Automated liquid handling systems for microfluidic applications

Advances in microfluidic research have improved the quality of assays performed in micro-scale environments. Improvement of liquid handling techniques has enabled efficient reagent and drug use while minimising waste. The requirements for the applied techniques vary with applications and a custom integrated liquid handling solution was developed to accomplish some of these applications with minimal changes to the system. It is desirable to employ this technology to neuroscience research that requires a fluidic system that can test theories of reinforcement learning in neuronal cultures. An integrated system is therefore required to implement transport and manipulation of media and drugs loaded in a microfluidic device. One requirement for such an integrated system for liquid handling is a transport mechanism to deliver reagents and nutrients to cultures. A liquid flow control system is required to allow precise and timely control of flow rates through a microfluidic device. This can be extended to enable more sophisticated drug delivery approaches like gradient generation, spatial drug distribution and high temporal resolution of the drugs delivered. Another requirement for an integrated system is a liquid loading system that is capable of inserting specified drugs into the flow line. Such a loading system would allow any number of drugs to be loaded during an experimental process to the microfluidic device containing cells as part of an assay. The integration of these systems will allow researchers take advantage of the combined systems. Software development process should also be undertaken to improve the modularity of the integrated system so that hardware changes have marginal effects on the system operation. The project scope was the development of these liquid handling systems as well as their integration in hardware and software to enable their spatio-temporal drug delivery to neuronal cultures in microfluidic devices. The approach was to optimise performance of custom liquid handling system which was developed to realise fast flow rate changes within 1 second interval. Macro- and micro-scale solutions have been investigated in order to realise effective off-chip liquid loading capabilities. Emphasis has been placed on ease of use, modularity, rapid prototyping and precision. A commercial autoloader was identified as a starting point for sequential drug delivery. This was characterised for suitability and the constraints with this setup was used to identify additional requirements for the development of a novel sequential liquid injection system. The design process of the novel liquid injection system was unable to realise a working system due to mechanical and operational challenges encountered. A modular on-chip liquid manipulation system has been investigated and proposed to realise the sequential injection requirements. Rapid prototyping techniques that can promote ubiquitous microfluidic applications have been identified and verified. An integrated liquid manipulation system has been developed using the commercial autosampler that enables sequential loading of agonists into the microfluidic device as well as reliable chemical signalling of the loaded drugs by switching flow rates of the inputs to the device. This system will be beneficial towards research of other cell types within other research fields requiring similar functionality

Modular integration and on-chip sensing approaches for tunable fluid control polymer microdevices

228 p.Doktore tesi honetan mikroemariak kontrolatzeko elementuak diseinatu eta garatuko dira, mikrobalbula eta mikrosentsore bat zehazki. Ondoren, gailu horiek batera integratuko dira likido emari kontrolatzaile bat sortzeko asmotan. Helburu nagusia gailuen fabrikazio arkitektura modular bat frogatzea da, non Lab-on-a-Chip prototipoak garatzeko beharrezko fase guztiak harmonizatuz, Cyclic-Olefin-Polymer termoplastikozko mikrogailu merkeak pausu gutxi batzuetan garatuko diren, hauen kalitate industriala bermatuz. Ildo horretan, mikrogailuak prototipotik produkturako trantsizio azkar, erraz, errentagarri eta arriskurik gabeen bidez lortu daitezkeenetz frogatuko da

Automated liquid handling systems for microfluidic applications

Advances in microfluidic research have improved the quality of assays performed in micro-scale environments. Improvement of liquid handling techniques has enabled efficient reagent and drug use while minimising waste. The requirements for the applied techniques vary with applications and a custom integrated liquid handling solution was developed to accomplish some of these applications with minimal changes to the system. It is desirable to employ this technology to neuroscience research that requires a fluidic system that can test theories of reinforcement learning in neuronal cultures. An integrated system is therefore required to implement transport and manipulation of media and drugs loaded in a microfluidic device. One requirement for such an integrated system for liquid handling is a transport mechanism to deliver reagents and nutrients to cultures. A liquid flow control system is required to allow precise and timely control of flow rates through a microfluidic device. This can be extended to enable more sophisticated drug delivery approaches like gradient generation, spatial drug distribution and high temporal resolution of the drugs delivered. Another requirement for an integrated system is a liquid loading system that is capable of inserting specified drugs into the flow line. Such a loading system would allow any number of drugs to be loaded during an experimental process to the microfluidic device containing cells as part of an assay. The integration of these systems will allow researchers take advantage of the combined systems. Software development process should also be undertaken to improve the modularity of the integrated system so that hardware changes have marginal effects on the system operation. The project scope was the development of these liquid handling systems as well as their integration in hardware and software to enable their spatio-temporal drug delivery to neuronal cultures in microfluidic devices. The approach was to optimise performance of custom liquid handling system which was developed to realise fast flow rate changes within 1 second interval. Macro- and micro-scale solutions have been investigated in order to realise effective off-chip liquid loading capabilities. Emphasis has been placed on ease of use, modularity, rapid prototyping and precision. A commercial autoloader was identified as a starting point for sequential drug delivery. This was characterised for suitability and the constraints with this setup was used to identify additional requirements for the development of a novel sequential liquid injection system. The design process of the novel liquid injection system was unable to realise a working system due to mechanical and operational challenges encountered. A modular on-chip liquid manipulation system has been investigated and proposed to realise the sequential injection requirements. Rapid prototyping techniques that can promote ubiquitous microfluidic applications have been identified and verified. An integrated liquid manipulation system has been developed using the commercial autosampler that enables sequential loading of agonists into the microfluidic device as well as reliable chemical signalling of the loaded drugs by switching flow rates of the inputs to the device. This system will be beneficial towards research of other cell types within other research fields requiring similar functionality

Printing cells and co-cultures for osteoarthritis models

PhD ThesisOsteoarthritis is a multifactorial disease characterised by the degradation of cartilage and bone tissue within the joint. Research into novel therapies is currently limited by the throughput and replicative accuracy of early-stage in vitro disease models. Biofabrication represents an emerging technology which allows for the selective deposition of cells and material in order to create complex cell-laden structures. The aim of this research was to characterise a combination of inkjet and valve-based drop-on-demand printing processes for the construction of osteoarthritis tissue models. An inkjet printing platform was characterised to deposit material at the picolitre-scale. Single cell printing was achieved, with biological analysis confirming that the printing process does not significantly affect cellular viability or function. A valve printing process was applied for the production of cellular aggregates that could be used for both in vitro osteoarthritis research and in vivo therapeutic applications. Reliable jetting performance was demonstrated, enabling material deposition at the nanolitre-scale. Cell printing was achieved across a concentration range of 1-20 million cells per mL, with biological analysis of printed cells revealing no significant effects on viability or function. No discernible impact on aggregate tissue structure was observed as a result of the printing process, confirming its suitability for the manufacture of tissue aggregates. A bioprinted co-culture tissue model comprised of mesenchymal stromal cell and chondrocyte cell types was successfully generated using a 3D insert culture format. Cell proliferation was maintained over a 14 day period, alongside an increase in tissue density and cellular organisation. In combination, this research has demonstrated the suitability of inkjet and valve printing processes to selectively deposit cells. Bioprinted aggregate and insert-based 3D tissue models were validated using the valve printing technique, providing an effective method to scale up the manufacture of in vitro platforms for osteoarthritis research applications.Versus Arthritis, the Engineering and Physical Sciences Research Council and the Centre for Doctoral Training in Additive Manufacturing and 3D Printin

Microfabrication Technology for Isolated Silicon Sidewall Electrodes and Heaters

This paper presents a novel microfabricationtechnology for highly doped silicon sidewall electrodesparallel to – and isolated from – the microchannel. Thesidewall electrodes can be utilised for both electricaland thermal actuation of sensor systems. Thetechnology is scalable to a wide range of channelgeometries, simplifies the release etch, and allows forfurther integration with other Surface ChannelTechnology-based systems. Furthermore, thefabrication technology is demonstrated through thefabrication of a relative permittivity sensor. The sensormeasures relative permittivity values ranging from 1 to80, within 3% accuracy of full scale, including waterand water-containing mixtures

Velocity-independent thermal conductivity and volumetric heat capacity measurement of binary gas mixtures

In this paper, we present a single hot wire suspended over a V-groove cavity that is used to measure the thermal conductivity ($k$) and volumetric heat capacity ($\rho c_p$) for both pure gases and binary gas mixtures through DC and AC excitation, respectively. The working principle and measurement results are discussed

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

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