1,721,024 research outputs found
Real-time online in situ monitoring and statistical design strategies for haematopoietic stem cell bioprocessing
Get PDFIn vitro erythropoiesis of cord blood haematopoietic stem cells (HSCs) to produce
fully enucleated red blood cells could provide an alternate resource for the erythrocyte.
However, haematopoietic processes are highly complex and dynamic; defining process
requirements to produce reproducible cells of high purity and yield is not an easy task. One
major obstacle is the lack of knowledge in process characteristics. Design of experiments
(DOE) is proposed as a tool to unveil process complexities that exists in HSC cultures.
Characterisation and optimisation of in vitro erythropoiesis as a single-step culture is first
performed via a simple DOE experimental strategy. The optimised DOE culture produced
significantly better results (higher growth and faster maturation) than other single-step
cultures. Subsequently, use of DOE to reveal in vitro process dynamics was attempted. This
study was much more challenging and the repeatability of DOE process models was
compromised in some cases. Process control of HSC culture bioprocesses is required for the
delivery of reliable cell culture products suited for clinical applications. The availability of a
convenient and economical online real-time process monitoring system can provide the
means to translate stem cell culture bioprocesses from the bench-side into manufacturing
production. The design and integration of such a system capable of simultaneous process
monitoring of multiple analytes (ammonia, pH and oxygen) is presented. Operational and
functional stability of this unique online real-time in situ monitoring platform was achieved.
Stability of oxygen and ammonia sensors was achieved for up to three and six days
respectively but biocompatibilities of both sensors require some improvements. Sensors of
pH were biocompatible but their stability in cell culture is required
Mathematical modelling of both chronic Lymphocytic Leukemia and Acute Myeloid Leukemia for their personalized treatment
Get PDFThis work is focused on developing models for two most common leukemia types which affect the different areas of haematopoiesis. These are the Chronic Lymphocytic Leukemia and the Acute Myeloid Leukemia. The first is a slow progressive disease which affects mostly the elderly and progresses in various tissues in the human body, whereas the second has an aggressive nature which progresses in the bone marrow. Our developed physiologically relevant models for both diseases are tested in hypothetical and semi-real patients with or without applied treatment and sensitivity analysis was performed for both. This kind of approach is still on the trial phase and is based mostly on data published in the literature.
In Chronic Lymphocytic Leukemia, two different models were developed. These models approximate the growth and the metastasis of cells within the human body. The former contained time delay differential equations (TDDE) with empirical terms and the latter described the disease with population balance models (PBMs) which use data mostly measurable from experiments. The most critical parameters for both models were identified by sensitivity analysis. The standard treatment protocol in CLL was also applied in a semi-hypothetical patient.
In Acute Myeloid Leukemia, the disease investigation was performed in different patients with multiclonal growth. In this model, the pharmacokinetics and the pharmacodynamics were directly connected because this disease is aggressive. Sensitivity analysis estimated the most important parameters under different treatment doses and protocols. Apart from the cancerous clonal growth, the neutrophil dynamic model was used in order to approximate the neutrophils in the peripheral blood in treated patients. This was seen to be affected by the growth of clones in the bone marrow.
We suggest future efforts should be focussed on incorporating patient biological characteristics, like clonal evolution, clonal interactions and interactions between the clones and their microenvironment. The incorporation of the disease biological characteristics will validate our work, improve the model predictability and achieve efficient treatment protocols minimizing the life-threatening effects that chemotherapy has in patients. Last but not least, developing validated and flexible mathematical models for the most common leukemia types, can be used for drug development purposes that would allow promising and more effective therapies in the future than the standard treatment protocols used nowadays.Open Acces
Mathematical modelling and experimental validation for optimisation and control of mammalian cell culture systems
Get PDFMonoclonal antibodies (mAbs) exhibit remarkable properties that make them suitable for
a wide range of diagnostic and therapeutic applications. The main manufacturing platform used
to produce mAbs is mammalian cell cultures due to their capacity for post-translational
modifications, which are essential for the mAbs functionality (Zhu, 2012). Unfortunately,
mammalian cell cultures present low yield, slow growth, and require expensive medium
components. Model-based techniques could be industrially applied to overcome these
limitations, e.g., implementing model-based optimisation strategies that identify feeding
regimes to maximise mAbs titre in GS-NS0 cultures (Kiparissides et al., 2011). However,
existing feeding strategies depend mainly on glucose and glutamate supply neglecting the
exhaustion of essential amino acids and cell’s energy requirements not only for proliferation
and maintenance but also for mAbs production.
In this work, cell and product compositions, and energy requirements for proliferation,
maintenance and production, have been considered in the development of a novel dynamic
predictive model for GS-NS0 cells producing cB72.3 mAbs, in modified DMEM medium
supplemented with 10% serum or in a serum-free CD-Hybridoma medium. The model
describes growth kinetics, nutrient metabolism, mAbs secretion and the adenosine triphosphate
(ATP) balance based on glucose/amino acids energy metabolic networks, in batch and fed-batch
cultures; and it successfully predicts the number cells, and the concentrations of ATP, glucose,
amino acids and lactate throughout the culture.
The successful coupling of growth kinetics equations and stoichiometric balances, and
the in vitro/in silico approach has enabled us to develop the first dynamic model that predicts
the intracellular ATP content in mammalian cell cultures. Additionally, this experimentally
validated model was utilised to design a tailor-made and low-cost supplemental medium and
implement an optimised fed-batch schedule that maximises the mAbs production and extends
the longevity of the culture. This integrated model-based approach has the potential to be
applied for media development, upstream optimisation and the development of control
strategies for a wide range of biopharmaceutical products.Open Acces
Integrated multi-scale mathematical models for biologics process development
No full textThe therapeutic monoclonal antibody (mAb) market is witnessing an unprecedented growth having nearly duplicated since 2012. Faster regulatory approvals and an increasing number of biosimilars are among the most important mAb market expansion drivers. As the market grows, so does competition making timelines more aggressive. Additionally, political pressure from governments and regulatory authorities over prices of life-saving biological drugs is also increasing. As a consequence, biopharmaceutical companies need to reduce costs while being faster, particularly in having drug substance ready for clinical trials. To this aim, reducing process development times and costs is crucial.
In this thesis, mathematical models integrating several cellular activities important for biomanufacturing are developed. A previously published model development framework is followed to ensure the development of predictive models. An integrated framework combining multivariate data analysis and biomarker identification techniques for cell culture understanding supports the development of predictive mathematical models for two industrially relevant cell lines, GS-NS0 and GS-CHO, capturing cell cycle, metabolism, energy production, mAb production and apoptosis. As the models herein suggested can capture population heterogeneity by describing the cell cycle and gene expression, their applications to bioreactor optimization is envisaged. To this aim, the models developed in this work can be combined with the description of product quality attributes and/or computational fluid dynamics descriptions of bioreactors. These are deemed important applications to accomplish significant reduction in process development time and costs.Open Acces
An integrated experimental and modelling approach for the study of apoptosis in GS-NS0 cell cultures
Get PDFMammalian cell cultures are nowadays adopted as industrial platforms for monoclonal antibody (mAb) manufacture. Although improving product quality and expression stability becomes predominant these days, enhancement in mAb production still remains one of the greatest challenges facing the industrial biotechnology community. One of reasons behind this is the synthesis of mAb is restricted by the decline phase occurring in the mammalian cell cultures as a result of cell death. Prolongation of culture viability via suppression of cell death appears as a potential strategy to improve mAb production and productivity. Understanding of cell death in the mammalian cell cultures is, therefore, crucial to the improvement of the strategy toward mAb production and productivity enhancement. Apoptosis, or programmed cell death, is found to be the major cause of culture viability loss in the mammalian cell cultures. It is a highly-organised physiological process of cell death that can be triggered through activation of the genetic programme underlying a signal cascade that governs particular morphological and physiological changes in response to extracellular and intracellular stimuli. In order to devise strategies to optimise degrees of apoptosis in the mammalian cell cultures, understanding of apoptosis is inevitable. However, to establish the understanding of apoptosis is an extremely challenging task given the complexity arising from the dynamic nature of apoptosis and its association with internal and external milieu, including other cellular processes and environmental conditions. This thesis presents a combined experimental and modelling approach for a study of apoptosis in GS-NS0 cell cultures, aiming to identify relevant metabolic stresses, evaluate their impact on apoptosis induction, and investigate kinetics of apoptosis in response to the identified stresses. The novelty of this study is a systemic view of apoptosis where apoptosis is studied in accordance with dynamic changes in culturing conditions, as well as cell proliferation and metabolic activities. Batch culture experiments of GS-NS0 cell line demonstrate interplay between apoptosis and other cellular processes, including cell cycle progression and metabolism. In addition, the batch cultures allow evaluating impact of metabolic stresses and culture conditions on induction of apoptosis. Fed-batch culture experiments help validate links between apoptosis and metabolic stresses as suggested based on the batch cultures, and also shed some light on feeding strategies for a delay in apoptosis induction. The first-principles mathematical model for apoptosis in the GS-NS0 cell cultures demonstrates a good reproduction of the experimental data. Model analysis reveals model characteristics through global parameter sensitivities and helps guide further model modification toward development of a predictive apoptosis mathematical model.Open Acces
Metabolomics in bone tissue engineering: a sensitive and robust tool for the evaluation of osteogenic differentiation in 2D and 3D cultures
Get PDFThe number of surgical procedures requiring bone grafts is constantly increasing every year. Current grafting strategies have drawbacks such as donor site morbidity, limited availability, high costs, limited efficiency and the transmission of pathogens. Tissue engineering can offer an alternative to current treatments with the production of engineered bone grafts combining biomaterials, stem cells and osteoinductive signals. To date, no single technique exists, that can independently evaluate the quality of mesenchymal stem cell (MSC) osteogenic differentiation in a specific and sensitive manner. Instead, low sensitivity, low specificity and expensive techniques have to be combined often with conflicting results. Evidence shows that MSC metabolism changes during osteogenic differentiation and such changes can be comprehensively examined with the use of metabolomics. Therefore, given the fact that metabolism dynamically receives and responds to both genetic and environmental signals and reflects the cellular phenotype, the application of metabolomics has been assessed as a robust and sensitive technique to evaluate osteogenic differentiaton. This thesis has utilised umbilical cord derived MSCs which are a promising stem cell source for tissue engineering purposes. Metabolomics have been used to study the physiology of undifferentiated MSCs and comprehensively examine MSC metabolic physiology during 2D osteogenic differentiation with two different osteoinductive agents. The results presented herein showed that metabolism of cells differentiated with the most potent agent, closely resembled metabolism of primary osteoblasts. Subsequently, metabolomics was sensitive enough to successfully evaluate the efficiency of novel RGD-functionalised 3D scaffolds to induce osteogenic differentiation. Finally, novel alginate hydrogels have been developed with the glycyl-L-histidyl-L-lysine peptide (GHK) peptide, showing that metabolomics can provide important information during biomaterial development, by indicating the efficiency of GHK hydrogels to promote osteogenesis, assessing the reproducibility of cellular phenotype and assisting in the delineation of mechanisms underlying the function of GHK.Open Acces
In vitro erythropoiesis in a 3D bone marrow biomimicry: reproducing physiologic biochemical and microenvironmental factors involved in red blood cell formation
Get PDFErythropoiesis studies have been hampered by the lack of culture systems that accurately reproduce the features of the bone marrow (BM). Existing knowledge has been obtained from 2D studies which do not reflect the complexity of the BM microenvironment, where haematopoietic stem cells (HSCs) and differentiating erythroid cells are exposed to complex stimuli, including cell-cell and cell-extra cellular matrix interactions and soluble factors. These limitations have been overcome by addition of high concentrations of exogenous cytokines, serum and/or stroma feeder layers, which introduce non physiologic signals to the cells.
In this study, a 3D BM biomimicry was developed, using collagen-coated polyurethane (PU) scaffolds, to expand and differentiate cord blood mononuclear cells (CBMNCs) under serum-free, near-physiologic conditions. This system mimics BM cellular heterogeneity, sustaining complex stromal cell populations and in situ cytokine production. The dynamic evolution of the erythropoiesis was studied with a focus on the role of the hypoxic environment under near-physiologic cytokine concentrations (SCF and EPO only). A combined hypoxia/nomoxia schedule, in this 3D BM biomimicry, was proposed to enable the establishment of erythroid progenitor/precursor populations and a specialized microenvironment, including stroma cells and endogenous cytokine production, and to recreate physiologic erythropoiesis. It was also verified that addition of dexamethasone was not required at any stage of the 3D culture. Furthermore, CBMNC recharge of cultured scaffolds successfully enhanced erythropoiesis with increased production of biconcave shaped enucleated red blood cells (RBCs), simultaneously with the maintenance of a progenitor pool. In parallel, RGD-modification was proposed as a xeno free, defined alternative to collagen coating in PU scaffolds. Preliminary studies using RGD-modified scaffolds did not show a significant improvement of the microenvironment in erythropoiesis when compared with the results of the previously established CBMNCs single cultures.
In summary, this thesis reports the development of a reproducible, robust and near physiologic erythropoiesis model that resembles the physiologic microenvironment and allows for in vitro study of erythropoiesis and sustained RBC production.Open Acces
Recombinant biosynthesis of bacterial cellulose in genetically modified Escherichia coli
No full textBacterial cellulose (BC) exhibits unique properties such as high purity compared to plant-based cellulose; however, commercial production of BC has remained a challenge, primarily due to the strain properties of cellulose-producing bacteria. Herein, we developed a functional and stable BC production system in genetically modified (GM) Escherichia coli by recombinant expression of both the BC synthase operon (bcsABCD) and the upstream operon (cmcax, ccpAx). BC production was achieved in GM HMS174 (DE3) and in GM C41 (DE3) by optimization of the culture temperature (22 degrees C, 30 degrees C, and 37 degrees C) and IPTG concentration. BC biosynthesis was detected much earlier in GM C41 (DE3) cultures (3 h after IPTG induction) than those of Gluconacetobacter hansenii. GM HMS174 (DE3) produced dense fibres having a length of approximately 1000-3000 mu m and a diameter of 10-20 mu m, which were remarkably larger than the fibres of BC typically produced by G. hansenii
Molecular dynamics modelling of skin and hair proteins
Get PDFThe binding free energy is one of the most important and desired thermodynamic properties in simulations of biological systems. The propensity of small molecules binding to macromolecules of human bio-substrates regulates their sub-cellular disposition. This subject is fundamental in transdermal permeation and hair absorption of cosmetic actives. Biomechanical and biophysical properties of hair and skin are related to keratin as their major constituent. A key challenge lies in predicting molecular and thermodynamic basis as the result of small molecules interacting with alpha helical keratin at the molecular level. In addition, elastic properties of human skin which are directly related to the interactions of keratin intermediate filaments remain a challenging subject.
Molecular dynamics (MD) simulations provide a possibility of observing biological processes within atomistic resolution providing more detailed insight into experimental results. However, MD simulations are limited in terms of the achievable time scales. Hence, in this thesis MD simulations were employed in order to provide better understanding of the experimental results conducted in parallel and to overcome the main limiting factor of MD – the simulation time. For this purpose, thermodynamic and detailed structural basis have been delivered for small molecules interacting with keratin explaining and validating experimental data. On the top of this the fast free energy prediction tool has been built within all-atom force field by a use of steered molecular dynamics alone. Within the coarse grain approach, the force field was developed for the application of elastic properties of human skin enabling orders of magnitude faster than all-atom force fields simulations. The application of the coarser representation enabled assessing the influence of the natural moisturizing factor composed of small molecules on the elastic properties of the outermost human skin layer. In this work, MD results reached excellent agreement with the experimental data.Open Acces
Materials design & modification for a three dimensional hollow fibres bioreactor for the production of blood cells
Get PDFOne of the World Health Organization’s biggest concerns is meeting blood demand while ensuring safety and despite the efforts of world blood banks, the gap between supply through donations and demand continues to widen (Nolan, 2017). Hence, a practical and cost-effective alternative to conventional blood donation is essential to meet the demand and reduce patient risks. Previous attempts to recruit blood stem cells to produce blood cells in platforms have achieved limited success specifically in areas of cell-platform interaction, perfusion, and cell harvest. The PhD thesis presented here is aimed at bringing the Bone Marrow (BM) mimicry Bioreactor (BR) developed and patented by BioBlood project closer to physiological representation of the natural human BM niche which hosts blood cells production. This is achieved by focusing on modification and optimization of the synthetic materials used in the bioreactor. Firstly, the polyurethane (PU) scaffold which mimics the BM microenvironment and modulates cell expansion and fate. Secondly, the alumina hollow fibres (HF) representing the vascular system of BM which regulate nutrients and cellular constituents while harvesting mature blood cells. To augment PU bio-functionality and optimize signalling/interaction between cells and scaffold, a novel protocol of RGD surface modification of PU was developed targeting enhancing: cell adhesion, cellular infiltration, and differentiation into blood cell lineages. Adhesion of human umbilical stem cells (hUSC) was improved by more than 85% in RGD-modified PU scaffolds, whereas cell penetration was increased by 4-folds. Alumina hollow fibres’ (HF) structural and filtration characteristics, on the other hand, were improved to support a higher yield and purity of harvested RBC through manipulation and optimization of fabrication parameters. HF improved purity of harvested RBC from 30% to 80% and supported a 1.6 fold increase in cellular density when incorporated in a PU-bioreactor. Combining the two optimized materials in the 3D bioreactor (BR) set-up envisioned to support increased production and selective harvesting of clinically relevant quantities of red blood cells.Open Acces
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