomimetic vessel network into the cardiac tissue. Finally, they removed the sacrificial core ink and seeded the inner surface of their SMC-laden vessels with ECs via perfusion and evaluated their performance.

Not only did these printed biomimetic vessels display the characteristic double-layer structure of human blood vessels, but after five days of perfusion with a blood-mimicking fluid, the cardiac OBBs started to beat synchronously -- indicative of healthy and functional heart tissue. The tissues also responded to common cardiac drugs -- isoproterenol caused them to beat faster, and blebbistatin stopped them from beating. The team even 3D-printed a model of the branching vasculature of a real patient's left coronary artery into OBBs, demonstrating its potential for personalized medicine.

"We were able to successfully 3D-print a model of the vasculature of the left coronary artery based on data from a real patient, which demonstrates the potential utility of co-SWIFT for creating patient-specific, vascularized human organs," said Lewis, who is also the Hansjörg Wyss Professor of Biologically Inspired Engineering at SEAS.

In future work, Lewis' team plans to generate self-assembled networks of capillaries and integrate them with their 3D-printed blood vessel networks to more fully replicate the structure of human blood vessels on the microscale and enhance the function of lab-grown tissues.

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Space-trekking muscle tests drugs for microgravity-induced muscle impairment

A gentle rumble ran under Ngan Huang's feet as a rocket carrying her research -- live, human muscle cells grown on scaffolds fixed on tiny chips -- lifted off, climbed, and disappeared into the sky to the International Space Station National Laboratory. These chips would help Huang better understand muscle impairment, often seen in astronauts and older adults, and test drugs to counter the condition.

Now, the results are back. Reporting in a study published July 25 in Stem Cell Reports, Huang's team showed that space-travelling muscle had metabolic changes that indicate impaired muscle regeneration and gene activities associated to age-related muscle loss called sarcopenia. But drug treatment partially prevented microgravity's adverse effects.

"Space is a really unique environment that accelerates qualities associated with aging and also impairs many healthy processes," says Huang, an associate professor at Stanford University. "Astronauts come back with muscle atrophy, or a reduction of muscle function, because the muscle isn't being actively used in the absence of gravity. As space travel becomes more common and available to civilians, it's important to understand what happens to our muscle in microgravity."

To understand the effects of microgravity on muscles, the researchers launched muscle chips -- bioengineered packages of oriented muscle cells on patterned biomaterials that mimic the structure of real muscles -- into space to grow for seven days under astronauts' care.

When the researchers compared muscle cells grown in microgravity to those grown on Earth, they found impaired muscle fiber formation. They also discovered differences in their gene activity and protein profile. Genes related to mitochondrial function, which muscles get their energy from, were compromised, and genes associated with fat formation were boosted. These findings suggest microgravity can lead to dysfunctions in muscle regeneration.

Space-traveled muscles also have gene activities that somewhat resemble muscles with sarcopenia, which most commonly affects people ages 60 and older.

"We think our research on muscle chips in microgravity may have broader implications on sarcopenia," says Huang. "Sarcopenia usually takes decades to develop on Earth, and we think that microgravity may have some ability to accelerate the disease process in orders of days."

In a proof-of-concept experiment to test the muscle chip for drug screening, the astronauts spiked the chips with drugs to treat sarcopenia or enhance muscle regeneration. The treatment partially mitigated some of the negative effects of microgravity on the muscles, preventing a metabolic shift to fat formation. Looking into gene activity patterns, the drug-treated muscle in microgravity is more similar to samples from Earth than untreated samples in microgravity.

Because space research is labor and resource intensive, the current study is a one-time experiment, and a limited number of samples were allowed to board the rocket. The scientists are now deploying equipment that simulates microgravity to overcome some of those limitations and aid their research in space. Huang's muscle chips are scheduled to embark on another space journey in 2025 to continue the research on identifying drugs for treating microgravity-induced impairment in muscle regeneration.

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New drug-like molecule extends lifespan, ameliorates pathology in worms and boosts function in mammalian muscle cells

Having healthy mitochondria, the organelles that produce energy in all our cells, usually portends a long healthy life whether in humans or in C. elegans, a tiny, short-lived nematode worm often used to study the aging process. Researchers at the Buck Institute have identified a new drug-like molecule that keeps mitochondria healthy via mitophagy, a process that removes and recycles damaged mitochondria in multicellular organisms. The compound, dubbed MIC, is a natural compound that extended lifespan in C. elegans, ameliorated pathology in neurodegenerative disease models of C. elegans, and improved mitochondrial function in mouse muscle cells. Results are published in the November 13, 2023, edition of Nature Aging.

The impact of mitochondrial dysfunction in age-related disease

Defective mitophagy is implicated in many age-related diseases. It's tied to neurodegenerative disorders such as Parkinson's and Alzheimer's; it plays a role in cardiovascular diseases including heart failure; it influences metabolic disorders including obesity and type 2 diabetes; it is implicated in muscle wasting and sarcopenia and has a complex relationship with cancer progression. Even though interventions that restore mitophagy and facilitate the elimination of damaged mitochondria hold great promise for addressing these conditions, not one treatment has been approved for human use despite advances in the field.

What's MIC?

MIC (Mitophagy-Inducing Compound) is a coumarin, which are naturally bioactive compounds that have anticoagulant, antibacterial, antifungal, antiviral, anticancer, and antihyperglycemic properties (among others) as well as being an antioxidant with neuroprotective effects. Coumarin is found in many plants and is found in high concentrations in certain types of cinnamon, which is one of the most frequent sources for human exposure to the substance.

A new mechanism of action in mitophagy

The project started in a mouse model of Parkinson's disease where researchers in the laboratory of Julie Andersen, PhD, a senior author of the paper, were looking at known enhancers of mitophagy, including rapamycin. "Co-author Shankar Chinta, PhD, started screening natural compounds in neuronal cells and MIC came up as a major hit," she said. "Rather than taking MIC immediately into a mouse model we wanted to understand its impact on overall aging and identify its mechanism of action, so we took the work into the worm where we found that MIC is in a different class of molecules that enhance the expression of a key protein, TFEB."

In an effort spearheaded by Andersen and research scientist Manish Chamoli, PhD, lead author of the study, researchers found that MIC enhanced the activity of transcription factor TFEB, which is a master regulator of genes involved in autophagy and lysosomal functions. Autophagy is the intracellular recycling process whereby cells clean up damaged proteins; it derives its abilities from the lysosome. Researchers found that MIC robustly increased the lifespan of C. elegans while also preventing mitochondrial dysfunction in mammalian cells.

"This paper helps support the overall notion of TFEB being a key autophagy regulator that extends lifespan, "said Buck professor and Chief Scientific Officer Malene Hansen, PhD, who collaborated on the paper. She added, "Mitophagy is a selective and very significant form of autophagy. The field has recognized TFEB as a player when it comes to quality control in mitochondria. This study provides a possible translational route to induce mitophagy in a TFEB-dependent fashion."

A link to the brain/gut connection

Mechanistically MIC works upstream of TFEB by inhibiting ligand-induced activation of the nuclear hormone receptor DAF-12 (in worms)/FXR (in humans), which in turn induces mitophagy and extends lifespan. FXR is best known for its ability to act in the liver and gut to maintain lipid homeostasis, where it acts to regulate levels of TFEB as part of a feed-fast cycle, but recently TFEB was shown to also be present in brain neurons. This provided Andersen with the clue needed to piece together MIC's potential mechanism of action in the latter. "This study provides another piece of the puzzle when it comes to understanding the brain/gut connection in terms of health and disease," said Andersen.

FXR is regulated by bile salts which are formed in the gut microbiome. "The gut microbiome impacts the body's use of bile acids. Aging impacts our microbiome," said Chamoli. "If levels of bile acids aren't correct it hinders mitophagy. That's how FXR can impact neuronal health. Neurons have a lot of mitochondria which makes mitophagy important in terms of neurodegeneration," he said, noting that experiments are underway to explore neuronal FXR as a therapeutic target for Alzheimer's disease as part of a jointly funded grant shared by the Andersen and Lithgow labs.

MIC as a general geroprotective therapeutic

"There's a bottleneck in efforts to develop potential therapeutics in the field of geroscience, and the bottleneck is that we don't have enough molecules in the pipeline," said Gordon Lithgow, PhD, Buck Professor and Vice President of Academic Affairs and senior co-author. "MIC is a great candidate to bring forward given its therapeutic effect across multiple models and the fact that it is a naturally occurring molecule."

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