Progenitor cells in auricular cartilage demonstrate promising cartilage regenerative potential in 3D hydrogel culture

The reconstruction of auricular deformities is a very challenging surgical procedure that could benefit from a tissue engineering approach. Nevertheless, a major obstacle is presented by the acquisition of sufficient amounts of autologous cells to create a cartilage construct the size of the human ear. Extensively expanded chondrocytes are unable to retain their phenotype, while bone marrow-derived mesenchymal stromal cells (MSC) show endochondral terminal differentiation by formation of a calcified matrix. The identification of tissue-specific progenitor cells in auricular cartilage, which can be expanded to high numbers without loss of cartilage phenotype, has great prospects for cartilage regeneration of larger constructs. This study investigates the largely unexplored potential of auricular progenitor cells for cartilage tissue engineering in 3D hydrogels

Energy recovery from organic wastewater via microbial fuel cell technology: a novel approach

One promising technology that offers a potential breakthrough in terms of energy recovery from wastewater efforts is Microbial Fuel Cells (MFCs). This technology exploits the ability of microorganisms, usually bacteria, to oxidise organic matter contained in wastewater and harness the produced electrons to generate electricity. However, despite its promise, current bottlenecks such as long start-up time, low power output and limited understanding of microbial communities central to the process, prevent this technology to achieve its maximum potential. Through this DPhil project, we have expanded our understanding of the underlying science and mechanisms behind Microbial Fuel Cell technology, as well as pushing towards its applications as a simultaneous solution for wastewater treatment and energy crisis problems in industrial scale. In this project, two main Extracellular Electron Transfer (EET) mechanisms for S. oneidensis MR-1 – mediated electron transfer (MET) and direct electron transfer (DET) were evaluated and analysed for their contributions. The results confirm that electron transfer via mediator contributed 70% of power output, and genetic engineering of cells to include additional flavin-production gene from B. subtilis increased the power output by over two-fold. In addition, the in-situ transfer of flavin-overexpression genes into the bacterial cell using ultrasound in an MFC setup was achieved for the first time. This study has also demonstrated a significant scale-up to ultrasound gene transfer technology – with working volume of 300 mL, providing ~150X scale-up than those previously reported elsewhere. Furthermore, the ability of S. oneidensis MR-1 to utilise acetate as sole carbon and energy source in an MFC setup was demonstrated. A voltage of 0.032 0.011 V was generated across 1kΩ resistor with 20 mM sodium acetate as the sole carbon source, with maximum power output that reached 1.2 0.1 mW/m2. The acetate utilisation by S. oneidensis was also demonstrated when using anaerobic digester liquor as MFC substrates – with 16.2 4.1 mg/L of acetate content consumed within 5 days, resulting in ~11% coulombic efficiency. This is a novel finding – as there are no previous literatures that report successful utilization of pure culture S. oneidensis to degrade acetate from real AD liquor for electricity generation. Furthermore, this further supports claims that have been made by other researchers – that acetate utilization by MR-1 is not limited only under aerobic condition. Finally, the viable application of magnetic nanoparticles (MNPs) in MFC setup was demonstrated. The deployment of silica-coated Iron-Oxide Nanoparticles (ION) prevented oxidative nature of the iron core, while maintaining the magnetic property of the nanoparticles. The combined result of these characteristics enabled the use of nanoparticles to form engineered biofilm on the electrode surface without compromising its electricity production. A voltage of ~40 mV was achieved using E. coli – S. oneidensis MR-1 consortium to degrade glucose, with maximum power production of 39.8 ± 2.4 mW/m2. The biofilm composition was found to have shifted towards a community predominated by the favoured electrigen which is MR-1 strains, reaching 38.3 ± 7.0% of total cell population – around 5-fold higher compared to 7.4 ± 4.2% of that the control where the nanoparticles were not present. To the best of our knowledge, this is the first reported study of MNP application in MFC as coating agent for bacterial cell – for the purpose of selective biofilm formation of electrigen-enriched electrode. Future research trends should be focused on the advancement of electrode materials towards cheaper, more biocompatible, and higher effective surface area which promote better biofilm attachment, and further understanding of the biology within bacteria consortium that is often very complex – coupled with genetic engineering and modifications to implement capabilities across bacterial species for complex substrate degradation and enhanced electricity generation capabilities. This study has contributed majorly to some of these aspects through its novel result and findings, although further studies, sensitivity analyses and development are still required to reach our target end-state. In the future, we believe that the application of MFC should not be limited to wastewater treatment, but also form part of important integrations with other technologies such as biosensory systems, anaerobic digester as well as energy storage and chemical productions. All these milestones should be achieved as we advance our understanding of the science and underlying mechanisms behind the technology

Dissecting the role of Plasmodium sporozoite curvature in gliding motility

Plasmodium parasites are unicellular, mosquito-borne pathogens that cause malaria in vertebrates such as mammals, birds and reptiles. These Plasmodium parasites undergo a complex lifecycle, necessitating their adaptation to different environmental niches. Transmission of Plasmodium begins, when an infectious bite from a female Anopheles mosquito delivers sporozoites in the skin. While in the skin, sporozoites rely on a substrate dependent mode of locomotion known as gliding to actively penetrate host tissue, find and enter blood vessels. Next, sporozoites are passively transported within the bloodstream to the liver where they differentiate into liver stage parasites. Liver stage parasites release merozoites into the bloodstream and start the blood stage phase of infection. Subsequently, mixed bloodstage parasites are ingested by mosquitos and differentiate into various forms before becoming infectious sporozoites again. Infectious sporozoites are polarized crescent shaped cells that typically move in circles on two-dimensional substrates in vitro and in helices in three-dimensional substrates or in vivo. In this thesis, the hypothesis that the curvature/crescent shape of the sporozoites is important for energy efficient corkscrew gliding motility and aids in recognition of blood vessels is investigated. To test the hypothesis two approaches were adopted. The first approach was to use micropillar arrays made of PDMS a plastic polymer, as blood vessel shape mimics. The aim of using pillar arrays was to understand how sporozoite shape guides their physical interactions. Here the results show that sporozoites associate with pillars that have diameters approximately similar to blood vessels. This suggests sporozoite curvature evolved in part to permit their association to blood vessels. The second approach was to generate a genetically manipulated parasite with altered curvature. In studies done in Toxoplasma gondii, PhIL1 a protein of the parasite pellicle was found to be important in maintaining parasite morphology. Here, PhIL1 was found to be essential in the blood stages of the parasite. Also, overexpressing PhIL1, IMC1h and IMC1l showed the sporozoite pellicle was very stable and yielded no curvature change. Although a change in curvature was elusive, the proteins considered here could be used in discovery of other curvature related proteins

Building 3D architectures for cardiomyocytes

Pharmaceutical companies currently rely on animal models for drug screening. This is a very expensive, time-consuming process and in some cases has been shown to be a poor predictor of human cardiac toxicity. Animal cells and tissue are not identical to their human counterparts. Therefore, it is not until human clinical trials at the later stages of drug screening that unexpected reactions to the drug are identified (Burridge et al., 2014). It would be greatly beneficial if this process could be shortened by identifying the risks of a drug earlier in the screening stages-chip based screening using mature human cardiomyocytes (CMs) is a route to achieve this. Substrates used to support CM growth have been identified including high-throughput chip-based screening strategies (Hook et al., 2013) (Celiz et al., 2014b) but so far stem cell derived CMs on these substrates do not adequately recapitulate the adult human CMs in terms of maturity (Denning et al., 2016). Many factors can affect how a cell matures from the soluble extracellular signals around it to the chemistry, topography, architecture/shape and mechanics of the substrate on which it is supported (Nikkhah et al., 2012). Mature cardiomyocytes have been successfully grown on 3 polymers synthesised by UV polymerisation-it has been confirmed that polymers like these can be successfully processed by 2-photon lithography. Photo initiator concentration has been optimised to create a complete structure. Glycerol propoxylate triacrylate and Tricyclodecane dimethanol diacrylate were shown to provide a wide operating window. Many relevant structures for CM growth were chosen and designed on AutoCAD to demonstrate the potential application of this material in CM culture. The 3D design freedom of the lithography approach will be used to explore the relationship between architecture and cell maturity. This will then enable a platform to be created using various architectures on a chip which will be utilised to assess cardiomyocyte maturity. This enables structure fabrication with more accuracy compared to previous methods due to the sub-micron scale of 2-photon lithography (Maruo et al., 1997). Greater resolution means improved results as cells interact on the sub-micron scale (~1µm) (Guck et al., 2010)Various architectures used for cardiomyocyte culture can show which ones are the most suitable to guide cardiomyocytes to a mature adult form

Investigating interactions between rat adult cardiac myocytes and fibroblasts in the heart

No abstract available

Building 3D architectures for cardiomyocytes

Pharmaceutical companies currently rely on animal models for drug screening. This is a very expensive, time-consuming process and in some cases has been shown to be a poor predictor of human cardiac toxicity. Animal cells and tissue are not identical to their human counterparts. Therefore, it is not until human clinical trials at the later stages of drug screening that unexpected reactions to the drug are identified (Burridge et al., 2014). It would be greatly beneficial if this process could be shortened by identifying the risks of a drug earlier in the screening stages-chip based screening using mature human cardiomyocytes (CMs) is a route to achieve this. Substrates used to support CM growth have been identified including high-throughput chip-based screening strategies (Hook et al., 2013) (Celiz et al., 2014b) but so far stem cell derived CMs on these substrates do not adequately recapitulate the adult human CMs in terms of maturity (Denning et al., 2016). Many factors can affect how a cell matures from the soluble extracellular signals around it to the chemistry, topography, architecture/shape and mechanics of the substrate on which it is supported (Nikkhah et al., 2012). Mature cardiomyocytes have been successfully grown on 3 polymers synthesised by UV polymerisation-it has been confirmed that polymers like these can be successfully processed by 2-photon lithography. Photo initiator concentration has been optimised to create a complete structure. Glycerol propoxylate triacrylate and Tricyclodecane dimethanol diacrylate were shown to provide a wide operating window. Many relevant structures for CM growth were chosen and designed on AutoCAD to demonstrate the potential application of this material in CM culture. The 3D design freedom of the lithography approach will be used to explore the relationship between architecture and cell maturity. This will then enable a platform to be created using various architectures on a chip which will be utilised to assess cardiomyocyte maturity. This enables structure fabrication with more accuracy compared to previous methods due to the sub-micron scale of 2-photon lithography (Maruo et al., 1997). Greater resolution means improved results as cells interact on the sub-micron scale (~1µm) (Guck et al., 2010)Various architectures used for cardiomyocyte culture can show which ones are the most suitable to guide cardiomyocytes to a mature adult form

Functional Human Cell-Based Cardiac Tissue Model with Contraction Force Measurements

Sydämeen kohdistuvat lääkkeiden haittavaikutukset ovat yksi suurimpia syitä lääkeainekandidaattien hylkäämiselle sekä jo markkinoille vietyjen lääkkeiden poisvedoille. Nykyiset menetelmät lääkkeiden turvallisuuden ja tehokkuuden testaamiseen eivät ennusta lääkkeiden vaikutuksia ihmiselle riittävän tarkasti. Koska sydänten toiminnassa on lajikohtaisia eroja, eläinkokeissa ei välttämättä tunnisteta kaikkia ihmisen sydämelle haitallisia aineita. Tätä varten tarvitaan toiminnaltaan mahdollisimman hyvin ihmisen sydänkudosta vastaavia ihmissolupohjaisia sydänkudosmalleja. Tämän väitöskirjan tavoitteena oli kehittää toiminnallinen sydänkudosmalli, joka mallintaa aikuisen ihmisen sydäntä, sekä yhdistää tähän malliin sykintävoiman mittaus. Tässä työssä kehitetty sydänkudosmalli koostuu ihmisen rasvan kantasoluista (hASC) ja ihmisen napanuoran laskimon endoteelisoluista (HUVEC) muodostuvasta verisuoniverkostosta, jonka päälle kasvatetaan ihmisen indusoiduista kantasoluist (hiPSC) erilaistetut sydänlihassolut. Tämä sydänkudosmalli karakterisoitiin rakenteellisesti, geenien ilmentymisen tasolla ja toiminnallisesti. Sydänkudosmallien sykintävoimanmittaukseen kehitettiin yksi- ja kaksisuuntaisia pietsosähköisiä sensoreita. Tulosten perusteella yhteisviljelmä verisuonipohjan kanssa parantaa sydänlihassolujen kypsymistä edistämällä niiden järjestäytymistä ja morfologiaa, sarkomeerirakennetta ja solu-solu-liitoksia. Myös sydänlihassolujen geenien ilmentymisessä oli vastaavuuksia aikuisen sydämeen. Toiminnallinen karakterisointi tunnetuilla sydämeen vaikuttavilla lääkeaineilla osoitti mallin tunnistavan tarkasti aineiden vaikutuksia. Kehitetyt pietsosähköiset voima-anturit soveltuivat sydänmallien sykintävoiman mittaamiseen. Antureiden todettiin pystyvän mittaamaan sekä voimaa eri mekanismein lisäävien että sitä vähentävien aineiden vaikutuksia. Yhteenvetona voidaan todeta, että työssä kehitetty sydänkudosmalli jäljittelee sydänlihaskudoksen rakennetta ja toimintaa. Malli sopii testaamaan ihmisen sydämeen kohdistuvia akuutteja lääkeaineiden haittavaikutuksia ja sydänlääkkeiden tehoa. Kehitettyyn sydänkudosmalliin liitetyllä pietsosähköisellä voima-anturilla on potentiaalia voimaankohdistuvien lääkeainevaikutusten testaamiseen.Adverse cardiac effects are a major reason for drug attrition during drug development and for post-approval market withdrawals. Therefore, drug development would greatly benefit from tests that better predict human cardiac function. Due to intrinsic species-to-species differences in the functionality of the heart, nonclinical animal testing does not fully represent the effects of the drugs on human. Thus, there is a need for reliable, human cell -based standardised in vitro models for cardiotoxicity and drug efficacy testing. The aim of this thesis was to develop a functional human cell -based cardiac tissue model that mimics the adult human heart and to develop a contraction force measurement system for measuring the cardiac contractility of the cardiac tissue model. The cardiac tissue model that was optimised in this thesis consisted of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes that are cocultured with preformed human adipose stromal cells (hASC) and human umbilical vein endothelial cells (HUVEC) vascular-like networks. The model was characterized structurally, in gene expression levels, and functionally. For measuring the cardiac contraction force, piezoelectric cantilever sensors with single axis and dual axis sensor designs were developed. The cell culture method was adjusted according to the sensor designs. The functionality of the cardiac tissue model and contraction force measurement technology were confirmed by known inotropic drug exposures. The results show that the coculture with the vascular-like networks improved the cardiomyocyte maturity and the tissue like structure of the model. The cardiomyocytes in the model showed improved organization and morphology, well- developed sarcomeres, and cell-cell connections. The gene expression of the cardiac tissue model also showed characteristics of the adult human heart. The functional characterization with known reference compounds showed that the model had good predictivity with high correlation to human data. The developed piezoelectric contraction force sensors were suitable for measuring the contraction force of cardiac tissue constructs. Both positive and negative inotropic effects including different mechanisms were measurable in the model with the system. In conclusion, the developed cardiac tissue model mimics the myocardium structure and functionality. The model is suitable for testing cardiotoxicity and efficacy of acute drug-induced effects on human heart. Moreover, the functionality of the cardiac tissue model with the developed contraction force measurement system was shown on a proof-of-concept level