DESIGN AND DEVELOPMENT OF A MICROFLUIDIC DEVICE FOR THE ASSESSMENT OF FIRST-PASS METABOLISM

The aim of the thesis is to develop a microfluidic platform in order to mimic the first pass metabolism of oral ingested compounds. In the first part of the thesis, there is an introduction about the in vivo mechanism involved in the in process of first pass metabolism. First pass metabolism is strictly correlated to oral bioavailability of new developed drugs. The prediction of the dose of drug that reaches the blood flow and the target is fundamental. The organ involved in the first pass metabolism are principally the intestine, where a first metabolic process takes place, and the liver where the quote of drugs is metabolized again. New bioengineered in vitro model to assess first pass metabolism are explained, with a particular attention on 3D intestine and liver model. Furthermore, the first chapter is focused on the recent studies on organ-on-chip device that can recapitulate the in vivo physiology and microenvironment, with the relative steps of fabrications. To achieve the reproduction of the first pass metabolism on chip, we first focussed on the production of an innovative hepatic three dimensional tissues and then on the development of a organotypic intestinal tissues. In the chapter 2 it is presented the comparison of two kind of hepatic 3D model: spheroids and microtissues. The 3D-hepatic model chosen, was cultured into the new developed liver-on-chip device in order to have a perfusion culture. The chapter 3 is focused on the fabrication of an organotypic intestinal 3D tissues cultured in both in static and dynamic conditions. In particular a gut-on-chip microfluidic device was fabricated in order to obtain an air-liquid interface culture. The combination of the two hepatic and intestine model on chip, is addressed in chapter 4. In this last chapter a microfluidic biochip, can accommodate both hepatic microtissues and 3D human intestinal equivalent. By the selective communication of the two tissues recreated into the biochip, it is possible to simulate in vitro the mechanism of orally ingested drugs

Mathematical modelling of a liver hollow fibre bioreactor

A mathematical model has been developed to assist with the development of a hollow fibre bioreactor (HFB) for hepatotoxicity testing of xenobiotics; specifically, to inform the HFB operating set-up, interpret data from HFB outputs and aid in optimizing HFB design to mimic certain hepatic physiological conditions. Additionally, the mathematical model has been used to identify the key HFB and compound parameters that will affect xenobiotic clearance. The analysis of this model has produced novel results that allow the operating set-up to be calculated, and predictions of compound clearance to be generated. The mathematical model predicts the inlet oxygen concentration and volumetric flow rate that gives a physiological oxygen gradient in the HFB to mimic a liver sinusoid. It has also been used to predict the concentration gradients and clearance of a test drug and paradigm hepatotoxin, paracetamol (APAP). The effect of altering the HFB dimensions and fibre properties on APAP clearance under the condition of a physiological oxygen gradient is analysed. These theoretical predictions can be used to design the most appropriate experimental set up and data analysis to quantitatively compare the functionality of cell types that are cultured within the HFB to those in other systems

Development Of Scaffold Architectures And Heterotypic Cell Systems For Hepatocyte Transplantation

In vitro assembly of functional liver tissue is needed to enable the transplantation of tissue-engineered livers. In addition, there is an increasing demand for in vitro models that replicate complex events occurring in the liver. However, tissue engineering of sizable implantable liver systems is currently limited by the difficulty of assembling three dimensional hepatocyte cultures of a useful size, while maintaining full cell viability, an issue which is closely related to the high metabolic rate of hepatocytes. In this study, we first compared two designs of highly porous chitosan-heparin scaffolds seeded with hepatocytes in dynamic perfusion bioreactor systems. The aim was to promote cell seeding efficiency by effectively entrapping 100 million hepatocytes at high density. We found that scaffolds with radially tapering pore architecture had highly efficient cell entrapment that maximized donor hepatocyte utilization, compared to alternate pore structures. Hepatocytes showed higher seeding efficiency and metabolic function when seeded as single cell suspensions as opposed to pre-formed, 100µm aggregates. Seeding efficiency was found to increase with flow rate, with single cell and aggregate suspension exhibiting different optimal flow rates. However, metabolic performance results indicated significant shear damage to cells at high efficiency flow rates. To better maintain hepatocyte basement membrane and cell polarity, spheroid co-cultures with mesenchymal stem cells (MSC) were investigated. Hepatocytes and MSCs were seeded in three different architectures in an effort to optimize the spatial arrangement of the two cell types. MSC co-culture greatly enhanced hepatocyte metabolic function in agitated cultures. Interestingly, the effects of diffusion limitations in spheroid culture, coupled with shear damage and subsequent removal of outer hepatocyte layers produced a defined oscillation of urea production rates in certain co-culture arrangements. A mathematical model of urea synthesis in shear-exposed, co-culture spheroids reproduced the metabolic oscillations observed. This result together with culture observations suggests that MSCs can provide both physiological support and some direct shear protection to hepatocytes in perfused or shear-exposed culture environments. Finally, in order to reduce hepatocyte exposure to excessive shear forces in perfused scaffolds, a modular scaffold design based on polyelectrolyte fiber encapsulation was explored. Scaffolds with uniformly distributed, shear protected cells were achieved

Investigating the effect of a 3D physical microenvironment on hepatocyte structure, function, and adhesion signalling

Presenting cells with a two -dimensional (2D) substrate, as is the case with traditional cell culture, causes them to aberrantly flatten out, and lose their characteristic cell shape. With the case of liver cells, their cuboidal cell shape is vital to cell -specific functions, such as xenobiotic metabolism. Accordingly, culturing hepatocytes in 2D may produce results that do not accurately reflect the behavior of such cells in-vivo. Cells in vivo are in constant contact with the ECM across three dimensions whereas culturing cells in 2D monolayers will alter the geometry of the cell leading to cytoskeletal remodeling and aberrant polarisation. As the cytoskeleton is physically and biochemically linked to the nucleus, this change in cell shape will in turn change the gene expression profile of the cell, leading to differences in cell behaviours such as proliferation, differentiation, and tissue -specific function. Mammalian cells respond to changes in the chemical composition and dimensionality of their microenvironment through complex signalling events at adhesion sites along their membrane. Changes in the microenvironment can result in up/down regulation of integrins, and changes in signalling downstream of adhesion. Using a commercially available highly porous polystyrene scaffold, a method was developed to propagate cells continually in 3D. This model has been used to analyse how long -term growth under 3D conditions affects cytoskeletal organisation and whether adhesion signalling differs between 2D and 3D maintained cells. Cells maintained in 3D show significant cytoskeletal re - organisation and significant changes in cell morphology. 3D maintained cells generally adopt a more physiological morphology than 2D counterparts. These changes are amplified the longer the cells are maintained and propagated in 3D. In addition, these cells show a significant decrease in the phosphorylation of Focal Adhesion Kinase (FAK) and higher levels of α5β1.The differences in morphology and adhesion signaling between 2D and 3D maintained cells appear to lead to enhanced hepatic functionality. Under the conditions tested, 3D maintained HepG2s showed higher drug resistance to model xenobiotics, as well as generally higher levels of albumin, urea and glucose metabolism. 2D and 3D maintained cells also showed different levels of gene expression of key metabolic enzymes. As such, it could be argued that 3D propagation results in cells in vitro more closely reflecting the activity of their counterparts in vivo

Converging organoids and extracellular matrix::New insights into liver cancer biology

Primary liver cancer, consisting primarily of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), is a heterogeneous malignancy with a dismal prognosis, resulting in the third leading cause of cancer mortality worldwide [1, 2]. It is characterized by unique histological features, late-stage diagnosis, a highly variable mutational landscape, and high levels of heterogeneity in biology and etiology [3-5]. Treatment options are limited, with surgical intervention the main curative option, although not available for the majority of patients which are diagnosed in an advanced stage. Major contributing factors to the complexity and limited treatment options are the interactions between primary tumor cells, non-neoplastic stromal and immune cells, and the extracellular matrix (ECM). ECM dysregulation plays a prominent role in multiple facets of liver cancer, including initiation and progression [6, 7]. HCC often develops in already damaged environments containing large areas of inflammation and fibrosis, while CCA is commonly characterized by significant desmoplasia, extensive formation of connective tissue surrounding the tumor [8, 9]. Thus, to gain a better understanding of liver cancer biology, sophisticated in vitro tumor models need to incorporate comprehensively the various aspects that together dictate liver cancer progression. Therefore, the aim of this thesis is to create in vitro liver cancer models through organoid technology approaches, allowing for novel insights into liver cancer biology and, in turn, providing potential avenues for therapeutic testing. To model primary epithelial liver cancer cells, organoid technology is employed in part I. To study and characterize the role of ECM in liver cancer, decellularization of tumor tissue, adjacent liver tissue, and distant metastatic organs (i.e. lung and lymph node) is described, characterized, and combined with organoid technology to create improved tissue engineered models for liver cancer in part II of this thesis. Chapter 1 provides a brief introduction into the concepts of liver cancer, cellular heterogeneity, decellularization and organoid technology. It also explains the rationale behind the work presented in this thesis. In-depth analysis of organoid technology and contrasting it to different in vitro cell culture systems employed for liver cancer modeling is done in chapter 2. Reliable establishment of liver cancer organoids is crucial for advancing translational applications of organoids, such as personalized medicine. Therefore, as described in chapter 3, a multi-center analysis was performed on establishment of liver cancer organoids. This revealed a global establishment efficiency rate of 28.2% (19.3% for hepatocellular carcinoma organoids (HCCO) and 36% for cholangiocarcinoma organoids (CCAO)). Additionally, potential solutions and future perspectives for increasing establishment are provided. Liver cancer organoids consist of solely primary epithelial tumor cells. To engineer an in vitro tumor model with the possibility of immunotherapy testing, CCAO were combined with immune cells in chapter 4. Co-culture of CCAO with peripheral blood mononuclear cells and/or allogenic T cells revealed an effective anti-tumor immune response, with distinct interpatient heterogeneity. These cytotoxic effects were mediated by cell-cell contact and release of soluble factors, albeit indirect killing through soluble factors was only observed in one organoid line. Thus, this model provided a first step towards developing immunotherapy for CCA on an individual patient level. Personalized medicine success is dependent on an organoids ability to recapitulate patient tissue faithfully. Therefore, in chapter 5 a novel organoid system was created in which branching morphogenesis was induced in cholangiocyte and CCA organoids. Branching cholangiocyte organoids self-organized into tubular structures, with high similarity to primary cholangiocytes, based on single-cell sequencing and functionality. Similarly, branching CCAO obtain a different morphology in vitro more similar to primary tumors. Moreover, these branching CCAO have a higher correlation to the transcriptomic profile of patient-paired tumor tissue and an increased drug resistance to gemcitabine and cisplatin, the standard chemotherapy regimen for CCA patients in the clinic. As discussed, CCAO represent the epithelial compartment of CCA. Proliferation, invasion, and metastasis of epithelial tumor cells is highly influenced by the interaction with their cellular and extracellular environment. The remodeling of various properties of the extracellular matrix (ECM), including stiffness, composition, alignment, and integrity, influences tumor progression. In chapter 6 the alterations of the ECM in solid tumors and the translational impact of our increased understanding of these alterations is discussed. The success of ECM-related cancer therapy development requires an intimate understanding of the malignancy-induced changes to the ECM. This principle was applied to liver cancer in chapter 7, whereby through a integrative molecular and mechanical approach the dysregulation of liver cancer ECM was characterized. An optimized agitation-based decellularization protocol was established for primary liver cancer (HCC and CCA) and paired adjacent tissue (HCC-ADJ and CCA-ADJ). Novel malignancy-related ECM protein signatures were found, which were previously overlooked in liver cancer transcriptomic data. Additionally, the mechanical characteristics were probed, which revealed divergent macro- and micro-scale mechanical properties and a higher alignment of collagen in CCA. This study provided a better understanding of ECM alterations during liver cancer as well as a potential scaffold for culture of organoids. This was applied to CCA in chapter 8 by combining decellularized CCA tumor ECM and tumor-free liver ECM with CCAO to study cell-matrix interactions. Culture of CCAO in tumor ECM resulted in a transcriptome closely resembling in vivo patient tumor tissue, and was accompanied by an increase in chemo resistance. In tumor-free liver ECM, devoid of desmoplasia, CCAO initiated a desmoplastic reaction through increased collagen production. If desmoplasia was already present, distinct ECM proteins were produced by the organoids. These were tumor-related proteins associated with poor patient survival. To extend this method of studying cell-matrix interactions to a metastatic setting, lung and lymph node tissue was decellularized and recellularized with CCAO in chapter 9, as these are common locations of metastasis in CCA. Decellularization resulted in removal of cells while preserving ECM structure and protein composition, linked to tissue-specific functioning hallmarks. Recellularization revealed that lung and lymph node ECM induced different gene expression profiles in the organoids, related to cancer stem cell phenotype, cell-ECM integrin binding, and epithelial-to-mesenchymal transition. Furthermore, the metabolic activity of CCAO in lung and lymph node was significantly influenced by the metastatic location, the original characteristics of the patient tumor, and the donor of the target organ. The previously described in vitro tumor models utilized decellularized scaffolds with native structure. Decellularized ECM can also be used for creation of tissue-specific hydrogels through digestion and gelation procedures. These hydrogels were created from both porcine and human livers in chapter 10. The liver ECM-based hydrogels were used to initiate and culture healthy cholangiocyte organoids, which maintained cholangiocyte marker expression, thus providing an alternative for initiation of organoids in BME. Building upon this, in chapter 11 human liver ECM-based extracts were used in combination with a one-step microfluidic encapsulation method to produce size standardized CCAO. The established system can facilitate the reduction of size variability conventionally seen in organoid culture by providing uniform scaffolding. Encapsulated CCAO retained their stem cell phenotype and were amendable to drug screening, showing the feasibility of scalable production of CCAO for throughput drug screening approaches. Lastly, Chapter 12 provides a global discussion and future outlook on tumor tissue engineering strategies for liver cancer, using organoid technology and decellularization. Combining multiple aspects of liver cancer, both cellular and extracellular, with tissue engineering strategies provides advanced tumor models that can delineate fundamental mechanistic insights as well as provide a platform for drug screening approaches.<br/

Bioprinting of human pluripotent stem cells and their directed differentiation into hepatocyte-like cells for the generation of mini-livers in 3D

We report the first investigation into the bioprinting of human induced pluripotent stem cells (hiPSCs), their response to a valve-based printing process as well as their post-printing differentiation into hepatocyte-like cells (HLCs). HLCs differentiated from both hiPSCs and human embryonic stem cells (hESCs) sources were bioprinted and examined for the presence of hepatic markers to further validate the compatibility of the valve-based bioprinting process with fragile cell transfer. Examined cells were positive for nuclear factor 4 alpha and were demonstrated to secrete albumin and have morphology that was also found to be similar to that of hepatocytes. Both hESC and hiPSC lines were tested for post-printing viability and pluripotency and were found to have negligible difference in terms of viability and pluripotency between the printed and non-printed cells. hESC-derived HLCs were 3D printed using alginate hydrogel matrix and tested for viability and albumin secretion during the remaining differentiation and were found to be hepatic in nature. 3D printed with 40-layer of HLC-containing alginate structures reached peak albumin secretion at day 21 of the differentiation protocol. This work demonstrates that the valve-based printing process is gentle enough to print human pluripotent stem cells (hPSCs) (both hESCs and hiPSCs) while either maintaining their pluripotency or directing their differentiation into specific lineages. The ability to bioprint hPSCs will pave the way for producing organs or tissues on demand from patient specific cells which could be used for animal-free drug development and personalized medicine

Mesenchymal stromal cells on bioscaffold for liver bioengineering

Tissue bioengineering is the creation of functional tissues or whole organs by manufacturing body parts ex vivo, seeding cells on a supporting scaffold. The final goal of organ bioengineering is the use of the bioengineered organs as ‘replacement parts’ for the human body. The need for bioengineered livers is significant: currently, the only effective treatment for end-stage liver failure is orthotopic liver transplantation. However, the shortage of organ donors results every year in the death of many patients in the waiting list. Moreover, the advantage of this technology is the use of autologous cells that eliminates the need for post-transplant immunosuppression. In the present study, we decellularized pig livers and then repopulated them with allogeneic porcine mesenchymal stromal cells (pMSCs) to study the interaction between pMSCs and liver specific ECM. The final aim was to understand if ECM can influence and/or promote pMSCs toward differentiation into hepatocytes or hepatocyte-like cells without specific growth factor in culture medium. In our experimental project, porcine livers were obtained by a surgical technique similar to the one used for explant in a human cadaveric donor. Liver samples were cut and then decellularized through agitation with 0.15% SDS. The quality of the decellularization was evaluated both qualitatively and quantitatively, with histological staining and DNA quantification respectively. pMSCs were isolated from the porcine bone marrow (BM) and expanded in vitro. pMSC were characterized by assessment of morphology, proliferation capacity, immunophenotype and their differentiation ability. Then, pMSCs were used for seeding the scaffolds with static culture method. The repopulation of the recellularized scaffold was evaluated at 3, 7, 14 and 21 days after seeding with H&E stain, DAPI, MTT assay and SEM analysis, showing an increase in the cell number with increasing culture days. In order to determinate whether culture on liver ECM-scaffold could promote/address differentiation of pMSC towards hepatocyte, the transcriptional levels of some hepatic genes were tested. In particular, we evaluated six genes (ALB, AFP, HNF4a, Cyp1a1, Cyp7a1 and Krt18) associated to different phases of the hepatic development. A comparison with the expression profile was made with both porcine primary hepatocyte and pMSC. The observations obtained so far allow us to state that: i) our decellularization protocol is effective in the removal of the cells from native liver, respecting the parameters for decellularization without damage the structure of ECM; ii) pMSCs obtained from porcine BM have characteristic phenotypically and functionally comparable to those of their human counterparts and therefore they can be used as a model for experimental studies such as for liver ECM recellularization; iii) the static seeding strategy of pMSCs on the scaffold resulted to be effective in terms of ECM cell attachment, cell proliferation and migration inside the specimen, iv) the genic profile of cells seeded on ECM scaffold without any growth factors is more similar to pMSC suggesting that the only contact with liver specific ECM is not strong enough to induce a complete differentiation in HLCs. Despite this, we observed that Cyp7a1 gene, expressed in hepatocyte but not in MSC, was present in pMSC seeded scaffolds at each time points. In conclusion, we can observe that our results are in accordance with data reported in literature and sustain the possibility to use decellularizated organs as biological scaffold to create functional organs. We believe that our results may provide new insights toward a better understanding of early HLCs development on ECM-scaffolds. However, a more detailed decellularization process, a better cell differentiation capacity and a more detailed understanding of the interaction between cells and ECM could represent crucial steps in the progression of this research field