Oxygen Transport, Shear Stress, And Metabolism In Perfused Hepatocyte-Seeded Scaffolds With Radial Pore Architecture: Experimental And Computational Analyses

Several modalities have been proposed as treatments or temporary stop-gap for patients suffering from liver failure until a suitable organ is available. However there is still an urgent need for an off-the-shelf device that can accommodate clinically relevant cell numbers, be cultured at physiological oxygen tensions and, can be fully integrated into and heal the injured hepatic space. In this study we investigated the effects that convective and direct oxygenation had on hepatocyte functionality, morphology and viability while cultured in bulk 3D chitosan scaffolds and perfusion bioreactor systems. Cylindrical chitosan scaffolds with radial directed pore structures were fabricated by a thermal gradient directed from the center to the periphery. Capillary-like direct oxygenation was facilitated by embedding gas permeable silicone tubing throughout the scaffold body. Three iterations of bioreactor design and optimization produced a perfusion system that could enable direct oxygenation, accommodate high density hepatocyte seeding (8x10-7 to 1x10-8 cells), ensure adequate mass transfer and induce sustainable metabolic outputs for a least 7 days at a flow rate of 10 ml/min. A computational fluid dynamics model of the internal scaffold pore structure infused with spheroids that resembled hepatocyte aggregates was utilized to understand how varying flow rates (5, 10, 15, 20 and 25 ml/min) effected fluid flow profiles, shear stress imposed on the cells and oxygen consumption within the microenvironment. The results showed that the volumetric flow rate 15 ml/min at the scaffold’s central port inlet produced the best oxygen consumption profile with no damaging effects due to shear stress or eddies flow. The simulation was validated and showed good correlation to empirically derived data. Experimentally the flow rate of 15 ml/min induced the most favorable hepatic response out of the five experimental flow conditions and a static culture (only direct oxygenation). We also looked at how increasing cellular compactness, via reduced scaffold dimensions, would affect phenotypic expression and viability. It was discerned that increasing the cell packing density by 14% increased the rate of albumin and urea production by 79% and 40% respectively. In total the results show that the experimental measures conducted in this study enhanced hepatocyte metabolic performance, viability and morphological appearance

Investigating oxygen transport efficiencies in precision-cut liver slice-based organ-on-a-chip devices

Microfluidic ‘organ-on-a-chip’ devices hold great potential for better mimicking the continuous flow microenvironment experienced by tissue and cells in vivo, thereby ensuring realistic transport of nutrients and elimination of waste products. However, the mass transport of oxygen, which arguably is the most critical nutrient due to its inherently low solubility in water, is rarely assessed. To this aim, the suitability of various precision-cut liver slice (PCLS) microfluidic devices for the defined maintenance of oxygen mass transport were evaluated using COMSOL simulations, leading to the development of a novel, optimised design to provide defined in vivo oxygenation conditions within an organ-on-a-chip system. Simulations found that the proposed device was capable of maintaining 43% of the tissue slice volume within the physiological range of the liver against 18% for the best performing literature device. The optimal device architecture derived from the modelling was then fabricated and its operation confirmed with an LDH assay. These simulation results form the basis for a greater understanding of not just the challenges involved in designing organ-on-a-chip devices, but also highlight issues that would arise from the incorporation of additional organs, as research progresses towards complete human-on-a-chip model systems

Novel Approaches for Enhancing Cell Survival and Function in Vivo

FDA has approved several cell-based therapeutics and hundreds of cell therapy clinical trials are ongoing. Cells will be a significant type of medicine after small molecule and protein drugs. However, several obstacles need to be addressed to achieve the widespread use of cellular therapeutics. The first challenge is the low efficacy of cell transplantation due to low retention, survival, integration, and function of cells in vivo. The second challenge is producing a massive number of cells for clinical treatment with cost-effectively and reproducibly technologies. In this thesis, we proposed and investigated two approaches to address these challenges. To begin with, we engineered two novel biomaterials to deliver cells to enhance their in vivo retention and function. The first biomaterial is a recombinant fibrin matrix which significantly improved cell delivery efficiency and safety. The second biomaterial is a novel γγ’F1:pFN complex fibrin matrix, which enhanced cell culture and improved wound healing. In the second approach, we engineered injectable, microscale, 3D tissues to address the challenges. Brown adipose microtissues were prepared and injected to alleviate obesity and associated type 2 diabetes mellitus(T2DM). In addition, we showed a novel, scalable and cell-friendly cell culture technology (AlgTubes) for scalable microtissue manufacturing. Animal cells were used for preliminary study and can be used for food science to produce cultured meat. This technology has the potential to produce any cell therapy-related cell types in the future. Finally, we also systematically proposed engineering a physiologically relevant microenvironment for large-scale therapeutic cell and microtissue production. Advisors: Yuguo Lei &William H. Velande

HEPATOCYTE POLARITY AND FUNCTION ENHANCEMENT THROUGH SCALABLE COMPACTION

Ph.DPH.D. IN MECHANOBIOLOGY (FOS

Engineering a 3D Novel in vitro Perfusion System for Pancreatic Cancer Research

The Pancreatic Ductal Adenocarcinoma is a malignant aggressive disease corresponding to a low survival rate of 5 years, only between 5 to 7%. This is mainly due to the high complexity and density of the malignant tumour microenvironment which difficult the efficiency of potential treatments and furthermore, to the disease’s progression and inhibition of apoptotic pathways. Over the last years, Tissue Engineering has been gaining an even more prominent role regarding the construction of tri-dimensional (3D) in vitro culture systems that enable a better comprehension of the physicochemical properties and a more realistic recapitulation of the structure of tumoural tissues. In this study, it is described for the first time, the use of a perfusion bioreactor for the culture of pancreatic cancer cells in 3D porous polyurethane scaffolds previously coated with one of the most abundant proteins in the extracellular matrix, the fibronectin. This dynamic culture system allowed a higher proliferation of the tumoural cells as well as a greater cell viability when compared to static culture systems. The addition of a chemotherapy agent also showed a higher resistance by the cells cultured in the bioreactor in addition to a lower percentage of cells in apoptosis. The results obtained suggest the great potentiality of perfusion bioreactors in high throughput studies regarding the in vitro culture of cancer cells for the vascularization mimicry of in vivo systems, including drug and treatment screening for patients detected with pancreatic cancer

Bio-Engineered Pancreas with Human Embryonic Stem Cells and Whole Organ Derived Extracellular Matrix Scaffolds

According to Centers of Disease Control (CDC), 25.8 million Americans were diagnosed with diabetes in 2010, and more than 300 million people were affected worldwide. One potential future treatment for diabetes is transplantation of bioengineered pancreas capable of restoring insulin function. However, bioengineering of the complex pancreas function is a significant engineering feat. It calls for appropriate combinations of cells, with biomaterials that provide structural support and a suitable extracellular environment to maintain cell survival and function in vitro and in vivo. The first objective of this work is to investigate a suitable 3D bioscaffold to support pancreatic cell types. Our result demonstrated that perfusion-decellularization of whole pancreas effectively removes cellular material but retains intricate three-dimensional microarchitecture and crucial extracellular matrix (ECM) components. To mimic pancreatic cell composition, we recellularized the whole pancreas scaffold with acinar and beta cell lines and cultured up to 5 days. Our result showed successful cellular engraftment within the decellularized pancreas, and the resulting graft gave rise to higher insulin gene expression over individual ECM proteins. The second objective of this work is to evaluate the feasibility to repopulate the native organ-derived scaffolds with renewable cell types such as differentiating human pluripotent stem cells (hPSC). We developed an in-house bioreactor to support the regenerative reconstruction of pancreas. Our result demonstrated that hPSCs cultured and differentiated as aggregates are more suitable than the parallel adherent cultures for organ repopulation. Upon continued culture with chemical induction in bioreactor, the seeded PP aggregates grow within the 3D organ scaffolds with homogeneity and mature in situ into monohormonal C-peptide positive cells. The last objective of this work is to evaluate the matrix-specificity of organ-derived ECM. We evaluated this by developing a miniaturized ECM array composed of organ-specific matrices derived from decellularized pancreas, liver and heart. Interestingly, our result showed higher PP cell adhesion and differentiation on liver-ECM over pancreas- and heart-ECM, suggesting that the requirement for ‘like-to-like” basis for tissue engineering approaches may not always be the case. Overall, the findings from this dissertation represent a notable step toward bioengineering of pancreas as an alternative therapeutic solution for diabetes

Development of Hollow Fiber-Based Bioreactor Systems for 3D Dynamic Neuronal Cell Cultures

Adult central nervous system tissue does not retain the ability to regenerate and restore functional tissue lost to disease or trauma. The peripheral nervous system only has the capacity to regenerate when tissue damage is minor. Most in vitro research investigating the neurobiological mechanisms relevant for enhancing nerve regeneration has focused on culture of neuronal cells on a 2D surface under static conditions. We have performed studies enabling development of an advanced in vitro culture model based on hollow fiber-based bioreactors to allow high density neuronal cell networking with directed axonal outgrowth.The model neuronal-like PC12 cell line was initially used to compare neurite outgrowth after nerve growth factor stimulation between cultures under either static or dynamic conditions with 2D or 3D configurations. High density PC12 cell cultures with extensive neurite outgrowth in three dimensional collagen gels were only possible under the dynamically perfused conditions of a hollow fiber-based bioreactor. Analysis of neurite networking within cultures demonstrated enhanced active synapsin I+ synaptic vesicle clustering among PC12 cells cultured within the 3D dynamic bioreactor compared to cells cultured statically on a 2D surface. We further used two different hollow fiber-based bioreactor designs to investigate primary mouse neural stem cell differentiation within different injectable extracellular matrix hydrogel scaffolds cultured under dynamic conditions. HyStem, a cross-linked hyaluronan hydrogel, allowed structure formation with improved neuronal differentiation compared to collagen and Matrigel hydrogels.We have made further developments in order to create a new hollow fiber-based bioreactor device for controlling directed axonal growth. Excimer laser modification was utilized to fabricate hollow fiber scaffolds allowing control over axonal outgrowth from neurons within a 3D space. Incorporation of these scaffolds into a novel hollow fiber-based bioreactor design will produce a device for high density neuronal tissue formation with axonal outgrowth in a 3D configuration. Such a device will provide an advanced research tool for more accurate evaluation of neurobiological events and development of therapeutic strategies useful for enhancing nerve regeneration

Micro/nanofluidic and lab-on-a-chip devices for biomedical applications

Micro/Nanofluidic and lab-on-a-chip devices have been increasingly used in biomedical research [1]. Because of their adaptability, feasibility, and cost-efficiency, these devices can revolutionize the future of preclinical technologies. Furthermore, they allow insights into the performance and toxic effects of responsive drug delivery nanocarriers to be obtained, which consequently allow the shortcomings of two/three-dimensional static cultures and animal testing to be overcome and help to reduce drug development costs and time [2–4]. With the constant advancements in biomedical technology, the development of enhanced microfluidic devices has accelerated, and numerous models have been reported. Given the multidisciplinary of this Special Issue (SI), papers on different subjects were published making a total of 14 contributions, 10 original research papers, and 4 review papers. The review paper of Ko et al. [1] provides a comprehensive overview of the significant advancements in engineered organ-on-a-chip research in a general way while in the review presented by Kanabekova and colleagues [2], a thorough analysis of microphysiological platforms used for modeling liver diseases can be found. To get a summary of the numerical models of microfluidic organ-on-a-chip devices developed in recent years, the review presented by Carvalho et al. [5] can be read. On the other hand, Maia et al. [6] report a systematic review of the diagnosis methods developed for COVID-19, providing an overview of the advancements made since the start of the pandemic. In the following, a brief summary of the research papers published in this SI will be presented, with organs-on-a-chip, microfluidic devices for detection, and device optimization having been identified as the main topics.info:eu-repo/semantics/publishedVersio