The approach could revolutionize regenerative medicine, enabling the production of complex tissues and cartilage that would potentially support, repair or augment diseased and damaged areas of the body.
While bioprinting has advanced significantly over the last 15 years, the pursuit of morphological complexity and biological functionality in fabricated cellular constructs remains challenging. Criteria relating to the printing process, including cytocompatibility, the resolution of cell placement and structural complexity, and the maturation of biologically active tissues, must all be addressed if printed tissues are to play a major role in regenerative medicine. To date, no single fabrication approach has addressed the gamut of design challenge for synthetic cellularized structures, however progress has been made by appropriating a range of 3D printing methodologies, including extrusion, laser-induced forward transfer, and droplet-based ejection.
Printing high-resolution living tissues is hard to do, as the cells often move within printed structures and can collapse on themselves. But, led by Professor Hagan Bayley, Professor of Chemical Biology in Oxford’s Department of Chemistry, the team devised a way to produce tissues in self-contained cells that support the structures to keep their shape.
The cells were contained within protective nanoliter droplets wrapped in a lipid coating that could be assembled, layer-by-layer, into living structures. Producing printed tissues in this way improves the survival rate of the individual cells, and allowed the team to improve on current techniques by building each tissue one drop at a time tNAture So a more favorable resolution.
To be useful, artificial tissues need to be able to mimic the behaviors and functions of the human body. The method enables the fabrication of patterned cellular constructs, which, once fully grown, mimic or potentially enhance natural tissues.
Oxford’s new bioprinting approach complements existing methodologies by combining advantages of various fabrication routes into a single methodology to produce millimeter-scale constructs with defined cellular patterns at tissue-like densities. By broadening our existing approach for aqueous droplet printing in oil, they demonstrate a high precision cell printing approach with a droplet resolution of 1 nL. The printed cellular constructs submerged in oil were subsequently encapsulated in a thin layer of gel for transfer to aqueous medium. By this means, we have generated patterned constructs from two populations of cells, with high-resolution 3D features including layers and channels under 200 μm in width, within robust cubic-millimetre-scale structures. The incorporated cells initially displayed high viability (90% average) and were present at tissue-like densities of 10 million cells mL−1, i.e., the same magnitude as the highest reported densities for a droplet-based bioprinting processes. Cell proliferation occurred over several days along with an overall increase in cell viability (over95% average). Significantly, ovine mesenchymal stem cells (oMSCs) in printed constructs responded to transforming growth factor-β3 (TGF-β3) and underwent differentiation to form cartilage-like structures. These data demonstrate that fundamental biological processes can remain intact after printing, which suggests that the approach presented here will be useful for complex tissue fabrication.
Currently, the constructs are limited to two cell types only, but in the future, the printer could be adapted to include a multi-nozzle dispenser increasing the number of patternable cell types.
Dr Alexander Graham, lead author and 3D Bioprinting Scientist at OxSyBio (Oxford Synthetic Biology), said: ‘We were aiming to fabricate three-dimensional living tissues that could display the basic behaviors and physiology found in natural organisms. To date, there are limited examples of printed tissues, which have the complex cellular architecture of native tissues. Hence, we focused on designing a high-resolution cell printing platform, from relatively inexpensive components, that could be used to reproducibly produce artificial tissues with appropriate complexity from a range of cells including stem cells’.
The researchers hope that, with further development, the materials could have a wide impact on healthcare worldwide. Potential applications include shaping reproducible human tissue models that could take away the need for clinical animal testing.
Over the coming months they will work to develop new complementary printing techniques, that allow the use of a wider range of living and hybrid materials, to produce tissues at industrial scale. Dr Sam Olof, Chief Technology Officer at OxSyBio, said: ‘There are many potential applications for bioprinting and we believe it will be possible to create personalized treatments by using cells sourced from patients to mimic or enhance natural tissue function. In the future, 3D bio-printed tissues maybe also be used for diagnostic applications – for example, for drug or toxin screening.’
Dr Adam Perriman from the University of Bristol’s School of Cellular and Molecular Medicine, added: ‘The bioprinting approach developed with Oxford University is very exciting, as the cellular constructs can be printed efficiently at extremely high resolution with very little waste. The ability to 3D print with adult stem cells and still have them differentiate was remarkable, and really shows the potential of this new methodology to impact regenerative medicine globally.’
The printing approach produces millimeter-scale cubic constructs with patterned cells that are biologically active. Cellular constructs of this size are desirable for high-throughput screening assays as they size-compatible with 96-well plates. Furthermore, the production of tens or potentially hundreds of mm-scale constructs could be achieved during a single print run.
For the manufacture of cellular constructs for surgical implantation centimeter-scale structures will be required. In the present work, only microliter volumes of cells were printed which is advantageous for high-value cells, such as low-yield primary cell samples or gene-edited cell-lines, however, this print volume can be expanded to hundreds of microliters and potentially milliliter quantities, therefore, allowing printing on a centimeter-scale. However, the current encapsulation method, which is necessary for stabilizing the structure during phase transfer, would likely need to be adapted to convey the required rigidity to larger constructs. In addition, the fabrication of centimeter-scale structures in a practicable manner would require the rate of printing to be significantly increased by generating droplets at higher frequencies (up to 20 Hz is feasible with the current hardware) and the simultaneous use of multiple droplet generators. Alternatively, larger structures could be attained by assembling mm-scale printed structures in a modular fashion and allowing the cells to mature into a single tissue over time. Regardless of the fabrication method, any constructs thicker than the oxygen diffusion limit (100 to 200 μm) must include microchannels or vasculature to sustain the tissue. Potentially, this might be achieved by incorporating artificial channels formed from sacrificial gel droplets. Similar approaches have been used to make extrusion-printed vascularized tissue.
The versatility and robust nature of their approach provides a new set of tools for bottom-up tissue engineering at a low cost. Our droplet-based bioprinter is relatively inexpensive, costing approximately £7,500 to set up. The device is therefore at the low-cost end of commercial bioprinters, which are valued from ~£5,000 (e.g. Inkredible, CELLINK) to £160,000 (e.g. 3D Bioplotter, EnvisionTEC). Furthermore, the capability of the printer has allowed the fabrication of patterned cellular constructs with high-resolution features (Nature Scientific Reports – High-Resolution Patterned Cellular Constructs by Droplet-Based 3D Printing
Bioprinting is an emerging technique for the fabrication of living tissues that allows cells to be arranged in predetermined three-dimensional (3D) architectures. However, to date, there are limited examples of bioprinted constructs containing multiple cell types patterned at high-resolution. Here we present a low-cost process that employs 3D printing of aqueous droplets containing mammalian cells to produce robust, patterned constructs in oil, which were reproducibly transferred to culture medium. Human embryonic kidney (HEK) cells and ovine mesenchymal stem cells (oMSCs) were printed at tissue-relevant densities (107 cells mL−1) and a high droplet resolution of 1 nL. High-resolution 3D geometries were printed with features of ≤200 μm; these included an arborised cell junction, a diagonal-plane junction and an osteochondral interface. The printed cells showed high viability (90% on average) and HEK cells within the printed structures were shown to proliferate under culture conditions. Significantly, a five-week tissue engineering study demonstrated that printed oMSCs could be differentiated down the chondrogenic lineage to generate cartilage-like structures containing type II collagen.
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