Harvard Researchers Create Stronger, Thicker, 3D Printed Vascular Tissues That Live Up to Six Weeks


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Jennifer A. Lewis

As spectacular new scientific processes like bioprinting begin to overshadow many of the other innovations in 3D printing that are currently also transforming many different industries, it’s easy to look ahead to so many fantastic predictions for the future–such as the fabrication of organs for transplants—causing us to lose sight overall of how difficult today’s tasks actually are; for instance, researchers today may have accomplished what was once unimaginable in terms of creating cellular structures via 3D printing—but once the excitement settled, they realized it was very difficult to keep them alive.

Whether they are contained within the human body or outside of it, 3D printed cells require an enormously complex system of nutrition. Great headway is being made on this front though, and we’re following all of it, with recent strides being made with innovations like new bioprinting technology and the actual creation of blood vessels.

And while so much is happening on the bioprinting front it’s often challenging to keep up, one thing is for sure: this is a fascinating area of discovery. Now, researchers at the Wyss Institute for Biologically Inspired Engineering at Harvard University and the Harvard John A. Paulson School for Engineering and Applied Sciences (SEAS) have developed a method for bioprinting stronger structures in the form of thick vascularized tissue. These constructs are made up of quite the recipe too: human stem cells, an extracellular matrix, and circulatory channels lined with endothelial blood vessel cells.

All of these new processes have been outlined and just published in the Proceedings of the National Academy of Sciences, in the paper ‘Three-dimensional bioprinting of thick vascularized tissues,’ by David B. Kolesky, Kimberly A. Homan, Mark A. Skylar-Scott, and Jennifer A. Lewis.

UntitledAlthough this is certainly an area of great study and ongoing work by many researchers, until now the methods for the ‘scaling up of human tissues’ made from a range of cells have simply been too weak for sustainability in the lab. Lewis and her team report that they have been able to make this new fabricated tissue stronger by ten times, offering a viable foundation for continuing in the area of tissue engineering.

“This latest work extends the capabilities of our multi-material bioprinting platform to thick human tissues, bringing us one step closer to creating architectures for tissue repair and regeneration,” says Jennifer A. Lewis, Sc.D., senior author on the study.

Through vascular ‘plumbing’ which includes both the living cells and extracellular matrix, the scientists are able to create tissue which is sustainable—in fact, they have now been able to offer these structures as viable and functioning for a stunning and unprecedented timeframe of up to six weeks so far. And the secret is, of course, all in the materials.

“Central to the fabrication of thick vascularized tissues is the design of biological, fugitive, and elastomeric inks for multimaterial 3D bioprinting,” states the team in their paper.

Regarding the ink and the matrix, they go on to state that with their new approach, arbitrarily thick tissues can be bioprinted successfully as the extracellular matrix does not require UV curing and can also be ‘readily expanded’ to other biomaterials too, such as fibrin and hyaluronic acid.

So far, the team has been successful in bioprinting with tissue that is one centimeter thick. They were able to pump bone growth factors through the structures (actually lined with the same endothelial cells found in our blood vessels) allowing for development of cells in a four-week duration.

This new bioprinting process is accomplished with a customized silicone mold used as a vehicle for housing and ‘plumbing’ the 3D printed tissue. The researchers were able to print the network of vascular channels, and then next layer live stem cells over that. With each layer, the structure grows and in this process, becomes strong enough to ‘live.’

“This research will help to establish the fundamental scientific understanding required for bioprinting of vascularized living tissues. Research such as this enables broader use of 3D human tissues for drug safety and toxicity screening and, ultimately, for tissue repair and regeneration,” explains Zhijian Pei, National Science Foundation Program Director for the Directorate for Engineering Division of Civil, Mechanical and Manufacturing Innovation, which funded the project.

Strength and sustainability in the inks is what makes a substantial difference, allowing for a network of intersections with vascular pillars, microvessels, and post-printing, a cellular liquid which fills in any open regions and links the entire structure together. The result is a structure of soft tissue with the required blood vessels. Both an entry and exit are available on the ‘chip,’ allowing—in an absolutely crucial process–for the proper nutrients necessary to keep the cells alive.


From the researcher’s paper: (A) Schematic illustration of the tissue manufacturing process. (i) Fugitive (vascular) ink, which contains pluronic and thrombin, and cell-laden inks, which contain gelatin, fibrinogen, and cells, are printed within a 3D perfusion chip. (ii) ECM material, which contains gelatin, fibrinogen, cells, thrombin, and TG, is then cast over the printed inks. After casting, thrombin induces fibrinogen cleavage and rapid polymerization into fibrin in both the cast matrix, and through diffusion, in the printed cell ink. Similarly, TG diffuses from the molten casting matrix and slowly cross-links the gelatin and fibrin. (iii) Upon cooling, the fugitive ink liquefies and is evacuated, leaving behind a pervasive vascular network, which is (iv) endothelialized and perfused via an external pump. (B) HUVECs growing on top of the matrix in 2D, (C) HNDFs growing inside the matrix in 3D, and (D) hMSCs growing on top of the matrix in 2D. (Scale bar: 50 µm.) (E and F) Images of printed hMSC-laden ink prepared using gelatin preprocessed at 95 °C before ink formation (E) as printed and (F) after 3 d in the 3D printed filament where actin (green) and nuclei (blue) are stained. (G) Gelatin preprocessing temperature affects the plateau modulus and cell viability after printing. Higher temperatures lead to lower modulus and higher HNDF viability postprinting. (H) Photographs of interpenetrated sacrificial (red) and cell inks (green) as printed on chip. (Scale bar: 2 mm.) (I) Top-down bright-field image of sacrificial and cell inks. (Scale bar: 50 µm.). (J–L) Photograph of a printed tissue construct housed within a perfusion chamber (J) and corresponding cross-sections (K and L). (Scale bars: 5 mm.)

Also, employing one of the greatest benefits of 3D printing, the researchers can customize these silicone chips, or molds, to be made in any variety of shape, width, and composition, meaning they can fabricate other cell types.

“Jennifer and her team are shifting the paradigm in the field of tissue engineering based on their unique bioprinting approach. Their ability to build living 3D vascularized tissues from the bottom-up provides a potential way to form macroscale functional tissue replacements that can be surgically connected to the body’s own blood vessels to provide immediate perfusion of these artificial tissues, and thus, greatly increase their likelihood of survival. This would overcome many of the problems that held back tissue engineering from clinical success in the past,”  said Wyss Institute Founding Director Donald Ingber, MD, PhD.

With prefabricated vasculatures, these researchers are able to improve the functioning of the bioprinted cells at their most central point—allowing them to exert influence over what the cells do with substances like growth factors.

Reflecting on this very successful study and research project, the scientists stated that with the ability to make these 3D printed tissues, the ‘exploration of emergent biological phenomena’ is made possible.

“Our 3D tissue manufacturing platform opens new avenues for fabricating and investigating human tissues for both ex vivo and in vivo applications,” concludes the team.

Below is a short movie showing the 3D bioprinting of the thick, vascularized tissues. What do you think of these new bioprinting discoveries? Discuss in the 3D Printed Thicker Vascular Tissues forum over at 3DPB.com.

[Source: Medical Press]


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