Process engineering of liver cells for drug testing applications

Dissertação para obtenção do Grau de Doutor em BioengenhariaThe primary culture of human hepatocytes is a requirement in drug development tests. This application is currently hampered by two problems: the limited proliferation of the hepatocytes and the rapid loss of liver-specific phenotype of these cells, when cultured in vitro. This thesis aimed at minimizing this latter issue by cultivating hepatocytes, as spheroids, in fully controlled bioreactors. The state of the art of the primary cultures of hepatocytes is reviewed in Chapter 1, after a brief introduction to the liver physiology the drug development process. The improvement of the bioreactor cultures of hepatocyte spheroids was initially done using freshly isolated rat hepatocytes; the effects of alginate microencapsulation, perfusion culture and their synergy on the maintenance of the hepatocyte spheroids liver-specific phenotype were assessed in Chapters 2 and 3; it was concluded that the perfusion culture and alginateencapsulation had a positive synergic effect on such hepatic phenotype. The perfusion bioreactor developed in Chapter 3 was used in Chapter 4 for the extended culture of freshly isolated human hepatocytes, as spheroids, from three different donors. These cultures responded to repeated dose drug treatments as expected from mature and differentiated hepatocytes, in up to 4 weeks culture time. In Chapter 5, human embryonic stem cell-derived hepatic progenitors were cultured as spheroids and further differentiated into hepatocyte-like cells; the differential expression of hepatic genes between this spheroid population and a monolayer differentiated hepatocyte-like cell population showed a more efficient differentiation under spheroid culture. The bioengineering improvements of this thesis, as well as the future work, were discussed in Chapter 6. This thesis has led to the establishment and validation of primary cultures of hepatocyte spheroids, in perfusion bioreactors, which can be used for long-term, repeated dose tests in drug development

Optimization of primary endothelial culture methods and assessment of cell signaling pathways in the context of inflammation

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012.Cataloged from PDF version of thesis.Includes bibliographical references.Tissue engineering is a potentially valuable tool for clinical treatment of diseases where host tissues or organs need to be replaced. Progression of engineering metabolically complex organs and tissues has been severely limited by the lack of established, functional vasculature. The thesis work described herein focused on methods of establishing and studying specific endothelial cell types in vitro for potential applications in establishing functional microvascular architecture. To achieve these objectives, a model system of primary liver sinusoidal endothelial cells (LSEC) was initially studied due to the high metabolic requirements of the liver, as well as the unique phenotype that they possess. We were able to demonstrate that free fatty acids were able to rescue LSEC in culture, promote proliferation, and maintain their differentiated phenotype. Our work with lipid supplementation in serum-free conditions provides flexibility in engineering liver tissue with a functional vasculature comprised with relevant endothelial types encountered in vivo. Following up our work with LSEC, we explored the human dermal microvascular endothelial cell (HDMVEC) system to understand the signaling mechanisms involved in sprouting angiogenesis. Engineered tissues that are implanted will require integration with host vasculature. We established a method to collect large signaling data sets from a physiologically relevant in vitro culture system of HDMVEC that permitted angiogenic sprouting. We were able to find statistically significant data regarding how angiostatic cues like Platelet Factor 4 can modulate angiogenesis signaling pathways. Our results from working with both types of endothelial cell systems provide insight into potential methods for establishing specialized microvasculature for engineered tissues, both in propagation of differentiated endothelial cells in vitro and promotion of tissue/organ survival following their implantation.by Ta-Chun Hang.Ph.D

A platform to restore intra-tissue flow in live explant assays

Tissue resection during first-line surgery is a standard strategy in the clinic for several life-threatening diseases, such as cancer. In case of malignancy, despite the benefits from surgery, cancer often becomes treatment-resistant and metastasises, limiting therapeutic options and patient survival. Due to tumour heterogeneity, treatment personalisation can improve patient outcomes, however tools based on native tissue samples, used for patient-specific drug screening remain very limited. This is primarily due to the diffusion-limited mass transport in static culture conditions, where tissue viability is rapidly reduced due to ischemia. Our aim is to develop a platform that restores intra-tissue flow through native tissue specimens to prolong their preservation ex vivo. Flow of culture media around tissue specimens has been commonly used for sample preservation. However, the efficacy of most currently available platforms has been limited, as ex vivo specimen perfusion is not facilitated in these technologies. As fluid is allowed to travel around specimen periphery, intra-tissue flow is hydraulically disadvantaged and benefits from culture media renewal only affect cells within 200 μm from explant surface. In this thesis, a novel system is presented that comprises a channel-based device with a suitably-designed constriction to block peri-fusion (i.e. flow around the tissue) and facilitate specimen entrapment and perfusion. Using a syringe pump, device efficacy to facilitate intra-tissue flow was investigated, showing that the induced perfusion occurred through both the vasculature and the interstitium. The effects of perfusion on specimen maintenance and function were also investigated. It was showed that healthy mouse liver and cancerous mouse and human omental specimens were better preserved under perfused conditions in the developed apparatus for 48h. Intra-tissue flow was also effective to inhibit cell metabolism after a 2h-specimen perfusion with a metabolic poison, suggesting this system may have great potential for predictive, live explant assays.Open Acces

Characterizing targeted drug delivery and endothelial cell dynamics using in vitro blood vessel models

Endothelial cells form the inner lining of blood vessels and regulate key blood vessel functions including host defense reactions, vascular smooth muscle tone, angiogenesis, and tissue fluid homeostasis. The occurrence of a pathological condition can lead to inflammation, a protective and tightly regulated response involving host cells, blood vessels and proteins. This process is promoted by circulating cytokines and other chemical mediators such as tumor necrosis factor-alpha (TNF-α), interleukins, thrombin, a few examples. Inflammation can be acute or chronic in nature and is characterized by specific cell receptor expression patterns on the endothelial layer and an increase in endothelial cell-cell gaps. Upregulation of intercellular adhesion molecule-1 (ICAM-1) on endothelial surface occurs during inflammation, and ICAM-1 plays an important role in leukocyte adhesion and recruitment. Understanding the dynamic nature of receptor expression and endothelial cell gap formation during inflammation would provide fundamental physiological information and is also essential for evaluating drug and other biomolecules targeted binding and uptake by endothelial cells.The far reaching accessibility combined with heterogenic behaviour make microvascular endothelium an attractive target for targeted drug delivery. Dynamic and complex processes governing the targeted drug particle binding and distribution on blood microvasculature are still partially understood. Part of this work focuses on the characterization of particle delivery in microcirculation on an ICAM-1 coating based blood vessel mimicking microfluidic device. In microvasculature the vessel size is comparable to that of red blood cells (RBCs) and the existence of blood cells largely influences the dispersion and binding distribution of drug loaded particles. Various factors that influence particle distribution and delivery such as the vessel geometry, shear rate, blood cells, particle size, particle antibody density were considered in this study. Better understanding of the pathologically challenged local endothelial cell layer microenvironment can help us engineer drug carriers decorated with specific biomolecules which can improve the pharmacokinetics and pharmacodynamics of drugs. In this study we also developed a biomimetic blood vessel model by culturing confluent, flow aligned, Endothelial cells in a microfluidic platform, capable of being treated with inflammatory mediators locally. Primary bovine aortic endothelial cells (BAOECs) were grown on semi-permeable membrane with pores that separates an upper and lower channel made of polydimethylsiloxane (PDMS). This dual channel design allowed localized direct TNF-α treatment on the endothelial cell layer leading to expression of surface ICAM-1. This model simulated spatially controlled healthy and pathologically challenged endothelial cells in the same channel and thus has the ability to investigate the microenvironment of locally activated endothelial cells. We characterized endothelial cell culture in this platform and and performed real-time in situ characterization of localized pro-inflammatory endothelial activation. Anti-ICAM-1 coated particles (210 nm and 1 µm diameter) of different antibody coating densities were used as imaging probes and availability of ICAM-1 was probed. This allowed the investigation of spatial resolution and accessibility of ICAM-1 molecules on endothelial cells for targeted binding studies. Anti-ICAM-1 coated particles specifically bound to TNF-α activated BAOECs in an antibody coating density, FSS and particle size dependent manner. F-actin remodelling was also observed in TNF-α treated and downstream sections of the channel. This work has developed a more realistic in vitro vascular model that can independently integrate various factors to effectively mimic a complex physiological endothelial cell microenvironment and has been applied to study endothelial cell microenvironment under localized inflammatory triggering. Inflammatory responses in endothelial cells are characterized by an increase in vascular permeability by formation of intercellular gaps. The biomimetic blood vessel model developed by culturing confluent, flow aligned, and endothelial cells in a microfluidic platform (described prior) was utilized in characterizing the dynamic nature of vascular permeability under inflammation. BAOECs were subjected to in vivo levels of prolonged flow and then treated with thrombin, a serine protease. Thrombin induced profound increase of endothelial cell monolayer permeability in a rapid and reversible way. Endothelial cells cultured in the upper channel were exposed to media mixed with thrombin and a tracer molecule. Tracer molecule samples were collected real time and analysed using spectroscopy, and the dynamic nature of the process was studied. The remodelling of F-actin in BAOECs after thrombin treatment was also characterized for different time points. Better understanding of the dynamics involved in increased vascular permeability can help engineer strategies to enhance targeted drug delivery through these para-cellular gaps

Development of biomedical devices for the extracorporeal real-time monitoring and perfusion of transplant organs

The goal of this Thesis is to develop a range of technologies that could enable a paradigm shift in organ preservation for renal transplantation, transitioning from static cold storage to warm normothermic blood perfusion. This transition could enable the development of novel pre-implantation therapies, and even serve as the foundation for a global donor pool. A low-hæmolysis pump was developed, based on a design first proposed by Nikola Tesla in 1913. Simulations demonstrated the theoretical superiority of this design over existing centrifugal pumps for blood recirculation, and provided insights for future avenues of research into this technology. A miniature, battery-powered, multimodal sensor suite for the in-line monitoring of a blood perfusion circuit was designed and implemented. This was named the ‘SmartPipe’, and proved capable of simultaneously monitoring temperature, pressure and blood oxygen saturations over the biologically-relevant ranges of each modality. Finally, the Thesis details the successful implementation and optimisation of a combined microfluidic and microdialysis system for the real-time quantitation of creatinine in blood or urine through amperometric sensing, to act as a live renal function monitor. The range of detection was 4.3μM – 500μM, with the possibility of extending this in both directions. This work also details and explores a novel methodology for functional monitoring in closed-loop systems which avoids the need for sensor calibration, and potentially overcomes the problems of sensor drift and desensitisation.Open Acces

Modelling and genetic correction of liver genetic diseases

The urea cycle is a set of biochemical reactions that converts highly toxic ammonia into urea for excretion. Deficiencies in any of the genes of the cycle can be life-threatening, with liver transplantation currently being the only definitive treatment. However, the scarcity of donor organs dictates the investigation of alternative treatments, which requires appropriate disease models, in vitro and in vivo, that faithfully recapitulate the disease pathology. Recent advancements in the field of genome engineering make interventions in the genetic code less challenging, thereby assisting in the generation of such tools, as well as raising the potential for genetic correction of these conditions. The research conducted in this thesis centres around two broad aims: the investigation of disease models and genetic correction of inherited liver disorders. Induced pluripotent stem cells (iPSC) hold great potential both for disease modelling and as a source of cells for cell therapy. However, their generation through cell reprogramming is sometimes challenging and inefficient. Therefore, in PAPER I we sought to optimize the reprogramming procedure by introducing modifications to the currently existing protocols, and managed to increase the reprogramming efficiency. IPSC could theoretically differentiate into any cell type, including hepatocytes. In order to assess the level of differentiation of the hepatocyte-like cells (HLC) generated from stem cell sources, comparisons with authentic primary liver tissues are necessary. To this end, in PAPER II we created gene expression profiles of fetal and mature (post-natal) liver tissues from a significant number of individuals. The dataset can serve as an accurate and simple assessment tool to evaluate and compare HLC, generated in different laboratories, to authentic human liver tissues. If HLC resemble the functions observed in mature primary hepatocytes, they could be used as in vitro disease models. In addition, programmable nucleases can be applied to either correct or introduce disease-causing of interest in the genome. In PAPER III, we generated iPSC from a patient with a pathogenic variant in the ornithine transcarbamylase (OTC) gene, the most common UCD, corrected the genetic defect and differentiated the cells into HLC. The correction was molecularly, as well as phenotypically confirmed by the restoration of urea cycle function. The thesis also focuses on the investigation of in vivo disease models of UCD. Specifically, in PAPERS IV and V we created liver-humanized mice with hepatocytes from patients with UCD, OTC deficiency (OTCD) or carbamoyl phosphate synthetase 1 deficiency (CPS1D). Highly repopulated animals faithfully recapitulated the clinical manifestations of the disease observed in patients, including hyperammonemia which is considered a hallmark of these UCD. Furthermore, in PAPER V, we investigated the efficacy and safety of ex vivo gene editing of primary OTCD hepatocytes. Ureagenesis was restored in vitro in edited cells, as well as in vivo as mice liver-repopulated with genetically engineered cells partially or completely reversed all markers of the disease investigated. Finally, extensive gene expression and deep sequencing analysis revealed no unspecific mutagenesis effected by the programmable nucleases, pointing out the safety of the application. In conclusion, the research work conducted in this thesis demonstrates the prospects that iPSC and humanized mice possess for the generation of models of liver genetic diseases, in vitro and in vivo. Furthermore, the emergence of genome editing technologies further enhances the aforementioned potentials, as well as raises possibilities for the treatment of liver genetic defects through genome manipulation

The application of loop mediated isothermal amplification for the detection of the sexually transmitted pathogens Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, and Trichomonas vaginalis, at the point of care.

The purpose of this multi-partnered project was the production of a fully integrated POC system, combining automated nucleic acid extraction in a centrifugally operated microfluidic disk (the LabDisk), with loop mediated isothermal amplification (LAMP) and optical detection, capable of detecting the sexually transmitted pathogens Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium and Trichomonas vaginalis in clinical urine and swab samples. LAMP is a novel nucleic acid amplification method, designed to amplify target nucleic acid in a highly specific and rapid manner, under isothermal conditions. The work detailed in this thesis presents the development of a rapid total nucleic acid extraction process, based on the capture of target nucleic acid by magnetic silica beads, optimised for use on the LabDisk platform. The extraction process was capable of the purification of target nucleic acid from a clinical sample within 5 minutes, and was robust when challenged with a range of inhibitory compounds potentially encountered in samples for STI testing. The system was capable of tolerating N. gonorrhoeae (1 x 105 CFU/ml) urine suspensions containing samples containing 50% total blood volume, 1x108 E. coli cells per ml, and 10mg/ml of BSA, without any effects on the downstream amplification time of the N. gonorrhoeae specific LAMP assay. A freeze dried lysis buffer pellet was developed, that was able to increase the sample volume, thereby decreasing the time to detection, whilst minimising the stored fluid volume on the LabDisk. LAMP assays were designed for the detection N. gonorrhoeae and M. genitalium, and the limits of detection and specificity of the assays were evaluated. The N. gonorrhoeae ORF1 assay was able to detect a minimum of 20 copies of the N. gonorrhoeae genome per reaction, whilst the M. genitalium pdhD assay was capable of detecting 16 genome copies. The tolerance of the ORF1 LAMP assay to urea, and blood, was found to be 1.8M, and 20% reaction volume, respectively. The increased tolerance of the LAMP assay to these inhibitors in comparison to PCR demonstrates the suitability of LAMP when processing urine samples for STI’s. To our knowledge this is the first application of LAMP technology for the detection of these organisms, and the first attempt at commercialising a fully integrated molecular diagnostics system based on LAMP

From Benchtop to Beside: Patient-specific Outcomes Explained by Invitro Experiment

Study: Recent analyses show that females have higher early postoperative (PO) mortality and right ventricular failure (RVF) than males after left ventricular assist device (LVAD) implantation; and that this association is partially mediated by smaller LV size in females. Benchtop experiments allow us to investigate patient-specific (PS) characteristics in a reproducible way given the fact that the PS anatomy and physiology is mimicked accurately. With multiple heart models of varying LV size, we can directly study the individual effects of titrating the LVAD speed and the resulting bi-ventricular volumes, shedding light on the interplay between LV and RV as well as resulting inter-ventricular septum (IVS) positions, which may cause the different outcomes pertaining to sex. Methods: In vitro, we studied the impact of the heart size to IVS position using two smaller and two larger sized PS silicone heart phantoms derived from clinical CT images (Fig. 1A). With ultrasound crystals that were integrated on a placeholder inflow cannula, the IVS position was measured during LV and RV volume changes (dV) mimicking varying ventricular loading states (Fig. 1B). Figure 1 A Two small (blue) and two large PS heart phantoms (orange) on B benchtop. C Median septum curvature results. LVEDD/LVV/RVV: LV enddiastolic diameter/LV and RV volume. Results: Going from small to large dV, at zero curvature, the septum starts to shift towards the left; for smaller hearts at dV = -40 mL and for larger hearts at dV = -50 mL (Fig. 1C). This result indicates that smaller hearts are more prone to an IVS shift to the left than larger hearts. We conclude that smaller LV size may therefore mediate increased early PO LVAD mortality and RVF observed in females compared to males. Novel 3D silicone printing technology enables us to study accurate, PS heart models across a heterogeneous patient population. PS relationships can be studied simultaneously to clinical assessments and support the decision-making prior to LVAD implantation