December 6, 2019 PAP-Q4-19-CL-018
According to the American Transplant Foundation, in the United States alone, over 113,000 people are on the waiting list for an organ transplant needed to save their lives.1 Twenty of these people per day die due to the lack of available organs for transplant. Many more people do not even qualify as candidates for the organ transplant waiting list due to their age, or weight or because they have an active infection, heart disease, cancer or other illness. Overall, liver and kidney diseases, which can often be overcome with organ transplantation, cause more deaths than Alzheimer’s disease, breast cancer or prostate cancer.
Bioprinters are similar to the 3D printers used to produce plastic and metal components. A number of academic groups and specialty biotech firms are already pursuing development of 3D bioprinting technologies for use in the construction of cartilage, bone, skin, blood vessel, muscle, brain, heart, kidney, liver, cornea, lung and other tissues.2,3 Bioprinted organoids and organ-like structures are already aiding drug discovery and development efforts.4,5 3D printed organs also show potential for use by surgeons to rehearse surgeries in advance.6 However, scientists, engineers and programmers see a convergence of cell biology, physics and bioinformatics that will enable the production of fully functional organs that could be used in transplantation to make organs widely available to all in need.
Accomplishing this will be no small feat, but it is envisioned that the process will first involve establishing the blueprint for the organ in silico using bioimaging and computer-aided design (CAD drawings). Next, the organ will be assembled according to the blueprint by delivering the bioinks — protein scaffolds, cells and other materials — via a bioprinter using advanced software systems. At that point, the nascent organ could be placed in a bioreactor to mature into a final, functioning product.
Key to the process will be the bioinks. If stem cells or the patient’s own induced pluripotent stem cells (iPSCs) can be effectively deployed, then safety and functionality are likely to be improved. Additionally, the use of natural, high-performing “scaffolds” (like collagens, laminins and elastins) that can “talk” to the cells and interact in a way to facilitate the proper maturation of the organ will likely be essential to delivering consistently high-performing bioprinted organs that can be produced at commercial scale.
It is expected that bioprinted tissues will sustain several advantages over conventional tissue-engineering methods for organ manufacturing.7 Perhaps most importantly, 3D bioprinting will allow a more precise placement of components and a maturation process that will enable the construction of configurations similar to those of actual human organs.
Some of the earliest work on bioprinting took place at Wake Forest Institute for Regenerative Medicine, where researchers developed the first 3D bioprinters (Integrated Tissue and Organ Printing System (ITOP)) and bioprinted the first bladder, which was implanted in a human patent in 2006. In 2016, they reported successfully implanting ear, bone and muscle structures into animals that matured into functional tissue and sprouted new systems of blood vessels.8
The number of groups focused on bioprinting has exploded since then. In the last 12–14 months alone, there have been reports on the bioprinting of a miniature heart and a vascularized heart, both from patient cells,9,10 ligaments and tendons, also from patient cells,11 artificial blood vessels with programmable rigidity control,12 a hydrogel model of a functional, lung-mimicking air sac,13 3D corneal transplants comprising corneal endothelial cells,14 a spinal cord implant that restored neural functioning,15 brain tissue with a functional neural network16 and tubules that simulate kidney function and can help reintroduce beneficial molecules into the bloodstream.17
Researchers have also constructed a 3D bioprinter that can function upside down in a zero-gravity environment for astronauts to potentially use — along with stem cells, plasma and a plant-matter mixture — to produce skin and bone tissue during the ESA Mars mission if there is a need to treat injuries.18
Bioprinted organs are highly complex structures with specific vasculature for blood and oxygen flow comprising multiple cell types that communicate with one another. Developing 3D printing methods that enable manipulation of the biological materials without damage or degradation and result in functioning organs remains a significant challenge.
The choice of printing method is generally dictated by the bioink formulation, the desired printing speed and the structural design.19 The three main methods of bioprinting include extrusion-based, inkjet-based and laser-assisted bioprinting. In extrusion-based bioprinting, structural and cellular layers are printed in alternating fashion. With this technique, however, there is no special control of cellular deposition, and shear stress can impact cell viability. Inkjet and laser-guided bioprinting provide greater spatial control, but the heat generated in these processes can damage the cells, leading to reduced cell viability. Inkjet printing is rapid but requires inks with low viscosity, while laser-assisted printers can tolerate highly viscous inks.
Other methods include stereolithography, which provides high resolution but is a slow process and can impact cell viability. Fused-deposition modeling produces porous structures but is a high-heat process not suitable for cellular formulations. Selective laser sintering is useful for making complex structures, but also involves heat and is not suitable for cell-based bioinks.
Several research groups have been focused on developing improved methods for organ and tissue bioprinting.
A new process developed by scientists at Vienna University of Technology that relies on two-photon polymerization allows for the creation of very fine structures with high precision at a speed of greater than one meter per second and is compatible with cellular mataterial.20 The SWIFT (sacrificial writing into functional tissue) technique created by researchers from Harvard’s Wyss Institute for Biologically Inspired Engineering and John A. Paulson School of Engineering and Applied Sciences (SEAS) enables 3D printing of vascular channels into living matrices composed of stem cell–derived organ building blocks (OBBs), yielding viable, organ-specific tissues with high cell density and function.21
The suspended layer additive manufacturing (SLAM) technique developed at the University of Birmingham uses low-viscosity biopolymers in a self-healing fluid gel matrix to generate soft materials in very fine detail,22 while the new open-source technology referred to as stereolithography apparatus for tissue engineering (SLATE) from Rice’s Brown School of Engineering allows the recreation of the complex vascular networks that mimic the body’s natural passageways for blood, air, lymph and other vital fluids.23 Meanwhile, the freeform reversible embedding of suspended hydrogels (FRESH) extrusion-based bioprinting technology from scientists at Carnegie Mellon University allows fabrication of collagen scaffolds capable of replicating the structure and function of tissues and organs.24
Scientists at the Friedrich-Alexander-University Erlangen-Nürnberg focused on the bioprinter itself, modifying an off-the-shelf 3D desktop printer that can be installed within a single day, fits into standard laminar flow hoods and is customizable and affordable.25
Bioinks for 3D bioprinting can contain a variety of different ingredients. Some may be based only on polymers of proteins intended to form the scaffold for the tissue or organ (cells are added later). Others may contain the cells and components that aid growth and maturation (scaffold-free approach). Yet others may include a combination of structural and cellular materials (cell–scaffold-based approach).26
Regardless, all biomaterials in a bioink must be printable and have appropriate mechanical, thermal and other physical properties, plus be biocompatible and retain their bioactivity after printing.27
The structrucal framework can be produced using structural bioinks containing synthetic polymers, including polyethyleneglycol (PEG), gelatin methacrylol (GelMA) and Pluronic® or natural proteins, such as collagen, gelatin, hyaluronic acid, silk, alginate, agarose, fibrin, fibronectin, elastin, laminin and other decellularized extracellular matrix (ECM)-based materials. 26–29 Other acellular materials can also be used to provide structural support and sites for cell attachment and to impart porosity. Examples include chitosan, nanocellulose and poly(lactic acid) (PLA), and polycaprolactone (PCL), among others.30
Cell-encapsulating hydrogels allow the creation of living tissue structures with precise control over the attachment and spacial distribution of the cells and other biomolecules in the scaffold.30 Natural biopolymers are preferable for 3D bioprinting of organs and tissues because they can communicate with cells and readily undergo reorganization of the ECM as needed.26
Sacrificial bioinks are deposited and then removed to create channels that enable the formation of vascular networks. They are often water soluble or degrade under specific conditions.27
Functional bioinks contain compounds that help direct the formation of the tissue or organ. They often contain growth factors and compounds that enable cell differentiation. Tissues that are implanted before they are fully developed often require external support, which is provided through the use of supportive bioinks that generate protective lattices produced from polymeric materials, such as poly(lactic-co-glycolic acid) (PLGA).26,27
Much research to date has focused on collagen, but there is growing recognition that optimized combinations of different proteins will be needed to mimic the multifaceted ECM. Future bioinks may enable 4D bioprinting — or the printing of biomaterials that respond to external stimuli in some way, such as changing their shape, structure or function.27
Given the rapid progress and high level of funding taking place in the bioprinting field, market research firm IDTechEx predicts that the value of the global market for 3D bioprinting — including hardware, software, ingredients and services — will reach $1.9 billion by 2028.31 Notably, this value only includes bioprinting that involves the deposition of living cells in a spatially controlled manner in the absence of any pre-existing scaffold.
Initial revenues will be generated by applications in product development and testing for cosmetics, consumer goods and drugs, while, in the longer term, regenerative medicine, cell-based biosensors and food production will become important. Regenerative medicine has the potential to be the largest application for 3D bioprinting in the future.31
Production of recombinant proteins for the formulation of bioinks used in the 3D printing of tissues and organs is an important strategic growth area for iBio. The iBio plant-based FastPharmingTM Expression System is ideally suited to the production of bioinks, maturogens and other biologics for use in 3D bioprinting. Green plants are grown hydroponically in chemically defined media in specially designed, climatically controlled indoor grow rooms. Following a short growth period, the plants are exposed to a mild vacuum with leaves submerged in a solution of agrobacteria containing the engineered vector with the appropriate viral replicon. The plants absorb the agrobacteria through their stoma to equilibrate pressure when the vacuum is released. With this method, gram quantities of target protein in the plant leaves per kilogram of fresh plant tissue can be produced within approximately 8 weeks.
iBio’s plant-based production platform offers a better safety profile than animal systems, and the automated hydroponics system presents reduced variability compared with soil-grown plant-made pharmaceuticals (PMPs). Additionally, the platform facilitates customization of glycosylation, allowing better glycosylation controls compared with bacteria (which do not glycosylate proteins), yeast (which hyperglycosylates) and Chinese hamster ovary or myeloma mammalian lines (which fail to precisely mimic human glycosylation patterns).
Due to the cost-effectiveness of our plant-based platform combined with its high yields, inherent scalability and tighter process control, the FastPharming System has great potential for facilitating the development and commercialization of bioprinted tissues and organs. Simplification of protein production allows rapid manufacture of molecules for R&D work. Processes can then be readily scaled to GMP quantities for clinical testing and commercial production without the scale-up issues that plague other, bioreactor-requiring production methods.
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Kent, Chloe. “The future of bioprinting: A new frontier in regenerative healthcare.” Medical Device Network. 10 Jun. 2019. Web.
Mazzocchi, Andrea, Shay Soker and Aleksander Skardal. “3D bioprinting for high-throughput screening: Drug screening, disease modeling, and precision medicine applications.” Applied Physics Reviews. 6: 011302 (2019).
Higgins, J. William et al. “Bioprinted pluripotent stem cell-derived kidney organoids provide opportunities for high content screening.” bioRxiv. https://doi.org/10.1101/505396 (2018).
“TAU scientists print first ever 3D heart using patient’s own cells.” Tel Aviv University. 16 Apr. 2019. Web.
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Scott, Clare. “Wake Forest Researchers Successfully Implant Living, Functional 3D Printed Human Tissue Into Animals.” 3D Print.com. 16 Feb. 2016. Web.
Coxworth, Ben. “Scientists create world’s first patient-specific vascularized 3D-printed heart.” New Atlas.com. 15 Apr. 2019. Web.
Coxworth, Ben. “Fully-formed, tiny heart is 3D bioprinted from human donor’s cells.” New Atlas.com. 10 Sep. 2019. Web.
Ahmed, Rawal. “Bioprinted Tubules Simulate Kidney Functions & Diseases.” 3D Printing.com. 5 Mar. 2019. Web.
Ahmed, Rawal. “Bioprinting Technique Could Create Artificial Blood Vessels.” 3D Printing.com. 31 Oct. 2018. Web.
Organ bioprinting gets a breath of fresh air. Unviersity of Washington. 2 May 2019. Web.
Zeldovich, Lina. “Biomedical Engineers 3D Print Corneal Transplants to Treat Eye Diseases.” AABNE News. 4 Mar. 2019. Web.
Zeldovich, Lina. “3D-Printed Implant Heals Severed Spinal Cord.”AABNE News. 11 Feb. 2019. Web.
Ahmed, Rawal. “Researchers Bioprint Brain Tissue Creating Functional Neural Network. ” 3D Printing.com. 20 Oct. 2018. Web.
Ahmed, Rawal. “Bioprinted Tubules Simulate Kidney Functions & Diseases. 3D Printing.com. 5 Mar. 2019. Web.
Ahmed, Rawal. “Mars Crew 3D Printing Skin & Bone Tissue in Space.” 3D Printing.com. 10 Jul. 2019. Web.
“3D Bioprinting: Bioink Selection Guide,” MilliporeSigma. n.d. Web.
Bioprinting Living Cells Extremely Fast and at Very High Resolution in a 3D Printer. Vienna University of Technology. 26 Oct. 2019. Web.
Skylar-Scott, Mark A. et al. “Biomanufacturing of organ-specific tissues with high cellular density and embedded vascular channels.” Science Advances. 6 Sep. 2019. Web.
Vialva, Tia. “University of Birmingham researchers develop SLAM 3D bioprinting method,” 3D Printing Industry. 3 Oct. 2019. Web.
Papadopoulos, Loukia. “New Breakthrough Method for 3D Bioprinting Organs Conceived.” Interesting Engineering. 5 May 2019. Web.
Lee, A. et al. “3D bioprinting of collagen to rebuild components of the human heart.” Science.. 365: 482–487 (2019).
Kahl, Melanie et al. “Ultra-Low-Cost 3D Bioprinting: Modification and Application of an Off-the-Shelf Desktop 3D-Printer for Biofabrication.” Front. Bioeng. Biotechnol. 31 Jul. 2019. Web.
“Give me the best bioink! | A short guide to the currently available bioinks.” Biogelx. 29 Oct. 2018. Web.
Gungor-Ozkerim, P. Selcan et al. “Bioinks for 3D bioprinting: an overview.” Biomater. Sci. 6: 915–946 (2018).
“Five Types of Bioinks.” Cellink. 26 Apr. 2017. Web.
Chameettachal, Shibu et al. “Tissue/organ-derived bioink formulation for 3D bioprinting,” Journal of 3D Printing in Medicine. 5 Mar. 2019. Web.
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Tsao, Nadia. 3D Bioprinting 2018 - 2028: Technologies, Markets, Forecasts. Rep. IDTechEx. 2018. Web.
Robert Erwin has been President of iBio, Inc. since 2008. He previously was Managing Director of Bio-Strategic Directors, CEO of Large Scale Biology Corporation, and Chairman of Icon Genetics AG. He is currently Chairman of Novici Biotech and a Director of Oryn Therapeutics. He was a member of the Cancer Policy Forum of the Institute of Medicine and a clinical advisor to the California Institute for Regenerative Medicine. Mr. Erwin received B.S. and M.S. degrees in Zoology and Genetics from Louisiana State University.