Development of multivalve-based bioprinting technology

Pluripotent stem cells (PSCs) are the most favourable sources of cells for tissue engineering applications due to their unique potency and self-renewal characteristics however they are quite fragile and can be directed to differentiate erroneously by the application of external forces. A novel multi-nozzle valve-based bioprinting platform was developed that was able to position droplets of bio-ink – such as cells in suspension – with high spatial accuracy and low impact. Volumes as low as 2 nL were successfully dispensed. Several different versions of the machine were created before the final machine was made integrating improvements and solutions to problems encountered during development. A complete evaluation of cell compatibility was carried out in order to quantify the response of cells to the bioprinting process. In the first ever study of this kind, the viability and pluripotency of human embryonic and induced pluripotent stem cells was investigated post-printing and were found to be almost completely unaffected by the bioprinting process. Many cells require a 3D culture environment in order to maintain their in vivo functions. A hybrid bioprinted-hanging-droplet technique was used to create uniform spheroid aggregates of programmable sizes from PSCs which could be used to direct PSC differentiation or as building blocks for tissue generation. Hydrogels can also be used to recreate the 3D in vivo cellular environment using the bioprinter. Alginate and hybrid polypeptide-DNA hydrogels were used, the latter for the first time with a bioprinting platform. Complex 3D structures could be created in a layer-by-layer approach with programmable heterogeneous properties throughout. Cells were added to the hydrogel precursor solution and used to bioprint 3D structures. The cells were found to be functional and highly viable while being encapsulated throughout the 3D structure of the bioprinted hydrogel which will allow the future creation of more accurate human tissue models. PSCs were successfully directed to differentiate into hepatocyte-like cells. It was shown that the bioprinting process did not interrupt or alter the pre-programmed differentiation of the cells which means that these cells can be patterned in 3D using the bioprinter while differentiating, greatly speeding up the creation of mini-liver tissue. Hepatic stellates and HUVECs were co-cultured with the hepatocyte-like cells in various ratios in an attempt to improve their hepatic function. However, no clear improvement in cytochrome P450 activity was observed indicating that further optimisation is required in this area

Development of multiwave-based bioprinting technology

Pluripotent stem cells (PSCs) are the most favourable sources of cells for tissue engineering applications due to their unique potency and self-renewal characteristics however they are quite fragile and can be directed to differentiate erroneously by the application of external forces. A novel multi-nozzle valve-based bioprinting platform was developed that was able to position droplets of bio-ink – such as cells in suspension – with high spatial accuracy and low impact. Volumes as low as 2 nL were successfully dispensed. Several different versions of the machine were created before the final machine was made integrating improvements and solutions to problems encountered during development. A complete evaluation of cell compatibility was carried out in order to quantify the response of cells to the bioprinting process. In the first ever study of this kind, the viability and pluripotency of human embryonic and induced pluripotent stem cells was investigated post-printing and were found to be almost completely unaffected by the bioprinting process. Many cells require a 3D culture environment in order to maintain their in vivo functions. A hybrid bioprinted-hanging-droplet technique was used to create uniform spheroid aggregates of programmable sizes from PSCs which could be used to direct PSC differentiation or as building blocks for tissue generation. Hydrogels can also be used to recreate the 3D in vivo cellular environment using the bioprinter. Alginate and hybrid polypeptide-DNA hydrogels were used, the latter for the first time with a bioprinting platform. Complex 3D structures could be created in a layer-by-layer approach with programmable heterogeneous properties throughout. Cells were added to the hydrogel precursor solution and used to bioprint 3D structures. The cells were found to be functional and highly viable while being encapsulated throughout the 3D structure of the bioprinted hydrogel which will allow the future creation of more accurate human tissue models. PSCs were successfully directed to differentiate into hepatocyte-like cells. It was shown that the bioprinting process did not interrupt or alter the pre-programmed differentiation of the cells which means that these cells can be patterned in 3D using the bioprinter while differentiating, greatly speeding up the creation of mini-liver tissue. Hepatic stellates and HUVECs were co-cultured with the hepatocyte-like cells in various ratios in an attempt to improve their hepatic function. However, no clear improvement in cytochrome P450 activity was observed indicating that further optimisation is required in this area

Multicomponent patterning of nanocomposite polymer and nanoparticle films using photolithography and layer-by-layer self -assembly

In this dissertation, the fabrication, characterization, and application examples of 3D multicomponent nanocomposite micropatterns (MNMs) with precise spatial arrangements are described. The ability to produce such small-scale 3D structures with versatility in composition and structure is a new development based on the integration of nanoscale layer-by-layer (LbL) self-assembly and microscale photolithographic patterning, enabling construction of surfaces with microscale patterns that possess nanotopographies. The techniques used here are analogous to surface micromachining, except that the deposition materials are polymers, biological materials, and colloidal nanoparticles used to produce 3D MNMs. A key feature of the resulting 3D MNMs is that the physical and chemical properties of the multilayer nanofilms may be finely tuned using the versatile LbL assembly process, which makes them attractive for many applications requiring polymeric structures with small features. The work presented here involves development of techniques for the fabrication, characterization, and applications of 3D MNMs, and evaluation of the process parameters involved in the developed techniques. These results clearly demonstrate the feasibility of the polymer 3D MNMs for biotechnological applications; specifically, they have potential as tailored surfaces for direct comparison of cell-material interactions on a single substrate, and for co-culture systems. In reality, the approach described here may enable study of and control over cell-biomaterial and cell-cell interactions in a whole new fashion. The techniques developed in this work represent a major advancement of nanoscale engineering through the integration of nanoscale LbL self-assembly and microscale photolithographic patterning for constructing 3D MNMs with varying physical and chemical properties in precise spatial arrangements. A major finding of this work, related to the applicability of the developed techniques, is that most of the seemingly harsh processes involved in constructing the 3D MNMs have minimal or no deleterious effects on the biological models used here. The exception is the resist developer (MF319), which due to its highly basic nature, results in disintegration of nanofilms exposed to it directly. Nevertheless, the methods developed here are not limited by the photoresists and resist developers used here; biocompatible photoresists and aqueous base developers could potentially be used. This work has pursued the development of organic and inorganic nanofilm scaffolds which can eventually be combined to achieve functionality desired for specific applications. It is anticipated that the 3D MNMs developed in this work will provide general platforms for studying biological processes, which will not only impact stem cell research in general but also provide useful information in support of biomedical device development, and tissue engineering. Although the intended purpose for developing 3D MNMs is to produce novel bioactive systems, their applicability is more general and may find use in a broad range of applications including electronics, photonics, optoelectronics, and chemical and biochemical sensors