Bioprinting is back in the news again as a team of scientists at Scotland’s Heriot Watt University and Roslin Cellab reported the February 4 issue of the journal Biofabrication described processes to create 3D stem cell spheroids using a new "valve-based" printing technology.
But researchers have been trying to create 3D tissues for awhile using bioprinting approaches. Academic laboratories and companies are using several technologic adaptations using the principles of bioprinting to develop tissues for regenerative medicine or replacements for damaged tissues.
While 3D printing is routinely applied to the direct digital manufacture (DDM) of a wide variety of plastic and metal items, scientists have been working on extending its principles to constructing organs and tissues using bioprinters. Bioprinters artificially construct living tissue by outputting layer-upon-layer of living cells where required, thus building an organic object in multiple thin layers.
3D Models with 100% Human Tissue
Organovo, a San Diego-based bioprinting company, bases its technology on inventions from the laboratory of its scientific founder, Gabor Forgacs, Ph.D., currently director of the Frontiers of Integrative Biological Research Program at the University of Missouri, Columbia. His laboratory has been conducting research in the physical mechanisms of early development using both experimental and modeling approaches.
In March 2008, the company reported that it had bioprinted functional blood vessels and cardiac tissue using cells obtained from a chicken. Their work relied on a prototype bioprinter with three print heads, the first two of which output cardiac and endothelial cells respectively while a third dispensed a collagen scaffold—termed “bio-paper”—to support the cells during printing.
Since then, the company has developed its NovoGen MMX in collaboration with Invetech, the first bioprinter developed for commercial-scale bioprinted tissue manufacture. Last December, the company announced that it had partnered with Autodesk, a cloud-based design and engineering software, to create the first 3D design software for bioprinting.
The software, which will be used to control Organovo's bioprinter, will represent, the company says, a major step forward in usability and functionality for designing three-dimensional human tissues, and has the potential to open up bioprinting to a broader group of users.
To create a bioimprinted tissue or organ, the NovoGen MMX works by first laying down a layer of a water-based hydrogel. The second step of the process involves layering “bio-ink” spheroids composed of cells, to build layers, along with additional hydrogel to hold the bioprinted ink(s) in place. The bio-ink spheroids fuse together and the hydrogel dissolves or is otherwise removed, thereby leaving a final bioprinted tissue. The bioprinted construct is then cultured under conditions that allow it to develop into a specific tissue.
Michael Renard, executive vice president of commercial operations at Organovo, told GEN that the system is unique in being able to produce completely “human” 3D tissues, without artificial scaffolds or any other long-lasting biomaterials in the final composition. “We are the only commercial enterprise working with this bioprinting technology to build functional human tissues. What distinguishes us is that we are creating 100% human tissue, without decellularized supports, scaffolding or introduced collagens. We are taking cells, printing them, then growing them and letting the native biology help to define and ultimately create the completely human tissue.”
While still a development stage business, Organovo has successfully met its first goal, developing disease models for drug discovery for pharmaceutical partners, currently including Pfizer and United Therapeutics.
Organovo is also pursuing a second line of business as it intends to market a variety of 3D bioprinted tissues to pharma companies and CROs for drug safety and efficacy testing. “Principally,” Mr. Renard says, “Human cells growing in two dimensions are currently used, followed by companies moving to animals to mimic a three-dimensional model.” And, he notes, “Animals don’t always have a great human predictive value.”
As for industry acceptance of the bioprinted 3D models, “We believe the market is aware that better models are needed earlier in the drug development process. Scientists say if you can show me a bio-relevant and predictive model, I will be open to adopting it. When we bring the characteristics of our tissues to scientists and show them the architecture, alignment, production of extracellular matrix, cellular and biochemical metabolism, they are very enthusiastic.”
The bioprinted tissues also maintain their expected biochemical activity longer than many 2D models, as indicated by specific enzyme and gene expression activity indicative of tissue specific metabolic function. This kind of information serves as a compelling set of evidence to share and allow scientists to evaluate a new product category.
Valve-Based Cell Printing
And in the February 4 issue of Biofabrication, scientists at Herriot-Watt University and Roslin Cellab, a stem cell technology company, reported that they had developed a new technique allowing them to deposit consistently-sized droplets containing living cells that survived the process and that could then develop into mature cell types. Previously, human embryonic stem cells (hES cells) had proven too fragile to survive the bioprinting process.
The investigators said they had set out to develop controllable and less harmful bioprinting processes, in order to preserve cell and tissue viability and functions. They reported the development of a valve-based cell printer validated, they said, to print highly viable cells in programmable patterns from two different bio-inks with independent control of the volume of each droplet (with a lower limit of 2 nL or fewer than five cells per droplet). Human hESCs were used to make spheroids by overprinting two opposing gradients of bio-ink: one of hESCs in medium, and the other of medium alone.
The Heriot-Watt and Roslin Cellab scientists developed a printing system driven by pneumatic pressure and controlled by the opening and closing of a microvalve. The researchers could precisely control the amount of cells dispensed by changing the nozzle diameter, the inlet air pressure or the opening time of the valve
The valve-based cell-printing processes delivered cells, the researchers said, in specific patterns in volumes as low as 2 nL or less than five cells per droplet. The investigators say they will subject the printed hES cells to directed differentiation protocol to produce human hepatocyte-like cells.
These innovations may bring bioprinted, fully human tissues developed for clinical use and regenerative medicine closer to reality.