In memory of Jemma Redmond
My journey in this industry started over ten years ago as a young wide-eyed regenerative medicine master’s student at NUI Galway, that suddenly became an “extrusion 3D bioprinting maximalist and deep technology entrepreneur” during my bioengineering British Heart Foundation-funded Ph.D. research scholarship at Imperial College London. Imperial is ranked third in Europe and seventh in the world1, but most importantly it is a vibrant hub of deep technology innovation that inspires students, researchers, and professors to become entrepreneurs. After watching the industry grow over the years it is finally becoming clear to me that extrusion 3D bioprinting technologies are mostly useful for entry-level tissue engineering research and development rather than realistic applications in regenerative medicine. More advanced multiplexed 3D biofabrication technologies (electric, magnetic, microfluidic, volumetric, & acoustic) in combination with non-animal derived or enhanced biomaterials (human, synthetic, 4D self-assembly, & cyborganic) are needed to overcome extrusion 3D bioprinting’s many limitations, fix a stagnating industry, and finally create realistic applications for all of regenerative medicine.
I always advise anyone in the industry to read the publications and proof surrounding the different technologies’ potential in real tissue formation before buying. Especially when it comes to using stem cells that are more sensitive to biomechanical changes and biomaterial properties. Not only do they need to remain viable it is essential that they maintain their multi/pluripotent characteristics during and after the 3D biofabrication process to ever create real 3D tissues and organs. Some basic technologies have many limitations & cannot create viable high throughput tissues or organs but are important for entry into the tissue engineering sector. Organ-on-a-chip technology which is within the realm of 3D biofabrication dominates in terms of creating high throughput 3D tissue models for important applications such as drug discovery and potential animal model replacement. Some of the limitations of extrusion 3D bioprinting in regenerative medicine applications include low cell viability, slow speeds, cell death from deformation caused by shear stress, & lack of cellular properties needed for complex tissue formation after the process. Newer advanced technologies can overcome these limitations and have better chances of success in regenerative medicine.
Ourobotics: early years of extrusion bioprinting
I remember years ago when I was walking through the bioengineering labs of Imperial College London I was becoming frustrated at trying to make fully structural 3D vessels with all cell layers for a novel bioreactor to study atherosclerosis under pulsatile & transmural flow conditions. In the early years of my Ph.D. in 2011-2012 copolymer synthesis, biomaterial molding, hydrogel formation, aorta decellularization, hollow fibers, & electrospinning were not giving me the results I wanted as it was not as easy to fully merge cells and materials in one instant process. It usually required long complex yet limited 2D or 3D scaffolding and the addition of cells after the process, not creating what I needed. What did I do back in 2013? I became obsessed with open-sourced 3D printing technology and littered my apartment in Hammersmith with 3D printer machines, electronics, syringes with biomaterials, designs, and other accessories. Then I took my RepRap 3D printer from home and my ideas for bioprinter modifications and co-supervised students2 during the Rio Tinto Sports Innovation Program3 (thanks to Dr. Southgate) to modify it into what we know now as 3D extrusion bioprinting. The goal of the student’s project was to 3D print a pressure-sensitive electroconductive wheelchair mat with sensors for novel Paralympics sports equipment.
Now in 2021, Puredyne (ViscoTec GmbH) has been released ultimately creating the extrusion bioprinting solution I needed years ago with an affordable bioprinting module that can be connected to and used on almost any 3D printer4. During my time in making extrusion-based technologies whilst exploring other electric fields and microfluidic technologies, I interacted with fellow RepRap bioprinting pioneer Prof. Miller (Co-Founder of Volumetric Inc) after watching his videos of 3D bioprinting sugar with a modified RepRap for regenerative medicine applications in 20125. Prof. Miller is a pioneer in this field from basic technologies using a RepRap to more advanced technologies such as volumetric bioprinting. Back then I was shocked at how simple & cheap it was to create a basic extrusion 3D bioprinter compared to other tissue engineering technologies2. The thought crossed my mind in 2013 what if it could be merged with biomaterials and cells and sold to researchers at an affordable price whilst being connected to a database of CAD files?
A year or so later, I started working with an incredibly talented & entrepreneurial engineer, Jemma Redmond, building our company Ourobotics. In 2015 Ourobotics released an advanced robotic arm 3D bioprinting machine, “The Revolution”6, with a capacity for ten biomaterial syringes, artificial intelligence retooling, an incubator enclosure, with one grant submitted for a GMP-compliant machine with Imperial College London. Our research with others into combining & designing multiplexed 3D biofabrication inventions using electrohydrodynamic jetting technology was so advanced that we even decided to open-source the basic extrusion 3D bioprinting machine as “The Renegade” for 900 EUR in 2016 7.
Cellink (BICO), Allevi, and Regemat were all developing extrusion 3D bioprinter machines around the same time. Together all start-ups pioneered the era of desktop 3D bioprinting and disrupted a market of overpriced bioprinter machines costing upwards of 200,000 EUR. My first start-up’s exploration into advanced technologies happened when Jemma and I met Prof. Suwan Jayasinghe, University College London. He told us in 2015 that 3D bioprinting alone would not create the tissue & cellular structures needed for realistic regenerative medicine applications. We were skeptical, but we realized we were nowhere near as experienced in this industry as he was, so we listened attentively.
He was adamant that his inventions8 like 3D Bioelectrospraying (integrated with microfluidics9), Electrohydrodynamic Jetting of Cells10, and Cell Electrospinning11 were far superior, proven in many publications to work, more sensitive to cells by maintaining their properties and could be merged with existing 3D bioprinting technology for regenerative medicine studies. I was familiar with electrospinning and its limitations of not being able to use cells in the process due to issues with the voltage and polymers. However, what Prof. Jayasinghe had invented was proven to work in 3D using the electric field for both cells and biomaterials at a nanoscale resolution in one process; it just was not in a commercial setting. Not only has it been published to make many viable 3D tissues for in vitro and in vivo research9, 12-17 it even has utility for encapsulation of nanomaterials for gene therapy18. He was right, and we just didn’t know it back then.
Even if we were set on robotic arm 3D bioprinting, it didn’t stop me from reading his hundreds of publications8, his patent on electrohydrodynamic jetting of cells10, and starting our start-up’s designs, prototypes, and experimentations for what we described as the “The Medusa”. During my research, we experimented with multiple novel ways for merging 3D Bioelectrospraying, Cell Electrospinning, & Electrohydrodynamic Jetting technologies with the X, Y, Z motions and robotics of extrusion 3D bioprinting. Those with more extensive engineering backgrounds, including my father Gerard Gray, Aidan Hickey, and others, also helped me co-invent, design, and prototype these technologies of newer multiplexed 3D biofabrication systems. Our technologies were first demonstrated as a poster and oral presentation at the NC3RS conference in London in December 201519,20 The Annual World Congress of Smart Materials in Singapore in 2016 and during my time as visiting lecturer for the Innovation Design Engineering masters at Imperial College London with workshops on 3D biofabrication for multiple sectors such as health, food, architecture, and textiles or on using SLA 3D printing for organ-on-a-chip technology.
The then CTO Dr. Comerford was even inventing an interactive 3D model database platform for all of biofabrication. We also met with Dr. Vaidyanathan from Imperial College London and explored ideas that we could one day 3D biofabricate deep brain neural stimulation devices to treat Parkinson’s. Ourobotics was voted in the top 10 3D bioprinters of 201521 and won many awards after being funded by SOSV as a Hax alumni. However, tragically the world lost the engineering genius that was Jemma Redmond in 201622 before we could start to make an impact with our advanced biofabrication technologies & industry vision mapped out sitting together in labs at Imperial College or on whiteboards at the office in Cork.
As a result of losing a friend and co-founder, I mostly walked away from the start-up industry, other than one company that started as FluxBio23 and later changed following co-founder separation. I owe a debt of gratitude to my supervisors and mentors at the bioengineering department of Imperial College London (Prof. Weinberg, Prof. Bull, Prof. Moore) and my career mentor Prof. Kamm from MIT. Their original inspiration that propelled me into the start-up industry, constant support during a hard time for me with the loss of Jemma, and endless encouragement are responsible for my journey from a bioengineering Ph.D. student to a serial deep technology entrepreneur.
I reconsidered my view of the 3D biofabrication start-up industry in 2020 when I met a young entrepreneurial Ph.D. candidate Paul Jochems from Utrecht University. Friso Smit of the Utrecht Science Park made the connection to Paul and Jan Zuidema of the Provincie Utrecht/ROM that supported and encouraged my work on helping to build start-ups. At that time I was working between both organizations and they knew start-ups were not my primary focus in life, but I do owe them a lot for encouraging me to help other new entrepreneurs. Both Friso & Jan have been two of the greatest career mentors I have been fortunate to have over the years, like many professors before them. Paul’s research had created a novel biofabricated 3D intestinal model with all cellular structures making it more physiologically relevant with plans to connect it to a future artificial intelligence clinical trial database in the start-up setting. His passion and drive reminded me of when I had a crazy start-up idea at Imperial and why I got into biotechnology entrepreneurship in the first place. Since helping Dr. Jochems build his start-up GUTSBV24 as a co-founding advisor in 2020, he has been awarded approximately 220,000 EUR, was selected to participate in the national venture challenge in the Netherlands, & is now fundraising. I expect big things for Paul in this industry based upon his strong technical background and natural entrepreneurial talents.
As for 3D bioprinting, years later, low and behold, Prof. Jayasinghe was right; extrusion 3D bioprinting did not work due to its many predicted limitations. In extrusion 3d bioprinting, cells suffered from deformation due to shear stress, low cell viability, slow speeds, limited tissue formation, & cellular behavior needed after the process, and ultimately higher probability of cell death. Extrusion 3D bioprinters cannot make high throughput tissue structures, so now it is clear there is a need for more advanced tissue engineering technologies to elevate the potential and hype surrounding 3D bioprinting. With over 150 publications8, some of the proven advantages of Prof. Jayasinghe’s technologies include higher cell viability [85%+] compared to extrusion bioprinting’s low cell viability; faster speeds (10nm/s+), higher cell density, precision with nano-scale resolution (< 50nm) when compared to extrusion (>5μm), inkjet (< 5-50μm), and laser-assisted bioprinting (>50μm), with non-contact biofabrication potential & no need for support structures.
The technology can handle more significant volumes of multiple cell types and biopolymers to control individual cell droplets and thread deposition, faster processing, cell separation into streams. It has been proven to maintain the dynamic metabolism of cells, allows cells to carry out all their expected cellular behavior, can be used to control the release of cellular/therapeutic agents at a specific site over the desired period, & allows for deeper cell penetration into the scaffolds. It can encapsulate cells, genes, proteins, & most notably, after the biofabrication process, the cells maintain their metabolic, morphological, and pluripotent characteristics. Something that is essential for cells in forming complex tissues that extrusion 3D bioprinting fails to achieve due to speed, resolution, cell deformation, & shear stress. Publications have proven Prof. Jayasinghes technologies can create functional 3D whole embryo models, 3D skin repair models, 3D mesenchymal progenitor and embryonic stem cell studies with pluripotency maintained, 3D cardiac repair patches, 3D lung models, 3D neural models, and more. It can dispense whole human blood as a diagnostic tool, be used as a gene therapy delivery system, encapsulate sperm cells, and has been coupled with microfluidics to control cell numbers in living residues and create multi-compartmental living structures. In a 2021 review, Prof. Jayasinghe’s advanced technologies were listed as important emerging technologies in the 3D biofabrication review article25.
Ourobionics: 4D biofabrication (electric, microfluidic and cyborganic)
Fast forward to 2020, and Ourobionics has started to form based upon John Zandbergen hearing all about the hard work, successes, failures, inventions & visions first mapped out by Jemma and me for Ourobotics before 2016. I first sat down with John in early 2020 at Cafe Hemingway in Utrecht and told him about my first start-up’s early-stage R&D work on merging Prof. Jayasinghe’s advanced patented technologies with 3D bioprinting. His eyes lit up with excitement surrounding its potential to change the commercial industry. He enjoyed that the first start-up’s name was derived from “Ouroboros”, meaning cyclical renewal (close to regenerative medicine) and he thought it was best to live up to the original start-up’s vision that was ahead of the curve in 2015. John has over 20+ years of corporate experience and even some hands-on experience in the bioprinting industry from the commercial side. That was when Ourobionics was first co-founded in stealth mode, prior to BV formation.
He saw the many limitations of the technologies & frustrations of clients in the sector. I introduced John to those with the essential technologies that can transform a stagnating industry and a couple of months later, Prof. Jayasinghe joined as Ourobionics B.V. co-founding Chief Scientific Officer. I connected him with the industry experts needed to build a strong management team with a new vision to go beyond bioprinting & into high throughput tissues with embedded sensor technologies & 4D biofabricated cyborganic optogenetic stem cell neural implant devices for the next generation of regenerative medicine and future human-machine interfacing. Even more than that, John started to build a stronger expert team of co-founders and management within the company26 with extensive corporate, marketing, financial, microfluidic gradient bioprinting, magnetic levitation bioprinting, acoustic levitation, cyborganic electroconductive biosensor materials27, and 4D self-assembly biomaterials28.
Prior to meeting John, I met Dr. Mohammad Albanna of Humabiologics Inc. who had developed human alternatives to animal biomaterials. In my personal opinion, we need more human versions of biomaterials to create physiologically relevant 3D tissues and organs. Dr. Albanna is an expert in the field of regenerative medicine and strangely enough, he too experimented with merging other similar technologies in 2012. It was as though we were working on the same technologies on different continents but didn’t manage to meet. Mohammad is quite an inspirational person to meet and he too reminded me why I should be there to help start-ups build and grow. Eventually, John and Mohammad formed a distribution partnership in 2021 but I see bigger things between both company’s long term.
In order to be fully scalable in the 3D biofabrication sector more than one technology will be needed for complete regenerative medicine applications. It is why Ourobionics is working with much more than the internal microfluidic, electric field, and magnetic field technologies through other strategic collaborations. One technology is not sufficient for what we need to achieve so more multiplexing of newer unreleased hardware technologies and a vision towards 4D bioprinting with co-founders patented technologies is needed for realistic applications in regenerative medicine. Why 4D Bioprinting? Well, the results of 3D Bioprinting are static & do not recapitulate the true nature of tissues that are more dynamic29. 4D Bioprinting has emerged to solve these issues and could transform the industry. It is such a new and complex emerging sector that I will explore all aspects of the technologies in my next article. To get these multiplexed advanced 3D and 4D biofabrication technologies into the hands of all researchers and developers at an affordable price, Ourobionics has launched their Beyond Bioprinting Ambassador Program this month for their first platform “The Chimera”.
Overall, extrusion 3D bioprinting has dominated the market for the past five years. Still, the trend is picking up in the industry, with many researchers switching to advanced technologies such as melt electrowriting, acoustic, laser bioprinting, two-photon, and volumetric. At present organ on a chip technology has set the gold standard for high throughput 3D tissues and organs but in time 3D biofabrication can achieve the same results and reduce the need for animal experiments. One of the early companies to work on melt electrowriting is RegenHu (Prof. Paul Dalton was the first to publish about the invention of MEW technology30,31), with more improvements made in 2020 from Prof. Malda’s group publication on Cell Electrowriting let by Dr. Castilho32. The paper referenced all of Prof. Jayasinghes work from years prior as he was the original co-inventor of the patent on electrohydrodymanic jetting of cells.
The “Cell Writing” process incorporated a heating component and x,y,z motions to his decades of work10. It is now evident that what Prof. Jayasinghe told me years ago about 3D bioprinting needing electrohydrodynamic jetting technologies for cells was correct and we are now at a pivotal moment for the industry that wants to expand beyond extrusion-based bioprinting and create realistic applications. Meanwhile, Aspect Biosystems has stimulated many industry advances over the years in reducing the problems of cell deformation from shear stress with their novel microfluidic print head. Poietis have a GMP-compliant system that combines extrusion, laser, and microvalve technologies, demonstrating the need for multiplexing of multiple technologies to create tissues faster.
Other light-based technologies such as two-photon lithography from Nanoscribe Gmbh (BICO) provide next-level capabilities for scaffold-based resolution but much needs to be done on biomaterial development to overcome cells behavior and important characteristics needed for tissue formation in resins that are not entirely suited for their natural environment. Then in 2020, Xolo launched the world of volumetric 3D printing with the release of their machine33 which brought superior speeds to 3D fabrication. The technology has many incomparable advantages to other photolithography technologies that will be important for the scaffolding component of tissue engineering. More work is needed to determine how the resins and lasers interact with cells and if it leads to realistic high throughput tissue formation, but I do believe in the technology’s potential. Xolo has speeds as fast as 100 µm/s and creates 3 cm objects in 2-4 minutes so if the biomaterial component can become more favorable to tissue formation the speeds will ensure stronger tissue formation. In volumetric technology no support structures are needed, the print speed is not dependent on geometry, no layering, has a higher resolution, surface smoothness & transparency, can use many materials. It allows for total design freedom: you can print objects in objects, flow-cells, lens arrays, etc.
Readily 3D has made some progress34 on a project to biofabricate a pancreas with Prof. Malda’s group, so progress is being made to test its limitations and actual realistic potential for cellular applications. From an industry perspective, 3D Systems saw the potential of advanced technologies by acquiring Volumetric Inc35 after acquiring basic extrusion bioprinting company Allevi & BICO (Cellink) acquisition of two-photon lithography company Nanoscribe GmbH. Much is still unknown about the technology’s full potential in regenerative medicine or what will happen in terms of cellular limitations. I do believe in volumetric as it already has a strong use case for an important part of tissue engineering which is scaffolding and Prof. Millers’ extensive industry experience on the application side of things. New limitations need to be tested to determine its full utility in realistic cellular regenerative medicine applications. The advantages of the technologies already have enormous benefits for tissue engineering from a scaffolding perspective, but more proof is needed on the cellular side. It is clear we are on the cusp of what I will term “Bioprinting 4.0”.
Mimix Biotherapeutics has also developed an advanced biofabrication technology in the form of sound-induced morphogenesis (SIM) biofabrication technology using sound waves to create well-defined biological patterns36. The technology of Mimix similar to the technologies of Prof. Jayasinghe has published scientific support37 and can be used for micro vascularization and patient-specific tissues. In time, more publications and the use of these electric, magnetic, acoustic, two-photon, and volumetric technologies will prove their potential in the industry. This year TissueLabs also released a masked SLA system. I remember I first worked with SLA technology when FormLabs first released their Form 1 on kickstarter38 for 2,300 EUR and now you can pick up an SLA machine on amazon for 200 EUR. FormLabs were industry pioneers and the Form 1 paved the way for many in the 3D printing industry. It was perfect for me when running workshops connected to my lectures for the IDE masters to create organ-on-a-chip devices or other materials for part of the 3D biofabrication process, but cells are not entirely happy in the resins.
Both DLP and SLA offer an improved resolution of the scaffolding but due to the known cytotoxicity of photopolymers more advances on the biomaterial side for realistic applications in the industry. Dr. Levato of Utrecht University recently led a fantastic paper on the advances in biomaterials for light-based technologies with the potential for creating 3D tissue structures. The bioresin that they co-developed opens up endless possibilities39. It’s clear that there is some traction in the world of advanced technologies and the race is on to enhance the 3D biofabrication and regenerative medicine industry. We just need to focus in on the level of published proof surrounding the technology and the reality of it in creating tissue structures that can be implanted in the body. At some point there should be standards surrounding minimal cellular viability or cellular properties otherwise we will not get the applications originally promised within the 3D bioprinting space.
Animal Vs. human Vs. synthetic biomaterials
The next stage in the evolution of the biofabrication industry is happening as we speak, where researchers and developers now realize they need much more advanced affordable technologies than overpriced extrusion 3D bioprinters. In parallel to the emergence of more advanced technologies, more affordable machines are coming onto the market and making them more accessible. The majority of the core structures of extrusion 3D bioprinting have minimal patent protection, and now companies like Felix Printers, Brinter, and TissueLabs are allowing researchers to get more access to entry-level 3D bioprinter machines at affordable prices. I am a fan of this movement so that anyone can gain entry-level experience in the world of tissue engineering. The Brinter Core concept is one of my favorite examples of a machine with the right modular capabilities at the right price point for clients that want to get entry into biofabrication. These affordable modular bioprinting hardware solutions give researchers more access to the world of tissue engineering via 3D bioprinting, which is very important for the future of industry growth.
Companies can no longer claim the marketing gimmick of being able to 3D bioprint an organ; it is an entry-level technology that at best, can create limited, low throughput tissue structures. It serves a purpose, but it’s essential to be realistic that more technological advancement is needed at all levels to achieve what is required at a clinical level for regenerative medicine. It was a fairytale used to promote the sale of entry-level technology machines in an industry that needed more time to grow with advanced technologies. In 2021 extrusion 3D bioprinting is now where it belongs, the perfect entry-level technology to the world of regenerative medicine and tissue engineering. A recent, more robust report evaluates the market saturation of low-cost extrusion 3D bioprinting technology40.
What is even better is that thanks to PureDyne it is open to any researcher with a 3D printer. Even at the end of this year, Carnegie Mellon University hosts a workshop where anyone can make an extrusion 3D bioprinter in 3 days and bring it home with them41. It is a pivotal moment for change in the industry towards advanced and transformative technologies.
It is not just a matter of extrusion 3D bioprinting being relegated to entry-level technology there is a shift happening where animal biomaterials like collagen, gelatin, gelma, and matrigel are being phased out for more human-like 3D models. At present, most 3D cell culture utilizes animal-derived materials, which means that the final 3D tissue model tends to lack many of the physiological characteristics necessary. Additionally, they can be plagued by batch to batch variation, lack of reproducibility, or the chemokines, proteins, and other factors impacting the cellular behavior and limiting tissue formation. Fluid Form have improved what can be done with these biomaterials using their FRESH method but more needs to be done to remove the limitations of the animal component of the materials. The European Commission42 has launched an initiative to reduce and remove animal testing, so now more physiologically relevant human models are needed. To do this, we need to switch from animal-based products to human, plant, or synthetically derived biomaterials. Thankfully, researchers now have access to more synthetic or human forms of collagen.
Humabiologics Inc. is one example of a company that has even created “Humamatrix43″, which is a human-based alternative to Matrigel with some great supporting scientific data. They also have a vast array of cost-efficient products such as human collagen gelatin and gelma. In my personal opinion, there is almost no need for animal-derived products with human options available to make human models in the lab. At the end of the day we want to make human 3D tissues, not animal-based tissues but again there are even more issues surrounding our use of animal-based serum in cell culture too. Also, on the market are some revolutionary non-animal derived biomaterial alternatives such as Manchester Biogel, Jellatech, AxolBio, BiogelX, denovoMatrix, Jellagen, and more44.
The technologies needed to replace and improve the standard of animal-based products are based upon recombinant polymers, plant-based scaffolds, micro-organism-derived scaffolds, and synthetic polymers. These advanced synthetic and human biomaterials are essential for creating 3D human models to replace animal experiments. The benefits of these biomaterials include limited supply chain issues, a reduction in infection, ability to be tailored towards specific cell types and models, highly reproducible, no batch to batch variation, physiologically relevant, and non-immunogenic. The importance of these non-animal derived biomaterials is that they can help bridge the gap between early-stage research and development and clinical applications by improving the reproducibility and standardization of 3D models similar to human tissue and organs.
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- Build your own bioprinter. https://www.eventbrite.com/e/3d-bioprinting-open-source-workshop-registration-205342203057?aff=efbneb.
- News, E.P. MEPs demand EU action plan to end the use of animals in research and testing. https://www.europarl.europa.eu/news/en/press-room/20210910IPR11926/meps-demand-eu-action-plan-to-end-the-use-of-animals-in-research-and-testing (2021).
- Humamatrix. https://www.ourobionics.com/humamatrix.
- Animal Free Scaffolds. https://nc3rs.org.uk/moving-animal-free-scaffolds-construction-3d-organotypic-models.